Symposium Organizers
Jose A. Garrido Technische Universitaet München
Erika Johnston Genzyme
Carsten Werner Leibniz Institute of Polymer Research
Thomas Boland The University of Texas-El Paso
SS1: Bioelectronics
Session Chairs
Monday PM, November 30, 2009
Ballroom A (Hynes)
9:30 AM - **SS1.1
Designing and Characterizing the Bioelectronic Interface.
Andreas Offenhaeusser 1
1 Inst. Bio- & Nanosystems - Bioelectronics (IBN2), Forschungszentrum Jülich, Jülich Germany
Show AbstractThe interfacing of man-made electronics with bio-systems like DNA, redox proteins, enzymes and cells not only allows us to learn about molecular processes in biology, but also paves the way to using it in derived sensory devices. Some of these have already had a profound impact on clinical diagnostics. Technological approaches also can be inspired by biological systems potentially leading to new cognitive and sensory approaches to information processing.Very important for a functional bioelectronic interface is the bio-functionalization of solid surfaces, the detailed characterization of interactions between biosystems and their substrates, as well as the experimental realization of bioelectronic hybrid systems, e.g. biological systems communicating with electronic substrates.The detection of biomolecules for biotechnology and medical diagnostics is subject of intense studies in recent years. For certain applications like the genetic testing at the ‘point of care’ fast, cheap and miniaturised analytical systems are required. Detecting the presence and activity of biomolecules by electronic means is of growing interest due to its potential to simplify and miniaturize biosensors or medical devices. One route to achieve label-free electronic detection of biomolecules is to detect the intrinsic molecular charge of biomolecules by a field-effect transistor.Another aspect of molecular bioelectronics is the development of active bio-inorganic components for the investigation and control of charge transport phenomena in and across biomolecules. Promising biological respectively bio-inorganic hetero-structures shall provide the basis for the development of conceptual electronic and sensing components. We aim in particular for the integration of biomolecules like proteins into electronic junctions.Another effort is directed to the coupling of neurons with electronic devices: the interfacing of neurons with semiconductor devices provides a challenging approach to mimic brain functions by bioelectronic information processing. The possibility of recording the neuronal functions by a transistor element, and eliciting neuronal activity by electrical and neurotransmitter stimuli, represents the two fundamental functions of neuroelectronic devices.The realization of this approach requires several steps: (1) development and fabrication of electronic devices for the low noise registration of cellular signals, (2) development and fabrication of electronic devices for the stimulation of cells, and (3) the effective coupling between the cellular systems and the electronic devices.
10:00 AM - SS1.2
The Development of Silicon Carbide Based Electrode Devices for Central Nervous System Biomedical Implants.
Christopher Frewin 1 2 , Alexandra Oliveros 1 , Christopher Locke 1 , Maj-Linda Selenica 3 , Edwin Weeber 2 3 , Justin Rogers 3 , Stephen Saddow 1 2
1 Electrical Engineering Department, University of South Florida, Tampa, Florida, United States, 2 Florida Center of Excellence for Biomolecular Identification and Targeted Therapeutics (FCoE-BITT), Universtiy of South Florida, Tampa, Florida, United States, 3 Molecular Pharmacology and Physiology Department, Universtiy of South Florida, Tampa, Florida, United States
Show AbstractBrain machine interface (BMI) technology has demonstrated to be a therapeutic solution for assisting people suffering from damage to the central nervous system (CNS), and can provide an interface for robotic prosthetic limb control to assist those who have suffered the loss of an extremity. Although there are non-invasive neural interface techniques available, implantable electrode devices allow direct targeting of specific neurons or neural groups, can interact within real time, and allows two-way communication between the BMI and the CNS. These devices have one major flaw in that they are recognized by glial cells, the support cells that regulate and protect the CNS, as being foreign material which leads to an immune response cascade process called glycosis. Many materials, ranging from hard semiconductors such as silicon to soft polymers like polyimide, have been used to construct microelectrode neural prosthetics, but to date each material has had some negative reaction that prevents this technology from moving into clinical trials.Cubic silicon carbide (3C-SiC) is wide band-gap semiconductor material that may provide an excellent platform for the generation of the neural prosthetic interface component of a BMI system. 3C-SiC offers many advantages that make it ideal for the construction of a planar neural prosthetic. It possesses excellent material strength, better elasticity than other semiconductors, and chemical inertness. We have recently reported on the biocompatibility of 3C-SiC with immortalized cells, and have extended this work by demonstrating neural cell action potential instigation via an electrode type device. Biocompatibility assessment of 3C-SiC was accomplished using in vitro methodology. 96 hour MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assays were performed to determine neural cell viability. Atomic force microscopy (AFM) was used to quantify attached cell morphology and determine lamellipodia/ filopodia interaction with the surface of the semiconductor. It was seen that neurons show excellent viability, cell morphology, and good lamellipodia/ filopodia permissiveness when interacting with 3C-SiC. A neuronal activation device (NAD), based on the planar Michigan microelectrode probe, was constructed from 3C-SiC with the goal of activating an action potential within a neuron. In order to illicit an action potential, neurons were seeded on the NAD device and then they were subjected to a biphasic square pulse signal. Successful action potential activation was recorded through the use of Rhod-2, a Ca2+ sensitive fluorescent dye. Based on the results explained before, 3C-SiC has proven to be an excellent material platform to interact with neurons.
10:15 AM - SS1.3
Electronic Control of Epithelial Cell Gradients using an Electrochemical Transistor.
Magnus Berggren 1 , Karl Svennersten 2 , Maria Bolin 1 , Agneta Richter-Dahlfors 2 , Edwin Jager 1
1 ITN, Linkoping University, Norrkoping Sweden, 2 Neuro, Karolinska Institutet, Stockholm Sweden
Show AbstractThe density and spreading of epithelial cells grown along the surface of conducting polymers is controlled by the electrochemical state expressed at the polymer surface. Here we report electrochemical surface switches and transistors based on poly(3,4-ethylenedioxythiophene) (PEDOT) doped with tosylate. Along the reduced electrode of planar PEDOT surface switches we find healthy cells that adhere and grow to form tight junction tissues, whereas along the oxidized electrodes the cells typically do not adhere at a high density and, if they do so, they quickly enter apoptosis. Along the channel of PEDOT-based electrochemical transistors, potential gradients are generated. The scalar and absolute value of the potential gradient is controlled by the drain and gate voltage, respectively. Epithelial cells were seeded along the channel of such transistors. By changing the gate and drain voltage, we can control the formation of tissue gradients. This is the first time cell systems are grown directly along the channel of a transistor. Thus, the special potential and electrochemical characteristics of a transistor channel has been utilized to regulate tissue gradients entirely by electronic means.
10:30 AM - SS1.4
Fabrication of Low Impedance Nanoporous Gold Electrode Arrays for Neural Interfaces.
Erkin Seker 1 , Yevgeny Berdichevsky 1 , Matthew Begley 2 3 4 , Michael Reed 3 , Kevin Staley 5 , Martin Yarmush 1
1 Center for Engineering in Medicine, Surgery, Massachusetts General Hospital, Harvard Medical School, Shriners Hospitals for Children, Boston, Massachusetts, United States, 2 Mechanical and Aerospace Engineering, University of Virginia, Charlottesville, Virginia, United States, 3 Electrical and Computer Engineering, University of Virginia, Charlottesville, Virginia, United States, 4 Materials Science and Engineering, University of Virginia, Charlottesville, Virginia, United States, 5 Neurology, Harvard Medical School, Boston, Massachusetts, United States
Show AbstractNeural interfaces are essential in the stimulation and recording of neural activity in order to study the nervous system. Traditional neural interfaces are composed of highly conductive and biocompatible microwire electrodes. While microwire electrodes are still in use, the challenge of expanding them to multiple electrode arrays remains. Microfabrication technology alleviated this issue by enabling facile micro-patterning of electrodes and reducing electrode footprint to capture electrical activity of a few neurons. As the size of electrodes decreased, a high signal-to-noise ratio (mostly attained by lowering electrode impedance) became even more important. Impedance is inversely proportional to the electrode surface area, which can be enhanced by nano-textured surfaces, while keeping the electrode footprint the same. Strategies to lower impedance include using porous silicon, dendritic conductive polymers, and most commonly platinum black as the electrode surface coating, where a combination of delamination, reliability, and process integration issues is present. In this paper, we present nanoporous gold (np-Au) as an alternative approach to address these issues. Np-Au can simply be produced by selectively leaching silver from a gold-silver alloy, where gold atoms rearrange themselves into an interconnected open pore network. Some desired properties of this material are large surface area-to-volume ratio, corrosion resistance, high conductivity, and well-studied thiol-based surface chemistry. Using conventional microfabrication techniques, we fabricated 64 highly-adherent np-Au electrodes with 30 μm-diameter electrode tips on microscope slides. Electrodes were created by consecutively sputtering 50 nm-thick chrome and 120 nm-thick gold, followed by simultaneous deposition of gold and silver. By varying the deposition time, we obtained various film thicknesses (~100 nm to 300 nm) to study the effect of increased surface area on impedance. We also evaluated the effect of alloy compositions that led to different porosities. A 2 μm-thick SU8 layer was patterned over the electrodes as an insulator exposing the electrode tips. Impedance measurements across 0.5 to 20 kHz of np-Au electrodes and planar gold control electrodes immersed in artificial cerebrospinal fluid (ACSF) revealed at least an order of magnitude impedance reduction for porous electrodes (i.e., at 1kHz, 900 kΩ impedance for conventional Au electrodes was reduced to 65 kΩ for np-Au electrodes). We further demonstrated the functionality of np-Au electrodes by successfully recording field potentials from rat hippocampal slices cultured on electrodes. This paper presents details of the microfabrication process, preliminary results from electrode impedance measurements, field potential readings from rat hippocampal slices, and electrode porosity analysis. We expect that this technology will facilitate the conventional microfabrication of low-impedance electrodes for neural interfaces.
10:45 AM - SS1.5
Controlling Neuronal Growth and Synaptic Connections on Au Surfaces by Directed Assembly of Extracellular Matrix Proteins.
Cristian Staii 1 , Chris Viesselman 2 , Jason Ballweg 2 , Steven Hart 4 , Justin Williams 3 , Erik Dent 2 , Susan Coppersmith 4 , Mark Eriksson 4
1 Physics and Astronomy, Tufts University, Medford, Massachusetts, United States, 2 Anatomy, University of Wisconsin-Madison, Madison, Wisconsin, United States, 4 Physics, University of Wisconsin-Madison, Madison, Wisconsin, United States, 3 Biomedical Engineering, University of Wisconsin-Madison, Madison, Wisconsin, United States
Show AbstractStudying how individual neuronal cells grow and interact with each other is of fundamental importance for understanding the functions of the nervous system. However, the mechanism of axonal navigation to their target region and their specific interactions with guidance factors such as membrane-bound proteins, chemical and temperature gradients, mechanical guidance cues, etc. are not well understood. Here we describe a new approach for controlling the adhesion, growth and interconnectivity of cortical neurons on Au surfaces. Specifically, we use Atomic Force Microscopy (AFM) nanolithography to immobilize extracellular matrix proteins at well-defined locations on Au surfaces. These surface-immobilized proteins act as a) adhesion proteins for neuronal cells (i.e. well-defined locations where the cells “stick” to the surface), and b) promoters/inhibitors for the growth of neurites. Our results show that protein patterns can be used to confine neuronal cells and to control their growth and interconnectivity on Au surfaces (1). We also show that AFM nanolithography presents unique advantages for this type of work: 1) high degree of control over location and shape of the protein patterns, 2) the procedure is carried out in aqueous solutions (protein buffers), such that the proteins are very likely to retain their folding conformation/bioactivity, and 3) minimum protein feature size can be reduced down to several tens of nm (typically ~50nm).(1) C. Staii et. al, Biomaterials 30,3397-3404(2009).
11:30 AM - **SS1.6
3D, Electrically Controlled, Nanostructured Polymeric Surfaces for Biosensing, Drug Delivery and Cell-Sheet Engineering.
Janos Voros 1
1 Laboratory of Biosensors and Bioelectronics, ETH Zurich, Zurich Switzerland
Show AbstractMicro- and nanotechnology brought new exciting possibilities in life sciences. In this talk, novel approaches using three dimensional structures that consist of nanoobjects and nanolayers will be presented. A common property of these 3D films is that they consist of molecules that respond to changes in their electrochemical environment. Also the application potential of the presented dynamic biointerfaces will be discussed: A key challenge in high-throughput screening is to make the methods compatible with membrane proteins. Membrane proteins are fragile and difficult to handle, but also highly important drug targets. The combination of microfabrication, and DNA-assisted self-assembly enabled the arraying of functional biomembranes opening the possibility for their high-throughput screening.[1,2] In addition, three dimensional multilayers of vesicles can be created via zirconium ion mediated self-assembly for biosensing applications. Furthermore, nanopore supported, polyelectrolyte multilayer enforced lipid bilayers serve as a new platform for ion-channel monitoring with the hope to achieve the stability and reproducibility required by the high-throughput screening application.Materials that can response to external electrical stimuli are interesting for a variety of biomedical applications: We have developed electrochemically active polyelectrolyte multilayers that can act as nanoactuators using low potentials in a liquid environment.[3] Multilayers can also be used as a sacrificial film for the harvesting of intact cell-sheets. This enables the creation of controlled 3D cell cultures that are expected to better represent the in vivo situation for e.g. drug testing experiments.[4] Furthermore, the combination of such electro-active multilayers with lipidic vesicles enables the local delivery of drugs for novel dynamic cell culture experiments.References[1] M. Bally, K. Bailey, W. Leifert, J. Vörös, T. McMurchie; G-Protein Coupled Receptor array technologies: Site Directed Immobilisation of liposomes containing the H1-histamine or M2-muscarinic receptors; Proteomics, 2009. (In press) [2] A. Binkert, P. Studer, J. Vörös; A microwell array platform for picoliter membrane protein assays; Small, 2009. (In press) [3] D. Grieshaber, J. Vörös, T. Zambelli, V. Ball, P. Schaaf, J.C. Voegel, F. Boulmedais; Swelling and Contraction of Ferrocyanide Containing Polyelectrolyte Multilayers upon Application of a Potential; Langmuir, 24(23), 13668-13676, 2008.[4] O. Guillaume-Gentil, Y. Akiyama, M. Schuler, C. Tang, M. Textor, M. Yamato, T. Okano, J. Vörös; Polyelectrolyte coatings with a potential for electronic control and cell sheet engineering. Advanced Materials, 20 (3); 560-565, 2008.
12:00 PM - SS1.7
In-situ Optical and Electrochemical Characterizations of S. oneidensis at Single-Cell Level: A ``Bottom-Up” Approach for Microbial Fuel Cell Studies.
Xiaocheng Jiang 1 , Jinsong Hu 1 , Charles Lieber 1 2 , Justin Biffinger 3 , Lisa Fitzgerald 3 , Bradley Ringeisen 3
1 Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts, United States, 2 School of Engineering and Applied Science, Harvard University, Cambridge, Massachusetts, United States, 3 Chemistry Division, US Naval Research Laboratory, Washington, District of Columbia, United States
Show AbstractMicrobial fuel cells (MFC) are a promising technology for sustainable energy production. While significant improvement of current and power density has been achieved in recent years, even under the best laboratory conditions the power densities remain very low (<500 μW/cm3). Additionally, the fundamental factors and limits determining charge transport and power extraction still remain unclear. Here, we report one approach to investigate these problems from the basic level (“bottom up”), which exploits in-situ optical imaging and electrochemical measurement to monitor and characterize individual cell behavior of Shewanella oneidensis MR-1 interacting with arrays of nanoelectrodes. Well-defined, optically-transparent nanoelectrode chips were developed via lithographic methods; coupled with our specifically designed polydimethylsiloxane static/flow chambers and high-resolution phase-contrast microscopy, we were able to perform highly controlled electrochemical measurement while simultaneously resolving each cell on different electrodes. Short-circuit current was recorded in real time during cell adhesion to and conditioning of electrodes by individual MR-1 cells. These experiments represent the first measurement of how much current a single electrochemically active bacterium can produce. The effects of culture/measurement condition and electrode surface modification on electron transfer between cell and electrode were also examined and discussed. The results from these studies provide an alternative insight for the intimate cell-electrode interaction in MFCs and are expected to advance significantly our fundamental knowledge of key factors affecting power extraction from MR-1 and other cellular systems.
12:15 PM - SS1.8
A 1.5-Microliter Microbial Fuel Cell For On-chip Bioelectricity Generation.
Fang Qian 1 , Mary Baum 1 , Qian Gu 1 , Daniel Morse 1 2 3
1 , Institute of Collaborative Biology, University of Califonia, Santa Barbara, California, United States, 2 , California NanoSystems Institute, University of California, Sanra Barbara, California, United States, 3 , Department of Molecular, Cellular, and Developmental Biology, University of Califonia, Sanra Barbara, California, United States
Show AbstractMicrobial fuel cells (MFCs) offer the possibility of harvesting electricity from organic substrates and renewable biomass through catalytic conversion by microorganisms. Continued progress towards high-performance portable MFCs will require efforts in optimization of small-scale devices, as well as a better understanding of bacteria/anode coupling. In this regard, we developed a miniaturized dual-chamber MFC that allows on-chip bacterial culture and harvest of bioelectricity. The MFC contains a well-defined 1.5 microliter anode chamber and 4 microliter cathode chamber, with provision for microfluidic deliveries of growth medium and catholyte. Different anode materials including gold and carbon were incorporated for bacterial growth and electron coupling. After inoculation of electrogenic Shewanella oneidensis strain MR-1, current generation was observed on an external load for up to two weeks, dependent on periodic replenishment of organic substrates. A maximum current density of 1300 A/m3 and power density of 15 W/m3 were achieved using a flat gold anode. Electron microscopic studies confirmed large-scale, uniform biofilm growth on the anodes, and suggested that the small chamber volume led to enhanced cell/anode interaction. Our result demonstrates a versatile platform for studying the electron transfer at the interface of electrogenic cells and a variety of anode materials in MFCs, and suggests the possibility of powering nanodevices using on-chip bioenergy.
12:30 PM - SS1.9
Interfacial Energetics of Photosynthetic Reaction Centers on Wide Bandgap Semiconductors.
S. Schoell 1 , J. Howgate 1 , M. Hoeb 1 , G. Hartwich 2 , M. Brandt 1 , M. Stutzmann 1 , Ian Sharp 1
1 , Walter Schottky Institut, Technische Universität München, Garching Germany, 2 , FRIZ Biochem GmbH, Neuried Germany
Show AbstractPhotosynthetic reaction centers (RCs) are highly efficient for light-to-energy conversion with an internal quantum yield of nearly 100%. Understanding charge transfer processes in organic-inorganic heterostructures composed of photosynthetic reaction center proteins on semiconductor substrates is of fundamental interest for design and fabrication of future electro-optical bio-inorganic devices. Additionally, RCs can enhance the photoelectronic response of semiconductors when immobilized on transistors, enabling the development of organic-inorganic photosensors. Wide band gap semiconductors such as GaN, AlGaN compounds, and SiC are promising substrate materials for studies of the interfacial electronic properties because the bulk Fermi level can be varied over a wide energetic window by doping. Here, we investigate the electronic properties of immobilized RCs isolated from rhodobacter sphaeroides on hydroxylated (0001) 6H-SiC and GaN. Kelvin probe measurements on RC-modified surfaces reveal an induced photovoltage of ~0.4 V on 6H-SiC and ~0.24 V on GaN upon illumination with near infrared light, indicating oriented attachment of the RCs as well as retained activity following immobilization on the semiconductor surfaces. The net charge separation arising from selective RC orientation is further exploited for optical tuning of AlGaN/GaN high electron mobility transistor characteristics. Photo-electrochemical measurements were performed to quantify energetic alignment and charge transfer levels at the hybrid interface. We thus show that SiC and GaN are useful substrate materials for fundamental studies of charge transfer processes at the organic-inorganic interface as well as promising materials for future applications in the field of bio-molecular electronics.
12:45 PM - SS1.10
Biosensor for Dielectric Spectroscopy of Mitochondria and for Monitoring Ion Activities.
Divya Padmaraj 1 2 , Rohit Pande 1 , Wanda Zagozdzon-Wosik 1 , Lei-Ming Xie 2 , Dorota Pijanowska 4 , John Miller 3 , Piotr Grabiec 5 , Bohdan Jaroszewicz 5 , Jarek Wosik 1 2
1 Electrical and Computer Engineering Department, University of Houston, Houston, Texas, United States, 2 Texas Center for Superconductivity, University of Houston, Houston, Texas, United States, 4 Institute of biocybernetics and biomedical engineering, Polish academy of sciences, Warsaw Poland, 3 Physics Department, University of Houston, Houston, Texas, United States, 5 , Institute of electron technology, Warsaw Poland
Show AbstractWe developed a BioMEMS device to study cell- mitochondrial physiological functionalities. The pathogenesis of many diseases including obesity, diabetes, heart failure as well as aging has been linked to functional defects of mitochondria. This is understandable as the mitochondria produces up to 90% of ATP, and plays a critical role in cell signaling and apoptosis. The synthesis of ATP is determined by the electrical potential across the inner mitochondrial membrane (IMM) and by the pH difference due to proton flux across it. Therefore, electrical characterization by E-fields with complementary chemical testing was used here. Mitochondrial ion channels present in the IMM control specific ion fluxes, and maintain ion homeostasis, matrix volume, IMM potential etc and thus serve a central role in cell growth and death related processes. Defects in ion channels (Channelopathies) are being attributed to many diseases like cancer, neurodegeneration, etc. Complete physiological roles of various ion channels and their interactions are still unknown, hindering the development of targeted therapeutic agents. The BioMEMS device was fabricated as an SU-8 based microfluidic system with gold electrodes on SiO2/Si wafers for electromagnetic interrogation. Ion Sensitive Field Effect Transistors (ISFETs) were incorporated for proton studies important in electron transport chain, together with monitoring Na+, K+, Ca++ ions for ion channel studies. ISFETs are chemically sensitive MOSFET devices, their threshold voltage is directly proportional to the electrolytic H+ ion variation. These ISFETs (sensitivity ~55 mV/pH for H+) were further realized as specific ion sensitive CHEMFETs by depositing a poly-HEMA layer sandwiched between the gate and a final specific ion sensitive membrane. Electrodes for dielectric spectroscopy studies of mitochondria were designed as 2- and 4-probe structures for optimized operation over a wide frequency range. In addition, to limit polarization effects (which masks actual impedance for high conductivity solutions at low frequencies), a 4-electrode set-up with unique meshed pickup electrodes (7.5x7.5 µm2 loops with 4 µm wires) was fabricated. An electrical model was developed for the mitochondrial sample, and its frequency response correlated with impedance spectroscopy experiments of sarcolemmal mitochondria. Using the mesh electrode structure, we obtained a reduction of 83.28% in impedance at 200 Hz. COMSOL simulations of selected electrical structures in this sensor were compared with experimental results to better understand the physical system. The simultaneous measurement of membrane potential, ion concentrations and pH would enhance diagnostics and studies of mitochondrial diseases.
SS2: Biosensing Technologies
Session Chairs
Monday PM, November 30, 2009
Ballroom A (Hynes)
2:30 PM - **SS2.1
Biosurface Science.
George Whitesides 1
1 Department of Chemistry & Chemical Biology, Harvard University, Cambridge, Massachusetts, United States
Show AbstractThe interface between synthetic and biological systems is an important component of functional biomaterials. Self-assembled monolayers (SAMs), combined with microfluidics, provide a set of technologies that is uniquely suited for studying the interactions of synthetic surfaces with biomolecules, cells, and organisms. SAMs provide methods of tailoring the composition and properties of the surfaces perpendicular to the plane of the supporting gold film; soft lithography makes it possible to pattern the film in the plane; surface plasmon resonance and quartz crystal microbalance are very useful in measuring interactions with the surface; electrochemistry is useful to modify these surfaces; microfluidics makes it possible to “engineer” the composition of flowing buffer. This talk will describe current work involving the engineering of surfaces and microsystems to provide tools for biology, with a particular emphasis on tools for cell biology.
3:00 PM - SS2.2
Layer-by-Layer Assembly for Increased Sustainability of Cell-Based Sensors.
Nancy Kelley-Loughnane 1 2 , Svetlana Harbaugh 3 , Molly Davidson 2 , Latha Narayanan 3 , Carter East 1 , Jorge Chavez 1 , Morley Stone 1
1 711th Human Performance Wing/RHPB, Air Force Research Laboratory, Wright Patterson Air Force Base, Ohio, United States, 2 , UES, Inc., Dayton, Ohio, United States, 3 , Henry Jackson Foundation, Dayton, Ohio, United States
Show AbstractRiboswitches are regulatory RNAs that are located at the 5'-untranslated region of selective mRNAs. These genetic control elements recognize and bind small molecules, and in turn, regulate gene expression. Application of synthetic riboswitches in cells provide detection systems that are self-replicating, self-powering, and posses internal signal amplification. We have selected as our model a theophylline-responsive riboswitch that was placed upstream of the gene encoding a new green fluorescent protein from Amphioxus (GFPa1). In order to build a cell-based sensor and to enhance cell viability, we have encapsulated E. coli cells harboring the riboswitch construct. We chose a layer-by-layer (LbL) technique based on the assembly of composite layers. We absorbed alternating layers of positively and negatively charged polyelectrolytes onto naturally occurring charged cell surfaces. Zeta potential measurements indicated successful LbL-deposition which included a full reversal of the surface charge upon addition of four layers of polyelectrolytes- creating two bilayers. Cell viability and fluorescence studies confirmed that encapsulated cells were both viable and functional. Encapsulated cells harboring the riboswitch showed a marked increase in fluorescence intensity due to GFPa1 expression in the presence of theophylline. Differences in the surface structure between uncoated and encapsulated cells were visualized in air and in fluid using atomic force microscopy. Current studies focus on the immobilization on a glass surface structured by microcontact printing to facilitate the use of encapsulated cells harboring riboswitch in biosentinel applications. Encapsulation of living cells harboring genetic control elements is a promising approach to create stable and controllable cell-based biosensors.
3:15 PM - SS2.3
Biomimetic Metallic Electrodes for Intracellular Electrical Measurements.
Piyush Verma 1 , Nick Melosh 1
1 Materials Science and Engineering, Stanford University, Stanford, California, United States
Show AbstractInterfacing living matter to electronics with the ability to monitor and deliver spatio-temporal signals to cells or cell networks is promising for various fundamental biophysical studies and also for applications such as high resolution neural prosthetics, on-chip electrically addressed artificial neural networks and arrayed on chip patch-clamps. Developing an inorganic nanostructure that can specifically and non-destructively incorporate into biological membranes is the key to such an interface. We report an approach towards this interface by functionalizing a nanoscale metallic post to mimic a transmembrane protein to directly insert into the lipid membrane and form a tight seal. These post-electrodes were formed by evaporation and lift-off onto conductive bottom electrodes, with 5-10 nm thick hydrophobic bands around the edge of the post formed by molecular self assembly. We recently reported AFM measurements of these posts inserting into lipid bilayers and showed that different molecular functionalizations adhered within the hydrophobic lipid core with different strengths depending on their molecular mobility. Here we describe nanoscale electrical measurements with these post-electrodes on red blood cells to determine the leakage current at the electrode-membrane interface.
3:30 PM - SS2.4
Improved Limits of Detection for Localized Surface Plasmon Resonance Sensors by Directed Binding Exclusively to the Most Sensitive Regions.
Laurent Feuz 1 , Peter Joensson 1 , Magnus Jonsson 1 , Fredrik Hoeoek 1
1 Department of Applied Physics, Chalmers University of Technology, SE-41296 Gothenburg Sweden
Show AbstractWe present a Au/TiO2- based localized surface plasmon resonance (LSPR) sensor enabling material specific surface chemistries to control analyte binding exclusively to the most sensitive Au regions of the sensor. The so obtained selective capturing increases the initial response of the sensor by a factor of around five for low (nM) analyte concentrations compared to the case when molecules are allowed to bind on the entire substrate (both Au and TiO2). The highly localized nanoplasmonic activity was obtained by combining colloidal lithography and dry-etching of TiO2-Au-TiO2 coated glass slides. In this way, short range ordered nanoholes with diameters of around 100 nm and depths of around 50 nm were obtained. Furthermore, both the top surface and the bottom of the holes consist of TiO2, while there is a 40 nm high ring of Au exposed within the holes – the latter being the most sensitive region of the sensor. The key issue for exclusive analyte binding to the Au regions at the walls of the holes is selective surface chemistry. The purpose of the selective surface chemistry is to (i) prevent non-specific adsorption of molecules to the insensitive part of the sensor (TiO2), and (ii) promote selective binding of target molecules to the sensitive regions of the sensor (Au). This was achieved by first densely grafting Au-specific thiolated poly(ethylene glycol) (PEG) chains on Au followed by exposure of poly(L-lysine)-graft-PEG, which under these conditions binds solely to TiO2. By including biotin endgroups to either one of the PEG-based systems, either Au, TiO2 or both can be turned into the bioactive area. We show that for low concentration (nM) NeutrAvidin solutions, adsorption occurs in the mass-transport limited regime. This, in turn, leads to higher adsorption rates and significantly lower detection limits for selective binding to the Au regions compared to binding to the whole surface (both Au and TiO2). We attribute this to molecules being effectively repelled from the insensitive areas of the sensor, leading to more efficient diffusion towards the sensitive biomolecular-capturing regions of the sensor. This conclusion was supported by finite element simulations, demonstrating excellent agreement with the experimental observations. In summary, these results demonstrate the paramount importance of having control over the surface chemistry to reach low limits of detection using nanoscale biosensor elements.
3:45 PM - SS2.5
Multiscale Nanoporous Structures for Sensing and Diagnostics.
Shalini Prasad 1 , Manish Bothara 2
1 EE, Arizona State University, Tempe, Arizona, United States, 2 , Portland State University, Portland, Oregon, United States
Show AbstractCurrent trends in sensing and diagnostics is towards developing hybrid devices that incorporate nanomaterial for enhancing device performance. These devices and systems have a broad impact ranging from personalized medicine in health care, environmental sensing and building multifunctional sensors for military applications. The overarching objective of the research work is to develop a new class of portable, bio-analytical tools with improved functionality and performance capabilities by utilizing the electrical effects on cellular and sub cellular species in micro and nanoscale domains. There are two key ideas underlying this research work. The first is to design and manufacture structures comprising of nanoscale-confined spaces integrated on to multi-scale architecture platforms. This model architecture has been engineered to harness the principle of macromolecular crowding for biomolecule binding and detection by monitoring perturbations in the electrical bi-layer in tailored nanoscale confined spaces. Enhanced performance metrics in biomolecule detection have been demonstrated in developing electrical immunoassays. We have demonstrated picogram/ml sensitivity in detection of specific cardiovascular disease biomarkers, cancer biomarkers from human serum samples with a dynamic range of response varying from pg/ml to mg/ml and response time within 120 seconds.
4:30 PM - **SS2.6
Supramolecular Bio-Functional Interfacial Architectures.
Wolfgang Knoll 1
1 , Austrian Institute of Technology, Vienna Austria
Show AbstractThis presentation summarizes some of our efforts in designing, synthesizing, structurally and dynamically characterizing, and functionally optimizing bio-inspired interfacial architectures. We explore different materials including synthetic polymer brushes and hydrogels, mono- and multilayers from proteins and DNA, and lipid bilayer membranes. The experimental methods employed for a detailed investigation of the structure/function relationship of these biomaterials include, in particular, evanescent wave techniques based on surface plasmons and optical waveguide modes, as well as electrochemical techniques.
5:00 PM - SS2.7
Nanomonitor Technology for Glycosylation Analysis.
Manish Bothara 1 , Vinay Nagraj 2 , Seron Eaton 2 , Srivatsa Aithal 3 , Gaurav Chatterjee 3 , Peter Wiktor 2 , Shalini Prasad 3
1 Electrical and Computer Engineering, Portland State University, Portland, Oregon, United States, 2 Center for Bioelectronics and Biosensors, The Biodesign Institute at Arizona State University, Tempe, Arizona, United States, 3 Electrical Engineering, Arizona State University, Tempe, Arizona, United States
Show AbstractChanges in protein glycosylation have great potential as markers for the early diagnosis of cancer and other diseases. The current analytical tools for the analysis of glycan structures need expensive instrumentation, advanced expertise, is time consuming and therefore not practical for routine screening of glycan biomarkers from human samples in a clinical setting.We are developing a novel ultrasensitive diagnostic platform called ‘NanoMonitor’ to enable rapid label-free glycosylation analysis. The technology is based on electrochemical impedance spectroscopy where capacitance changes are measured at the electrical double layer interface as a result of interaction of two molecules.The NanoMonitor platform consists of a printed circuit board with array of electrodes forming multiple sensor spots. Each sensor spot is overlaid with a nanoporous alumina membrane that forms a high density of nanowells. Lectins, proteins that bind to and recognize specific glycan structures, are conjugated to the surface of nanowells. When specific glycoproteins from a test sample bind to lectins in the nanowells, it produces a perturbation to the electrical double layer at the solid/liquid interface at the base of each nanowell. This perturbation results in a change in the impedance of the double layer which is dominated by the capacitance changes within the electrical double layer. The nanoscale confinement or crowding of biological macromolecules within the nanowells is likely to enhance signals from the interaction of glycoproteins with the lectins leading to a high sensitivity of detection with the NanoMonitor as compared to other electrochemical techniques. Using a panel of lectins, we were able to detect subtle changes in the glycosylation of fetuin protein as well as differentiate glycoproteins from normal versus cancerous cells. Our results indicate that NanoMonitor can be used as a cost-effective miniature electronic biosensor for the detection of glycan biomarkers.
5:15 PM - SS2.8
Functional Branched Monolayers on Hydrogen Terminated Silicon Surfaces for Biosensing Applications.
Trevor Mischki 1 , Olga Mozenson 1 , Danial Wayner 1 , Greg Lopinski 1
1 Steacie Institute for Molecular Sciences , National Research Council Canada, Ottawa, Ontario, Canada
Show AbstractFunctional organic monolayers formed directly on hydrogen terminated silicon surfaces are promising platforms for biosensing applications, offering significant advantages compared to conventional functionalization strategies involving silanization of oxide covered surfaces. In contrast to the Si-O-Si link the Si-C bond is stable over a wider range of conditions. For electrically based biosensors based on field effect transistors (BioFETs), the direct functionalization approach is also expected to reduce the large undesirable pH response that reduces the sensitivity of oxide based devices [1]. The challenge is to develop approaches for the stable, controlled immobilization of biomolecules (including inhibition of non-specific binding) while maintaining a low density of electrically active defects at the silicon-monolayer interface. Functional monolayers produced by photochemical or thermal reactions of undecylinic acid/decene mixtures have been used to successfully immobilize biomolecules and shown to function as reasonable gate dielectrics in BioFET devices exhibiting low threshold voltages, minimal leakage and reduced pH dependence (~10mV/pH unit) as compared with ~30mV/pH unit typically observed on functionalized oxide surfaces. However, the density of alkyl chains in these monolayers is insufficient to prevent oxidation of the silicon substrate for extended exposure in aqueous buffers. Previously we have demonstrated that branched structures formed by reaction of alkyl Grignards with undecylinic ethyl ester monolayers exhibit enhanced stability in aqueous buffers [2]. However, these initial branched monolayers were methyl terminated, lacking a synthetic handle to enable the attachment of biomolecules. Utilizing functional groups compatible with Grignards such as alkenes or protected alcohols facilitates the preparation of reactive (N-hydroxysuccinimide (NHS), maleimide or isocyanate surfaces). XPS and surface photovoltage measurements indicate minimal increase in oxidation and electrically active defects upon extended exposure to water. Using the protected alcohol-isocyanate route, patterned mixed monolayers containing varying compositions of polyethylene glycol (PEG) and PEG-biotin have been used to demonstrate specific immobilization of avidin and streptavidin. Maps of the surface potential as measured with a scanning Kelvin probe are shown to correlate well with fluorescence images. Another approach, involving oxidation and NHS activation of alkene terminated monolayers, has been used for immobilization of oligonucleotides. Electrochemical impedance measurements illustrating the use of these branched functional monolayers in biosensing applications will also be presented. [1] D. Landheer, G. Aers, W.R. McKinnon, M.J. Deen, and J.C. Ranuarez,J. Appl. Phys., 98, 044701 (2005). [2] X. Bin, T.K.Mischki, C. Fan, G.P. Lopinski and D.D.M. Wayner, J. Phys. Chem. C, 111, 13547 (2007)
5:30 PM - SS2.9
Hydroxyalkylphosphonate Self-Assembled Monolayers as Interface Systems for Label-free DNA Detection and for Applications in DNA Microarrays.
Anna Cattani-Scholz 1 , Daniel Pedone 1 , Florian Blobner 2 , Stefan Neppl 2 , Peter Feulner 2 , Samira Hertrich 5 , Bert Nickel 5 , Luisa Andruzzi 5 , Manish Dubey 3 , Jeffrey Schwartz 3 , Gerhard Abstreiter 1 , Marc Tornow 4
1 Walter Schottky Institute, Technical University Munich, Garching Germany, 2 Physics, Technical University Munich, Garching Germany, 5 Physics, Ludwig-Maximilians-University, Munich Germany, 3 Chemistry, Princeton University, Princeton, New Jersey, United States, 4 Institute of Semiconductor Technology, Technical University Braunschweig, Braunschweig Germany
Show AbstractWe investigated hydroxyalkylphosphonate monolayers as a novel platform for the biofunctionalization of silicon-based field effect sensor devices. This included a detailed study of the thin film properties of organophosphonate films on Si substrates using several surface analysis techniques, including AFM, ellipsometry, contact angle, X-ray photoelectron spectroscopy (XPS), X-ray reflectivity, and current-voltage characteristics in electrolyte solution. Our results indicated the presence of an almost complete, dense monolayer on the native oxide with thickness ca. 1 nm. The monolayer was biofunctionalized with 12-mer peptide nucleic acid (PNA) receptor molecules in a two-step procedure using the heterobifunctional linker, 3-(maleimido)propionic acid N-hydroxysuccinimidyl ester. Successful surface modification with the probe PNA was verified by XPS and contact angle measurements, and hybridization with complementary single strand DNA was determined by fluorescence measurements. This protocol was translated to 100 nm wide p-doped Si nanowire field effect devices fabricated from SOI, thereby constituting the first successful example of phosphonate monolayer synthesis and derivatization on a silicon-based, nanoelectronic device. The PNA-functionalized nanowire devices allowed for label-free detection of DNA hybridization. A decrease in wire resistance equivalent to a surface potential change of 1.5 mV was measured upon injection of 1 µM DNA electrolyte buffer solution (1).Furthermore we investigated the optimization of the organic interfacial layer for DNA microarrays applications by modifying the alkylphosphonate monolayers with a dense layer of poly(ethylene glycol) (PEG) spacers before PNA immobilization. Fluorescence hybridization experiments carried out in the presence of complementary and non-complementary DNA showed that PNA-PEG functionalized surfaces were effective for hybridization of cDNA and minimized nonspecific adsorption of the analyte (2). Spatial patterns prepared by PDMS micromolding showed selective hybridization of fluorescently labeled DNA at PNA-PEG functionalized regions with a dramatic reduction in adsorption to probeless PEG-functionalized regions.[1]A. Cattani-Scholz, D. Pedone, M. Dubey, S. Neppl, B. Nickel, P. Feulner, J. Schwartz, G. Abstreiter, M. Tornow, Organophosphonate based PNA-functionalization of silicon nanowires for label-free DNA detection, ACS Nano 2008, 2, 1653–1660; Winner of a 2008 CeNS Publication Award, Center of Nanoscience (CeNS), LMU, Munich; Highlighted “In Nano” (ACS Nano 2, 1507 (2008). [2] A. Cattani-Scholz, D. Pedone, F. Blobner, G. Abstreiter, J. Schwartz, M. Tornow, L. Andruzzi, Novel PNA-PEG-Modified Silicon Platforms as Functional Bio-Interfaces for Applications in DNA Microarrays and Biosensors, Biomacromolecules, 2009, 10 (3), 489–496.
5:45 PM - SS2.10
SERS Spectroscopy For Studying The Interface Between Self-Assembled Semiconductor Quantum Dots And Biomolecules.
Lucia Quagliano 1 , John Lombardi 2
1 Istituto ISMN, Consiglio Nazionale delle Ricerche, Roma Italy, 2 Department of Chemistry and Center for Analysis of Structures and Interfaces (CASI), City College of New York, New York, New York, United States
Show AbstractMotivated by the sensitivity of surface enhanced Raman spectroscopy (SERS) to very small amounts of material and by our illustration of an enhanced Raman sensitivity of molecules adsorbed on self-assembled semiconductor quantum dots (1, 2), we explore the application of the SERS technique for directly probing the interface between semiconductor quantum-dots and biological molecules. The objective is the development of SERS spectroscopy as a fundamentally new method for studying biologically inspired systems in which nanostructured materials, especially semiconductors, play an important role. This will provide new and fundamental insights into the role of interfaces.SERS spectroscopy is a very sensitive technique that employs rough substrates with structures on the scale of nanometers to enhance the Raman signal produced by adsorbed species. We have observed the SERS spectra of tyrosine molecules adsorbed on self-assembled InAs/GaAs quantum dots, grown by molecular beam epitaxy. These spectra provide detailed molecular information on chemical composition, molecular structure, orientation on the surface, and order. This demonstrates that SERS can be used to directly probe the adsorption of molecules on MBE-grown semiconductor quantum-dots. This work shows that SERS has extremely high sensitivity to identify adsorbed species and provide insight into the nature of interaction of adsorbate with substrate. Thus, the SERS technique can provide a useful and versatile technique to probe interfacial structures.1- Lucia G. Quagliano, J. Am. Chem. Soc. Vol. 126 (2004), p. 73932- L. G. Quagliano, R. Livingstone, N. Perez-Paz, M. Munoz, M. C. Tamargo, F. Jean-Mary and J. R. Lombardi, Nanosensing: Materials and Devices II, SPIE proceedings 2005, M. Saif Islam, A. K. Dutta eds, 6008-0A
Symposium Organizers
Jose A. Garrido Technische Universitaet München
Erika Johnston Genzyme
Carsten Werner Leibniz Institute of Polymer Research
Thomas Boland The University of Texas-El Paso
SS3: Lipid and DNA Technologies
Session Chairs
Tuesday AM, December 01, 2009
Ballroom A (Hynes)
9:30 AM - **SS3.1
Model Systems of Polysaccharide-Rich Cell Coats Based on Supported Lipid Membranes.
Natalia Baranova 1 , Patricia Wolny 1 2 , Anthony Day 3 , Ralf Richter 1 2
1 Biosurfaces Unit, CIC biomaGUNE, Donostia - San Sebastian Spain, 2 , Max-Planck-Institute for Metals Research, Stuttgart Germany, 3 Wellcome Trust Centre for Cell Matrix Research, Faculty of Life Sciences, University of Manchester, Manchester United Kingdom
Show AbstractMany living cells produce a carbohydrate-rich pericellular coat that plays a crucial role in the protection of the cell and which is also vital in structuring and communicating with the cell’s environment. Examples of such self-organizing supramolecular structures are the hyaluronan-rich matrices that are found around chondrocytes (in cartilage), oocytes (during ovulation) or endothelial cells, and the mucosal films in the airway or in the digestive system.What are these coats made of and how do they exert their function? An outstanding feature is their dynamic self-organization into large, hydrated matrices. The supra-molecular level of organization that results from the assembly of glycans and proteins into soft, hydrated, gel-like networks gives rise to new qualities and functions, which differ from those characterizing the individual constituents. The highly hydrated nature of these coats, and the complex structure and dynamics of the living cell make them difficult to probe in their native environment or to determine the coat’s structure with high resolution methods. Therefore, to understand structure/function inter-relationships of these coats, it is vital to move from living cells to simplified model systems.We have recently developed a new method to create in vitro model systems of the pericellular coat at the solid-liquid interface (Richter et al. (2007) JACS 127:5306). The method is based on the end-grafting of hyaluronan to a supported lipid bilayer. These model systems are well-controlled and capture characteristic properties of the pericellular coat, including its dimensions and hydration. Thanks to a toolbox of in situ surface-sensitive techniques (QCM D, ellipsometry, microinterferometry, AFM, among others), the dynamics of coat reorganization and relevant physico-chemical properties can be quantified, and related to polymer physics theory.We employ such model systems to capture how hyaluronan binding proteins, including aggrecan (a cartilage proteoglycan) and TSG-6 (a protein secreted in response to inflammatory stimuli) remodel pericellular coats, thereby affecting their mechanical properties, permeability and stability. Ultimately, this work aims to gain novel insight into the relationship between the pericellular coat’s composition, supramolecular structure and biological function.
10:00 AM - SS3.2
Selective Formation of Supported Lipid Bilayers on Step-controlled Sapphire Surfaces.
Toshinari Isono 1 , Kenji Yamazaki 1 , Toshio Ogino 1
1 , Yokohama National University, Yokohama Japan
Show AbstractWe studied control of biointerfaces using step-controlled sapphire surfaces that exhibit unique properties about the surface charge and hydrophilicity. These properties are very important factors for construction of solid-bio interfaces. A supported lipid bilayer is often used as an artificial cell membrane system. In the natural cell membranes, phase-separated structures named “raft” enhance the interaction between membrane proteins and assembly for efficient signaling. In this study, we controlled formation and phase separation of the supported lipid bilayers on well-defined sapphire surfaces. Single crystalline sapphire (0001) surfaces were used as substrates to support lipid bilayers. By a high temperature annealing, the sapphire surfaces are covered with bunched steps accompanied with crossing steps and flat terraces. These surfaces were cleaned using a mixture of sulfuric acid and hydrogen peroxide. We call this surface an oxidized surface. Two domains with different hydrophilicity and charge density from each other coexist on the oxidized surface. Center regions of the terraces (domain A) are relatively hydrophobic and weakly charged, and the others (domain B) hydrophilic and negatively charged. To adjust the surface chemical properties, the oxidized surfaces were treated by phosphoric acid. We call it an etched surface, where the domain A was relatively hydrophilic. The supported lipid bilayers were formed on both the oxidized and etched surfaces by the vesicle fusion method. The formed lipid bilayers were observed by atomic force microscopy in a buffer solution.When electrically neutral lipids were used, the bilayers and monolayers simultaneously formed on the oxidized surfaces: the lipid bilayers selectively formed on the domain B with a high hydrophilicity, and the monolayers, on the other hand, formed on the domain A with a low hydrophilicity. On the etched surfaces, the lipid bilayers selectively formed on the domain A because its hydrophilicity is suitable for formation of the lipid bilayers. When charged lipids were used, the bilayers selectively formed on the oppositely charged region of the etched surfaces.To form the phase-separated structures, we used two types of lipid molecules. One is electrically neutral and exhibits a fluid phase at room temperature. The other is positively charged and exhibits a gel phase. When a mixture of these lipids was used, the fluid bilayers formed on the domain A and the gel bilayers on the domain B on the etched surfaces owing to the surface charge. In conclusion, we succeed in control of the phase-separated structures as well as the selective formation of supported lipid membranes using our designed sapphire surfaces.
10:15 AM - SS3.3
Self-spreading Lipid Bilayer as Nanofluidic Medium for Micro- and Nanostructured Biosurface Fabrication.
Kazuaki Furukawa 1 , Yoshiaki Kashimura 1
1 , NTT Basic Research Laboratories, Atsugi Japan
Show Abstract Self-spreading is a lipid wetting process at a solid-liquid interface, which induces the spontaneous formation of a supported lipid bilayer on a solid surface from a lump of randomly oriented lipid molecules. When we used hydrophilic and hydrophobic patterned surfaces we found that self-spreading occurs only on the former [1,2]. We also showed that a self-spreading lipid bilayer could be used as a molecule-carrying medium, which led us to propose a new type of microchannel device [1]. We demonstrated that fluorescence resonance energy transfer (FRET) efficiencies could be quantitatively determined using the device [3]. In addition, we found that the self-spreading continued through a sub-100-nm gap [4]. Since the self-spreading lipid bilayer is about 5 nm thick, it can or must be treated as a nanofluidic medium. In this presentation, we discuss the kinetics of self-spreading on a patterned surface. Analyses of our experiments provided two interesting results. One is that the self-spreading speed increases when the bilayer proceeds into the line patterns. This capillary effect-like behavior indicates an additional attractive interaction in the line patterns, which is probably due to the interaction between the bilayer and the wall. The other is that the front edge is always normal to the spreading direction even for curving lines. This can be attributed to the line tension at the spreading front edge. The results reveal that the line tension effect is probably stronger than the bilayer-wall interaction effect. We also describe the self-spreading observed when using a more complex surface pattern, where the self-spreading behavior also becomes more complex. Our findings open both a new route for fabricating micro- and nanostructured biosurfaces and a new experimental setup for studying the interactions of molecules, most interestingly membrane proteins, embedded within a supported membrane. This work was supported by JSPS KAKENHI 20310076.References: [1] K. Furukawa et al., Lab Chip 6, 1001-1006 (2006).[2] K. Furukawa et al., Langmuir 23, 367-371 (2007).[3] K. Furukawa et al., Langmuir 24, 921-926 (2008).[4] Y. Kashimura et al., Jpn. J. Appl. Phys. 47, 3248-3252 (2008)
10:30 AM - SS3.4
Modification of Phospholipid Bilayer Interfaces with Functionalized Cyclodextrins.
Suzanne Balko Barber 1 , Carlos Marques 1 , Marc Schmutz 1 , Tatiana Schmatko 1 , Olivier Felix 1 , Christophe Fajolles 2 , Angelika Klaus 2 , Jean Daillant 2 , Mayeul Collot 3 , Jean-Maurice Mallet 3
1 , CNRS - Institut Charles Sadron, Strasbourg France, 2 LIONS, CEA IRAMIS, Gif Sur Yvette France, 3 , Ecole Normale Supérieure, Paris France
Show AbstractIn the last 20 years, liposomes and PEG-coated liposomes, a.k.a “Stealth Liposomes,” have been studied extensively for drug-delivery and specific targeting, both clinically and experimentally. Other methods of pharmaceutical formulation using “helper” molecules to increase solubility or efficacy, such as cyclodextrins, have been employed. Cyclodextrins are a class of cyclic oligosaccharides, which are known to form host-guest complexes with polymer chains and other molecules, including the well-known a-cyclodextrin-PEG complex. α-cyclodextrin consists of 6 glucose molecules, while two other common forms are β-cyclodextrin (7 glucose) and γ-cyclodextrin (8 glucose). All three types of cyclodextrins have a conical toroid shape, giving rise to a smaller and larger opening due to the position of their primary and secondary hydroxyl groups in the ring, respectively. Recently, synthetic schemes have been proposed to modify the structure of cyclodextrins to produce “modified cyclodextrins.” Typically these modified cyclodextrins have included the addition of a lipidic structure to the cyclodextrin ring, thereby allowing the cyclodextrin molecule to be anchored to a lipid monolayer [Roux et. al. Eur. Biophys. J. 2007; Collot et. al. Tetrahedron Lett. 2007; Angelova et. al. J. Coll. & Inter. Sci. 2008; Darcy Eur. J. O. C. 2008].It is the marriage of two technologies, the use of liposomes and the host-guest complex properties of cyclodextrins anchored to a liposome bilayer, we wish to utilize to produce functionalized liposomes. Presently, we have investigated the incorporation of a modified β-cyclodextrin (m-β-CD) in both giant unilamellar vesicles (GUVs), typically greater than 5 mm in diameter, and small unilamellar vesicles (SUVs), typically less than 100 nm in diameter. In GUVs, we focus on the interactions of the m-β-CD incorporated into phase separating mixtures of DOPC, sphingomyelin (SM) and cholesterol (Chol). We study the effects that m-β-CD may have on phase segregation via fluorescence and confocal microscopy. In SUVs, we probe the effects of the m-β-CD with TEM of individual liposomes and also the effects on bilayers formed by vesicle fusion of SUVs onto a planar substrate with AFM. To confirm the incorporation of the m-β-CD into the bilayers for AFM and complement the work with fluroescence and confocal microscopy, we also performed vesicle fusion using quartz microbalance measurements (QCM).
10:45 AM - SS3.5
Nanoporous Xerogel Thin Films as Scaffolds for Lipid Bilayer Membranes.
Barbara Nellis 1 2 , Joe Satcher 2 , Subhash Risbud 1
1 Chemical Engineering and Materials Science, University of California - Davis, Davis , California, United States, 2 PLS, Lawrence Livermore National Laboratory, Livermore, California, United States
Show AbstractSupported phospholipid bilayers are typically used as models of the cell membrane. However, supports consisting of smooth, solid substrates result in disadvantageous interactions such as an inability to access the lower membrane leaflet as well as hindering bilayer mobility due to strong surface interactions. According to previous studies, the membrane and the solid supports are separated by only a few nanometers of water which results in transmembrane proteins experiencing non-physiological interactions with the surface. Nano-porous xerogel thin film substrates synthesized using sol-gel chemistry were explored as an alternative support for lipid membranes in this study. Previous work has demonstrated that silica xerogels can act as supports for two-phase DOPC/DSPC lipid membranes and can affect the phase behavior of these systems. Novel, non-silica xerogel thin film materials have been developed in an attempt to more realistically create a biologically relevant system. In order to better characterize and understand the nature of the surface-bilayer interactions, several oxide and organic nano-porous xerogel surfaces have been investigated as a scaffold for lipid vesicle fusion and bilayer formation. The surface topography of the different substrates has been analyzed using contact and tapping mode atomic force microscopy (AFM) and bilayer formation has been observed with fluorescence microscopy and lipid mobility has been measuring using Fluorescence Recovery After Photobleaching (FRAP). Results indicate that xerogel roughness, xerogel thin film surface chemistry, and vesicle preparation conditions can affect the ability of vesicles to rupture and fuse to the support to form a continuous bilayer. This work will contribute to a better understanding of vesicle fusion on nano-porous substrates as well as present novel xerogel materials for bilayer formation.
11:30 AM - SS3.6
Switch DNA Biosensors for the Label-free Detection and Sizing of Protein Targets on a Chip.
Ulrich Rant 1 , Wolfgang Kaiser 1 , Jelena Knezevic 1 , Erika Pringsheim 1 , Makiko Maruyama 1 , Paul Hampel 1 , Kenji Arinaga 1 2 , Gerhard Abstreiter 1
1 Walter Schottky Institut, Technische Universitaet Muenchen, Garching Germany, 2 , Fujitsu Laboratories Ltd., Atsugi Japan
Show AbstractWe introduce a chip-compatible scheme for the label-free detection of proteins in real-time that is based on the electrically driven conformation-switching of DNA oligonucleotides on metal surfaces. The switching behavior is a sensitive indicator for the specific recognition of IgG antibodies and antibody-fragments, which can be detected in quantities of less than 1 amol on the sensor surface. We show how the dynamics of the induced molecular motion can be monitored by measuring the high-frequency switching response as well as by time-resolved fluorescence measurements. When proteins bind to the layer, the increase in hydrodynamic drag slows the switching dynamics, which allows us to determine the size of the captured proteins. We demonstrate the identification of different antibody fragments by means of their kinetic fingerprint. The switchDNA method represents a generic approach to simultaneously detect and size target molecules using a single analytical platform.
11:45 AM - SS3.7
Enhancement and Optimization of Electrostatics for DNA Surface Hybridization
Ian Wong 1 , Nicholas Melosh 1
1 Materials Science & Engineering, Stanford University, Stanford, California, United States
Show AbstractThe hybridization of DNA at surfaces is widely utilized in biosensors, microarrays, biomolecular patterning and self-assembly. However, the physical mechanisms that govern this phenomenon are poorly understood, particularly when strands are immobilized in dense arrays where electrostatic and steric crowding dominates. We have recently demonstrated that these effects can be harnessed in conjunction with applied voltages to enhance hybridization density, allowing significantly elevated signal-to-noise ratios. Moreover, these voltages allow for reversible hybridization and melting of DNA at surfaces, which can be used for error-correction, drug delivery and gene therapy. These results are validated by a numerical model based on polyelectrolyte brush theories that we use to elucidate electrostatic mechanisms as well as optimize hybridization density and kinetics over a wide range of applied voltages, DNA grafting densities and ion concentrations. Finally, we examine the role of these electrostatic effects when uncharged peptide nucleic acids (PNA) are used.
12:00 PM - SS3.8
Label-free Detection of Virus DNA/RNA from Complex Background using PNA Probes.
Peng Li 1 , Philip Paul 1 , Piero Migliorato 1
1 Engineering Department, University of Cambridge, Cambridge United Kingdom
Show AbstractThe detection of virus DNA/RNA, especially from complex background, is very important for the identification of infectious pathogens in many clinical applications. The current technique depends on optical methods which require expensive labelling and complicated read-out instrumentation. Most of the alternative techniques demonstrated in academic research are not robust enough to interface with prevailing sample pretreatment procedures such as Polymerase Chain Reaction (PCR). In this work we showed a novel electrochemical detection method, which utilizes the intrinsic charge property of DNA molecules to detect and sub-type pathogenic virus through a label-free electrochemical sensor. In the demonstration system Peptide Nucleic Acid (PNA) probes were designed to recognize feature sequences of the avian influenza virus and its hemagglutinin and neuraminidase subtypes. The PNA probes were modified with thiol linkers and immobilized on an array of gold electrodes, together with spacer molecules in a mixed Self Assembled Monolayer (SAM) structure. Through a customized multiplexed asymmetric PCR protocol, the DNA target and virus RNA were amplified, with excess single stranded DNA in the amplicon. The PCR product, without any further purification, was applied on the developed sensor surface and the hybridization of PCR amplified DNA strand was subsequently measured as a change of charge transfer resistance using Electrochemical Spectroscopy Impedance (EIS). Through optimization of the device structure, probe density and assay conditions, a detection limit of 10 aM has been successfully achieved.The results shown in this work demonstrate a platform technology for the detection of DNA or RNA from real-world samples, illustrated using H5N1 influenza virus as an example. Compared with the current real-time PCR method, this new approach is more robust, cost-effective, efficient and has the potential to be integrated into portable instruments for in-field applications.
12:15 PM - SS3.9
Surface Modification of Diamond Aimed to Detect SNPs.
Yoko Ishii 1 , Shinya Tajima 1 , Hirofumi Arai 1 , Yuichiro Ishiyama 1 , Yuto Nonaka 1 , Hiroshi Kawarada 1
1 science and engineering , Waseda University, Shinjyuku-ku, Tokyo, Japan
Show Abstract1."Superiority of the diamond surface as SNPs detection"The diamond has superiority as the biosensor, such as its physiochemical stability, biocompatibility, and simple chemical-modification[1,2]. Here, we observed possibility of SNPs detection on the carboxyl-terminated diamond surface. In the previous study, we enabled four kinds of surface termination on the diamond surface, such as hydrogen-, oxygen-, amine- and fluorine- termination[3]. The carboxyl group functionalized on the diamond surface was negatively charged under our experimental conditions (pH=7) and was able to directly immobilize various biological molecules by covalent bond with the amino group. By the negative charge of carboxyl group, non-specific adsorption of target DNA is hard to occur on the carboxyl-terminated surface. When target DNAs approach near the diamond surface, the repulsion force exists between negatively charged target DNA and carboxyl terminal surface. It is indicated that incomplete hybridization by a mutated base pair and non specific adsorption of target DNA can be suppressed by this repulsion force[3]. Therefore this surface has possibilities of detecting SNPs.2."Precise detection mismatched pairs on carboxyl-terminated diamond surface"A polycrystalline diamond was treated a mixed solution of H2SO4 + HNO3 (9:1) at 90 degree for 24 hours. And the diamond was treated 0.1 M NaOH and 0.1 M HCl at 90 degree for 2 hours. As a result, the carboxyl groups formed on the diamond surface. The C1s spectrum of X-ray photoelectron spectroscopy (XPS) showed that the surface coverage of the carboxyl group was 3%. 21 mer amino-modified various sequence probe DNAs were directly immobilized on the surface without linker molecule. Then, Cy5 labeled target DNA was hybridized with these probe DNAs for 1hour. According to the calculation of the Gibbs energy by nearest neighbor method, the maximum discrimination between complementary DNA and single mismatched DNA appeared in the case of G/C and C/C mismached base pair which has much higher Gibbs energy than that of G/C. Experimental result by fluorescence observation indicated that, fluorescence intensity ratio between G/C and C/C was 10:1. Very accurate and sensitive detection was achieved. Meanwhile, the minimum discrimination is expected in the case of C/G and G/G pair having relatively close Gibbs energy to that of C/G. Although the detection was thought to be difficult, the fluorescence signal ratio between C/G and G/G was 10:3. These experiments yield that the fluorescence intensity ratio reflects the Gibbs energy of each DNA duplex.In conclution, the repulsion force between carboxyl group on the surface and negatively charged DNA is advantageous for the discriminative detection of mismatched pairs depending on Gibbs energy.[1]K.S.Song, H.Kawarada et al, Phys.Rev.E:74, 4, 041919, 2006[2]J.H.Yang, H. Kawarada et.al, Jpn.J.Appl.phys: 45, 42, L1114, 2006[3]S. Kuga, H. Kawarada et al, J.Am.Chem.Soc:130, 40, 13251, 2008
12:30 PM - SS3.10
Development of a Rapid SERS Assay for Viral DNA using Colloidal Gold.
Patrick Johnson 1 , Hao Zhang 1 , Mark Harpster 2 , William Wilson 2 , Keith Carron 3 , Bryan Ray 3
1 Chemical & Petroleum Engineering, University of Wyoming, Laramie, Wyoming, United States, 2 Arthropod-Borne Animal Diseases Research Laboratory, USDA/ARS, Laramie, Wyoming, United States, 3 , DeltaNu, Inc., Laramie, Wyoming, United States
Show AbstractRapid and accurate detection is the key to control virus spreading. Current methods for virus detection have disadvantages such as time-consuming, expensive, not very sensitive or not portable. Recent advances in the field of surface enhanced Raman scattering (SERS) spectroscopy have demonstrated the utility of this technology in diverse research disciplines ranging from surface chemistry and electrochemistry to forensics and the development of protein/nucleic acid sensor technologies. Our long-term goal is to develop a SERS based diagnostic tool for viremia that can be easily adapted for cost effective, portable and compact Raman spectroscopy in a point-of-care facility or field setting. In this work, an indirect and a direct capture model assays using colloidal Au nanoparticles are demonstrated for SERS spectroscopy detection of viral DNA. The DNA sequence targeted for the indirect capture was derived from the West Nile Virus (WNV) RNA genome and selected on the basis of exhibiting minimal secondary structure formation. Upon incubation with colloidal Au, hybridization complexes containing the WNV target sequence, a complementary capture oligonucleotide conjugated to a strong tethering group and a complementary reporter oligonucleotide conjugated to methylene blue (MB), a Raman label, anchors the resultant ternary complex to Au nanoparticles and positions MB within the required sensing distance for SERS enhancement.
SS4: Cell / Surface
Session Chairs
Tuesday PM, December 01, 2009
Ballroom A (Hynes)
2:30 PM - **SS4.1
Healing and Regeneration Moves the Biointerface into Three Dimensions.
Buddy Ratner 1
1 , University of Washington, Seattle, Washington, United States
Show AbstractMillions of medical devices made of synthetic or modified natural materials all trigger a similar reaction upon implantation, the foreign body reaction (FBR). Biocompatibility, for materials that pass routine cytotoxicity assays, is largely associated with a thin, avascular, non-adherent foreign body capsule. Surface modifications have been minimally successful in addressing this FBR. Most all surface chemistries seem to produce similar healing responses. Based on studies over the past 10 years at the University of Washington, a class of 3D biomaterials will be described that readily integrates into tissue and stimulates spontaneous reconstruction of tissue. The material is made by sphere-templating of synthetic polymers. All pores are identical in size and interconnected. Studies from our group have shown optimal healing (as suggested by induced vascularity and minimal fibrosis) for spherical pores of approximately 30 micron size. The integrative healing effect noted is independent of biomaterial – similar results are observed with sphere-templated silicone rubber and pHEMA hydrogel. In addition, surface chemical modification of the hydrogel with carbonyl diimidazole (CDI), or immobilization on the hydrogel of collagen I or laminin did not change the healing response. Also, good healing results have been seen upon implantation in skin (subcutaneously, percutaneously), heart muscle, sclera, skeletal muscle, bone and vaginal wall. This talk will describe these sphere-templated materials, and the concept of a 3D biointerface mechanically driving the reaction. The role of the macrophage in the reaction will be discussed.
3:00 PM - SS4.2
Molecular Presentation of Substrate-tethered FGF-2 Elicits Switch-like Signaling in Neural Stem Cells.
Xingyu Liu 1 , Dengke Ma 2 3 , Shawn Lim 4 , Kenneth Lin 1 , Guo-li Ming 2 3 5 , Hongjun Song 2 3 5 , Hai-Quan Mao 1 6
1 Department of Materials Science and Engineering, Johns Hopkins Univ, Baltimore, Maryland, United States, 2 Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States, 3 Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States, 4 Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States, 5 Institute of Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States, 6 Whitaker Biomedical Engineering Institute, Johns Hopkins Univ, Baltimore, Maryland, United States
Show AbstractIn the stem cell niche, many signaling molecules including fibroblast growth factors (FGFs), Wnt and Shh, execute their functions through anchorage with either the extracellular matrix (ECM), or cell membrane components from self or adjacent cells. Change or even loss of function occurs when these molecules are presented in their soluble forms; furthermore, accumulating evidence suggests that nano-scale organization of these signaling proteins is crucial to their functions. However, numerous efforts aimed at understanding how the stem cell niche regulates the local concentration and presentation of signaling cues in vivo—and consequently engaging and instructing cell fate decisions—were performed by tracking the effect of supplemented soluble factors in culture medium in vitro. Here, we developed a quantitative platform that allows control of FGF-2 molecular presentation locally as either monomers or clusters when tethered to a polymeric substrate. In striking contrast to a continuous MAPK activation pattern revealed from soluble FGF-2, substrate-tethered FGF-2 enables a switch-like activation pattern. Consequently, cellular decisions in rat adult neural stem cell (rANSC) behaviors including proliferation, differentiation and scattering were made according to this bimodal switch. we also demonstrate that the combination of the ligand concentration and its cluster size in the stem cell microenviroment), rather than concentration alone, serves as the more common determinants to govern its biological consequences.
3:15 PM - SS4.3
Endothelial Cell Attachment and Proliferation Studies on Modified Metal Stent Surfaces.
Vipul Dave 1 , Charito Buensuceso 2 , David Colter 2 , Jonathon Zhao 1 , Robert Falotico 1
1 Therapeutics and Advanced Research, Cordis Corporation, Johnson & Johnson, Warren, New Jersey, United States, 2 , Advanced Technologies and Regenerative Medicine, Johnson & Johnson, Somerville, New Jersey, United States
Show AbstractDrug eluting stents have transformed the practice of interventional cardiology and are the treatment of choice for patients with symptomatic coronary artery disease undergoing percutaneous intervention. There are risks of stent thrombosis (sub-acute thrombosis and last stent thrombosis) due to late reendothelialization, poor stent deployment, and premature anti-platelet therapy discontinuation. If stent surface modifications can enhance cell attachment and proliferation, then it will improve healing, minimize thrombus formation, reduce long term dual antiplatelet therapy, and improve stenting in high risk patients. In this study, cobalt chromium (CoCr) flat coupons and coronary stents were modified using different methods (low energy excimer laser processing, electron beam irradiation, immobilized covalently-bound heparin coating, and non-absorbable and absorbable polymer coatings). Human coronary artery endothelial cell (HCAEC) attachment and growth kinetics were investigated on unmodified and modified metal surfaces. Results showed that HCAECs attach to CoCr coupons and surface-modified CoCr coupons. No change in cell number was observed when cells were grown on CoCr coupons, heparin coated coupons and electron beam treated coupons throughout the 72h study period. A decrease in cell number was observed for excimer treated and polymer coated coupons. HCAEC seeding on CoCr stents indicated that cells attached and proliferated on the stents over a ten day study period. This research showed that in vitro screening method of testing endothelial cell attachment and proliferation on modified coronary stent surfaces can be used to gain insight for developing next generation drug eluting stents with improved endothelialization behavior.
3:30 PM - SS4.4
Immobilized Trypsin as a Surface Coating to Prevent Reactive Cell Adhesion on Silicone Surfaces.
Anil Kumar Achyuta 1 , Kyle Stephens 1 , Hilton Pryce Lewis 2 , Shashi Murthy 1
1 Dept of Chemical Engineering, Northeastern University, Boston, Massachusetts, United States, 2 , GVD Corporation, Cambridge, Massachusetts, United States
Show AbstractThe occlusion of silicone shunts used to treat hydrocephalus is a major clinical challenge. This occlusion results from adhesion and proliferation of reactive cells such as glial and vascular cells into the lumen of the catheter and on valve components. This presentation will examine the adhesive behavior of astrocytes, microglia, fibroblasts and endothelial cells on poly (dimethylsiloxane) (PDMS) surfaces immobilized with a proteolytic enzyme, trypsin, in vitro as a potential means to reduce hydrocephalus shunt occlusion. The covalently conjugated trypsin retains its proteolytic activity in its tethered form as evidenced by reduced human astrocyte adhesion in a dose dependent manner after 24 hours of culture in vitro. Unmodified PDMS surfaces exhibit significantly high adhesion of all four cell types namely normal human astrocytes, human microglia, human dermal fibroblasts and human umbilical vein endothelial cells (HUVECs) compared to the trypsin-immobilized surfaces (p < 0.0001). Immunofluorescence of cellular fibronectin determined that the PDMS surfaces immobilized with trypsin inhibited surface remodeling of all cell types. Qualitative adsorption studies revealed that unmodified PDMS surfaces support the adsorption of serum components compared to trypsin-immobilized surfaces (p < 0.0001).
3:45 PM - SS4.5
Silicon-Nanowire Array as Three-Dimensional Nanostructured Substrates Toward Efficient Capture of Circulating Tumor Cells.
Shutao Wang 1 , Hao Wang 1 , Ken-ichiro Kamei 1 , Kuan-Ju Chen 1 , Jing Sun 1 , David Sherman 1 , Jing Jiao 2 , Hong Wu 2 , Christian Behrenbruch 1 , Hsian-Rong Tseng 1
1 Department of Molecular and Medical Pharmacology, Crump Institute for Molecular Imaging, Los Angeles, California, United States, 2 Department of Molecular and Medical Pharmacology, Institute for Molecular Medicine , Los Angeles, California, United States
Show AbstractDuring the progression of metastasis, cancer cells from the solid tumor detach from the primary tumor, enter the blood stream, and travel to different tissues of the body. These break-away cancer cells in the peripheral blood are known as circulating tumor cells (CTCs).[1] In addition to conventional diagnostic imaging and serum marker detection CTC counting has recently been used for examining early-stage cancer metastasis, predicting patient prognosis, and monitoring the therapeutic outcomes of cancer.[2] However, isolation of CTCs has been technically challenging due to the extremely low abundance (a few to hundreds per mL) of CTCs among a high number (109 cells/mL) of haematologic cells in the blood.[3] Although several technology platforms for isolating/counting CTCs have been developed with strategies that involve either immunomagnetic beads or microfluidic devices,[3-5] they suffer from low CTC-capture yield, low capture efficiency and lack of single cell accessibility for down stream molecular analysis. By introducing a three-dimensional (3D) nanostructured substrate – specifically, a silicon-nanowire (SiNW) array coated with anti-EpCAM, a commonly used CTC-capture agent – we can reliably capture cancer cells from artificial CTC blood samples.[6] Taking EpCAM-positive cell line MCF7 as model system, we compared the capture efficiency of SiNW substrates with that of flat ones. SiNW substrates exhibited an efficient cell capture that can be up to 10 times more than the flat substrate, likely due to enhanced nanoscaled local topographic interactions between CTCs and substrate. Also we carried out the control experiments, including two EpCAM-negative cell lines (HeLa cell line and Daudi cell line) and different surface modification control, and demonstrated that this 3D nanostructured substrate is real specific for EpCAM-positive cells. Finally, we were able to capture cancer cells from the artificial blood samples. It is believed that this kind of nanostructured surfaces will bring an ideal candidate platform to early cancer diagnosis and therapy monitoring.References.[1] P. S. Steeg, Nat. Med. 2006, 12, 895.[2] M. Cristofanilli, G. T. Budd, M. J. Ellis, A. Stopeck, J. Matera, M. C. Miller, J. M. Reuben, G. V. Doyle, W. J. Allard, L. W. M. M. Terstappen, D. F. Hayes, New Eng. J. Med. 2004, 351, 781.[3] V. Zieglschmid, C. Hollmann, O. Böcher, Crit. Rev. Clin. Lab. Sci. 2005, 42, 155. [4] W. J. Allard, J. Matera, M. C. Miller, M. Repollet, M. C. Connelly, C. Rao, A. G. J. Tibbe, J. W. Uhr, L. W. M. M. Terstappen, Clin. Cancer Res. 2004, 10, 6897.[5] S. Nagrath, L. V. Sequist, S. Maheswaran, D. W. Bell, D. Irimia, L. Ulkus, M. R. Smith, E. L. Kwak, S. Digumarthy, A. Muzikansky, P. Ryan, U. J. Balis, R. G. Tompkins, D. A. Haber, M. Toner, Nature 2007, 450, 1235.[6] S. T. Wang, H. Wang, K. Kamei, K. J. Chen, J. Sun, D. J. Sherman, J. Jiao, H. Wu, C. P. Behrenbruch, H. R. Tseng, Angew. Chem. 2009, revised.
4:30 PM - **SS4.6
Roles of Oligo- and Polysaccharides in Modulation of Biointerfaces.
Motomu Tanaka 1
1 Institute of Physical Chemistry and BIOQUANT, University of Heidelberg, Heidelburg Germany
Show AbstractIn nature, cell-cell and cell-tissue contacts are madiated by various hydrated biopolymers. In my talk, I will introduce some of our recent studies that physically model the active roles of "soft" biopolymers (carbohydrates) in fine-adjustment of contacts at biological interfaces by specular and off-specular neutron and X-ray scattering at defined relative humidity as well as in bulk water. The planar geometry of the biomembrane models enables us to identify the in-plane and out-of-plane momentum transfers at various angles quantitatively, which can be used to determine the influence of carbohydrates on structural and mechanical properties of membranes.References[1] M. Tanaka and E. Sackmann, Nature 2005, 437, 656-663.[2] M. Tanaka, MRS Bulletin 2006, 31, 513-520.[3] R. Oliveira, E. Schneck, B. Quinn, O. Konovalov, K. Brandenburg, U. Seydel, T. Gill, C. Hanna, D. Pink, M. Tanaka, Comptes Rendus Chimie, 2009, 12, 209-217.[4] E. Schneck, F. Rehfeldt, R. Oliveira, C. Gege, B. Demé, M. Tanaka, Phys. Rev. E 2008, 78, 061924.
5:00 PM - SS4.7
Chemomechanics of Cell-Material Interaction Kinetics Over Large Ranges of Length and Time Scales.
John Maloney 1 , Ranjani Krishnan 2 , Adam Zeiger 1 , Krystyn Van Vliet 1 2
1 Materials Science and Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States, 2 Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States
Show AbstractTissue cells are inhomogeneous, dynamic objects. To understand the cell as a material, it is necessary to explore how intracellular properties and extracellular cues are integrated to regulate cell behavior. We have investigated biomolecule and cell behavior in the context of chemomechanics, here defined as the rate-dependent coupling between intra- and extracellular environments and a cell's mechanical properties. A survey over different length and time scales extends the scope of our knowledge of cell chemomechanics. First, at the biomolecular level, we have studied the interactions between cell-surface integrin receptors and extracellular matrix ligands as a function of extracellular pH, which is known to be acidic in the tumor and wound healing environments. We use nanosecond-scale molecular dynamics simulation to gain an atomistic understanding of how pH affects integrin conformation and integrin-ligand interactions, and complement simulations with experimental tools such as atomic force microscope (AFM) enabled molecular force spectroscopy. These molecular-scale studies are then connected to cell-scale measurements of migration speed and adhesion strength. The combination of these results gives us a multiscale understanding of how an extracellular chemical property such as pH can affect complex, coordinated cell behaviors. Second, at the cellular level, cell rheology regimes can be elucidated and cell deformability characterized by optical stretching, in which dual opposing laser beams are used to stretch a whole cell in suspension by photonic pressure alone. This technique relies on photon momentum transfer at points of changing refractive index, specifically at the cell-liquid interface, to exert a stress without applying physical contact. At a time scale of seconds, the passive time-dependent response of the biopolymeric cytoskeleton under load is coupled to active actomyosin-induced contraction and expansion. The animate nature of the cell thus becomes evident. Third, from the cellular level to the cell-cell and cell-matrix level, the mechanical properties of the cell membrane, cell cytoskeleton, and whole cell can be determined by using AFM-enabled indentation. Cell membrane and cytoskeleton mechanical properties influence cell behaviors such as mobility, proliferation, and adhesion. Specifically, by investigating the influence of various macromolecules added to the cytosol, we can investigate how changes to the internal cellular environment influence these properties and behaviors over a time scale of days to weeks. The integrated understanding of cell chemomechanics over this wide range of length and time scales allows us to begin to build a model of the cell and its environment as controllable materials that can be engineered to produce desirable outcomes such as particular differentiated states, or to suppress undesirable behaviors such as tumor metastasis.
5:15 PM - SS4.8
Fabrication of Living Yeast Cellosomes by Polyelectrolyte Mediated Assembly and Magnetically Responsive Templates.
Rawil Fakhrullin 1 3 , Javier Garcia-Alonso 2 , Vesselin Paunov 1
1 Department of Chemistry, University of Hull, Hull, North Humberside, United Kingdom, 3 Department of Biochemistry, Kazan State University, Kazan, Tatarstan, Russian Federation, 2 Department of Biology, University of Hull, Hull United Kingdom
Show AbstractWe have developed a new method for fabrication of living multicellular structures that could show how colonial organisms evolved in nature and be used in tissue engineering. These structures, termed cellosomes, were made by layering yeast cells on to templates of aragonite (rod-shaped) and calcite (rhombohedral) microcrystals. We coated yeast cells with a layer of an anionic polyelectrolyte and the templates with a layer of cationic polyelectrolyte to promote their irreversible binding and adhesion. The templates were pre-coated with magnetite nanoparticles so they could be manipulated with an external magnetic field. We used this to extract the cell-coated templates by magnetic separation which is very useful for selective sorting and separation of the cellosomes from the excess of single cells. The templates were dissolved with ethylene diamine tetra acetic acid to give rod- and rhombohedral-shaped, hollow 3D cellosomes. We also pioneered a similar technique for templating of air micro-bubbles with yeast cells to produce spherical cellosomes. We analyzed the obtained hollow structures with Scanning Electron Microscopy to study their arrangement in the cellosome “membrane” and also used fluorescence microscopy after treating them with fluorescein di-acetate to find that the yeast cells were still active in these assemblies and remained viable for more than two weeks. These cellosomes resemble primitive multicellular organisms like Volvox to a certain degree, so we could speculate that nature has used a similar assembly mechanism in evolution. The technique, works not only with yeast cells but also with many other kinds of cells. It could be applied with stem cells and opens new possibilities for novel ways of engineering tissues where their shape can be directed by the shape of the micro-template. Our method also works for fabrication of living cellosomes of various shapes and from different types of cells, to produce symbiotic colonies of cells which is the next step in the design of "artificial" living multicellular organisms. We also report a related technique for polyelectrolyte mediated magnetization of living cells which leads to deposition of an integrated layer of magnetite nanoparticles on the surfaces of yeast cells. We show that these “magnetic yeast” cells preserve their viability and can be manipulated by external magnetic field. We demonstrate the usefulness of these materials by fabricating magnetic yeast cells that have been genetically modified to express their Green Fluorescent Protein (GFP) gene whenever the cells repair damaged DNA. We report a simple microfluidic device in which these cells were directed by external magnetic field and used to simultaneously detect genotoxicity and cytotoxicity.
5:30 PM - SS4.9
Nanomechanical Probes of Individual Corneal Epithelial Cells.
Scott Perry 1 , Joelle Payne 1 , Francis Limpoco 1 , Natalia Dolgova 2 , Benjamin Keselowsky 2 , Greg Sawyer 3 , Howard Ketelson 4
1 Materials Science and Engineering, University of Florida, Gainesville, Florida, United States, 2 Biomedical Engineering, University of Florida, Gainesville, Florida, United States, 3 Mechanical Engineering, University of Florida, Gainesville, Florida, United States, 4 , Alcon Research, Ft. Worth, Texas, United States
Show AbstractLiving human corneal epithelial cells have been probed in vitro via atomic force microscopy, revealing the frictional characteristics of the outer surfaces of mature corneal cells in media. Measured shear stresses of 0.40 kPa are among the lowest reported values for aqueous lubrication. This is perhaps not surprising, yet the ability to demonstrate the lubricity of single epithelial cell surfaces in contact with a microsphere probe highlights the opportunities for conducting interfacial/solution studies at the cellular level. The mechanical properties of individual epithelial cells have been further probed through nanometer scale indentation measurements. An elastic foundation model, based on experimentally verifiable parameters including cell diameter and thickness, is used to fit the indentation data, producing an effective elastic modulus of 16.5 kPa and highlighting the highly compliant nature of the cell surface. The elastic foundation model is found to more accurately fit the experimental data, to avoid unverifiable assumptions, and to produce a modulus significantly higher than that of the widely used Hertz-Sneddon model. The results provide a baseline understanding of the mechanical properties of individual corneal cells and again highlight the opportunities for probing the details of mechanobiology related to the function of the eye surface.
5:45 PM - SS4.10
Interfacial Energy Dependence on Cell-Penetrating Behavior of Monolayer-Protected Nanoparticles.
Jeffrey Kuna 1 , Francesco Stellacci 1
1 DMSE, MIT, Cambridge, Massachusetts, United States
Show AbstractRecent work on highly water-soluble nanoparticles synthesized to contain a mixture of hydrophobic and hydrophilic patches on the surface has shown that they can penetrate cell membranes without poration of the cell membrane (Verma et. al., Nature Materials 7, 588-595 (2008)). At certain compositions, the surfaces of such particles phase separate into nanometer wide domains of alternating composition. This surface structuring is critical to cell-penetrating behavior and has already been implicated in having drastic effects on interfacial properties (Centrone et al., PNAS 105, 9886-9891 (2008)). However, the interfacial energy of nanoparticles in solution remains an experimentally difficult quantity to measure. To analyze the interfacial energies of cell-penetrating particles, nanoparticles synthesized with identical surface chemistries to the cell-penetrating particles but with varying ligand lengths were produced and assembled into smooth films using electrostatic assembly. The interfacial energies of these films were characterized and related to the cell-penetration capability of the constituent nanoparticles. The interfacial energies of the particles in water and hydrophobic solvents contribute to an understanding of the passive mechanism by which such nanoparticles and other nano-structured objects (e.g. cell-penetrating peptides) penetrate membranes.
SS5: Poster Session: Biosurfaces and Biointerfaces I
Session Chairs
Wednesday AM, December 02, 2009
Exhibit Hall D (Hynes)
9:00 PM - SS5.1
Control of Pseudomonas aeruginosa Biofilm Formation on Medical Implants Through Surface Nanostructuring.
Bin Yu 1 , Dongyang Li 2 1 , Randall Irvin 3 , Elisabeth Davis 3
1 Biomedical Engineering, University of Alberta, Edmonton, Alberta, Canada, 2 Chemical and Materials Engineering, University of Alberta, Edmonton, Alberta, Canada, 3 Med Microbiology and Immunology , University of Alberta, Edmonton, Alberta, Canada
Show AbstractThe development of implanted medical devices has made great contributions to modern medicine. However, the formation of infectious biofilms on medical implants is still an unsolved problem, which may cause chronic infection that is difficult to control, since bacteria within a biofilm are extremely resistant to antibiotics and the human body immune system. Attempts were previously made to enhance the antibacterial activity of implanted materials through incorporation with Ag, an antibacterial agent. However, adding Ag to metallic implant materials decreases their corrosion resistance and thus generates other problems. Surface nanocrystallization is an effective approach to enhance the corrosion resistance of passive materials. In this study, Ag powder was added to nanocrystallized surface of 304 stainless steel achieved using a process combining severe plastic deformation and annealing treatment. It was demonstrated that the corrosion resistance of the Ag-containing nanocrystallized surface exhibited significantly higher corrosion resistance than Ag-containing microcrystalline surface. The nanocrystalline steel surfaces incorporated with Ag also showed an enhanced antibacterial capability during bacterial cell binding assay tests. The adhesive forces of the nanocrystalline surfaces with different grain sizes from 32nm to 110nm were measured using an atomic force microscope. The binding behavior of Pseudomonas aeruginosa bacterial cells to the surfaces was also investigated. It was demonstrated that both the adhesive force and bacterial binding of the Ag-containing nanocrystallized surface of 304 stainless steel were reduced as the grain size decreased.
9:00 PM - SS5.10
Development of Functional Membranes for the Bio Processing Unit Prepared by Radiation-induced Graft Polymerization
Tadashi Okobira 1 2 , Ken Youkawa 1 , Tomoko Kagenishi 1 2 , Tomonori Kawano 1 , Kazuya Uezu 1
1 Life and Environment Engineering, The Kitakyushu University, Kitakyushu, Fukuoka, Japan, 2 , Fukuoka Industry, Science & Technology Foundation, Fukuoka, Fukuoka, Japan
Show AbstractIn these days, biosensors are being developed rapidly, and they are beginning to be applied to biological controls, environment measurement, and other related areas. However, the conventional technology has difficulties in order to perform real-time multi signal responses and recognition of spatial information. Therefore, novel technologies overcoming such difficulties are required. We are proposing a novel concept “bio-processing unit (BPU)” by making use of orchestrated sensing and signal processing capabilities of living cells from various organisms (cellular composites) packed in a system powered by organic and inorganic chemistry, microbial engineering, genetic engineering, optics and electric physiology. In this work, we prepared the two functional membranes; “release material which can respond cell” and “produces stimulation for cell to be able to respond”.An epoxy-group-containing monomer, glycidyl methacrylate (GMA) was grafted onto a porous hollow-fiber membrane made of polyethylene, by radiation-induced graft polymerization.The degree of GMA grafting defined by the weight increase of the membrane was set at 80-100%. Two kinds of functional groups, N-methyl-D-glucamine (NMG) and Glycine-Glycine-Histidine peptide (GGH) were introduced to the epoxy group of the graft chain, respectively. GMA was polymerized on the pore of the membrane to attach the polymer brush.Small amount of boron in washing water sometimes causes serious problem for the semiconductor industry. It is well known that NMG captures boron selectively. NMG membrane was given a function of substrate exchange type. Boron solution (0.05, 0.1, 0.5, 1.0 mg/L) was prepared with boric acid, and pH of boron solution was adjusted 7.0 by adding HCl and NaOH solutions. NMG membrane caused desorption of the lantern when we penetrated boron solution after having penetrated lantern solution to NMG membrane. The desorption amount of the lantern depended on the concentration of penetrated boron. Therefore, it was considered that NMG membrane could use as the small amount boron sensor.On the other hands, GGH membrane was given a function of substrate reaction type. It was reported that copper-binding GGH reacted with tyramine and hydrogen peroxide in water solution, and generated radical oxygen. We made membrane having the function to generate superoxide by immobilized copper-binding GGH to use this phenomenon. Copper-binding GGH membrane, phosphoric buffer solution (50mM), hydrogen peroxide (0.15mM), tyramine (1.25mM), and chemiluminescence reagent (CLA 3μM), were added in a test tube to measure the luminescence intensity. In the case of copper-binding GGH membrane the strong luminescence was occurred. This luminescence was caused as the generated active oxygen reacted with CLA.
9:00 PM - SS5.11
Development of Silane-coupling Silicon Substrate for Device Fabrication using Protein.
Megumi Fukuta 1 , Heiji Watanabe 2 , Ichiro Yamashita 1
1 Graduate School of Materials Science, Nara Institute of Science and Technology, Nara Japan, 2 Department of Material and Life Science, Graduate School of Engineering, Osaka University, Osaka Japan
Show AbstractIn the field of nano-electronic device fabrication, proteins are fixed on substrates and serve as scaffolds and/or tem-plates for making nano-functional structures. Ferritin has abilities to accommodate homogenous metal core and to make two-dimensional array by self-assembly manner. We have made devices making use of these properties[1.2]. Salt and alkali ions of the buffer contaminate substrates, inhibiting the sensing and fabrication of nano-electronic devices. To avoid such undesirable effects, buffer solutions have been replaced by pure water or alkali-metal-ion-free buffers[1.3]. Since the buffer replacement is time-consuming and causes several problems, a new method of salt-free ferritin fixation using buffer solutions is needed. We analyzed correlation between substrate properties, adsorption of ferritins, and buffer components to satisfy the requirement. The analysis showed that the low coverage of silane on silicon substrate caused metal contamination. It was also shown that silane-coupled silicon substrate such as amino-silane and mercaopto-silane which ionizes in the solution, adsorbs Na, Cl and phosphate ions of PBS buffer solution. Nonionic silane that has electron-donating ability or electron-accepting ability such as phenyl and trifluoromethyl-silane, adsorbs buffer components. After throughtout anlysis and investigations, it was found that the high coverage methyl-silane-coupled silicon substrate achieved adsorption of ferritin without buffer components adsorption even if the protein solution contains metal ions or salts. Accordingly, we thought that methyl-silane-coupled substrate is suited for substrate of device using ferritin. [1] K. Yamada et al., Jpn. J. Appl. Phys., 46(11), (2008) 7549-7553. [2] S. Kumagai et al., J. Photopolys. Sci. Technol., 18(4), (2005) 495-500. [3] A. Miura, et al., J. Appl. Phys., 103, (2008) 074503.
9:00 PM - SS5.13
Biogenic Silica Incorporated Bio Sensors for Ultra Sensitive Protein Detection.
Gaurav Chatterjee 1 , Vindhya Kunduru 1 , Kai-Chun Lin 2 , B. Ramakrishna 2 , Shalini Prasad 1
1 Electrical Engineering, Arizona State University, Tempe, Arizona, United States, 2 School of Materials, Arizona State University, Tempe, Arizona, United States
Show AbstractWe have explored the impact of nanoscale confinement on biomolecule detection. Recent research has indicated that improved detection parameters such as ultrasensitivity, selectivity can be achieved in electrical detection by employing size based confinement techniques in nanomaterials.We have previously observed that nanoporous membranes, while improving biomolecule detection sensitivity, had various issues regarding diffusion, transport and selectivity. We address those issues using biogenic silica as the nanoporous material.Biogenic silica (diatoms) are single celled algae with rigid cell walls made of amorphous silica. They offer great symmetry, hierarchy and porosity, combined with excellent physical and electrical isolation. These properties make them amenable to nanoporous biosensors. We have designed the physical geometry utilizing them into protein detection.We have incorporated a nanoporous biogenic silica membrane onto a microelectronic platform. The membrane offers excellent nanoconfinement and aids in electrical detection of protein concentrations. By interfacing the diatom membrane with the microelectronic platform we have generated arrays of nanoscale confined spaces into which we have trapped size matched biomolecules. We have demonstrated that such a multi scale architecture system can be engineered to function as an electrical biosensor using the principle of electrochemical impedance spectroscopy. We have demonstrated the functionality of the structure as an electrical biosensor by using the inflammatory marker C-reactive protein as the test protein. We have observed that the detection capabilities of the diatom based biosensor to be far improved as compared to the detection performance with an alumina membrane. We have demonstrated limit of detection in the pg/ml regime up to the μg/ml regime. The detection range is within the clinically relevant regime. We have demonstrated detection of CRP from purified as well as serum samples and have identified that the hierarchical porous structure of the diatom to b provide better performance metrics as compared to manufactured alumina membranes.
9:00 PM - SS5.14
Fabrication of Metallized Nanopores in Silicon Nitride Membranes.
Ruoshan Wei 1 , Daniel Pedone 1 , Gerhart Abstreiter 1 , Ulrich Rant 1
1 Walter Schottky Institut, Technische Universitaet Muenchen, Garching Germany
Show AbstractNanopore translocation experiments have evolved into powerful means to study single molecules. Engineered solid state pores hold considerable advantages over their biological counterparts with respect to stability and adjustability. With the aim of creating electrically gateable pores and to make a chemical functionalization feasible, we studied a novel device concept which involves the coating of the pore with a metallic film. Here we report on the device fabrication and characterization of the metallized pores through various techniques including SEM, TEM, S-TEM and AFM. Pores featuring diameters from 20 to 80 nm are prepared in Si3N4 membranes using electron beam lithography. Subsequently, thin metal films of gold with titanium as adhesion layers are evaporated onto the pre-structured nanopores. We systematically investigated the coating behaviour of the inner pore walls with metal depending on the film thickness and evaporation parameters.For slow metal evaporation rates (<1 Å/s) we observe a linear relationship between evaporated film thickness and the resulting reduction of the pore diameter. For higher evaporation rates (5 Å/s) we find that the shrinking of the pore is enhanced and depends on the initial pore size. We ascribe both effects to an increased diffusion of gold atoms into the pore. Interestingly, for fast evaporation we also observe the formation of self assembled gold islands which decorate the pore opening. Furthermore our results show that pores metallized with very thin films (10 nm) exhibit a discontinuous gold film at the pore opening while thicker films resulted in grainy pore edges. Our observations suggest an intriguing growth process of gold inside the pore: after wetting of the pore walls with gold, further metal deposition closes the pore at its distal opening. Subsequently, the pore is filled with metal from the bottom up.
9:00 PM - SS5.15
Terminal Phosphate Group Influence on DNA - TiO2 Nanoparticle Interactions.
Zachary Rice 1 , Nathaniel Cady 1 , Magnus Bergkvist 1
1 Nanobiology, College of Nanoscale Science & Engineering, Albany, New York, United States
Show AbstractImmobilization of DNA/RNA, onto various metal and metal oxide surfaces is of great importance for the development of future microarray, gene mapping, DNA sequencing, nanoparticle targeting, and sensor applications etc. Attachment of DNA to solid interfaces typically occurs through either electrostatic interactions or covalent bonds between a derivatized surface and functional groups introduced in the nucleic acid termini. Direct immobilization methods through electrostatic charge or semi-covalent attachment could circumvent modification of both substrate and nucleic acids terminal groups; however this binding interaction is reversible and is more pronounced for shorter oligomers. Previously, we and others have demonstrated that alkanephosphates and terminal phosphate groups present on nucleic acids play an important role in the interaction with group IV metal oxides such as zirconium and hafnium, providing a stable linkage to the surface. Titanium dioxide (TiO2), which is frequently employed in various nanoscale applications belong to the same group and similar interactions with phosphate is expected. Various adsorption studies have demonstrated binding of nucleic acids to TiO2 surfaces, although the influence of terminal phosphate vs. electrostatic interaction (via the DNA/RNA backbone) on the surface interaction is somewhat unclear. The research presented here investigated the effect of nucleic acid length, presence of terminal phosphates, and differences between dsDNA and ssDNA on their binding to TiO2 nanoparticles. TiO2 nanoparticles (75 nm) were used to study the adsorption of Lambda DNA, and shorter (21bp) ssDNA and dsDNA oligomers with/without a 5’ phosphate group. Initial adsorption of DNA to nanoparticles was calculated via UV absorption and the binding affinity was evaluated by observing the amount of DNA removed during successive washing steps. Results showed that all types of nucleic acids (Lamda DNA, ssDNA and dsDNA) initially bind to nanoparticles, independent of molecular weight single/double strandedness, or phosphorylation state. The total amount of DNA initially adsorbed to nanoparticles (ng/particle) was similar for all nucleic acids and scaled with amount of DNA added. However, after washing it was observed that only high molecular weight lambda DNA remained strongly associated with the titanium dioxide particles. These results show that nucleic acid interaction with TiO2 nanoparticles mainly depends upon the chain length where electrostatic interactions via the phosphate backbone dominate over the terminal phosphate group interaction. These results provide valuable data for future applications based on DNA-nanoparticle constructs including nanoelectronics, photovoltaics, and biotemplated synthesis of semiconducting materials.
9:00 PM - SS5.16
Exploiting Phosphate Dependent DNA Immobilization on HfO2 and AlGaN for Integrated Biosensors.
Nicholas Fahrenkopf 1 , Vibhu Jindal 1 , Neeraj Tripathi 1 , Serge Oktyabrsky 1 , Fatemeh Shahedipour-Sandvik 1 , Natalya Tokranova 1 , Magnus Bergkvist 1 , Nathaniel Cady 1
1 College of Nanoscale Science and Engineering, University at Albany, Albany, New York, United States
Show AbstractA significant challenge for traditional DNA microarrays used for high throughput analysis and sequencing is the bulk of the optical detection and readout components. In this work we propose using the semiconductor materials hafnium oxide and aluminum gallium nitride as the foundations for transistor based DNA biosensors. Since DNA has an intrinsic negative charge along its backbone, the immobilization and hybridization of probe DNA can be detected providing a biosensor chip that can be fabricated with a much smaller footprint. In cutting edge CMOS technology HfO2 has emerged as the gate dielectric of choice over traditional silicon dioxide in order to provide significant advancement of gate performance in field effect transistors. As a result, the technology exists to fabricate “gate-less” FETs with HfO2 as the material in the gate region, and therefore a platform for FET-based DNA sensing. The delocalized two dimensional electron gas in high electron mobility devices such as AlGaN/GaN heterostructures, have been used to create a variety of sensors. These sensors are particularly attractive for biosensing because of their high sensitivity. In both cases a key concern for the construction of the biosensors is the immobilization of the probe biomolecule on the surface of the sensor. Conventionally, reactive organosilane or gold-thiol interactions are utilized to link biomolecules to the surface of a sensor; however this can complicate the fabrication of the device. In previous works our group has shown that single strand DNA can be directly immobilized on both HfO2 and AlGaN surfaces without the need for complex surface chemistry or crosslinking strategies. In addition, we have shown that the immobilization of the single stranded DNA onto HfO2 is influenced by the terminal phosphate group, in agreement with literature that shows that phosphates and phosphonates show strong attachment to III-V metal oxides. In our current work, we have shown that surface-immobilized DNA is available for hybridization and that hybridization is sequence specific making this immobilization method attractive for biosensing applications using HfO2 or AlGaN. We have also explored the direct immobilization of ssDNA to these surfaces. We have found that the immobilization of ssDNA on the AlGaN surfaces is much more stable for 5’ or 3’ phosphorylated ssDNA than non-phosphorylated ssDNA. Further, we present the results of x-ray photoelectron spectroscopy, Fourier transform infrared spectroscopy, and electrical characterization of FET devices before and after the direct immobilization of the probe DNA.
9:00 PM - SS5.18
Functionally Graded Polycaprolactone Scaffolds: Fabrication via Twin Screw Extrusion/Spiral Winding Process and in vitro Assessment for Tissue Engineering.
Seher Ozkan 1 , Dilhan Kalyon 2 , Xiaojun Yu 2
1 Material Science Group, Global R&D, International Specialty Products Inc. , Wayne, New Jersey, United States, 2 Chemical, Biomedical and Materials Engineering Department, Stevens Institute of Technology, Hoboken, New Jersey, United States
Show AbstractThe engineering of biomimetic tissue relies on the ability to develop biodegradable scaffolds with functionally graded physical and chemical properties. Another major challenge is to translate current laboratory-based manufacturing processes into large scale production methods that are reproducible and economical. In this study, a twin-screw-extrusion/spiral winding (TSESW) process was developed to enable the radial grading of porous scaffolds (discrete and continuous gradations) that were composed of polycaprolactone (PCL), β-tricalciumphosphate (β-TCP) nanoparticles, and salt porogens. Scaffolds with interconnected porosity, exhibiting myriad radial porosity, pore-size distributions, and β-TCP nanoparticle concentration could be obtained. Their compressive properties were characterized and in vitro cell proliferation studies in conjunction with MTS, ALP and ALZ red assays were conducted for 56 days using human fetal osteoblast cells. We have also demonstrated in a parallel study the use of a twin screw extrusion/spiral winding (TSESW) process to generate protein encapsulated tissue engineering scaffolds. Bovine serum albumin (BSA) was distributed into PCL matrix using both wet and hot melt extrusion methods. The encapsulation efficiency and the time-dependent release rate, as well as the tertiary structure of BSA (via circular dichroism), were investigated as a function of processing method and conditions. Within the relatively narrow processing window of this demonstration study it was determined that the wet extrusion method gave rise to greater stability of the BSA on the basis of circular dichroism data. Histological analysis and confocal microscopy analysis showed that, after 56 days of culturing, a noticeable amount of new ECM was observed in the inter-pore space across the entire scaffold cross-section, confirming that the interconnectivity of the porous structure of the scaffolds has allowed the cells to proliferate and penetrate into the volume of the entire scaffold. The rate of proliferation of human fetal osteoblast (hFOB) cells and the rate of mineral deposition were found to be greater for wet extruded scaffolds, presumably due to the important differences in surface topographies (smoother scaffold surfaces upon wet extrusion). Overall, these findings suggest that the twin screw extrusion/spiral winding (TSESW) process offers significant advantages and flexibility in generating a wide variety of non-cytotoxic tissue engineering scaffolds with controllable distributions of porosity, physical and chemical properties and protein concentrations that can be tailored for the specific requirements of each tissue engineering application.
9:00 PM - SS5.19
The Effects of Viscoelastic Polymer Substrates on Dental Pulp Stem Cell Differentiation.
Chungchueh Chang 1 , Vladimir Jurukovski 1 , Marcia Simon 2 , Miriam Rafailovich 1
1 Materials Science and Engineering, SUNY at Stony Brook, Stony Brook, New York, United States, 2 Oral biology and Pathology, SUNY at Stony Brook, Stony Brook, New York, United States
Show AbstractDental Pulp Stem Cells (DPSCs) are known to differentiate in either bone, dentine, or nerve tissue through different environment signals. In this study we explored whether differentiation could occur in the absence of chemical induction and through mechanical stimuli only. This was accomplished by coating siliocn wafers with Polybutadiene (mw=205,000 mw/mn=1.49) films 20 and 200 nm thick. Scanning force microscopy in the SMFM mode indictaed that the modulus of the thinner film was a factor of 4 higher than that of the thick film, which was bulk-like or G=1MPa. DPSC were then plated on both substrates and cultured with and without dexamethasone induction media. The modulus of the cells was measured on the seventh day and the result showed that in the absence of dexamethasone incudtion media, the cells cultured on the thinner, harder films had a module that was 4 times higher than those cultured on the softer films. Cells cultured with dexamethasone induction media were 1.5 times harder on the thinner films. After 21 days of incubation, SEM analysis indicated that the cells on the thin substrate had produced large amount of calcium phosphate deposites, while those culture on the thicker substrate did not produce any. Cells cultured in the induction media produced biomineralized dpeosites on both types of films, but the amount was larger on the thinner substrates. GIXD was performed on the substrate, where we observed crystalline peaks corresponding to crystalline forms of hydroxyapatite. Our research all indicated that once the cells were induced, either by chemical or mechanical signals to biomineralize, the cells continued to biomineralize even after removal to another substrate. The Real-Time Polymerase Chain Reaction (RT-PCR) assays were also performed on the cells after 21 days of incubation and we found that the expression of osteocalcin, a protein associated with biomineralizaion, was increased for all samples that were cultured on the harder thin films, again confirming the ability of substrate mechanics to induce cell differentiation into osteoblasts.All research was supported in part by the NSF-MRSEC program.
9:00 PM - SS5.2
Endotoxin Detection using an Electrochemical Method and The Effect of Nanoscale Confinement.
Bothara Manish 3 , Sutapa Barua 1 , Srivatsa Aithal 1 , Lilian Gong 4 , Gaurav Chatterjee 1 , Amrita Malik 5 , Kaushal Rege 2 , Shalini Prasad 1
3 Electrical and Computer Engineering, Portland State University, Portland, Oregon, United States, 1 Electrical Engineering, Arizona State University, Tempe, Arizona, United States, 4 , Wellesley College, Wellesley, Massachusetts, United States, 5 , UC Santa Cruz, Santa Cruz, California, United States, 2 Chemical Engineering, Arizona State University, Tempe, Arizona, United States
Show AbstractThe goal of this project is to design a nanotextured, electrical, label-free detection system for detection of low doses of endotoxins. Endotoxins are large, heat-stable lipopolysacchrides, which are the major component of cell walls of gram negative bacteria. Endotoxin detection have broad food safety and disease diagnostic application.We have developed a label free electrochemical based endotoxin sensor, which detects the endotoxin using Electrochemical Impedance Spectroscopy. In Electrochemical Impedance Spectroscopy the impedance between the electrodes at different frequency points is measured. This frequency response changes as a function of the nanolayers on the electrode, we use this property to detect endotoxin binding to the detector surface. Nanoporous alumina membranes which have been used in water filtration systems, have been engineered to bind and detect endotoxins. We have designed a multi scale architecture detection platform comprising of a base microelectronic platform on to which the nanoporous alumina membranes are overlaid. We have adopted layer-by-layer chemistry to immobilize endotoxins onto the detector surface. The endotoxin in the solution is immobilized on the surface of the electrode for detection using a sandwich of polymers PAA (anionic polymer)-and a cationic polymer. Various cationic polymers that have been synthesized have been evaluated to identify the polymer which demonstrates maximum affinity to endotoxins. These polymers are oppositely charged to that of the endotoxin which makes the endotoxon bind to the nanotextured alumina surface coated with the cationic polymer. The nanoporosity of the alumina membrane which was functionalized with these polymers contributes towards enhancing the sensitivity of detection. This increased sensitivity can be attributed to the increase in the surface area and increase in the number of binding sites within the membrane. We have been able to correlate endotoxin binding to specific impedance changes which can be used to detect and quantify the presence of endotoxins.
9:00 PM - SS5.20
Electrical Control of Cell-Adhesion and Cell-Growth Gradients on a Conducting Polymer Surface.
Alwin Wan 1 , Daniel Brooks 2 , Abdurrahman Gumus 1 , Claudia Fischbach 2 , George Malliaras 1 3
1 Materials Science and Engineering, Cornell University, Ithaca, New York, United States, 2 Biomedical Engineering, Cornell University, Ithaca, New York, United States, 3 , Centre Microelectronique de Provence, Gardanne France
Show AbstractOrganic electronic materials are uniquely suited for bioelectronics research, where their properties facilitate the study and understanding of the biological/electronic interface. In particular, the electrochemically-tunable properties of conducting polymers make them promising candidates as “active” growth substrates for cell-culture studies. We report the use of a thin film of the conducting polymer poly(3,4-ethylenedioxythiophene) (PEDOT) doped with p-toluenesulfonate (Tosylate, TOS) as an “active” growth substrate for normal and cancerous cell lines. A redox gradient is established in the PEDOT:TOS film with an applied bias, and cells are cultured on the polymer surface. The results show that electrochemically altering the properties of the polymer substrate affects the adhesion and growth characteristics of both normal and malignant cells, as well as the preferential adsorption of relevant adhesion proteins to the polymer surface.
9:00 PM - SS5.21
Cytocompatibility of a-C:H:N Films Deposited on Polymeric Fibrous Scaffold.
Atsuko Toriu 1 , Haruki Matsuo 2 , Kazuhiro Nonaka 2 , Yasuharu Ohgoe 2 , Kenji Hirakuri 1 , Akio Funakubo 2 , Yasuhiro Fukui 2
1 , Tokyo Denki University - Tokyo, Tokyo Japan, 2 , Tokyo Denki University - Hatoyama, Hatoyama Japan
Show AbstractAn extracellular matrix and scaffold engineering from polymeric materials have been widely useful for tissue engineering. However, the polymeric scaffold materials have to be improved cytocompatibility because of very low cellular adhesion. On the other hand, hydrogenated amorphous carbon (a-C:H) film coatings have attracted attention for biomedical applications due to their good biocompatibility including cytocompatibility, chemical stability and high anti-corrosion. Therefore, a-C:H film coatings is one of the surface modification technology for polymeric scaffold materials. As addition of nitrogen to a-C:H films improves biomedical property, it is also expected to enhance cytocompatibility. In this study, we focused on cellular adhesion property of a-C:H:N film deposited on a segmented polyurethane (SPU) fibrous scaffold to improve the cytocompatibility of scaffold material for tissue engineering.a-C:H:N film was deposited on SPU fibrous scaffold by using a convenient r.f. plasma CVD method. The r.f. power was constantly kept at 100 W for decomposition of source hydrocarbon gas (CH4) and nitrogen gas (N2) as a function of gas ratio between the CH4 and N2 in 1 to 0, 4 to 1, 3 to 2, 2 to 3 and 1 to 4. The a-C:H:N film was deposited under the following condition as total gas pressure (CH4 and N2) of 100 Pa and deposition time of 5 minutes. The a-C:H:N films were uniformly deposited on each fiber of the SPU fibrous scaffold.Characteristics of the a-C:H:N films deposited on the SPU fibrous scaffold were evaluated by using a X-ray photoelectron spectrometer with Mg Kα radiation (XPS), an Ar-ion laser Raman spectrometer (Raman), scanning electron microscope (SEM) and the contact angle measurement with pure water of 2μL. To estimate of cytocompatibility, the mouth fibroblasts (NIH-3T3) cells in the D-MEM solution were cultured in each sample for 4 days.As a result of the cell culture, the number cells of cells on the scaffold with the a-C:H:N is higher than that of the a-C:H film. In the C-N peak intensity of XPS spectra, the bonds and hydroxyl functional group increased with the nitrogen concentration. It was found that the introduction of nitrogen gas leaded the addition of nitrogen atom to the films and cytocompatibility was improved by the C=N arrangement on the surface. The a-C:H:N film coating is expected as a surface modification technique for coating polymeric scaffold materials.
9:00 PM - SS5.24
Comparison of Cell Response to Different Sources of Type 1 Collagen.
Tighe Spurlin 1 , Anne Plant 1
1 Biochemical Sciences Division, National Institute of Standards and Technology, Gaithersburg, Maryland, United States
Show AbstractThe majority of cell-based assays performed to elucidate cellular pathways and responses to drugs and toxins have been performed in cells on plasma treated polystyrene dishes or ill-defined extracellular matrix (ECM) protein coatings. In many studies, cell responses have been shown to be sensitive to surface topography, protein coverage, surface chemistry, extracellular matrix composition, supramolecular structure, and mechanical features. Type I collagen gels have been widely used as model ECM systems for cell studies, since collagen is a highly prevalent ECM in vivo. However, gels are fragile structures that are difficult to characterize and use with quantitative optical microscopy. We have previously shown that thin films of Type I collagen fibrils can be used to alleviate the difficulties associated with collagen gels. Thin films of collagen fibrils have been shown to provide cells with an ECM environment that induces cell responses that are indistinguishable from those seen in cells on the thicker gels in terms of spreading, proliferation, integrin ligation, and tenascin-C expression. Thin films of collagen are highly reproducible, but we have up to now only used one source of Type I collagen to prepare them. To determine if different sources of Type I collagen can be used to create a robust, reproducible, and standardized biomimetic surface for cell assay applications, we prepared films using type I collagen from different manufactures and characterized them with atomic force microscopy and optical microscopy. Results from this series of studies illustrate that collagen films created using the same protocol but different comparable sources of collagen show fibril content that ranges from 43.4 % ± 1.32 % to 6.6 % ± 0.6 %. In initial studies we have observed that vascular smooth muscle cell spread area can differ dramatically from 2043 μm2 ± 121 μm2 to 6616 μm2 ± 237 μm2 depending on the source of the Type I collagen used. The findings suggest that different Type 1 collagen preparations are not identical, and highlight the importance of carefully characterizing and reporting the characteristics of the surfaces that cells are grown on during biological assays.
9:00 PM - SS5.25
A Microfluidic Chip for Analysis of Mechanical Forces Generated During Cell Migration.
Xiaoyu Zheng 1 , Xin Zhang 1
1 , Boston University, Brookline, Massachusetts, United States
Show AbstractCell migration is a microscopic in vivo process where specific cells crawl in order to partake in crucial physiological functions relating to embryonic development, wound healing, and tissue development. Abnormalities of cell migration result in pathologies such as tumor metastasis, angiogenesis, chronic inflammation, and various immune response dysfunctions. The mechanism behind cellular migration and the role of intracellular proteins in the instigation of cell directionality remains poorly understood without effective biomedical device available. The development of microfluidic biochips technologies enable detection, sample preparation and treatment on one single chip. We are reporting the design and fabrication of a novel microfluidic trip for guiding and quantifying cell migrations. The chip featured micropillar arrays imbedded in a multichannel microfluidic chip, where cell migration can be guided by utilizing the characteristics of laminar flow. Non-blending layers of fluid injected through the multi-channel device simulated a wounded edge across a monolayer of cells by limiting flow of trypsin, a serine protease, to half of the main channel, promoting cell migration in a desired direction. Control over cell directionality allows for the measurement and analysis of mechanical forces generated during cell migration in relation to migratory responses from intracellular protein inhibition. The micro-fluidic chip template was designed and manufactured using photolithography techniques. Polydimethylsiloxane (PDMS) served as the bulk material of the two compromising chip layers (channels and pillars), which were subsequently aligned and adhered to form the device. It was confirmed through both computer simulation and experimentation that the through optimized arrangement of the chip design, this device can effectively hold laminar flows of trypsin and cell media. Thus, this microfluidic device allows the user to simultaneously acquire force data during cell migration and observe migratory patterns to ultimately gain a better understanding of the underlying mechanisms of cell migration and directionality.
9:00 PM - SS5.26
The Effects of Viscoelastic Polymer Substrates on Adipose-derived Adult Stem Cell Differentiation.
Chungchueh Chang 1 , Marcia Simon 2 , Miriam Rafailovich 1
1 Materials Science and Engineering, SUNY at Stony Brook, Stony Brook, New York, United States, 2 Oral biology and Pathology, SUNY at Stony Brook, Stony Brook, New York, United States
Show AbstractAdipose tissue is an abundant, accessible, and replenishable source of adult stem cells that can be isolated from liposuction waste tissue by collagenase digestion and differential centrifugation. These adipose-derived adult stem cells are multipotent, differentiating along the adipocyte, chondrocyte, myocyte, neuronal, and osteoblast lineages. Adipose-derived adult stem cells have potential applications for the repair and regeneration of acute and chronically damaged tissues. Adipose-derived adult stem cells were plated on different thicknesses of spun-cast polybutadiene (PB) thin films and cultured in standard media. After 14 days incubation, the moduli of the cells were measured, and no significant differences were found between cells cultured on hard (20nm) and soft (200) substrates. On the other hand, SEM analysis indicated that the cells incubated on the hard substrates deposited a thick layer of carbonatious materials with embedded hydroxyapatite deposits, even in the absence of induction media. Cells cultured on the soft substrates did not have any deposites in the absence of induction media. Those cultured in osteogenic culture media produced biomineralized deposits on all substrates. GIXD was also performed on the substrates and despite the presence of the calcium phosphate deposits, no crystalline peaks corresponding to hydroxyapatite were observed, indicating that some forms of amorphous materials were being deposited.All research was supported in part by the NSF-MRSEC program.
9:00 PM - SS5.27
Probing Bioadhesive Performance for Delivering Active Materials to Targeted Biological Surfaces.
Chung-Yi Chiang 1 , Selina Moses 1 , Tao Wu 1
1 Molsaic IV, Johnson and Johnson, Skillman, New Jersey, United States
Show AbstractIn cosmetic science, surface delivery and retention of active materials is of great interest in technology research and product development. Consumer products such as lotions, make-ups, nail polish, lipsticks and sunscreens are complex materials delivery systems applied by consumers every day. Functionalized particles or formulized actives are employed in these products to provide desired effects such as smoothness, brightness, covering and protection. The surface property of the active material is often altered to provide required compatibility with the delivery vehicle and to impart desired durability on the targeted biological surfaces, such as skin and hair. Traditional cosmetic technology generally involves delivering particles in wax, polymer, oil and solvent, which sometimes result in decreased surface permeability, premature dryness and allergic reactions. To solve these issues, natural and biocompatible adhesives such as proteins (e.g. gelatin) and carbohydrates (e.g. starch) have been commonly used in cosmetic products. New adhesives and formulations are continually researched and developed to improve the clinical performances and properties of consumer products. On the other hand, commercialized measurement tools and benchmarking methods are highly demanded in cosmetic science to investigate and optimize the delivery efficacy and retention performance. Traditional methods such as spectroscopy to evaluate the delivery and retention usually lack of direct measurements on mechanical retention of delivered materials. In this presentation, methods utilizing atomic force microscopy (AFM) to measure the molecular binding/retaining forces between delivered cosmetic materials and targeted biological surfaces (skin and hair) will be demonstrated. Depending on the end-use purpose, the force measurement is carried out at ambient temperature in air or liquid, using the fluid cell to create the desired environment to simulate the product applications. Various bio-adhesives and formulations are also used to model the delivery systems and to measure the adhesive forces against various surfaces relevant to consumer applications.
9:00 PM - SS5.28
In vitro Antimicrobial Susceptibility of S. aureus to Lysostaphin-coated Hernia Repair Meshes.
Rohan Satishkumar 1 , Yuliya Yurko 4 , John Shipp 3 , Amy Lincourt 2 , Todd Heniford 2 , Alexey Vertegel 1
1 Bioengineering, Clemson University, Clemson, South Carolina, United States, 4 , Maine Medical Center, Portland, Maine, United States, 3 , VI MedRX, LLC, Charlotte Amalie Virgin Islands (U.S.), 2 , Carolinas Medical Cenyter, Charlotte, North Carolina, United States
Show AbstractHernia repair is one of the most common interventions in general surgery. Although hernia repair with mesh has been shown to decrease the rate of hernia recurrence, an ideal material for hernia repair has not been developed yet, and there is still a significant risk of morbidity. One of the most important issues is mesh infection. The reported incidence of mesh-related infection following hernia repair has been 1%-18% in different studies. In a study of mesh-related infections [1], 63% of the organisms isolated were methicillin-resistant S. aureus. Currently, there is a constant need for new antistaphylococcal drugs owing to the development of antibiotic-resistant strains. Natural antimicrobial enzymes have recently attracted much attention as promising candidates for antibiotic-free treatment. Lysostaphin is an antibacterial enzyme which specifically cleaves cell walls of S. aureus, thereby lysing the bacteria. Antimicrobial coating of medical devices/implants has recently emerged as a potentially effective method for preventing infections. Previously, lysostaphin-coated catheters were shown to be effective against S. aureus in vitro [2]. The goal of the proposed study is to evaluate in vitro antimicrobial activity and cytotoxicity of lysostaphin-coated meshes (LCSM) for hernia repair. Lysostaphin was successfully attached to polypropylene mesh using physical adsorption and incubation in fetal calf serum using a patent pending protocol. Enzyme-coated samples were then incubated with a suspension of S. aureus containing ~ 10^8 colony forming units per mL at 37C, and the rate of bacterial lysis was measured by monitoring optical density of bacterial suspension at 600 nm. Rapid decrease of turbidity was observed for all LCSMs. In vitro cytotoxicity of the samples was evaluated using human 3T3 fibroblast cell culture by monitoring intracellular levels of a proinflammatory cytokine, IL-8, using a commercially available IL-8 ELISA kit. IL-8 levels in fibroblasts in contact with LCSMs was not significantly different from those in contact with uncoated mesh (p <0.05). An in-vivo study with 6 Lewis rats was performed to assess safety/toxicity and determine antibacterial lysostaphin activity post-implantation. LSCMs were implanted subcutaneously. At 8, 12, 24, and 48 hours, mesh was procured and incubated with ~108 colony forming units of S. Aureus. The residual antibacterial activity was determined by monitoring optical density of the bacterial suspension. In animals, no adverse effects to LSCM were observed. LSCM retained considerable antibacterial activity up to 48 h post-implantation. These preliminary studies demonstrate that lysostaphin-coated meshes can potentially be used as antibacterial materials for hernia repair.References1. Cobb WS et. al, Am Surg 2003; 69: 7842. Shah A et. al, Antimicrob Agents Chemother., 2004; 48, 2704
9:00 PM - SS5.29
HGF Adhesion on Modified Surfaces of Zirconia Based Framework Cores.
Alejandro Pelaez-Vargas 1 2 3 , Luis Restrepo 3 , Maria Fernandes 4 , Derek Hansford 2 , Fernando Monteiro 1
1 , INEB - Instituto de Engenharia Biomédica, Divisão de Biomateriais, Universidade do Porto, Portugal and Departamento de Engenharia Metalúrgica e Materiais, FEUP - Faculdade de Engenharia, Universidade do Porto, Portugal., Porto Portugal, 2 Department of Biomedical Engineering, The Ohio State University, Columbus, Ohio, United States, 3 CES-LPH Research Group. School of Dentistry, Universidad CES, Medellin Colombia, 4 Laboratório de Farmacologia e Biocompatibilidade Celular. Faculdade de Medicina Dentária, Universidade do Porto, Porto Portugal
Show AbstractIntroduction:Zirconia ceramics, in combination with CAD/CAM techniques, present an appealing choice for partially fixed restorations involving molar, due to their high mechanical performance and aesthetic appearance. However, restorations in anterior areas are a challenge because the pigments, additives and porcelains used may modify any roughness features and the biological response of the restorations. With this in mind, our objective is to compare the adhesion, morphology and proliferation of Human Gingival Fibroblast (HGF) cells cultured on surface modified zirconia based framework cores (ZBFC).Methods:Thirty-six ZBFC were produced using CAD/CAM technology (Procera) and divided into three groups (n=12): (AZBFC) Aluminum Oxide Veenering Porcelain (NobelRondo) on ZBFC; (PZBFC) Pigment Ceramic Layer (NobelRondo) on ZBFC; and ZBFC without modification as a control group. AZBFC samples were prepared by a multilayer technique including baseline, dentin porcelain, enamel porcelain, and glass layers application. In the PZBFC group, a single body stain Zirconia layer was applied on ZBFC. All thermal cycles followed the manufacturer instructions. SEM/EDS and AFM were used for chemical and surface characterizations. In vitro biological evaluation used HGF cells, cultured according to a standardized protocol for four days. Morphology and proliferation were evaluated by optical microscopy. Results:Elemental analysis by EDS showed differences among the surfaces of the three groups evaluated in agreement with different applied layers. AFM analysis showed the following order of roughness values: ZBFC>PZBFC>AZBFC. HGF presented a normal morphology on all surfaces, at 4h, 24h and 4 days. HGF proliferation using MTT assay did not reveal any statistically significant differences at intergroup analysis and showed statistical significant differences for 24h and 4 days at intragroup analysis.Conclusions:These preliminary studies revealed that ZBFC modified with body stain or alumina veneering porcelain did not induce morphological or proliferative changes on HGF cultures.Acknowledgment:The PhD grant FCT/SFRH/BD/36220/2007 is acknowledged.
9:00 PM - SS5.3
Silver Adsorbed Biodegradable PHBV Matrix.
Chandra Bhan 1 , Almaz Gebregeorgisa 1 , Sonia Arneja 1 , Lindsay Moore 1 , Dharmaraj Raghavan 1
1 Chemistry, Howard University , Washington, DC 20059, District of Columbia, United States
Show AbstractOsteomyelitis treatment involves surgical intervention of dead bone tissue and regulated dosage of antibiotics. The primary objective of the study is to formulate antibiotic loaded biodegradable polymeric film that favors bone tissue growth and inhibits bacterial infection. In this study, an attempt was made to functionalize biocompatible poly hydroxyl butyrate valerate (PHBV) films, so as to attach silver ions to the film. The selection of silver ion was based on well established antimicrobial characteristics. The synthesis of functionalized PHBV involves a 2-step process of initial activation, followed by graft polymerization of methacrylic acid to render the PHBV film hydrophilic. The density of the carboxylic acid groups grafted on PHBV film was quantified using a spectrophotometric method at 633 nm by measuring the complexed Toluidine Blue O. The grafted PHBV film and methacrylic acid solution was alkali treated, washed, and equilibrated with silver nitrate solution, and the adsorption of silver ion was followed by Fourier transform infrared spectroscopy (ATR-FTIR). Preliminary FTIR results show a shift of the carbonyl peak and a reduction in the intensity due to adsorption of Ag+ ions.
9:00 PM - SS5.30
Carbon Nanotube Nano-probe for Neural Recording.
Huan-chieh Su 1 , Chia-Min Lin 2 , Chang-Hsiao Chen 2 , Yung-Chan Chen 3 , Wei-Lun Hsu 4 , Shih-Rung Yeh 4 , Weileun Fang 2 , Da-Jeng Yao 2 , Hsin Chen 3 , Yen-Chung Chang 4 , Tri-Rung Yew 1
1 Department of Materials Science and Engineering, National Tsing-Hua University, Hsinchu Taiwan, 2 Institute of NanoEngineering and MicroSystems, National Tsing-Hua University, Hsinchu Taiwan, 3 Department of Electrical Engineering, National Tsing-Hua University, Hsinchu Taiwan, 4 Department of Life Science, National Tsing-Hua University, Hsinchu Taiwan
Show AbstractA carbon-nanotube (CNT) nano-probe as an electrode is proposed for applications in neural recording. The nano-probe consists of CNTs synthesized on a Si microstructure by chemical vapor deposition (CVD). The improvement of CNT characteristics by plasma treatment to modify CNT surface will be presented. The electrochemical characterization in phosphate buffered saline (PBS) of CNT nano-probes reveals low impedance at 1 kHz and high specific capacitance. The biocompatibility test by neuron culturing on CNT nano-probes will be also presented. In addition, the CNT nano-probe has been employed to record the neural signals of a crayfish nerve cord for in vitro recording. Our findings here suggest that CNT nano-probes are durable and suitable for future biological applications.
9:00 PM - SS5.31
Use of an In Vitro Glial Scar Assay to Assess Neurocompatibility of Vapor Deposited Silicone Coatings.
Anil Kumar Achyuta 1 , Vadim Polikov 3 , Aleksandr White 2 , Hilton Pryce Lewis 2 , Shashi Murthy 1
1 Dept of Chemical Engineering, Northeastern University, Boston, Massachusetts, United States, 3 Dept of Biomedical Engineering, Duke University, Durham, North Carolina, United States, 2 , GVD Corporation, Cambridge, Massachusetts, United States
Show AbstractVapor deposited silicone coatings are attractive candidates for providing insulation in neural probes owing to their excellent ability to coat small, three-dimensional substrates conformally. Biocompatibility assessment of these coatings is an essential part of the materials design process for this application but current techniques are limited to rudimentary cell viability assays or animal muscle implantation tests. This work demonstrates how an in vitro model of glial scar formation can be utilized as a platform to assess the biocompatibility of vapor-deposited silicone coatings on micron-scale metallic wires. Stainless steel microwires were coated with two novel insulating thin film silicone polymers: a homopolymer coating made from trivinyl-trimethyl-cyclotrisiloxane (V3D3), and a copolymer coating made from V3D3 and a spacer molecule, hexavinyl disiloxane (HVDS). Both coatings, namely polyV3D3 and polyV3D3—HVDS, respectively, were synthesized by initiated chemical vapor deposition (iCVD) technique. A multi-cellular confluent layer of astrocytes and microglia was obtained by culturing mechanically dissociated midbrain cells from E-13/14 Fischer 344 rats on poly-D-lysine coated well plates. The confluent monolayer of astrocytes and microglia was disrupted by placing segments of silicone-coated microwires following 10 days of culture, in vitro. The cells were allowed to grow for 7 days in the presence of coated microwires prior to immunocytochemistry. The microglial proximity to the microwires was shown to be a function of amount of fibronectin adsorbed on the coating material surface where polyV3D3—HVDS adsorbed least amount of fibronectin compared to stainless steel and polyV3D3 (p < 0.05). Consequently, the relative number of microglia within a close proximity of polyV3D3—HVDS coatings was diminished compared to the control and polyV3D3 (p < 0.05). In addition, the astrocyte reactivity on polyV3D3—HVDS coatings was lower (p < 0.05) compared to its silicone cohort and stainless steel control and was therefore determined to be relatively more neurocompatible.
9:00 PM - SS5.34
Chinese Hamster Ovary Cells Grow Unimpeded on Bare Hydrogen and Oxygen Terminated Diamond Surfaces.
Nick Smisdom 1 , Ilse Smets 2 , Oliver Williams 3 , Michael Daenen 4 , Sylvia Wenmackers 3 , Ken Haenen 5 , Milos Nesladek 5 , Jan D'Haen 5 , Patrick Wagner 5 , Jean-Michel Rigo 1 , Marcel Ameloot 1 , Martin vandeVen 1
1 Biomed Res. Inst. , Hasselt University, Diepenbeek Belgium, 2 PHL-Bio, PHL University College, Diepenbeek Belgium, 3 Institute for Materials Research, Hasselt University, Diepenbeek Belgium, 4 IMO & Dept Industrial Sciences and Technology, Hasselt University & XIOS Hogeschool, Diepenbeek Belgium, 5 IMO & Division IMOMEC, Hasselt University , Diepenbeek Belgium
Show AbstractChinese Hamster Ovary (CHO) cells were cultured on hydrophobic hydrogen terminated (HT) and oxygen terminated (OT) diamond surfaces. The influence of these otherwise bare surfaces on cell morphology, density, viability and proliferation were investigated. Microwave plasma-enhanced chemical vapor deposited thin diamond films were grown on silicon. The influence of nano- and polycrystalline diamond was compared with cells grown on glass control substrates. For a total of 7 days cell morphology and density were followed using epi-, dark-field and confocal microscopy with a Zeiss LSM 510 META. Cell viability was evaluated using flow cytometry (living/necrotic/apoptotic cells). Cells were also assessed via several biochemical assays: the MTT cell proliferation assay (proliferation and metabolic activity), the Bradford assay (total protein content), the [3H]-thymidine cell proliferation assay (proliferation). It can be concluded that compared with glass control substrates for these CHO cells bare nano- and microcrystalline (NCD and MCD) diamond surfaces affect growth and viability only in a minor way if at all. Scanning electron and optical microscopy as well as biochemical analysis show that cell density nor morphology were hindered by the grain size and surface termination. Differences between the mean parameter values obtained for control glass substrates and the diamond substrates, i.e. HT/OT-NCD and HT/OT-MCD were insignificant (one-way ANOVA). Grain size nor surface termination had a significant influence after 5 days (two-way ANOVA). Therefore these diamond surfaces seem easiest to grow and functionalize for the development of non-invasive biosensor interfaces. Funded by the Research Council of UHasselt, tUL, K.U.Leuven (GOA/2006/02), a Ph.D grant of the Inst. for the Promotion of Innovation through Science and Technology in Flanders (IWT-Vlaanderen), IAP P6/27 Functional Supramolecular Systems (BELSPO), FWO-onderzoeksgemeenschap Scanning and Wide Field Microscopy of (Bio)-organic Systems.
9:00 PM - SS5.35
Detection of Nucleoside Triphosphate on Phenylboronic Acid-modified Diamond Surface by Optical and Potentiometric Methods.
Hirofumi Arai 1 , Shinya Tajima 1 , Ishiyama Yuichiro 1 , Kyosuke Tanabe 1 , Yoko Ishii 1 , Hiroshi Kawarada 1
1 Advanced Science and Engineering, Waseda University, Tokyo Japan
Show Abstract1. The Principle of Nucleoside Triphosphates Detection by Phenylboronic Acids on Diamond Surface Diamond is known as a physicochemically stable and biocompatible material. So, it has been applied to the substrate for biosensor [1]. In addition, surface conductive diamond films, undoped hydrogen-terminated (H-terminated) diamond, can be used as solution gate field effect transistors (SGFETs). The diamond SGFETs have a characteristic of highly sensitive detection of charged biomolecules because these molecules can be directly immobilized on the channel surface without linker molecules. In this study, we tried to detect cytidine triphosphate (CTP), a kind of nucleoside triphosphates (NTPs), which is captured by phenylboronic acids fixed on the diamond surface by fluorescence observation and potentiometric detection with SGFETs. As recognition molecule of CTPs, 3-carboxyphenylboronic acid (CPBA) was introduced onto the diamond surface. Phenylboronic acids have been used for the recognition of the biomolecules with cis-diol structures, like saccharides, by forming the structure of the boronic acid-biomolecule complexes [2, 3]. Then, NTPs have also a cis-diol and can bind to phenylboronic acids. Therefore, phenylboronic acids can be used for the recognition of NTPs. 2. Reproducible Detection of Nucleoside Triphosphates by Fluorescence and Solution Gate FET H-terminated diamond surface with partial amination (coverage: 15-20%) was treated in 10mM phosphate buffer solution (PBS, pH=7.4) with 1mM 3-CPBA, 0.4M EDC and 0.1M NHS for 2hours at room temperature, and 3-CPBA was immobilized directly onto the surface through peptide bond. For binding CTPs to the boronic acids, the surface were immersed in 10mM PBS (pH=7.4) with 100μM CTPs for 30min at 38°C. To dissociate CTPs from the boronic acids, the surface was washed in 1.2M HCl solution for 30min at room temperature. The results of the fluorescence observation indicated that CTPs were captured on the diamond surface and dissociated by washing treatment. By the potentiometric detection using SGFETs in 10mM PBS (pH=7.4) with 1mM NaCl, as a result of CTPs binding the gate potential was shifted positively by ~24mV. After the dissociation of CTPs from boronic acids, the negative shift with ~30mV was observed. These results are considered that the positive shift in the gate potential was induced by CTPs on the surface. We achieved the detection of CTPs on diamond surface by both optical and potentiometric methods. It suggests that other NTPs (ATP, UTP, GTP) can be recognized and the lower concentration of NTPs can be also detected by the same methods.[1] S.Kuga, H.Kawarada et al., J. am. Chem. Soc. 130, (2008) 13251. [2] L. I. Bosch, T. D. James et al., Tetrahedron 60, (2004) 11175. [3] A. Matsumoto, Y.Miyahara et al., J. Solid State Electrochem. 13, (2009) 165.
9:00 PM - SS5.36
AFM Force Spectroscopy on TAT Membrane Penetration.
Elizabeth Hager-Barnard 1 , Benjamin Almquist 1 , Nicholas Melosh 1
1 Materials Science and Engineering, Stanford University, Stanford, California, United States
Show AbstractCell penetrating peptides (CPPs) are a remarkable class of hydrophilic molecular agents that are able to transfer cargo across cell membranes. Despite the significant body of research on CPPs like TAT, a positively charged 9-mer with six arginine groups, their behavior remains controversial. Whether TAT actually lowers the energy barrier for membrane penetration or is transported via an indirect pathway is a crucial question to unraveling the mechanism of TAT translocation. Direct measurement of TAT-lipid mechanics during the actual translocation event is an ideal method to elucidate the interaction forces, mechanisms and timescales of membrane penetration. We used atomic force microscopy (AFM) on lipid bilayer stacks with TAT-functionalized probes to monitor both the TAT position within a single bilayer and the associated force with microsecond resolution. These AFM measurements directly quantify the mechanics of TAT penetration and show that TAT by itself does indeed alter the membrane structure over a timescale of 0.5ms. These results corroborate many of the conclusions from molecular dynamics simulations on TAT-lipid systems, which indicate that the arginine groups in TAT interact with lipid phosphate head groups to compress the bilayer and facilitate penetration.
9:00 PM - SS5.37
Electrically Triggered Release of Nanoparticles from Liposomes for Controlled Release Applications.
Dayane Tada 1 , Evin Gultepe 1 , Dattatri Nagesha 1 , Francisco Reynoso 1 , Srinivas Sridhar 1
1 Eletronic Material Research Institute, Department of Physics, Northeastern University, Boston, Massachusetts, United States
Show AbstractLiposomes are widely used as drug carriers and nanoparticles (NP) carriers for therapeutic applications in order to improve NP circulation and to avoid non-specific binding. Liposomes also enable sustained release of drugs at the disease site under physiological conditions. Here we present the controlled release from liposomes by application of electric fields. When an electric field is applied to lipid membranes it causes asymmetric tilting of the dipolar phospholipid headgroups, forming hydrophilic pores. This process, called electroporation, has been used to increase drug transport through tissues and into the cells. Although an electric field of at least 10V/cm is required to increased permeation of cellular membrane, we showed that an electric field twenty times lower is enough to induce NP release from liposomes.In the present work, magnetic cationic liposomes (MCL) were prepared by encapsulating Fe3O4 NP in liposomes composed by DMPC:DMTAP:CHOL. The electroporation of MCLs was analyzed through light scattering measurements of MCL suspension after current application (2mA AC; 20Hz; 0.5V/cm). The average diameter (145nm) as well as polydispersity of the MCL increased with time until it reached a maximum value (310nm), and then for longer times, smaller structures (90nm) were detected. These results can be explained by the fact that under electrical fields, membrane fluidity is increased resulting in pore formation and liposome swelling. After they reach a critical size, liposomes are completely disrupted and re-assembled in smaller structures of different composition. The NP release from liposomes induced by electrical field was determined after centrifugation of MCL under 3100rpm. At this speed, uncoated magnetic NP precipitate while coated NP stay in suspension. Precipitated NP were dissolved in acid and the iron content was determined using phenanthroline colorimetric method. We find that short time current (30sec) releases 30% of NP from the liposomes. This value increased with time and after 5 minutes, 90% of NP were released. The high release after 5 minutes was already expected since at this time the highest liposome size was detected which reflects the increased permeation of the membrane that facilitates the NP release. These results show that low electric fields can lead to the poration of liposomes inducing the release of nanoparticles from the interior. The NP release can be controlled by adjusting the time of current application. Our results show that electroporation of NP loaded liposomes is a useful method for controlled or on-demand release systems. Work supported by Nanomedicine Science and Technology (NSF-0504331).
9:00 PM - SS5.39
Understanding the Interaction Between Graphene Oxide-based Nanomaterials and Mammalian Cells.
Soo-Ryoon Ryoo 1 , Young-Kwan Kim 1 , Mi-Hee Kim 1 , Dal-Hee Min 1
1 Chemistry, Korea Advanced Institute of Science and Technology, Daejeon Korea (the Republic of)
Show AbstractCarbon based nanomaterials have shown much interest due to their unique structural and electrical properties such as mechanical strength, flexibility, electrical transport capability, young’s modulus, lightness and chemical inertness. Especially, graphene has the infinite possibilities to serve as novel nanoscale building blocks to create distinctive macroscopic materials. Due to their outstanding thermal and mechanical properties and high electrical conductivity, graphene sheets have been considered as a promising candidate for nanoelectronic devices, quantum computer, transparent electrode, and nanocomposite materials. Conductive substrate show great potential in biological applications such as tissue engineering, implants, drug delivery carriers, biochips for diagnostics and nano-devices for biological study. Substrates for immobilizing cells and tissues are valuable in use of biological and medical field study. The adhesion and spreading of mammalian cells is mediated by the binding of cell-surface integrin receptors to peptide ligands from the extracellular matrix (ECM) and the clustering of these receptors into focal adhesion complexes. Integrins play a critical role in the formation of focal adhesions, which attach cells to the extracellular matrix. It has been reported that the interaction between cells and ECM depends on the multiple substrate characters such as chemical composition, geometry, and topological aspects, ligand organization, and substrate stiffness. In addition, these factors of engineered substrates based on nanomaterials can affect and even lead to various cellular responses and cell physiology. Little is to investigate their properties that make the influence of carbon based nanomaterials including graphene sheet on living system. While the advancements in technology may be considerable, there is also concern about unintended effects of exposure to nanomaterials. In this work, the chemically modified graphene oxides were immobilized on the glass substrates and used as a substrate for mammalian cells as a model of biological system to examine their influence on cell adhesion, spreading pattern and proliferation by various assays. We believe our result could serve as a fundamental standard for biological investigation of chemically prepared graphene-based nanomaterials.
9:00 PM - SS5.4
Fiber-based Biosensors for Self-diagnosis of Bacterial Vaginosis.
Alexey Vertegel 1 , Vladimir Reukov 1 , Victor Maximov 1 , Oleksandr Burtovyy 2 , Igor Luzinov 2 , Konstantin Kornev 2 , Paul Miller 3
1 Bioengineering, Clemson University, Clemson, South Carolina, United States, 2 Materials Science and Engineering, Clemson University, Clemson, South Carolina, United States, 3 OB/Gyn, Greenville Hospital System, Greenville, South Carolina, United States
Show AbstractBacterial vaginosis (BV) is the most common vaginal infection in women of childbearing age. BV is an infection caused by a change of normal vaginal bacterial flora, characterized by profuse vaginal discharge. In the United States, 29% of women between the ages of 14 and 49 years and as many as 16 % of pregnant women have BV. In pregnant women, the presence of BV is strongly associated with preterm birth, a risk that is mitigated by early diagnosis and treatment with oral clindamycin. Thus, it is highly desirable to have a simple non-invasive self-diagnostic test that would enable a patient to monitor her BV status on a regular day-to-day basis. Such a test would be especially valuable because in ~50% of cases, BV patients have no noticeable vaginal discharge, thus leading to delayed diagnosis and potentially dire consequences later, especially for pregnant patients.One approach to the diagnosis of BV is based on the detection of the enzyme sialidase produced by bacteria that are not normally present in the vagina. A number of colorimetric tests, such as recently FDA-approved BVBlue, have been proposed for detection of sialidase. Use of these tests, however, requires patient’s visit to a doctor’s office.Our project focuses on the fabrication and testing of surface-modified fibers suitable for early colorimetric detection of BV. Such fibers can be incorporated into female panty liners or used for point-of-care analyses. More specifically, since most of colorimetric substrates for sialidase contain a negatively-charged D-N-acetylneuraminic acid moiety, we chemically modified the surface of the fibers by covalent attachment of a polycationic polymer, which serves as the anchor for electrostatic attachment of the negatively-charged sialidase substrate.Because of higher sialidase concentrations in BV discharge and increased amounts of discharge in most of the patients, such fibers were expected to change their color if BV is present. Since panty liners are used by women on a day-to-day basis, availability of such an affordable test would enable early detection and treatment of BV. Our preliminary experiments show that colorimetric sialidase substrate can be readily and with high yield immobilized on polymeric fibers surface-modified by covalent attachment of a cationic polymer. The fibers change color from white to bright blue in the presence of sialidase. Pilot study with human subjects showed 70% coincidence of the results obtained using unoptimized chemically modified fibers with the results of the Amsel test, currently used as a “gold standard” for BV diagnosis. Further research is needed to optimize the amount of the substrate attached to the fibers to achieve higher specificity of the test. More broadly, the results obtained during this study will serve as the springboard for the development of a novel family of active biosensors embeddable in everyday household items.
9:00 PM - SS5.42
Ion Pairing and Hydration in Polyelectrolyte Multilayer Films Containing Polysaccharides.
Thomas Crouzier 1 , Catherine Picart 2
1 UMR5235 DIMNP, Université Montpellier 2, Montpellier France, 2 LMGP, INPG, Grenoble France
Show AbstractThe internal structure and growth properties of polyelectrolyte multilayers are an intensive field of research since the past decade. Yet, little is known about the internal ion pairing within the films. In this study, thin films constituted of poly(Lysine) (PLL) as polycation and of the anionic polysaccharides hyaluronan (HA), chondroitin sulfate (CSA), and heparin (HEP) as polyanions with increasing sulfate contents have been investigated. Our aim was to study the influence of the COO- and SO3- groups on i) film growth ii) water content (film hydration) iii) ion pairing in polysaccharide-based thin films.Film buildup in physiological solutions was followed in situ by quartz crystal balance with dissipation monitoring (QCM-D) and infrared spectroscopy (ATR-FTIR). Based on calibration curves, ATR-FTIR allows an unambiguous quantification of the total polyelectrolyte adsorbed amounts and of the precise amount of each type of group (sulfate, carboxylate, ammonium) present in the dissacharide units. Thanks to the possibility to selectively cross-link carboxylate and ammonium ions via carbodiimide chemistry, the COO-/NH3+ and SO3-/NH3+ ion pairing was determined. PLLFITC diffusion in the film was also measured by fluorescence recovery after photobleaching (FRAP) measurement.HA and CSA-based films were the most hydrated ones with the water content estimated at 76 and 63% respectively and their growth was of exponential type. HEP based films were much less hydrated (20% hydration) and presented a close to linear growth curve. The monomer ratio (disaccharide/lysine) was very similar for all the films whatever the polyanion, and tended toward a plateau value at 0.5, indicating that there are 2 lysine molecules per disaccharide monomer. Similarities between films might be due to the similar structures of the polyanions governing PLL arrangement “around” each dissacharide monomer. We found that 46% of NH3+ groups from PLL are unpaired (i.e. extrinsically compensated by counterions) in HA-based films, 21% in CSA-based films and none in HEP ones. FRAP measurements of PLLFITC diffusion in the films confirmed that PLL diffusion was much higher in PLL/HA and PLL/CSA film than in PLL/HEP. These results also bring an explanation for the differences in growth mode: heparin being tightly interacting to PLL does not allow its diffusion. Thus the films grow more linearly than HA or CSA based films.Hydration, ion pairing and PLL diffusion in polysaccharide based films are interconnected properties that arise from the specific structures of the biomacromolecules constituting the films. These films can be viewed as mimicking the self-assembly processes occurring in vivo in natural extracellular matrices, including complex protein/GAG interactions via the positively charged lysine and arginine groups. This work thus contributes to a better understanding of glycosaminoglycans-based films and of the self-assembly properties of extracellular matrices.
9:00 PM - SS5.43
Plasma Enhanced CVD of Crosslinked Amino Acid Films for Biometallization.
Kyle Anderson 1 , Joseph Slocik 2 , Michael McConney 1 , Jesse Enlow 2 , Rachel Jakubiak 2 , Timothy Bunning 2 , Rajesh Naik 2 , Vladimir Tsukruk 1
1 Materials Science & Engr, Georgia Tech, Atlanta, Georgia, United States, 2 Materials and Manufacturing Directorate, Air Force Reseach Laboratory, Dayton, Ohio, United States
Show AbstractPlasma enhanced chemical vapor deposition (PECVD) has been used to fabricate uniform, ultrathin coatings of amino acids on a variety of different surfaces which allows for further modification of a surface through the reduction of inorganic nanoparticles. Dry tyrosine monomer was directly sublimed into the plasma reactor, which allowed extensive crosslinking of the monomer and subsequent deposition on a variety of substrates including silicon, nitrocellulose, quartz, polystyrene and polytetrafluoroethylene. This allowed for the formation of a robust polyamino acid coating through a solvent-free process. Once deposited, the highly crosslinked tyrosine formed reactive sites which facilitated the reduction of gold nanoparticles onto the surface from aqueous gold chloride solution. Micropatterning of the plasma deposited tyrosine is also shown to direct the formation of the gold nanoparticles on the surface, with a higher particle density occurring on the patterned tyrosine areas. This method of film deposition and biometallization shows that coatings suitable for particle reduction can be utilized on many types of substrates in varying applications.
9:00 PM - SS5.44
Fabrication and Characterization of Bioactive Layer-by-Layer (LbL) Film Using Hevea brasiliensis Latex.
Christiane Davi 1 , Luiz Fernando Dias Galdino 1 , Osvaldo Oliveira 2 , Primavera Borelli 3 , Mariselma Ferreira 1
1 CCNH, UFABC, Santo Andre, SP, Brazil, 2 IFSC, USP, Sao Carlos, SP, Brazil, 3 FCF, USP, Sao Paulo, SP, Brazil
Show AbstractThe control of interactions between living cells and modified surfaces is essential for tissue engineering and may be exploited in the fabrication of bioactive coatings. One possible method to produce such coatings is the layer-by-layer (LbL) technique, based on the spontaneous adsorption of oppositely charged materials onto a substrate, which has been used in various applications [1,2]. Natural rubber (NR) latex, a polydispersive system extracted from the Hevea brasiliensis, comprises 40-45% of 1,4 poly-cis-isoprene, 4-5% of non-rubber constituents such as proteins, lipids and 50% of water. NR has been used in bandages (Biocure®) and is a strong candidate for biomaterials. Proteins extracted from NR latex have been studied using the choriollantoic membranes model, with the results pointing to a strong angiogenic activity [3]. In this work we have studied and compared two different systems of LbL films using NR latex. Thin polymer films were produced by alternating bilayers of natural rubber latex (NR) (0.25 g.L-1) as a negative polyelectrolyte and PEI (0.1g/L, 25.000 g.mol-1 purchased from Sigma Aldrich) and PAH (0.5g/L, 60.000 g.mol-1, purchased from Alfa Aesar) as a positive one. After each layer deposition, the substrate/film system was rinsed in the washing solution and dried under a N2 flow. The same procedure was repeated to deposit 15 bilayers of the PAH/latex and PEI/Latex onto a quartz substrate and the growth of the multilayers was monitored using UV-VIS spectroscopy. This technique showed that the electronic absorbance of the films increased linearly with the number of bilayers, suggesting that the same amount of material was adsorbed at each deposition step and for both evaluated systems. However, higher absorbance values were observed for PEI/NR latex films. Morphological characterization of the films was evaluated by atomic force microscopy AFM (Digital Instruments) and the RMS (root mean square) roughness was calculated by Nanoscope III software. In the films using PEI as positive polyelectrolyte the roughness observed was higher than in the films using PAH. In addition, we evaluated the bioactivity of the films spreading human fibroblast cells (NHF) (10.000 cell/cm2) onto the films and using by MTT essay. These tests revealed that PAH/latex and PEI/latex films can accelerate NHF proliferation greater than control (polystyrene) up to the seventh day. In conclusion, we demonstrated a simple method to produce a cell interactive material from natural macromolecules such as natural rubber using the LbL technique.Acknowledgements: Capes, CNPq, Fapesp. [1] Oliveira Jr, O. N., Zucolotto, V., Ferreira, M., Mattoso, H. C., Riul, Jr, A., Supramolecular Engineer of Conducting Materials, Edit by Manoj Kumar Ram, Kerala, India, 11, 399-425, 2005.[2] G. Decher, Science, 277, 5330 (1997), 1232-1237.[3] Ferreira, M., Mendonça, R. J., Mulato, M., Coutinho Netto, J., Brazilian Journal of Physics, 2009 (in press.
9:00 PM - SS5.5
Computational Design of Oligothiophene Polymer Biomarkers for Fluorescent Labeling.
Bryan Wong 1
1 Materials Chemistry, Sandia National Laboratories, Livermore, California, United States
Show AbstractFluorescent techniques are widely used in cellular imaging and are progressively replacing radioisotopes in other medical diagnostics. In particular, there is a wide interest in using functionalized organic polymers as optical sensors in immunological labeling and as biological optical sensors. Polymers based on oligothiophene esters show great promise as biological probes since they possess high fluorescence efficiencies, good optical stabilities, easy binding affinities to biomolecules, lack of toxicity, and versatile color tunability in the entire visible range. From a practical point of view, one would like to use a computational design beforehand to predict the effect of functionalization on the optical properties of these biomarkers before they are used as probes in fluorescent experiments. In order to tailor these polymer systems with the desired optical properties, we recently utilized novel density functional theory calculations [B. M. Wong et al., Phys. Chem. Chem. Phys. 2009, 11, 4498] to predict their absorbance and fluorescence properties. In contrast to other density functional methods, our approach is capable of accurately predicting optical excitations in these polymers as a function of size and chemical functionalization. Our results allow a guided approach to optimizing the photophysical properties of these polymer biomarkers for use in fluorescent detection applications.
9:00 PM - SS5.6
Electrochemical Stability of Self-assembled Monolayers of PEG-alkanethiols on Gold and Platinum for Biosensor Applications.
Anna Cattani-Scholz 1 , Ruoshan Wei 1 , Felix Reimold 1 , Gerhard Abstreiter 1 , Ulrich Rant 1
1 Walter Schottky Institute, Technical University Munich, Garching Germany
Show AbstractThiolated molecules are routinely used for self assembled monolayers (SAM) on gold surfaces and have found wide applications as basic units in molecular electronics, sensing and recognition devices, actuators and molecular motors. However, the technological application of thiol films as interface systems can be limited due to SAM degradation when the sulfur Au bonds are exposed to ambient conditions or liquid media for many days. Although intense investigations have addressed degradation processes under various buffer conditions, little is known about the stability of thiol monolayers under applied electrochemical potentials, i.e., conditions which are common during the operation of SAM-functionalized electrical devices. To address this issue, we prepared alkanethiol, oligo-ethyleneglycol(EG)-terminated alkanethiol and mixed oligo(EG)-NTA-terminated alkanethiol monolayers on gold and studied their stability against the application of electrochemical potentials in buffered saline by impedance spectroscopy, AFM and XPS. In particular, the properties of the capacitance associated with the different monolayers were assessed by impedance spectroscopy while applying step-like voltage ramps over the range from -1V to 1V. The integrity of the monolayers was further tested by applying a long-term voltage stress of -0.3 V in buffer conditions. The results show different stabilities of the investigated monolayers depending on their chemical structure and film preparation method. In addition to the measurements on gold, the formation and stability of PEG-alkanethiol monolayers was investigated on platinum. We find that the layers remain stable over a wide potential range, although the thiol SAMs on Pt are not as resistant as their counterparts on Au surfaces to the application of high potentials. In conclusion our results show that Pt is an attractive alternative substrate material, yet also that there is a need for novel biofunctionalization strategies with improved electrochemical stabilities against thiolate desorption in technological applications where the molecular integrated system is based on the affinity of sulfur to the metal substrate.
9:00 PM - SS5.7
Biosensor Capture Kinetics Model of Nanocube-Augmented Carbon Nanotube Networks.
Jonathan Claussen 1 , Pradeep Nair 2 , Stephen Hodson 3 , Aeraj Haque 1 , Muhammad Alam 2 , David Porterfield 1 , Timothy Fisher 3
1 Agricultural and Biological Engineering, Purdue University, West Lafayette, Indiana, United States, 2 Electrical Engineering, Purdue Univesity, West Lafayette, Indiana, United States, 3 Mechanical Engineering, Purdue Univesity, West Lafayette, Indiana, United States
Show Abstract Au-coated Pd (Au/Pd) nanocubes connected by a network of single-walled carbon nanotubes (SWCNTs) are employed and exercised as an electrochemical biosensor. As previously reported, these in situ Au/Pd nanocube SWCNT networks are capable of ultrasensitive amperometric sensing of glucose, with a sensitivity, detection limit, and linear sensing range greater than similar nanomaterial-based glucose biosensors. The diffusion of glucose molecules to the Au/Pd nanocube surfaces, forced convection environment of the testing vial, and Brownian motion of the Au/Pd nanocubes are all likely factors contributing to the strong electrochemical performance of the Au/Pd-SWCNT biosensor. In an effort to elucidate the effects of these contributing factors, this work demonstrates an analytical biosensor capture kinetics model that models the analyte-biosensor mass transfer by molecular diffusion and convection due to both the fluid motion within the test vial and the Brownian motion of the Au/Pd nanocubes themselves. The biosensor capture kinetics model incorporates a quasi steady-state integrated incident flux equation to represent mass diffusion of glucose molecules to the surface of 1D planar, 2D nanowire, and 3D nanosphere surfaces in Cartesian, cylindrical, and spherical coordinates respectively. A Burgers vortex model predicts the biosensor diffusion boundary layer formed from fluid downwelling and upwelling within the vial center and boundaries due to the rotation of a magnetic stir bar. Finally the convection-diffusion equation simplified by Stokes flow is utilized to model the diffusion boundary layer of the Au/Pd nanocubes experiencing Brownian motion. Several key conclusions can be interfered from this biosensor capture kinetics model. First, a biosensor under 3D mass diffusion exhibits an analyte concentration flux at least one order of magnitude greater than that of a biosensor experiencing 1D or 2D diffusion in quiescent and convective fluid environments. Additionally, mass transfer by convection increases the concentration flux to the biosensor by impeding the advancement of the analyte depletion layer around the biosensor. Furthermore, the Brownian motion model of the Au/Pd nanocubes is shown to improve mass transfer to the biosensor surface, enabling a substantial increase in amperometric current signal output as compared to a similar electrochemical-based biosensor with immobilized Au/Pd nanocubes. In summary, the results of the biosensor capture kinetics model corroborate the high sensitivities and low detection limits previously observed experimentally by the Au/Pd nanocube-SWCNT biosensor.
9:00 PM - SS5.9
Label-free Electrochemical Impedance Detection of Ovarian Cancer Markers CA-125 and CEA.
Allison Whited 1 , K. Singh 1 , Raj Solanki 1
1 Physics, Portland State University, Portland, Oregon, United States
Show AbstractOvarian cancer is the fifth leading cause of cancer related deaths in women. It is difficult to detect due to its non-specific symptoms and lack of an adequate gynecological exam; as such, a majority of cases are diagnosed in the later stages of the disease, thus leading to a high mortality rate. CA-125 and carcinoembryonic antigen (CEA) are two biomarkers present in blood that can indicate the presence of ovarian cancer. They can also be used, both in conjunction with each other and independently, to determine the effectiveness of the treatment being used to treat the disease. A label-free multiplexed immunosensor was developed to detect both CA-125 and CEA in buffer solution at levels typically seen in patients with ovarian cancer . Electrochemical impedance spectroscopy was used to measure the increase in impedance when a binding event occurred between the target antigen and its specific antibody that was anchored to the surface of an interdigitated electrode array. Such a device could eventually be used in a clinical setting to aid in the diagnosis of ovarian cancer while it still in the early stages of progression and thus lead to an improved prognosis for the disease.
Symposium Organizers
Jose A. Garrido Technische Universitaet München
Erika Johnston Genzyme
Carsten Werner Leibniz Institute of Polymer Research
Thomas Boland The University of Texas-El Paso
SS6: Proteins at Surfaces
Session Chairs
Wednesday AM, December 02, 2009
Ballroom A (Hynes)
9:30 AM - **SS6.1
Bioinspired Immobilization Chemistries for Surface Modification and Biointerfacial Control.
Phillip Messersmith 1
1 Biomedical Engineering, Northwestern University, Evanston, Illinois, United States
Show AbstractMany biological organisms rely on temporary or permanent adhesion to surfaces for their very survival. One interesting example is the mussel, which glues itself to surfaces by secreting specialized protein adhesives. In this talk I will describe the molecular features of mussel adhesive proteins, which are enriched in the catechol containing amino acid, 3,4-dihydroxy-L-alanine (DOPA). Our experiments have revealed the catechol functional group of DOPA to be a highly versatile adhesive moiety, capable of strong interactions with both inorganic and organic surfaces. A secondary goal of our research is to exploit the special chemical properties of DOPA and its catechol analogs as coatings. Several approaches exploit catechol-surface interactions to enhance or prevent adhesion of proteins, cells and bacteria at surfaces. These coatings have a variety of potential uses in biosensing, prevention of environmental biofouling, and for control of biointerfacial phenomena at the surface of medical/diagnostic devices and nanoparticles.
10:00 AM - SS6.2
Polymer Brushes for Enzyme Immobilization: Impact on Enzyme Activity and Stability.
Sarah Lane 1 , Jeannie Yom 1 , Zhifeng Kuang 1 , Shafi Arifuzzaman 2 , Steve Diamanti 1 , Jan Genzer 2 , Barry Farmer 1 , Rajesh Naik 1 , Richard Vaia 1
1 , Air Force Research Lab, Wright-Patterson AFB, Ohio, United States, 2 Department of Chemical Engineering, North Carolina State University, Raleigh, North Carolina, United States
Show AbstractPolymer brushes offer a number of advantages over traditional self-assembled monolayers for the immobilization of biomolecules and cells. These advantages include multiple binding sites per chain, synthetic on-surface control of the brush thickness and the possibility of using responsive polymer brushes. As we have shown previously, post-polymerization activation of poly(hydroxylethyl methacrylate) (pHEMA) using N,N’-disuccinimidyl carbonate (DSC) allows versatile modification of the brush with amine containing molecules and biomolecules, including soft lithography patterning. Using these concepts immobilized arrays of enzymes, such as horseradish peroxidase (HRP), were assembled onto the DSC activated brush. Molecular dynamics simulations show that HRP most likely binds through lysine residues K149 or K232, which are near the heme pocket of the enzyme. Experimental analysis of the immobilized HRP indicated that its activity is approximately an order of magnitude less than free HRP in solution, consistent with other reports in the decrease of activity for HRP immobilized via lysine groups. On the other hand, immobilizing the enzyme increases its long-term stability relative to free enzyme. Furthermore, unmodified pHEMA is fairly resistant to nonspecific protein adsorption, making it an ideal base for protein and tissue patterning.
10:15 AM - SS6.3
Immobilization of Citrus Tristeza Virus antigen/antibody on Si and InP surfaces.
Alberto Luis Moreau 1 , Luis Peroni 2 , Jose Raimundo Reis 2 , Dagmar Stach-Machado 2 , Monica Cotta 1
1 Instituto de Fisica Gleb Wataghin, Universidade Estadual de Campinas, Campinas, SP, Brazil, 2 Instituto de Biologia, Universidade Estadual de Campinas, Campinas, SP, Brazil
Show AbstractBiosensors that are based in field-effect transistors have been widely studied in the literature. Most conductometric sensors rely on a layer of biological material immobilized at the semiconductor surface; the interaction with target molecules alters the charge distribution near the surface which in turn modulates current transport through the semiconductor. Thus, controlling the immobilization of specific targets on semiconductors surfaces is extremely important for biosensor development. Many biosensors are based on Si; however, III-V compounds represent another choice of material which can show improved sensitivity in such applications. However, functionalization of InP surfaces has not been fully explored so far.In this work, we study the immobilization of antigen CB22 (receptor) and antibody 37.D.09 (ligand) of Citrus Tristeza Virus (CTV) at both Si and InP surfaces, and compared the experimentally obtained results. This virus affects citrus plants and their early detection has a large economical impact in many countries; we are thus interested in building a sensor with high sensitivity and the immobilization is the first step towards this goal. The semiconductor surfaces were functionalized using AminoPropilTrietoxi-Silano (APTES), a reagent widely used to this purpose on Si surfaces; the biological material was immobilized directly on APTES using a wet chemical route. ELISA immunochemical assays have shown an efficient immobilization on both semiconductors, and also an efficient block mechanism of these molecules using casein. Once the immobilization is confirmed, topographical AFM images were carried out to verify the uniformity of the film in different stages.Using the same wet chemical route, antibodies were immobilized on AFM tips and interaction force measurements – in phosphate buffered saline solution – were carried out with antigens on the Si and InP surfaces. For both cases, smaller forces were observed when the active tips were blocked by the antigen added into the solution. Hence the InP functionalization/immobilization works as well as Si under these conditions; this simple procedure can thus be used for both semiconductors and AFM tips. The measured forces also indicate that the antigens and antibodies immobilized at the studied surfaces are still active for their specific biological interaction.
10:30 AM - SS6.4
A Novel High-sensitive Immunoassay Platform with Orientation-controlled Immobilization of Antibodies on Non-biofouling Polymer Brush.
Nobuyuki Tajima 1 2 , Ryosuke Matsuno 1 2 , Madoka Takai 1 2 , Kazuhiko Ishihara 1 2
1 Department of Materials Engineering, School of Engineering, The University of Tokyo, Bunkyo, Tokyo, Japan, 2 , Center for Nano-Bio Integration (CNBI), Bunkyo, Tokyo, Japan
Show AbstractWe have developed a novel immunoassay platform with enhanced sensitivity, achieved by the orientation-controlled antibodies and the non-biofouling polymer brush. The orientation of surface-bound antibodies was strictly controlled on Staphylococcal Protein A (SpA) immobilized on the polymer brush with the aid of tyrosinase. We synthesized a block-type copolymer brush of well-defined structure, poly(2-methacryloyloxyethyl phosphorylcholine (MPC)-block-2-aminoethyl methacrylate (AEMA)) (PMbA) from the substrate by surface-initiated living radical polymerization. The primal layer of this grafted polymer, PMPC, is expected to reduce the non-specific protein adsorption on the surface, since the non-biofouling ability of MPC polymer has already been well established. The second layer, PAEMA, is to offer the “smart” conjugation of SpA molecules with its orientation controlled by the tyrosinase catalyzed chemistry. In other words, PMbA grafted surface is designed to enhance the sensitivity by reducing the non-specific signal (“noise”) with the PMPC layer and increasing the specific signal (“signal”) with orientation control of the surface-bound antibodies. Though many researches have shown the “controlled orientation” of antibodies using SpA or similar ligands, most of them seem to ignore the orientation of these ligands themselves; if these ligands are mal-oriented, the orientation of antibodies on top of them are in no way controlled. In our study, SpA is first oxidized by tyrosinase to convert its tyrosine residues into active quinone groups and then immobilized onto the amino groups of the PMbA substrate through these quinone groups. The complete amino acid sequence of SpA molecule reveals that the tyrosine residue based immobilization method of SpA is expected to well conserve the SpA’s IgG binding region on the solution side. As the result of sandwich immunoassay, this system using tyrosinase and SpA showed the best secondary antibody/primary antibody ratio (s/p=8.1) among the system without SpA (s/p=1.6) and that with randomly oriented SpA (s/p=1.7). Assuming the secondary antibodies are immobilized through antigens, which are captured by the surface bound primary antibodies, this ratio correlates the primary antibodies’ antigen binding abilities. The calibration curve drawn with the amount of immobilized secondary antibodies against the various concentrations of antigen was greatly alleviated with the tyrosinase catalyzed SpA system compared to the other two systems. This tendency was also observed in the real-time QCM-D measurements. In addition, the expected non-biofouling ability of MPC polymer appeared even in our PMbA brush, attributing to the “blocking” ability throughout the assay. Thus, we conclude that we have successfully realized the novel, high-sensitive immunoassay platform with the orientation-controlled SpA on top of the non-biofouling polymer brush.
11:15 AM - **SS6.5
Molecular Understanding, Design and Development ofZwitterionic-based Biomaterials.
Shaoyi Jiang 1
1 Chemical Engineering & Bioengineering, University of Washington, Seattle, Washington, United States
Show AbstractAn important challenge in many applications ranging from biomedical devices to ship hulls is the prevention of nonspecific biomolecular and microorganism attachment on surfaces. For example, nonspecific protein adsorption degrades the performance of surface-based diagnostic devices and causes an adverse effect on the healing process for implanted biomaterials. Our goals are to provide a fundamental understanding of molecular-level nonfouling mechanisms using an integrated experimental and simulation approach and to develop biocompatible and environmentally benign ultra low fouling materials based on design principles. Over the last few years, we have demonstrated that zwitterionic and mixed charge materials are highly resistant to nonspecific protein adsorption from undiluted blood plasma and serum and to bacterial adhesion and biofilm formation. In addition to their excellent nonfouling properties, zwitterionic carboxybetaine-based materials have abundant functional groups for ligand immobilization while cationic zwitterionic precursors have self-sterilizing capabilities. Both simulation and experimental results show that the strong hydration of zwitterionic materials is responsible for their excellent nonfouling properties. At present, zwitterionic materials have been applied to a number of applications, including implantable medical devices, protein arrays, targeted drug/gene delivery carriers, antimicrobial coatings, and marine coatings.
11:45 AM - SS6.6
Evaluation of New Cationic Polymer Brush Layers as Antimicrobial Coatings.
Kenneth Carter 1 , Damla Koylu 1
1 Polymer Science and Engineering, University of Massachuesetts - Amherst, Amherst, Massachusetts, United States
Show AbstractTethering of polymer brushes on a solid substrate is a valuable method for modifying surface properties. Altering the surface charge is one property that can be altered by growing polymer brushes. We explore the synthesis of the positively charged surfaces by utilizing brushes consisting of various polymers and copolymers. Copolymerization techniques give the ability to control the number of functional groups in the product, hence controlling its structure, and introducing the desired properties into the polymer system. An intriguing property of these positively charged surfaces is their antimicrobial activity. In order to prepare the charged surfaces, polymerization reactions have been carried out and the surfaces characterized by a number of techniques including, IR, XPS and ellipsometry. Polymer brushes of glycidyl methacrylate and hydroxyethyl methacrylate were prepared by free-radical polymerization technique. These brushes were functionalized with chloroacetyl chloride for the introduction of chloromethyl groups, followed by quaternization with triphenyl phosphine and triethyl amine yielding positively charged surfaces. The surfaces were functionalized with lysozome and the antimicrobial properties of the charged and enzyme-functionalized surfaces were evaluated.
12:00 PM - SS6.7
Effect of Solid Surfaces on the Thrombosis.
Jaseung Koo 1 , Dennis Galanakis 2 , Miriam Rafailovich 3
1 Chemistry, Univer of Pennsylvania, Philadelphia, Pennsylvania, United States, 2 Pathology, Stony Brook University, Stony Brook, New York, United States, 3 Materials Science and Engineering, Stony Brook University, Stony Brook, New York, United States
Show AbstractAlthough fibrinogen is essential blood plasma protein in the process of hemostasis and coagulation, it can also be detrimental when it leads to thrombosis, or the formation of clots that lead to obstruction of blood in the circulatory system. But their molecular self-assembly is partly understood and is potentially relevant in tissue fibrinogen deposits and implantable medical devices. Here we investigated the effect of the surface of foreign materials on the fibrin or fibrinogen fiber assembly. We found that alpha-C chains are key domains to initiate the molecular self-assembly. If the alpha-C domains interact with surfaces (for example, on hydrophilic P4VP or P4VPh spun cast films), they will not be allowed to associate with another alpha-C domain in adjacent molecules. Hence the fiber did not form on the surfaces. However, on the hydrophobic surface, other domains such as D and E domains, involved in the surface interaction so that the alpha-C domains were repelled from the surfaces, hence free to interact the molecules. The adsorbed molecules have finite mobility and can self-assemble into the short, straight fibrils, of uniform diameter, d = 9 nm. alpha-C domains remain on the outside of fibrils and enable further interactions between fibrils, which eventually combine to form the large fibers. We demonstrate that fiber formation is a general phenomenon, occurring on most polymeric surfaces and even on coronary stents. If functional groups are added which bind the domains, fiber formation can be completely prevented.
12:15 PM - SS6.8
Amyloid Fibril Formation on Thiol Modified Surfaces.
Brad Moores 1 , Janet Simons 2 , Zoya Leonenko 1 2
1 Physics and Astronomy, University of Waterloo, Waterloo, Ontario, Canada, 2 Biology, University of Waterloo, Waterloo, Ontario, Canada
Show AbstractMany proteins are known to actively interact with both biologically relevant surfaces, as well as inorganic and synthetic surfaces which are widely used in nano- and bio-technology as biosensing platforms and in tissue engineering. Amyloid fibrils are insoluble aggregates of proteins in beta-sheet conformation and are implicated in at least 20 diseases for which no cure is currently available. The molecular mechanism of fibril formation, as well as the mechanism of interaction of fibril clusters with the surface of lipid membrane are currently unknown. The surface of lipid membrane is complex by biochemical composition and is also electrostatically non-homogeneous. Currently, the experimental data available for amyloid fibril formation both on lipid surfaces and artificial surfaces are limited. The goal of our study is to investigate how the physical properties of the surface affect binding of amyloid nanoclusters. To elucidate the effect of electrostatic interactions of amyloid peptides with surfaces we used Atomic Force Microscopy (AFM) and Kelvin probe force microscopy (KPFM) to study Amyloid beta (1-42) fibril formation on model surfaces. These surfaces are uniformly charged or possess periodicity of charges and hydrophobic functionality based on thiol self-assembly
12:30 PM - SS6.9
Atomic Force Microscopy Study of Protein Interactions with Microphase Separated Polyurethane Biomaterial Surfaces.
Lichong Xu 1 , Christopher Siedlecki 1 2
1 Surgery, Penn State University, Hershey, Pennsylvania, United States, 2 Bioengineering, Penn State University, Hershey, Pennsylvania, United States
Show AbstractMicrophase separation is an important characteristic of polyurethane (PU) biomaterials. An improved understanding of protein interactions with PU surface and correlation with microphase structure at the molecular scale is important for an improved understanding the mechanisms of biomaterial-induced thrombosis as well as the development of new biomaterials. In this paper, we utilized an array of atomic force microscopy (AFM) techniques to characterize the dynamic phase restructuring of PU material during hydration, the protein interactions with surfaces, and the immuno- identification of protein adsorption and function on surface. The goal of study is to better understand how the spatially-dispersed chemical functionalities present in these important biomaterials contribute to their success of the material in biomedical device applications.The microphase separation structure of PU in ambient and aqueous environments was visualized by AFM tapping images. Sequential in-situ AFM phase images showed that PU underwent phase restructuring and rearrangement, resulting in enrichment of hard domains at the surface after hydration. Force measurements were used to produce a 3-dimensional map of the micromechanical properties of polymer quantitatively demonstrating this rearrangement. A monoclonal antibody-modified probe was used to detect the platelet-binding activity of fibrinogen adsorbed on the surface. Both fibrinogen activity and platelet adhesion on the polyurethane surfaces were found to decrease with increasing hydration time. Taken together with the previous results, these observations suggest that water-induced enrichment of the more hydrophilic hard domains in PU changes the local surface physical and chemical properties in a way that influences the conformation of fibrinogen, changing the availability of the platelet binding sites in the proteins. Additional studies were undertaken to understand the nature of these interactions. The interactions of protein and PU surface were studied by tapping and force modes. A nanogold-labeled protein conjugate was used to visualize individual protein adsorption to the separate microstructures on PU surface, with preferential protein adsorption seen on the more hydrophobic soft segment regions. Force measurements using a protein-modified AFM probe correlated the mechanical properties and local adhesion forces for bovine serum albumin, and results showed that low adhesion forces were primarily associated with polar hard domain regions. Both results suggest that the microphase separation structure mediates local surface microenvironments that influence biological interactions with the surface.
SS7: Bacteria, Biofilms and Bio/Inorganic Interfaces
Session Chairs
Wednesday PM, December 02, 2009
Ballroom A (Hynes)
2:30 PM - **SS7.1
Bacteria on Surfaces: Benefits, Problems and Provisions.
Jens Rieger 1 2
1 Polymer Physics, BASF SE, Ludwigshafen Germany, 2 School of Engineering and Applied Sciences, Harvard University, Cambridge, Massachusetts, United States
Show AbstractThe relevance of biofilms in the clinical, industrial and personal field will be reviewed giving special attention to societal relevance as well as to scientific approaches to understand and manipulate biofilms. There is a number of existing approaches to fight biofilms which will be discussed with respect to their applicability and efficacy. Furthermore it will be shown how progress is made in this field through interdisciplinary approaches involving microbiology, chemistry, material sciences and physics.
3:00 PM - SS7.2
The Role of Organic/mineral Interfaces in the Structure Formation of the Coral Skeleton in Porites Lutea.
Roland Kroeger 1 , Elisabeth Brown 1 , Tim Rixen 3 , Jeff Gelb 2
1 , University of York, York United Kingdom, 3 , Center for Marine Tropical Ecology, Bremen Germany, 2 , Xradia Inc, Concord, California, United States
Show AbstractUnderstanding the mineral formation in biological systems is a fundamental requirement in order to obtain crucial information on the environmental impact of the crystallization as well as for ways to control this process e.g. for medical applications or for material synthesis. In this context the coral skeleton presents an important example for biomineralization, which is by far not yet understood.Hence, the focus of this work was on the detailed structure of the skeleton sidewall and the relevant structural components therein. We have investigated the microstructure of the coral sidewall of the genera Porites Lutea with respect to the relevant building blocks of the structure by X-ray nano computer tomography (XNCT) as well as scanning and transmission electron microscopy (SEM/TEM). The spatial density of the centers of calcification could be determined by XNCT to be in the range of 106 cm-2 and their three-dimensional arrangement inside the crystal phase could be determined. These centers consist of organic fibers embedded in aragonite compartments with a characteristic size of 20 – 30 µm constituting the coral thecae. SEM and TEM revealed the building blocks of the skeleton to be acicular aragonite crystals wrapped in an organic matrix. The first TEM investigations ever to be performed on these structures show a high degree of structural quality of flat crystal needles with diameters in the range between 10 nm and 500 nm with the maxima of the size distributions centered at 150 nm and 380 nm. In comparison with aragonite platelets found in the nacre of the abalone shell [1] the <010> growth is suppressed in the coral, whereas the rapid growth directions <100> are similar to the ones found for nacre. These observations indicate significant differences of the role of the organic tissue in the mineralization compared to shell forming marine organisms.[1] K. Gries, R. Kröger, C. Kübel, M. Schowalter, M. Fritz and A. RosenauerUltramicroscopy 109, 230-236 (2009).
3:15 PM - SS7.3
Imaging and Recognition of Bacteria by Broadband Electromechanical Response Using Artificial Neuron Networks.
Vladimir Reukov 1 , Maxim Nikiforov 2 , Gary Thompson 1 , Oleg Ovchinnikov 2 , Stephen Jesse 2 , Sergei Kalinin 2 , Alexey Vertegel 1
1 Bioengineering, Clemson University, Clemson, South Carolina, United States, 2 Materials Science and Technology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee, United States
Show AbstractScanning probe microscopy (SPM) techniques have become the mainstay of nanoscience and nanotechnology by providing easy to use, non-invasive structural imaging and manipulation on the nanometer and atomic scales. Here, we introduce a new SPM method – functional recognition imaging - that allows interpretation of multiple channel or spectroscopic data in terms of desired functionality, and demonstrate it on an example of rapid single cell identification using the detection of broadband frequency dependent electromechanical response. The method is demonstrated using Gram-positive bacteria Micrococcus luteus and Bacillus subtilis and Gram-negative bacteria Pseudomonas fluorescens deposited on a Poly-L-lysine (PLL)-coated mica substrate. The central concept of Functional Recognition SPM is direct recognition of local behaviors from measured spectroscopic responses using neural networks trained on examples provided by the operator. The operation of FR-SPM includes the steps of training a neural net using a set of examples, data acquisition, and feature recognition. To summarize, we demonstrate an approach for rapid recognition imaging based on the response recognition using trained neural network. The approach is demonstrated for rapid bacterial identification using dynamic electromechanical response, but can be used for other spectroscopic and multimodal SPM modes including force-distance curves, multiple harmonic detection, HarmoniX, and many others. Given the well-known scalability of principal component analysis for real-time operation, this approach can be used in real-time imaging. Furthermore, the standard strategies for biological recognition based on functionalized tips can be synergistically combined with this approach to increase selectivity. This analysis mode is ideally suited for differentiation and identification of cells with differing phenotype obviating the need for quantitative extraction of materials properties, as such can be broadly applied for cancer identification, biodetection, and multiple other areas. This work was supported by NIH grant# R21RR024449 and CNMS User program [CNMS2009-102]
3:30 PM - SS7.4
Modifying Bacterial Behavior Through Chemical and Mechanical Properties of Nanostructured Surfaces.
Allon Hochbaum 1 2 , Joanna Aizenberg 1 2
1 School of Engineering and Applied Sciences, Harvard University, Cambridge, MA, Massachusetts, United States, 2 Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA, Massachusetts, United States
Show AbstractBacteria are generally found in interface-associated communities, called biofilms, in the natural environment. In medical settings, bacteria are responsible for hospital-acquired infections of about 10% of patients (about 2 million per year) and account for several billion dollars of additional health-care costs. Biofilms formed at infection sites are particularly problematic because they protect the associated cells from environmental attacks such as antibiotics and human immune responses. Most materials are susceptible to bacterial adhesion and biofilm growth due to non-specific protein fouling or degradation of the surface, which masks designed chemical functionalities. Alternatively, structured surfaces disrupt bacterial aggregation and alter the dynamics of biofilm development by mechanical interactions with the cells. Substrates were fabricated by a fast polymer molding technique, whereby replicas of a microfabricated master were reproduced with a variety of polymers. The mechanical interactions were modified by varying the polymer type or curing conditions, or by changing the dimensions of the substrate features. Furthermore, a general route to arbitrary surface functionalization of these structures was developed to systematically investigate the effects of concurrent chemical and mechanical stimuli on bacterial adhesion and biofilm development. Bacteria spontaneously assemble on these substrates into long-range patterns and exhibit different biofilm growth dynamics than those on flat substrates. This phenomenon was observed with different species and is promising as a general route to long-term bacterial resistant surfaces. Moreover, these substrates systematically elucidate the chemical and mechanical signals that bacteria gather from synthetic surfaces, which may help design materials for specific microbial interactions.
3:45 PM - SS7.5
Heterogeneities of Nanoscale Interactions Mmeasured Between L. monocytogenes and Inert Surfaces.
Nehal Abu-Lail 1 , Bong-Jae Park 1
1 Chemical Engineering and Bioengineering, Washington State University, Pullman, Washington, United States
Show AbstractWith bacterial surfaces that are known to comprise many types of biopolymers, it is not surprising to observe a range of nanoscale adhesion affinities between bacterial surface and inert surfaces in contact. Since the heterogeneity in nanoscale bacterial adhesion measurements is a fact that researchers in this community have to deal with, implementing analytical and statistical tools to potentially describe the heterogeneity effects on the bacterial adhesion measurements can be useful. To investigate how heterogeneous are the nanoscale bacterial adhesion measurements, atomic force microscopy (AFM) was used to quantify the nanoscale adhesion force and energy measured between Listeria monocytogenes 1002 and a model surface of silicon nitride in water. Two dimensional maps of the distribution of adhesion forces and adhesion energies on the surface of many bacterial cells were investigated. Our results show that adhesion energies can describe surface bacterial adhesion events in a more comprehensive way when compared to adhesion forces. Our results also show the presence of a distribution in both the adhesion forces and adhesion energies across individual cell surface and also across the surfaces of cells that belong to the same strain and that have been cultured together. Such heterogeneities can be attributed to the distribution of a wide array of biopolymers on the bacterial surface and to other factors that include heterogeneities in surface charge density distribution on the bacterial surface. Finally, our results indicate that in order to obtain representative nanoscale AFM measurements of bacterial adhesion events of interests, measurements have to be collected between the inert surface of interest and as many locations as possible on the bacterial surface.
4:30 PM - SS7.6
Study of E. coli and C. xerosis Bacteria Colonization on Microdiamond and Nanodiamond Surfaces.
Jose Nocua 1 2 , Olga Medina 1 2 , Maria Arvelo 1 , Alberto Ortiz 1 , Javier Avalos 1 2 , Brad Weiner 1 3 , Gerardo Morell 1 2
1 Department of physic, University of Puerto Rico, Rio Piedras, Puerto Rico, United States, 2 , Institute for Functional Nanomaterials, Rio Piedras, San Juan, Puerto Rico, United States, 3 Department of Chemistry, University of Puerto Rico, Rio Piedras, San Juan, Puerto Rico, United States
Show AbstractThe high incidence and costs of post-surgery complications due to bacterial infection in catheters and implants in the human body require the development of coating materials that resist bacterial colonization. The bacteria resistant coating must also be inert and hard in order to withstand the harsh fluids of the human body and friction, and biocompatible to avoid an inflammatory body response. Diamond appears to meet these requirements, but its antibacterial properties are not well studied or understood. We have systematically studied the ability of E. coli and C. xerosis bacteria to survive and reproduce on microcrystalline diamond (MCD) and nanocrystalline diamond (NCD) surfaces. For comparison, stainless steel, cooper, silver and glass surfaces were also employed. The results strongly indicate that the NCD surface significantly inhibits bacterial growth. These results are discussed in terms of the surface energy, electric properties and nanostructure of the materials employed, and how they affect the interactions with the bacteria.
4:45 PM - SS7.7
Surface Modification During Bacteria Growth: Effect on Adhesion and Formation of Xylella Fastidiosa Biofilms.
Gabriela Lorite 1 , Carolina Rodrigues 2 3 , Alessandra Souza 2 , Monica Cotta 1
1 Instituto de Fisica Gleb Wataghin, Universidade Estadual de Campinas, Campinas, SP, Brazil, 2 Centro APTA Citros Sylvio Moreira, Instituto Agronômico, Cordeiropolis, SP, Brazil, 3 Instituto de Biociências, Universidade Estadual Paulista, Botucatu, SP, Brazil
Show AbstractBacterial biofilms are complex microbial communities that provide resistance against external factors. An essential step to biofilm formation is the adhesion of microbial cells to a solid surface. For this process, several factors like surface hydrophobicity, presence of extracellular polymers and growth conditions are determinant. In this work we study the adhesion mechanism of the bacteria Xylella Fastidiosa (XF) by considering the changes caused by different culture media on the surface and how these changes influence the bacteria surface adhesion for XF biofilms grown both in cell culture dishes and into the atomic force microscopy (AFM) liquid cell. Glass and polylisine-treated glass surfaces were used as substrates. Non specific (PW) and specific (XDM) medias were put into contact with the substrates; bacteria were later inoculated into the solution in some cases. AFM topographic images showed morphological changes in the surface, independent of the inoculation, indicating a thin film is formed on the surface due to the culture media. Contact angle measurements showed that both PW and XDM significantly modify the original surface hydrophobicity. For the inoculated samples, however, measurements of film thickness through AFM indentation have shown larger material deposition near the XF biofilms; this result suggests that there is a contribution from the bacteria to the observed morphology. The early stages of XF biofilm formation were observed in real time inside the AFM liquid cell, up to two days after bacteria inoculation. Topographic and phase images indeed suggest the presence of an extra material around the biofilm, possibly exopolymers produced during adhesion and biofilm growth. The influence of this material in the adhesion process will be discussed.
5:00 PM - SS7.8
Using Bacterial Growth to Template Catalytic Asymmetry
Bryan Kaehr 1
1 Advanced Materials Laboratory, Sandia National Laboratories, Albuquerque, New Mexico, United States
Show AbstractThe use of biological structures to template inorganic materials has become an increasingly widespread strategy to explore otherwise inaccessible micro and nano-scale architectures, under mild chemical conditions, for use as catalytic and device materials. For example, proteins, peptides, DNA, viruses, cells and multicellular structures have been employed as templates to develop inorganic particles, wires, and arrays. While most of these approaches have treated the biological structure as a static template, the development of strategies to recruit biological processes—accompanying, for instance, cell growth and differentiation—to position function/activity would greatly expand potential synthetic outcomes and functionality for a given biotemplate. We are pioneering an approach to develop metallic asymmetry on a bacterial cell template (E. coli) by exploiting the polar development of the cell envelope during growth and division. Metallization precursors (Au-NPs) bound to the cell envelope segregate to cell poles following insertion of new envelope material. Further, growth results in new template surfaces that can be targeted by additional precursors and/or bio-reductive chemistries and we employ this strategy to the synthesis of asymmetric catalytic micro particles comprised of gold and platinum that are capable of complex, ‘bacterial-like’ swimming behavior in the presence of chemical fuel. This work demonstrates a new route for Janus particle synthesis and should provide a foundation to realize catalytic and bio-electronic systems based on more complex biological architectures.
5:15 PM - SS7.9
Hydroxyapatite Fused-GFP Protein: Towards a Biomineralization Monitoring System.
Esra Yuca 1 2 3 , Urartu Seker 1 2 , Mustafa Gungormus 2 , Ayten Yazgan Karatas 1 , Mehmet Sarikaya 1 2 , Candan Tamerler 1 2
1 Molecular Biology and Genetics, Istanbul Technical University, Istanbul Turkey, 2 Genetically Engineered Material Science and Engineering Center, and Materials Science and Engineering, University of Washington, Seattle, Washington, United States, 3 Biology, Yildiz Technical University, Istanbul Turkey
Show AbstractFluorescent proteins with different emission wavelength and enhanced brightness are widely used as fusion partners in a variety of applications. In addition to discovery of naturally occuring fluoresent proteins from marine organisms, different variants became commonly used tools as biological markers and reporters. In our study, we used GFPuv, a variant of the Aequorea GFP (green fluorescent protein), as a fluorescent probe for monitoring hydroxyapatite mineralization. Hydroxyapatite (HA) is found in different size and shape in various mammalian hard tissues as the main inorganic mineral component. As previously reported, we have biocombinatorially selected HA binding peptides (HABP) and their binding affinities and structural properties have been fully characterized. The effect of the two heptapeptides, a strong (HABP1) and a weak (HABP2) peptides, on the mineralization reaction and crystal morphology were demonstrated. Here, we genetically fused, using recombinant DNA, these HABPs with GFPuv to monitor biomineralization in situ. The binding affinities of the fusion proteins were quantified using a quartz crystal microbalance (QCM) system. We then made a correlation between the results of the QCM-D and fluorescence microscopy (FM) experiments. The values of the binding constants, KsubD, for GFP-HABP1 and GFP-HABP2 were calculated as 6.6 and 429 µM, respectively. These values point out distinct molecular recognition property of the GFP-HABP1 molecular construct towards HA surfaces. Additionally, the GFPuv-HABP constructs were also tested with biological samples; for in vitro experiments, human acellular afibrillar cementum was used. The fluorescent images demonstrated that the GFPuv-HABP1 construct can recognize biological mineralization by the cells as well a synthetically-made HA. As we demonstrate here, the GFPuv-HABP1 fusion partner can serve as an efficient reporter to detect HA formation and as a real-time molecular agent to monitor biomineralization processes in vitro. This research is supported by NSF-MRSEC, NSF-IRES and TUBITAK-NSF programs.
5:30 PM - SS7.10
The Facile Fabrication of Controlled Thickness Silica Films on Solid Surfaces using a Bio-inspired Approach.
Carole Perry 1 , Akhilesh Rai 1
1 School of Science and Technology, Nottingham Trent University, Nottingham United Kingdom
Show AbstractThe fabrication of controlled composite coatings on solid surfaces presents a promising new method to fine-tune properties for applications in biosensors, biomedical devices, structural materials, membrane fabrication and electrical components including transducers. The fabrication methods typically used require harsh conditions of high temperature, pressure and pH that are not suitable for processes using biomolecules to promote the synthesis of networks of nanosized particles on the surface. In contrast, biological organisms can synthesize silica-based materials over many length scales under environmentally benign conditions. Herein we present our study on the fabrication of silica films on solid surfaces using a biological approach.Bovine serum albumin (BSA) and lysozyme, having different intrinsic properties, were used as model proteins for the fabrication of silica films on solid surfaces. Different amines such as poly(allylamine) (PAH), poly(ethyleneimine) (PEI) and octadecyl amine (ODA) were used to functionalize gold surfaces in order to achieve strong and uniform adsorption of protein at pH 7.2. BSA adsorbs strongly on PAH and PEI coated gold surfaces (hydrophilic) due to electrostatic interactions, while lysozyme shows a stronger affinity towards ODA coated surfaces (hydrophobic) via largely hydrophobic interactions. Further experimentation with silicatein, a special class of protein found in sponges immobilized on cystamine/cysteamine-glutardialdehyde functionalized gold surfaces via a covalent interaction with maintenance of protein functionality was performed. Uniform silica films were fabricated on the protein bound surfaces after treatment with silica precursors (tetramethoxysilane) under environmentally benign conditions. The thickness (ca. 10-100nm) and roughness (ca. 1-4nm) of films could be tuned by varying the amount of proteins adsorbed and/or varying exposure time to silica precursor. Microgram amounts of BSA and lysozyme that are inexpensive and readily available proteins and nanogram quantities of silicatein can be used to generate homogeneous films over cm2 length scales, making our methods accessible to technologists for the cost-effective fabrication of uniform silica films over multiple length scales on glass and gold surfaces as well as on Si (n and p type) wafers. This method might also be extended to fabricate others inorganic films (such as oxides of Ti and Zr) on different surfaces in an economical fashion.
SS8: Poster Session: Biosurfaces and Biointerfaces II
Session Chairs
Thursday AM, December 03, 2009
Exhibit Hall D (Hynes)
9:00 PM - SS8.1
Presence of Water Molecules inside Nanopores of Mesoporous Silica.
Yoshie Aoki 1 , Junko Hieda 2 , Maria Antoaneta Bratescu 2 , Nagahiro Saito 3 , Osamu Takai 2
1 , School of Engineering, Nagoya University, Nagoya Japan, 2 , Graduate School of Engineering, Nagoya University, Nagoya Japan, 3 , EcoTopia Science Research Institute, Nagoya University, Nagoya Japan
Show AbstractMesoporous materials are widely used in industrial field and biotechnology as an absorbent, a supporter of metal catalyst and biomolecules and membranes.Recently, theoretically was found that the internal water present inside the nano-scale space behaves different as compared with bulk water molecules. Hence, internal water inside the nanomaterials plays a very important role related with the properties of nanodevices and nanomaterials, experimental evidence of chemical and physical properties of water molecules inside nano-scale space is necessary. In this study, we aimed to investigate water molecules inside the nanopores of the mesoporous materials. For this purpose we used two experiments: Differential Scanning Calorimetry (DSC) and Coherent anti-Stokes Raman Spectroscopy (CARS). We used commercial mesoporous silica powders with 2 and 4 nm pore size (Taiyo Kagaku Co., Ltd.). The surface of the mesoporous silica particles was modified by long alkyl silane organic molecules (n-octadecyltrichlorosilane) which size longer than the nanopore size of the mesoporous silica. The outer surface of the mesoporous particles turned into a hydrophobic surface. The water molecules are adsorbed mainly inside the nanopore of the mesoporous silica material.In a CARS experiment we measured the stretching vibration transition of the OH bonds of water around 3500 cm-1 for both samples of mesoporous silica with 2 and 4 nm nanopores sizes, with and without outer functionalized surface, although the Raman signal of the OH vibration is a weak signal. The differences in the CARS signals between mesoporous silica with different pore sizes will be discussed.The heat of water desorption reaction measured by DSC showed that water desorption heat between the samples with and without alkyl silane functionalized silica surface is different.
9:00 PM - SS8.12
Preparation of Titanium Oxide Using a Biomimetic Approach to Control Structure.
Shaun Filocamo 1 , Robert Stote 1
1 , US Army NSRDEC, Natick, Massachusetts, United States
Show AbstractMetal oxides are a widely utilized class of materials for many applications. One such material, titanium oxide (titania), has found usage in the area of medicine, energy conversion, electronics and decontamination. Studies have shown that the function of the titania is related to the morphology of the oxide. Control of morphology is currently accomplished through synthetic procedures that require harsh reaction conditions (e.g. organic solvents, corrosive pH) and/or high energy expenditures (e.g. high temperature and pressure). This is in contrast with the environmentally benign processes developed by organisms in nature, which result in metal oxides with highly complex structures. Facilitated by enzymes or peptides and polyamines, thousands of intricate structures unique to each organism are formed repeatedly, at ambient temperature and pressure, and in aqueous solution at a neutral pH. Silaffin, a well-studied naturally-occurring peptide, has been shown to be active not only towards silicic acid to form silicon oxide, but also aqueous titanium oxide precursors. Unlike straight polyamines, which also can form titanium oxide from these precursors, the silaffin peptides produced the oxide with evidence of some structural control. The active amino acids in the silaffin structure were identified and used in the design of a new set of peptides which were tested against the titanium oxide precursor. The effects of the peptide primary and secondary structure on the morphology of the titanium oxide precipitates will be presented.
9:00 PM - SS8.13
Spider Silk as a Novel Humidity-Driven Biomimetic Muscle.
Vasav Sahni 1 , Ali Dhinojwala 1 , Todd Blackledge 2 , Ingi Agnarsson 2
1 Polymer Science, The University of Akron, Akron, Ohio, United States, 2 Biology, The University of Akron, Akron, Ohio, United States
Show AbstractSpider webs represent the confluence of intricate architecture and natural materials that have optimal mechanical properties. The web’s non-sticky, supporting threads (dragline silk) effectively absorb the forces of prey strikes and its sticky, spirally arrayed capture silk retain insects long enough for a spider to subdue them . Typical spider dragline silk tends to outperform other natural fibres and most man-made filaments. However, even small changes in the surrounding environment can have large effects on the mechanical properties of a silk fibre as well as on its water uptake. Absorbed water leads to significant shrinkage in an unrestrained dragline fibre and reversibly converts the material into a rubber. This process is known as supercontraction and may be a functional adaptation for the silk's role in the spider's web. Here, we characterize the response of dragline silk to changes in humidity before, during and after supercontraction. Also, we show that silk also exhibits powerful cyclic contractions, allowing it to act as a high performance mimic of biological muscles. These contractions are actuated by changes in humidity alone and repeatedly generate work 50 times greater than the equivalent mass of human muscle. Furthermore, because this effect already operates at the level of single silk fibers, only 5 µm in diameter, it can easily be scaled across the entire size range at which biological muscles operate. The simplicity of using wet or dry air to drive the biomimetic silk muscle fibers and the incredible power generated by silk offer unique possibilities in designing lightweight and compact actuators for robots and micro-machines, new sensors, and green energy production.
9:00 PM - SS8.14
Functionalized Clay Nanomaterials for Wound Healing Applications.
Christopher Vaiana 1 2 , Lawrence Drummy 1 , Richard Vaia 1 , Athanasios Bubulya 3 , Rajesh Naik 1 , Madhavi Kadakia 2
1 Materials & Manufacturing Directorate, Air Force Research Lab, Dayton, Ohio, United States, 2 Biochemistry and Molecular Biology, Wright State University, Dayton, Ohio, United States, 3 Biological Sciences, Wright State University, Dayton, Ohio, United States
Show AbstractThe ability to adsorb active biomolecules on the surface of nanoclay (montmorillonite) is being exploited for many applications such as drug delivery and wound healing. Natural layered aluminosilicates, such as montmorillonite, have demonstrated an inherent ability to induce blood clotting, and the addition of tissue regeneration promoters would be attractive for the development of a unique wound healing material. Here, we use the adsorption of Horseradish Peroxidase (HRP) onto montmorillonite particles as a model system of bio-functionalization of nanoclays. Using common biochemical and materials characterization techniques, we evaluate and quantify the binding and retained activity of HRP on the montmorillonite surface. Using this established model, we explore the functionalization of nanoclays with human Epidermal Growth Factor (hEGF). Efficient binding of hEGF is quantified using sensitive biochemical techniques. Primary cells culture is used as the basis for toxicity, metabolic activity, and wound-healing studies with the introduction of the nanoclay-hEGF system. This will provide a basic understanding of the ability to deliver growth factors using bio-derivatized nanoclays, and sets the stage for the exploration of a unique wound healing solution.
9:00 PM - SS8.15
Influence of Surface Treatment and Biomimetic Hydroxyapatite Coating on the Mechanical Properties of Hydroxyapatite/Poly(L-Lactic Acid) Fibers.
Fei Peng 1 , Montgomery Shaw 1 2 , James Olson 3 , Mei Wei 1
1 Department of Chemical, Materials, and Biomolecular Engineering, University of Connecticut, Storrs, Connecticut, United States, 2 Polymer Program, Institute of Materials Science, University of Connecticut, Storrs, Connecticut, United States, 3 , Teleflex Medical, Coventry, Connecticut, United States
Show AbstractPoly(L-Lactide) (PLLA) micro-fiber was coated with hydroxyapatite (HA) using a biomimetic method to form a bioactive composite for bone repair applications. To increase the HA content within the composite fiber, as well as enhance the bonding between the coating and the fiber substrate, PLLA fibers were treated by soaking in either NaOH or NaOCl solutions at different concentrations and temperatures. The PLLA fibers without soaking and soaked in de-ionized water (DIW) acted as controls. Homogenous but porous HA coatings were generated on all the studied PLLA substrates by immersing the fibers into a modified simulated body fluid (m-SBF) at 60oC for 1-2.5 h. The influence of surface treatments and coating variables on the mechanical properties of the PLLA and/or HA/PLLA fibers were investigated in depth. It was found that the NaOH or NaOCl treatments are effective in increasing the amount of HA coating deposited onto the fiber substrates and improving the bonding between them, but they also resulted in degraded mechanical properties of the PLLA fibers. In addition, such obtained HA coating had cracks and induced stress-raising defects. As the HA coating was not intact, its reinforcing effect mainly depends on its bonding to the PLLA substrate. As a result, the coated fiber was found not stronger than the uncoated fiber due to the limited bonding formed. The enhanced bonding in treated HA/PLLA fibers was not sufficient to compensate the mechanical strength reduction caused by cracks on the treated PLLA substrate and those within the HA coating as the stress-transition hinders.
9:00 PM - SS8.16
Structure and Reactivity at the Bioglass-Water Interface.
Antonio Tilocca 1 , Alastair Cormack 2
1 Department of Chemistry, University College London, London United Kingdom, 2 School of Engineering, Alfred University, Alfred, New York, United States
Show AbstractThe biomedical applications of materials based on bioactive glasses, such as the common 45S5(R) composition, rely on a sequence of rapid surface processes occurring immediately after exposure of the biomaterial to a physiological aqueous environment. Atomistic models of these processes are essential if a real understanding of these materials is sought. We have explored the properties of the surface of 45S5 bioactive glass using ab-initio Molecular Dynamics (Car-Parrinello, CPMD) simulations. The strength of different exposed sites, such as under-coordinated Si3c, modifier Na+ and Ca2+ cations, and small (2- and 3-membered) silicate rings was assessed on the basis of their interaction with a water molecule. Si3c are the strongest adsorption sites on the as-created surface, followed by modifier Na+ and Ca2+ cations, whose Lewis acidity can favor the penetration of water inside the surface towards the bulk, thus initiating the process of dissolution of the glass. Opening of small rings through water dissociation turns out to be hindered by a kinetic barrier, suggesting a possible role of these rings as nucleation sites in the bioactive mechanism. (Tilocca & Cormack, J. Phys. Chem. C 2008). These individual effects were further examined by modeling the fully hydrated surface of the glass: this represents a more realistic model incorporating most of the effects present in a macroscopic sample of the biomaterial implanted in a physiological environment. The CPMD trajectories of the extended interface between the glass and a film of liquid water provided atomistic insight into the initial stages relevant to the biological activity of these materials: following contact of the glass with an aqueous medium, the initial enrichment of the surface region in sodium cations establishes dominant Na+ - H2O interactions at the surface, which in turn promotes sodium leaching into solution: Ca2+ - H2O interactions are established only after the initial dominant fraction of sodium is lost. The Lewis acidity of the modifier cations is essential to neutralize OH- groups produced by water dissociation and protonation of non-bridging oxygen (NBO) surface sites. Small rings appear very stable even after exposure to liquid water. (Tilocca & Cormack, ACS Appl. Mat. & Interfaces, 2009) We discuss the potential implication of these findings for the overall bioactive process.
9:00 PM - SS8.17
A Novel Bioactive Ceramic Coating for Improved Fixation of Orthopedic Implant.
.. Aniket 1 , Ahmed El-Ghannam 1
1 Dept. of Mechanical Engineering and Engineering Science, The University of North Carolina at Charlotte, Charlotte, North Carolina, United States
Show AbstractCoating orthopedic implants with bioactive ceramics facilitates bone tissue integration, implant fixation and hence longevity. Various bioactive ceramics have been used for coating with limited success due to instability of the ceramic/metal interface. Silica calcium phosphate nano-composite (SCPC) is a novel bioactive resorbable ceramic that has the ability to bond to bone and expedite bone formation. In the present study, we coat medical grade Ti-6Al-4V implants with SCPC using electrophoretic deposition (EPD) and demonstrate the stability, uniformity and bioactivity of the ceramic coating. Flat Ti alloy discs were passivated in 34% HNO3. EPD was carried out in 2-7.5% (w/v) SCPC suspension in ethanol for 60-600 sec at 30-120V. The coated discs were treated at 600-800 °C under argon. Adhesion strength was measured and the fractured surface was analyzed using SEM-EDX. The coated discs were immersed in phosphate buffer for 7 days and the adhesion strength was evaluated thereafter. Foam Ti alloy discs were coated with SCPC using optimized EPD parameters, cold-mounted in epoxy and sectioned using diamond wheel. The thickness of the SCPC coating on the surface of the inner and outer pores was measured. A uniform 30 - 40 µm SCPC layer was detected on passivated Ti alloy surface after 3 min EPD coating in 5% (w/v) SCPC suspension using 50 V. Tensile tests showed that the adhesion strength between SCPC and passivated Ti alloy after thermal treatment at 800 °C was 47 ± 4 MPa. Although the adhesion strength was higher for samples treated at 800 °C than those treated at 700 °C or 600 °C, the difference was not statistically significant. Fracture surface analyses revealed that the failure was largely at the ceramic/polymer interface or within the ceramic layer, suggesting a strong ceramic/metal interface. SEM analyses of the cross section of Ti alloy foam showed that after 30 sec coating, the entire thickness of the porous structure was coated with a uniform 4 µm thick SCPC layer. The interface between the ceramic and metal appeared to be very intact. After 7 days immersion in physiological solution, SCPC enhanced the deposition of a biological hydroxyapatite layer on its surface. Mechanical testing showed that the fracture occurred at the interface between the precipitated hydroxyapatite and the SCPC layer at 6.4 ± 1.8 MPa.In conclusion, successful coating of a thin and uniform layer of bioactive SCPC on the surface of Ti alloy implant material was achieved using EPD. The SCPC layer was strongly adhered to the metal surface even after immersion in physiological solution. Moreover, the SCPC coating enhanced the bioactivity properties of the implant as indicated by the formation of a biological hydroxyapatite layer on the material surface. Therefore, SCPC coating has the potential to expedite bone bonding to the metallic implant, improve fixation and longevity.
9:00 PM - SS8.18
Hydroxyl Termination and Reactivity of HF-etched SiC Surfaces.
Sarit Dhar 2 , Oliver Seitz 1 , Sungho Choi 5 2 , Mathew D. Halls 3 , Yves J. Chabal 1 4 , Leonard C. Feldman 4 2
2 Physics and Astronomy Dept., Vanderbilt University, Nashville, Tennessee, United States, 1 Materials Science and Engineering Dept., University of Texas at Dallas, Richardson, Texas, United States, 5 Device Materials Research Center, Korea Research Institute of Chemical Technology, Daejeon Korea (the Republic of), 3 Materials Science Division, Accelrys Inc., San Diego, California, United States, 4 Inst. of Adv. Mat. Dev. and Nanotech, Rutgers University, Piscataway, New Jersey, United States
Show AbstractThe hydrogen-termination of oxidized silicon in hydrofluoric acid results from an interesting etching process that is now well understood and accepted. This surface has become a standard for studies of surface science and an important component in silicon device processing for microelectronics, energy and sensor applications. The present work shows that HF etching of oxidized silicon carbide leads to very different surface termination, whether it is carbon- or silicon-terminated. Specifically, the silicon carbide surfaces are hydrophilic with hydroxyl-termination, resulting from the inability of HF to remove the last oxygen layer at the oxide/semiconductor interface. The final surface chemistry and stability critically depend on the crystal face and surface stoichiometry, with implication for surface functionalization, and therefore for biomedical applications.To characterize the chemical nature of the SiC surfaces after HF etching, we combine macroscopic probes (CA= contact angle), elemental analysis (NRA=Nuclear Reaction Analysis) and spectroscopic investigations (XPS= X-ray photoelectron spectroscopy and IRAS= Infrared Absorption spectroscopy) with first principles Density Functional Calculations. Starting from both carbon- and silicon-terminated 4H SiC oxidized with 18O, and etched with DF, NRA reveals that the last layer of oxide (~ 1 monolayer of oxygen) remains on the surface upon etching. Moreover, on C-SiC, a monolayer of deuterium is also observed. XPS shows that the C-SiC surfaces have a surface C-O bond, while the Si-SiC surfaces have a surface Si-O bond. IRAS unambiguously identifies hydroxyl groups (~ 1 monolayer) at the surface, accounting for the hydrophilicity of the surfaces (CA= 24o and 4o on C- and Si-faces, respectively). Moreover, IRAS reveals that the OH bonds on C-SiC are much more stable than on Si-SiC, preventing further chemical functionalization with silane-based organic molecules. Thus, while the surfaces are hydrophilic and hydroxyl terminated, they are resistant to chemical attacks. On the other hand, the Si-terminated SiC surfaces can be further functionalized to present either a hydrophobic head group or a carboxylic-terminated layer to the external environment. These findings are important for the use of silicon carbide in biomedical applications, such as for implants or for biosensors.
9:00 PM - SS8.19
Bone Cells Growth and Proliferation on TiO2 Nanotubes Modified by Plasma Discharge.
Meena Mahmood 1 , Rajesh Sharma 1 , Ashley Fejleh 1 , Philip Fejleh 1 , Alokita Karmakar 1 , Frank Hardcastle 1 , Yang Xu 1 , Enkeleda Dervishi 1 , Zhongrui Li 1 , Alexandru Biris 1
1 Applied Sciences, UALR/ Nanotechnology Center, Little Rock, Arkansas, United States
Show AbstractTitanium implants are commonly used for a large number of medical applications that range from dental, hip, or knee replacements. One of the major problems is the bio-compatibility of these Titanium surfaces and the lack of cellular proliferation on its surface, which could reduce its integration in the osseous system. We report the fabrication of a self-assembled vertical and ordered nanotubular TiO2 array by electrochemical anodization. The samples were plasma treated and further analyzed by electron microscopy, Raman spectroscopy and atomic force microscopy.Bone Cells MC3T3-E1 were grown in TiO2 Nanotube substrates and their proliferation was analyzed by optical microscopy. It was observed a good cellular proliferation on the TiO2 nanostructures, but which was significantly influenced by the chemical surface modification of the substrates. Oxygen and Nitrogen plasma treatments were found to significantly alter the proliferation of the bone cells over the TiO2 substrates. These findings can be explained by the introduction of various O and N groups onto the TiO2 surfaces during the plasma treatment.
9:00 PM - SS8.2
Advanced Solid State NMR Techniques for the Investigation of the Organic-Mineral Interfaces in Biomaterials.
Danielle Laurencin 1 , Gilles Guerrero 1 , Julien Almaric 1 , Christian Bonhomme 2 , Christel Gervais 2 , Mark Smith 3 , P. Hubert Mutin 1
1 Institut Charles Gerhardt, UMR 5253, CNRS - UM2 - ENSCM - UM1, Montpellier France, 2 Laboratoire de Chimie de la Matière Condensée de Paris, UMR 7574, UPMC University Paris 06, Paris France, 3 Department of Physics, University of Warwick, Coventry United Kingdom
Show AbstractOver the past 20 years, extensive research has been carried out in biomaterials science in order to improve the in vivo performance of implants and drug-delivery devices. In particular, much effort has been made to better control the surface morphology and chemistry of biomaterials, because of their known impact on the biological response,[1] and the functionalization of materials by active molecules such as drugs or antibacterial species has thus been widely investigated.[2,3] Early on, it was shown that the in vivo properties of functionalized biomaterials strongly depend on the mode of anchoring of the grafted active molecules at the surface. For example, the antibacterial effect of a coating persists longer if the grafted molecules are linked via strong covalent bonds and not simply adsorbed onto the material. Thus, it appears crucial to characterize in detail the mode of binding of active molecules at the surface of biomaterials, in order to rationalize and improve their properties. Because it is a site-specific probe, solid state NMR appears as one of the methods of choice to analyze the structure of biointerfaces. Here, we will show how thanks to recent advances in the technique, it is now possible to investigate in detail the surface structure of widely used biomaterials such as titanium oxide and hydroxyapatite. In particular, results of 17O NMR experiments carried out on TiO2 surfaces functionalized by 17O enriched phosphonic acids will be presented,[4] because they bring clear evidence of the formation of Ti-O-P bridges and of the presence of residual P=O and P-OH groups. Furthermore, we will present high-resolution 43Ca NMR studies of calcium phosphates and carboxylates, in order to demonstrate how they can now be used to look at Ca...C and Ca...H proximities in materials,[5,6] and help elucidate interface structures. References:[1] H. Schliephake, D. Scharnweber, J. Mater. Chem. 2008, 18, 2404.[2] P. Wu, D. W. Grainger, Biomaterials 2006, 27, 2450.[3] J. Amalric, P. H. Mutin, G. Guerrero, A. Ponche, A. Sotto, J. Lavigne, J. Mater. Chem. 2009, 19, 141.[4] F. Brodard-Severac, G. Guerrero, J. Maquet; P. Florian, C. Gervais, P. H.Mutin, Chem. Mater. 2008, 20, 5191.[5] D. Laurencin, A. Wong, J. V. Hanna, R. Dupree, M. E. Smith, J. Am. Chem. Soc. 2008, 130, 2412.[6] D. Laurencin, C. Gervais, A. Wong, C. Coelho, F. Mauri, D. Massiot, M. E. Smith, C. Bonhomme, submitted.
9:00 PM - SS8.20
Increasing the Potential of Bioactive Glass as a Scaffold for Bone Tissue Engineering.
Mohamed Ammar 1 , Max Kaplan 1 , Therese Quinn 2 , Sabrina Jedlicka 1 2 3
1 Materials Science & Engineering, Lehigh University, Bethlehem, Pennsylvania, United States, 2 Bioengineering Program, Lehigh University, Bethlehem, Pennsylvania, United States, 3 Center for Advanced Materials and Nanotechnology, Lehigh University, Bethlehem, Pennsylvania, United States
Show AbstractBioactive glass is known for its potential as a bone scaffold due to its ability to stimulate osteogenesis and differentiation of stem cells into bone cells. In an attempt to investigate if we can increase these potentials, we decorated the structure of the bioactive glass made by the sol-gel technique with 3 peptides sequences from different proteins known for their potentials to stimulate the osteogensis process (fibronectin, BMP-2 and protein kinase CKI). This material was tested with Human Mesenchymal Stem Cells (hMSCs) and MC-3T3 preosteoblasts to see the difference in the effect on uncommitted and committed cells. The bioactive glass sol with and without the peptides was dip coated onto glass cover slips, leading to a film of the material, surface decorated with the peptides of choice. The two cell types were seeded onto the materials in standard proliferation medium without additives for differentiation induction. Cells were also grown on tissue culture treated cover slips with and without differentiation induction media as positive and negative controls, respectively. The cells were grown on the materials for a total of five weeks, and were tested at four time points (weekly from week two) by immunocytochemical assays to investigate the levels of different osteogenic markers (alkaline phosphatase, osteopontin, osteocalcin and osteonectin) and by qRT-PCR to investigate the mRNA potential of the same proteins. The expression of the differentiation markers follows the anticipated path over the course of differentiation for all samples except the negative control. On the native bioglass samples, the cells ultimately begin expressing protein markers of differentiation; however, the differentiation is markedly slower than the positive control. On the peptide-decorated samples, the cells appear to differentiate at a rate that is equal to or faster than the positive control, indicating that the peptide effect is similar to that achieved by traditional BMP-2 soluble protein techniques. This supports our hypothesis that adding specific peptide sequences known for their effects in cells adhesion, proliferation and differentiation can increase the potential of the bioactive glass as a scaffold for bone tissue engineering.
9:00 PM - SS8.22
Selective Biofunctionalization all-Si (111) Surface Nanowires.
Muhammad Masood 1 2 , Songyue Chen 1 2 , Edwin Carlen 1 2 , Albert den Bereg 1 2
1 Faculty of Electrical Engineering, Mathematics and Computer Science, University of Twente, Enschede, Overijssel, Netherlands, 2 , MESA+ Institute of Nanotechnology, University of Twente, Enschede, Overijssel, Netherlands
Show AbstractAdvances in the alkylation of hydrogen-terminated silicon surfaces preclude the use of chemical attachment of organic monolayers to oxide passivated surfaces.1 This approach has several advantages for silicon nanowire (Si-NW) biosensors, including improvement in sensitivity by the removal of the intervening SiOx barrier that brings the ligand-ligate couple very close (~1 nm) to the sensing surface, as well as improving selectivity since only the Si-NW surfaces are functionalized with ligand biomolecules. Additionally, the electrically imperfect SiOx layer, which typically suffers from large trapped charge densities, is replaced with a direct Si-C covalent bond between the organic and inorganic phases.It is well known that the preferred silicon surface for the chemical attachment of organic monolayers is the Si(111) surface due to the atomic arrangement, which leads to densely packed layers.2 However, existing reports of the alkyl monolayer formation on Si-NWs have been conducted on Si(100) surfaces due to limitations in device technology. We present alkyl monolayer formation and characterization on our recently reported Si-NWs with all-Si (111) surfaces3 using a well-established attachment scheme.4 Si-C monolayers are formed on Si (111) surfaces by first etching the native oxide layer with 40% NH4F or 1% HF. Hydrogen terminated Si(111) surfaces have further been reacted with N-(5-hexynyl)phthalimide and UV irradiation. After deprotection with a methylamine solution, a terminal amine group (—NH2) was released for further conjugation.Surface preparation and modification quality have been characterized with x-ray photoelectron spectroscopy on planar Si(111) surfaces. Si-NW device sensitivity (ΔId/ΔVg |vds) is characterized with electrochemical gating measurements where the device body current is modulated with a reference electrode (Ag/AgCl) in electrolyte solution. Comparisons with sensitivity of Si-NWs with thermally oxidized surfaces (~10 nm) will be presented. The precise control of biosensing selectivity is demonstrated with the conjugation of streptavidin functionalized Au nanoparticles to the amine/biotin active Si-NW surfaces and subsequent high resolution scanning electron microscopy.References1. Y.L. Bunimovich, G. Ge, K.C. Beverly, R.S. Ries, L. Hood and J.R. Heath, Langmuir 2004, 20, 10630-10638.2. M.K. Weldon, K.T. Queeney, J. Eng Jr., K. Raghavachari and Y.J. Chabal, Surface Science 2002, 500, 859-878.3. S. Chen, J.G. Bomer, W.G. van der Wiel, E.T. Carlen and A. van den Berg, Materials Research Society (MRS2009), Spring Meeting, San Francisco, CA, U.S.A, 2009.4. J. M. Buriak, Chemical Reviews 2002, 102, 1271-1308.
9:00 PM - SS8.23
Osteoconductive HAp and TiO2 Coatings on Titaniumusing Hydro-process.
Dai Yamamoto 1 , Kensuke Kuroda 1 , Masazumi Okido 1 , Ryoichi Ichino 2
1 Department of Materials Science and Engineering, Nagoya University, Nagoya, Aichi, Japan, 2 EcoTopia Science Institute, Nagoya University, Nagoya, Aichi, Japan
Show AbstractHydroxyapatite (Ca10(PO4)6(OH)2, HAp) and titanium dioxide (TiO2, titania) are of interest for bone-interfacing implant applications, because of their demonstrated osteoconductive properties. In this study, they were coated on the titanium implants using hydro-processes and investigated the in vivo performance. HAp coatings were formed on cp titanium plates or rods by the thermal substrate method in an aqueous solution included 0.3 mM Ca(H2PO4)2 and 0.7 mM CaCl2. In the formation of carbonate apatite coating, CaHCO3 was added to the solution, and in HAp/gelatin and HAp/collagen composite coatings, acid-soluble collagen (Type I) was added. The coating experiments were conducted at 40-140 oC and pH = 8 for 15 or 30 min. Titania films were formed on the titanium implants by anodizing at < 100 V in 0.1 M H2SO4, H3PO4, and NaOH aqueous solutions at 25 oC. The properties for the coated samples were studied using XRD, EDX, FT-IR, and SEM. And the surface roughness of titania coatings was measured. In in vivo evaluations, the coated rod specimens were implanted in rats femoral for 2 weeks, the osteoconduction on them was evaluated. Two weeks postimplantation, new bone formed on the coated and non-coated titanium rods in the cancellous bone and cortical bone, respectively. Bone-implant contact ratio, RB-I, which was used for the evaluation of new bone formation, was significantly depended on the compound formed on titanium implants, and also the coating processes.
9:00 PM - SS8.24
Fabrication of Plastic DNA Arrays by de novo Inkjet Synthesis on SiO2 Functionalized Cyclic Olefin Copolymer.
Ishtiaq Saaem 1 , Kuosheng Ma 1 , Jingdong Tian 1
1 Biomedical Engineering, Duke University, Durham, North Carolina, United States
Show AbstractParallel synthesis, miniaturized analysis and interrogation of libraries of molecules are one of the most promising tools of modern biomedical research. One such critical application involves biomolecular assays and gene analysis using oligonucleotide arrays or DNA chips. However, such chips often suffer from low SNR, inaccuracies from feature-edge issues that often produce erroneous results and are too fragile for portable deployment. We try to alleviate these problems by improved synthesis on novel materials. With the foreseeable integration of microfluidics and microarrays, polymers stand to play a critical role. Polymeric, or plastic, biochips have several advantages in cost, durability, the ability to scale to industrial techniques and possibly serve as disposable point-of-care devices. We have developed a robust and efficient process of depositing SiO2 thin film on cyclic-olefin copolymer (COC) surfaces at low temperature using an RF sputtering technique. This method is suitable for functionalizing COC surfaces with a variety of controllable factors. For example, the thickness of sputtered SiO2 film can be controlled accurately by controlling sputtering conditions. The COC surface wettability can be patterned by selective deposition of SiO2 films. In addition, further modification of SiO2 can generate heterogeneous surface functional groups with different characteristics. Since there is no high temperature requirement in the process, this technique can be applied to COC materials of different Tg and potentially to other polymers as well. In our studies, we utilized an inkjet based in situ oligonucleotide synthesis platform that uses salvaged printheads from commercial printers. The platform utilizes standard four- step phosphoramidite chemistry in order to synthesize oligonucleotides on functionalized substrates. A sensitive pressurization system is used to ensure print quality and an on-board vision system enables substrate registration and synthesis monitoring. We then use this system to address accuracy of parallel oligonucleotide synthesis on standard glass substrates and COC. In our studies, we investigate both non-patterned and patterned glass and COC substrates to address concerns of cumulative synthesis errors arising from printing errors. Our analysis then comprises characterization of the synthesized oligonucleotides using functional assays, staining, yield estimation and comparison of products from nonpatterned and patterned substrates. Such an improved microarray synthesis platform could be crucial for developing more reliable and accurate medical diagnostic chips and for synthetic biology applications.
9:00 PM - SS8.25
Soft Substrate Fabricated by Silver Nanoparticles-Polypeptide Brushes for SERS Application.
Diyan Wang 1 , Yi-Cheng Lee 2 , Ying-Chih Chang 2 , Chia-Chun Chen 1 3
1 Department of Chemistry, National Taiwan Normal University, Taipei Taiwan, 2 The Genomics Research Center, Academia Sinica, Taipei Taiwan, 3 Institute of Atomic and Molecular Sciences, Academia Sinica, Taipei Taiwan
Show AbstractOf recent, new synthetic approaches have been intensively explored to prepare new SERS substrates using silver or gold nanoparticles and Polypeptide polymer. In this abstract, we have fabricated a soft surface-enhanced Raman scattering (SERS) substrate was fabricated based on a three-dimensional (3D) structure of biocompatible end-tethered poly(L-lysine) (“t-PLL”) with a brush-like configuration conjugated with silver nanoparticles (Ag NPs) (Ag NPs-t-PLL film). The films with different interval widths (W) between Ag NPs and diameters (D) of Ag NPs can be carefully adjusted under the conjugation procedures. The resulting film was then characterized by zeta potential, CD spectropolarimeter and scanning electron microscopy. Furthermore, the studies of SERS enhancements using Ag NPs-t-PLL film as a substrate were performed. The significant increases of SERS enhancements have been obtained as W/D was decreased from 0.9 to 0.2. Currently, the identification of cultivated E-coli and PC-12 cells were performed on Ag NPs-t-PLL films using SERS. The SERS spectra have been clearly obtained. The interpretation of the SERS results will be discussed.
9:00 PM - SS8.26
Fabrication of Cell Array Using Superhydrophobic Surfaces.
Jau-Ye Shiu 1 , ChiungWen Kuo 1 , Peilin Chen 1
1 Research Center for Applied Sciences, Academia Sinica, Taipei Taiwan
Show AbstractEver since the discovery of the importance of the surface roughness to the water repellent behavior of plant leaves, material scientists have developed various strategies to produce the so-called “superhydrophobic surfaces”, whose water contact angles are larger than 150 degree. It is generally believed that the water repellent properties of such type of superhydrophobic materials could reduce the water contact on the surfaces, therefore, minimizing the adsorption of particles and molecules on the surfaces. In the past few years, several potential applications of the superhydrophobic surfaces have been identified including the self-cleaning coating, fog condensation, contamination reduction, oxidation reduction, oil water separation, and rapid water spreading. However, there are very limited researches in exploring the applications of the superhydrophobic materials in the field of biology. Here we report the fabrication of the patterned superhydrophobic surfaces using the plasma treatment of fluoropolymers. It was found to the superhydrophobic surfaces could accumulate more ECM proteins than the flat surface made of the same materials. When the patterned superhydrophobic surfaces were used in the cell culture, it was observed that the cells attached preferentially on the roughened area allowing the formation of cell microarrays. The biocompatibility of the fluoropolymer was improved by converting fluoropolymer into superhydrophobic materials. It was also observed that the transfection efficiency of the CHO cells was greatly improved on the superhydrophobic surfaces. Therefore, we conclude that the patterned superhydrophobic surfaces could be used as cell microarray with the advantages of improved cell adhesion, nature separation of colony and enhanced transfection efficiency.
9:00 PM - SS8.27
Surface Modification of Iron Oxide/Silica Core/Shell Nanoparticles with Poly(styrene sulfonate) Brush for Cells Adhesion.
Maria Antoaneta Bratescu 1 , Nagahiro Saito 2 , Osamu Takai 1
1 Graduate School of Engineering, Nagoya University, Nagoya Japan, 2 Ecotopia Science Institute, Nagoya University, Nagoya Japan
Show AbstractRecently a great research effort is invested to magnetic resonance tracking of magnetically labelled cells as a non-invasive tool for dynamics and image of cells in tissues. As magnetic resonance agents, iron oxide nanoparticles (NPs) are advantageous due to biocompatible iron. In this research, we study the effect of the surface modification of the iron oxide/silica core/shell NPs on the cells adhesion. The present study consists in: (i) chemically preparation of the iron oxide/silica core/shell NPs, (ii) silica surface modification with poly(styrene sulfonate) (PSS) brush and (iii) growth of the fibroblast cells together with as prepared particles with and without silica modified surface.The precursors used for iron oxide NPs fabrication were FeCl3 6H2O, polyethylene glycol and urea in ultra pure water. The solution was refluxed at 100oC and then the obtained colloidal suspension was centrifuged followed by drying and calcination at 600oC in air. From TEM analysis, the mean diameter of the spherical iron oxide NPs was about 70 nm with a dispersion of ± 20 nm. XRD analysis showed that the crystal structure corresponds to α-Fe2O3 structure. Magnetic property was easy observed with a permanent magnet.To cover iron oxide NPs with silica a sonication assisted Ströber process was used. The solution of α- Fe2O3 NPs in anhydrous ethanol was sonicated with ammonia and tetraethyl orthosilicate. The resultant Fe2O3/SiO2 core/shell structures were obtained by calcination at 600oC in hydrogen atmosphere. The core/shell NPs have an average diameter of 200 – 300 nm.The NPs silica surface was modified by the PSS brush grafting. The reactive chains of the polystyrene macromolecules are terminated by a trichlorosilane endgroups (PS-SiCl3), which are able to form covalent bonds with the silanol surface groups of the silica surface. The presence in the FTIR absorbance spectrum of the -CH2 asymmetric stretching and the aromatic =C-H stretching bands show that the silica surface was modified with the PS-SiCl3 macromolecules. To complete surface modification a sulfonation reaction with acetyl sulfate was followed by neutralization with NaHCO3.Mouse embryonic fibroblast cells (NIH-3T3) were grown with the Fe2O3/SiO2 core/shell NPs. The growth rate was not much affected by the presence of the particles, but we observed a higher adhesion of the cells with the NPs which were functionalized with the PSS brush. To analyze adhesion chemistry of the fibroblast cells on the NPs with and without the PSS brush we used Coherent Anti-Stokes Raman Scattering microscopy method. The cells grown on a cover glass were observed by recording the CARS signal corresponding to the aliphatic C-H stretching vibration mainly arising from lipid membranes that are rich in C-H.
9:00 PM - SS8.28
Dual-scale Structures of Diamond-like Carbon (DLC) for Anti-biofouling Coating.
Yudi Rahmawan 1 2 , Kyung-Jin Jang 1 , Kwang-Ryeol Lee 2 , Myoung-Woon Moon 2 , Kahp-Yang Suh 1
1 Mechanical Engineering, Seoul National University, Seoul Korea (the Republic of), 2 Future Convergence Technology Laboratory, Korea Institute of Science and Technology, Seoul Korea (the Republic of)
Show AbstractThe textured surface with superhydrophobic nature was explored for an anti-biofouling template. Hierarchical structures composed of the nano-scale wrinkle covering on micro-scale polymer pillar patterns were fabricated by combining the deposition of a thin coating layer of biocompatible diamond-like carbon (DLC) and the replica molding of poly-(dimethylsiloxane) (PDMS) micro-pillars. The resulting surface assembly consists of micro-scale PDMS pillars covered by nano-scale wrinkles that are induced by a residual compressive stress and a difference in elastic moduli between DLC and PDMS without any external stretching or thermal contraction on the PDMS substrate. In addition to providing the mechanical conditions on wrinkle formation, one-step DLC coating also brings a chemical functionality of low surface energy or higher water wetting angle on the surface with hierarchical structure, which enhances superhydrophobicity. The as-prepared surfaces were shown to have extreme hydrophobicity (static contact angle > 160 deg.) owing to low surface energy (24.2 mN/m) and dual-roughness structures of the DLC coating. The wetting states on dual-scale structures are also discussed with a mathematical modeling derived by decoupling of nano- and microscale roughness contribution.It was explored that the hierarchical surfaces showed poor adhesion of the Calf Pulmonary Artery Endothelial (CPAE) cells for cultures of 7 days suggesting that the 3-dimensional (3-D) patterned superhydrophobic DLC coating exhibits excellent anti-biofouling properties against non-specific cell adhesion. In particular, the reduced filopodia extension during cell growth was caused by disconnected focal adhesions on the pillar pattern. This limited cell adhesion could prevent undesired growth and proliferation of biological species on the surface of biomedical devices such as stents, implants or even injection syringes.
9:00 PM - SS8.29
Comparative Adsorption of CdSe/ZnS Quantum Dot Nanoparticles on Collagen Versus Model SAM Surfaces.
Jung Park 2 1 , Jack Douglas 2 , Dharmaraj Raghavan 1 2 , Alamgir Karim 3 2
2 Polymers Division, National Institute of Standards & Technology, Gaithersburg, Maryland, United States, 1 Chemistry, Howard University , Washington, DC 20059, District of Columbia, United States, 3 Polymer Engineeering, University of Akron, Akron, Ohio, United States
Show AbstractThe adsorption of carboxylic acid terminated CdSe/ZnS core-shell nanoparticles on collagen extracellular matrix (ECM) versus model self assembled monolayers (SAMs) with different end-groups (–NH2 and –CH3) was investigated in a comparative study of nanoparticle-adsorption on these substrates. Characterization of the immobilized collagen surface by AFM revealed that its morphology (Rrms ≈ 1.66 nm) was rougher than the aminosilane SAM surfaces (Rrms ≈ 0.25 nm) and, moreover, the Rrms varied greatly with environment conditions (chemistry of underlying substrate and pH of solution). The kinetics of nanoparticles adsorption on these substrates was measured by surface plasmon resonance (SPR) imaging, and quartz crystal microbalance (QCM) over a range of nanoparticle concentrations (0.05 micromol/L to1.4 micromol/L). The adsorption data indicates that QD nanoparticle adsorption to substrate generally depends on solution pH used for deposition, the chemistry of the underlying substrate. Specifically, a positive correlation existed between the density of surface functional amine groups on the substrate of collagen and aminosilane SAM layer and the amount of QD adsorbed. X-ray photoelectron spectroscopy (XPS) data showed that larger amount of NH2 groups present on the aminosilane than the collagen layer, resulting in more QD adsorption on the aminosilane. We develop methods to characterize these different types of particle adsorption.
9:00 PM - SS8.3
Characterization of (bio)organic - Silica Interfaces: Combination of Solid State NMR and ab initio Calculations.
Nicolas Folliet 1 2 , Christel Gervais 1 , Thierry Azais 1 , Lorenzo Stievano 2 , Frederick Tielens 2 , Jean-Francois Lambert 2 , Florence Babonneau 1
1 Laboratoire de Chimie de la Matière Condensée de Paris, Université Pierre et Marie Curie-Paris6 and CNRS, Paris France, 2 Laboratoire de Réactivité de Surface, Université Pierre et Marie Curie-Paris6 and CNRS, Paris France
Show AbstractThe aim of this presentation is to highlight the latest experimental advances in solid-state NMR that can contribute to a better description of interfaces between amorphous silica and (bio-)organic molecules. The experiments can be combined fruitfully with a theoretical approach by modelling the possible adsorption geometries and calculating the corresponding spectroscopic data. For this purpose, a model for hydroxylated surface of amorphous silica has been proposed (1) that accounts for the experimentally encountered ring size distribution, Si-O-Si and O-Si-O angles, silanol density and distribution. Combination of the geometric and spectroscopic data (NMR and IR) obtained from periodic slab DFT calculations can then be used to investigate the reactivity of this surface towards a range of organic molecules, which opens new ways for a better understanding of organic/silica interfaces.Three systems will be discussed in this presentation:-Adsorption of glycine on high surface area silica. The 13C NMR response of glycine adsorbed on silica was used to identify the possible forms of glycine present at the surface, either molecular (zwitterionic or neutral) or crystalline forms. A DFT study of the adsorption of microsolvated glycine on a hydrophilic silica surface was used to calculate ab-initio NMR parameters (2), which can thus be compared to the observed experimental ones.-Encapsulation of ibuprofen in nanoporous silica. Ibuprofen molecules encapsulated in templated mesoporous silica with 3 nm pore diameter are submitted to strong confinement effects at room temperature clearly revealed by 13C and 1H NMR spectra (3,4). The high molecular mobility leads to high efficiency of NMR sequences issued from solution-state NMR. A decrease in temperature that lowers molecular mobility allows recovering efficiency in dipolar coupling based NMR sequences used to explore interactions of the molecules with the silica surface. -Silica coating on liposomes. Interaction between phospholipids (L-α-dipalmitoylphosphatidycholine, DPPC) and silica surface was investigated in liposils that consist in liposomes covered with a protective silica coating (5). The recently developed 1H-X-1H Double Cross Polarization experiment (6) with X = 29Si and 31P, was used to evidence proximities between DPPC molecules and surface sites. These experimental data were then confronted with DFT modeling. (1) F. Tielens et al., Chem. Mater. 20 (2008) 3336-3344.(2) D. Costa et al., Phys. Chem. Chem. Phys. 10 (2008) 6360.(3) T. Azaïs et al., Chem. Mater. 18 (2006) 6382-90.(4) T. Azaïs et al., Pure Appl. Chem., ASAP Article.(5) S. Bégu et al., J. Mater. Chem., 14 (2004) 1316.(6) N. Baccile et al., Chem. Mater. 19 (2007) 1343.
9:00 PM - SS8.30
Elimination of Quantum Dots Cell Uptake.
Zoraida Aguilar 1 , Hengyi Xu 1 2 , John Dixon 1 , Hua Wei 2 , Andrew Wang 1
1 , Ocean NanoTech, Springdale, Arkansas, United States, 2 Jiangxi-OAI Joint Research Institute, Nanchang University , Jiangxi China
Show AbstractThe nanotechnology industry is booming nowadays especially in the areas of biological research for clinical, environmental, and life sciences applications. Among the nanoparticles currently in use, quantum dot (QDs) nanoparticles have received considerable attention due to their advantages [1]. QDs have unique size-dependent physical properties such as broad absorption spectrum, precise small bandwidth emission wavelength, enhanced chemical and photochemical stability, controlled and enhanced endocytosis, enhanced cooperative binding activity, and easy introduction of multi-functionalities for targeted delivery and imaging [2-4]. It can provide special approaches for complex studies and play very important roles in the modern biomedical researches. However, non-specific uptake of QDs is a major concern because they can lead to false positives or false results. Hence, we report our preliminary studies on the elimination of cellular uptake of QDs by using Ocean’s blocking buffers. We will discuss preliminary results on studies conducted on HeLa cells and will include studies showing the different effects of size and surface coating on the QDs. We will discuss and compare the cell uptake between QDs of different wavelengths of emission.References1. L. Yang, H. Mao, Y. A. Wang, Z. Cao, X. Peng, X. Wang, H. Duan, C. Ni, Q. Yuan, G. Adams, M. Q. Smith, W. C. Wood, X. Gao and S. Nie, Small, 5, 235 (2009).2. N. L. Rosi and C. A. Mirkin, Chem. Rev., 105, 1547 (2005).3. I. L. Medintz, H. T. Uyeda, E. R. Goldman and H. Mattoussi, Nat. Mater., 4, 435 (2005).4. E. Katz and I. Willner, Angew. Chem. Int. Ed., 43, 6042 (2004).
9:00 PM - SS8.31
Effect of Micro-Structured Topography on Adhesion of Gastropods.
Ravikumar Vasudevan 1 , Alan Kennedy 2 , Ronald Baney 1
1 Materials Science and Engineering, University of Florida, Gainesville , Florida, United States, 2 Environmental Laboratory, Army Engineer Research & Development Center (ERDC), Vicksburg, Mississippi, United States
Show AbstractThe effect of topographical surfaces on the adhesion of marine fouling species has motivated us to apply this idea in a novel way to decrease adhesion of terrestrial gastropods. Previous studies on bio-adhesion of macro-organisms have focused on aquatic species such as barnacles. Novel cross patterned topographies have proved to be effective in inhibiting bio-adhesion of the terrestrial snail species, Otala lactea. Snails can enter a period of dormancy, called aestivation, in which they adhere to surfaces and be dispersed to new geographical areas. Control of terrestrial snails and other potentially invasive species is important, as indirect costs (e.g., agricultural damage) through human transport systems are huge. Additionally, federal agencies must abide by Executive Order 13112 which mandates measures to reduce the transport of non-native species. Military vehicles are an example of one such transport system. PDMSe (polydimethylsiloxane elastomer) was the material chosen for testing based on its success in inhibiting adhesion of aquatic species. The topographical features were made by replicating a silicon mold that was produced by photo-lithography and subsequent etching. The effect of basic patterned topographies including a screen pattern and cylindrical and ridge-like pillars with different arrangements was tested. Cross-shaped pillars of various amplitudes (e.g., 17, 59μm) were designed to provide stability to the flexible PDMSe features because, as ridges alone, the features would partially collapse. Topographies with cross-shaped pillars with different aspect ratios were also tested. In addition, the effect of the negatives of all topographies was investigated. For comparison, smooth PDMSe and other smooth materials of various elastic moduli were tested. Each sample was characterized using profilometry, scanning electron microscopy (SEM), atomic force microscopy (AFM) and contact angle measurements. The results indicate an 80% decrease in selection of the cross topographical surface for aestivation in comparison to the control surface (smooth PDMSe). An overall decrease in the selection of a surface for aestivation was observed, with a decrease in its elastic modulus. This is consistent with previous research on adhesion of aquatic species to surfaces with varying elastic moduli. Fractured surfaces obtained following aestivation were analyzed using SEM & AFM for morphological differences between the various types of surfaces. Chemical differences were characterized using FTIR (Fourier Transform Infra red). This study is funded by the US Army ERDC Environmental Quality Technology Program, under grant number 000-72370.
9:00 PM - SS8.32
Transfection of Cells from Nanoparticle-functionalized Metal Surfaces.
Anna Kovtun 1 , Sebastian Neumann 2 , Manuel Neumeier 1 , Henning Urch 1 , Rolf Heumann 2 , Manfred Koeller 3 , Matthias Epple 1
1 Institute of Inorganic Chemistry and Center for Nanointegration Duisburg-Essen, University of Duisburg-Essen, Essen Germany, 2 Chair of Biochemistry, Faculty of Chemistry and Biochemistry, University of Bochum, Bochum Germany, 3 Department of Surgery, BG Kliniken Bergmannsheil, University of Bochum, Bochum Germany
Show AbstractThe transfer of genetic information into living cells is of high interest for cell biology and in the treatment of genetically-caused diseases. The production of specific proteins by a cell can be regulated by introducing the corresponding DNA into the cell nucleus where it is transcribed into the corresponding protein ("transfection").Previously we have reported a method of efficient gene transfer by calcium phosphate nanoparticles and a way to build up layers of polymer-functionalized calcium phosphate nanoparticles on metallic surfaces by electrophoretic deposition. In this study we combined both procedures for the electrophoretic deposition of DNA-functionalized calcium phosphate nanoparticles on metallic surfaces. This helps to localize the application of nanoparticles in the living organism.We performed transfection on fibroblasts (NIH3T3). The cells were cultivated on a metallic surface (titanium or silica) coated with pcDNA3-EGFP-functionalized nanoparticles. The efficiency of transfection was monitored by the expression of the characteristic green fluorescence of EGFP (enhanced green fluorescent protein). After 48 h of incubation, we observed transfection which was shown by fluorescence microscopy, flow cytometry and western blot.A morphological analysis of the cell culture by scanning electron microscopy (SEM) showed an excellent adhesion of the cells to the substrate and also a large number of nanoparticles on the cell surface. This leads to cell transfection - probably due to the special method of cell adhesion on solid surfaces, thus giving the proof-of-principle for bioactive implant surfaces which influence the surrounding tissue with high selectivity on a genetic basis.
9:00 PM - SS8.33
A Facile One Pot Synthesis of Highly Monodispersed Cysteine Capped Cadmium Selenide Nanoparticles: Spectroscopic and Electrochemical Studies.
Oluwatobi Oluwafemi 1 2 , Opeoluwa Oyedeji 3
1 Physics, Nelson Mandela Metropolitan University, Port -Elizabeth, Eastern Cape, South Africa, 2 Chemistry, University of Zululand, Kwadlangezwa, Kwazulu-Natal, South Africa, 3 Chemistry, University of Kwazulu-Natal, Durban, Kwazulu-Natal, South Africa
Show AbstractRecently conjugation of semiconductor nanoparticles with biomolecules through biosynthesis and biological surface modification of semiconductor and metal nanoparticles with biomolecules such as starch, proteins and nucleic acids has introduce a new dimension into nanoparticle research. The novel hybrid material synthesis enables selective interaction with target molecules or biochemical species thereby extending the area of application from electronic or optical devices to the biological or chemical system such as the preparation of non radioactive biological labels or chemical opto–sensor.1-3 However, little is known about the electrochemical studies of these materials in aqueous solution. We report the synthesis of highly monodispersed; water soluble and biocompatible L-cysteine-capped CdSe nanoparticles in an alkaline medium, without the use of additional stabilizer. The nanoparticles were characterized by optical spectroscopy, IR, x-ray diffraction, transmission electron microscopy and high resolution transmission electron microscopy (HRTEM). FTIR study shows that CdSe nanoparticles are capped through mercapto-group of cysteine amino acid. While it’s free amino and carboxylate groups make it amenable to bio-conjugation HRTEM image show that the nanoparticles formed exhibit good crystallinity with lattice plane. The particle size as estimated by the UV lies in the size quantization regime which is in agreement with the TEM and XRD analysis. Electrochemical studies was investigated under anaerobic conditions by Cyclic, Osteryoung -square –wave and Differential pulse voltammetry using lithium perchlorate as the supporting electrolyte and glassy carbon as the working electrode, within the potential window of -2000 to 1500mV vs Ag/AgCl. The cyclic voltammetry of the cysteine capped CdSe nanoparticles shows clear oxidation and reduction peaks typical of an irreversible process as compared to the electrolyte. Increasing the scan rate from 5mVs-1 to 100mVs-1 resulted in increases in all peak currents without significant shift in potential indicating kinetic effect. Osteryoung -square -wave and Differential pulse voltammetry confirmed the cyclic voltammetry result.
9:00 PM - SS8.34
Antibody-Gold Nanoparticle Conjugates for Fast Detection of Orexin A.
Jorge Chavez 1 , Latha Narayanan 1 , Nancy Kelley-Loughnane 1 , Morley Stone 1
1 , Air Force Research Laboratories, Wright-PAtterson Air Force Base, Ohio, United States
Show AbstractThe synthesis of nanomaterials with well defined dimensions, shapes and surface chemistries is extremely important for the development of hybrid materials for sensor applications. When nanomaterials are combined with biomolecules that demonstrate high target specificity, sensors with superior performance can be designed. Antibodies bind a specific target with high affinities and can be utilized as recognition elements in sensing materials. Nanoparticles offer unique optical properties that include changes in their surface plasmon resonance based on their surroundings and aggregation. Therefore, our approach includes the immobilization of highly-specific antibodies as a sensing modality onto the nanoparticles, to obtain an optical response from the nanoparticles upon target binding. The main challenge involved in hybrid materials synthesis deals with the fragile nature of antibodies. Antibodies are often denatured by heat, high salt concentration and interactions with certain materials. Therefore, the methodology used to chemically couple these units must prevent the disruption of the biomolecule. In this work, we used Orexin A as an indicator of human alertness. This small peptide is involved in the control of sleep deprivation and wakefulness. Our goal is to design an approach that determines Orexin A levels in human samples. To this end, we have coupled gold nanoparticles (AuNPs) to an antibody to Orexin A to determine different levels of the target using the optical response of the AuNPs. Our preliminary results showed that a low density coating with a short PEG ligand was necessary to stabilize the system. Moreover, the antibody needed to be coupled to a thiolated PEG, in order to be properly immobilized on the surface of the AuNPs. Agarose and PAGE gel electrophoresis were used to confirm the successful immobilization of the antibodies. Current studies focus on the design of an in vitro essay to determine different levels of Orexin A.
9:00 PM - SS8.35
The Effect of the Inclusion of Protein-Modified Gold Nanoparticles in Polymer Matrices on the Behavior of Human Breast Cancer Cells.
Jinhwa Seo 1 , Hyojin Lee 2 , Jwamin Nam 2 , Kookheon Char 1
1 School of Chemical and Biological Engineering, Seoul National University, Seoul Korea (the Republic of), 2 Department of Chemistry , Seoul National University, Seoul Korea (the Republic of)
Show AbstractThe interaction between cellular membranes and a biomaterial matrix surface has been considered as one of the most important issues to alter cell functions including cell adhesion, proliferation, migration, and differentiation. Particularly, the formation of cytoskeletons of cancer cells on a matrix surface has received much attention to study the malignant progression and metastasis. In present study, we investigate the effect of the inclusion of protein-modified gold nanoparticles in polymer matrices on the behavior of human breast cancer cells. The human breast epithelial cell lines including cancerous cells (MCF-7) and metastatic cancerous cells (CAMA-1) have been cultured and analyzed on the polymer films containing gold nanoparticles with different proteins. Poly(allylamine hydrochloride) (PAH) and poly(acrylic acid) (PAA) were employed as the basic building blocks for polyelectrolyte matrices and two different proteins (fibronectin and ephrin) were introduced for the surface modification of gold nanoparticles. The surface roughness as well as bio-recognition feature of the polymer matrices was varied by either coating different proteins on the polymer surface or incorporating protein-grafted gold nanoparticles on flat polymer substrates. Cellular phenotypic changes in adhesion, morphology, cytoskeletal organization, and cell motility on different polymer matrices were analyzed for the two different human breast epithelial cancer cell types.
9:00 PM - SS8.36
Self-Assembled Monolayers of Firmly Grafted Silver Nanoparticles on Glass Surfaces: Safe Materials with Enhanced Antimicrobial Activity.
Giorgio Guizzetti 1 , Cesare Dacarro 4 , Giacomo Dacarro 2 , Frank Denat 5 , Yuri Diaz-Fernandez 3 , Matteo Galli 1 , Pietro Grisoli 4 , Piersandro Pallavicini 3 , Maddalena Patrini 1 , Nicholas Sok 5 , Angelo Taglietti 3
1 Dept. of Physics "Volta", University of Pavia, Pavia Italy, 4 Dept of Pharmacology, University of Pavia, Pavia Italy, 2 CILSOMAF, University of Pavia, Pavia Italy, 5 Inst. de Chimie Moléculaire, Université de Bourgogne, Dijon France, 3 Dept. of Chemistry, University of Pavia, Pavia Italy
Show AbstractAntibacterial coatings for implanted medical devices and medical materials are extensively studied, and the use of Ag nanoparticles (NP) as antibacterials has been one of the most studied nanotechnology issues in the last decade. Debate on the mechanism of Ag NPs antibacterial action is still ongoing, but the fact that it should involve Ag+ release and its subsequent interaction with bacteria is largely accepted. On the other hand, health and environmental risks connected to exposition to NPs is a growing concern both in industry and in the scientific community. We envisaged a new approach for preparing antibacterial coatings of Ag NP, based on the formation of a molecular self assembled monolayer (SAM) on the chosen surface (glass, quartz, silica gel or silicon with a native SiO_2 layer), capable of further reaction with Ag NP to obtain a grown SAM of firmly grafted nanoparticles. We obtained this through two patterns: A) formation of a covalent, thiol-terminated SAM on glass (1), to which Ag NP (stabilized by citrate anions) may be bound with a covalent-like interaction; B) formation on glass of a covalent SAM terminated with a Cu2+ complex of a macrocyclic ligand, that acts as a platform for the deposition of a monolayer of Ag NP stabilized by citrate, firmly bound to the surface by Cu2+-citrate coordinative interactions. Reproducible, stable NP monolayers are easily obtained and their chemical and physical properties have been indagated through UV-Vis spectroscopy, Atomic Force Microscopy, Neutron Activation Analysis. From these data, the total quantity of silver present as nanomaterial on the functionalized glass was calculated, and found coherent with a close packing of covalently attached NPs on the modified glass surface. Moreover, a precise evaluation of Ag+ release in solution by the monolayer has been carried out with ICP, showing that only a quantity < 1.5% of the total silver is released as ion, while consuming NP remain stably bound to the surface. This apparently small quantity is however capable of exerting a strong and prolonged antimicrobial activity, that has been verified against both Gram-positive and Gram-negative bacteria such as S. Aureus and E. Coli.(1) P.Pallavicini, G. Dacarro, M. Galli, M. Patrini, J. Coll. Interf. Sci., 332 (2009) 432.
9:00 PM - SS8.37
Zr(IV)-immobilized Affinity Beads Prepared by Surface Template Polymerization for Capturing Phosphorylated Proteins.
Kazuya Uezu 1 , Hidenobu Mizuki 1 , Yudai Ito 1 , Hisashi Harada 1 , Haruka Oshiumi 1
1 Life and Environment Engineering, The University of Kitakyushu, Kitakyushu Japan
Show AbstractThe development of new efficient methods for highly specific enrichment of phosphorylated proteins and peptides is one of the most active research fields in phosphorproteome analysis. Enrichment of phosphorylated proteins and peptides from complex peptides mixtures by immobilized metal affinity chromatography (IMAC) is a poplular way to perform phosphoproteome analysis. IMAC is originally based on the affinity of the phosphate group with metal ions (usually Fe(III) or Ga(III)) immobilized on a choromatographic support. However the specificity of those IMAC adsorbents is still not high enough. To characterize phosphoproteins more efficiently, it was necessary to develop novel affinity suface with higher selectivity for phosphoproteins.In this study, we focused on the interaction between Zirconium phosphonate (Zr-Phos complex) and phosphate groups is strong. Taking advatage of strong interaction, we have developed a novel IMAC adsorbent immobilized Zr(IV) on the polymer surface (Zr-Phos IMAC) by surface template polymerization with W/O/W emulsion. Phosphoric acid oleyl ester (DOLPA), sorbitan monooleate (Span80), divinyl-benzene (DVB), polystyrene, and toluene were employed as the functional monomer, emulsion stabilizer, matrix-forming monomer, porogen, and diluent, respectively.So far, we investigated the conformation of Zr-Phos complex on the polymer surface by using fluoride ion. The molecular model for the F-Zr-Phosphate complex was predicted by computational chemistry. From the geometry-optimized structure of the complex, the optimum molar ratio of F-Zr-Phosphate complex is 3:1:3. And the calculation results are in good agreement with the results of the F adsorption tests on the beads. Therefore, we expect that the binding site with phosphorylated proteins and peptides exist enough on the polymer surface. Because of the multicoordination effect, the interaction between Zr-Phos complex and phosphopeptide is much stronger.Model phosphoproteins were employed to investigated the performance of a novel Zr-Phos IMAC. The separation performance of the phosphoproteins (Phosphorylase a , phosphorylase b, β-casein, bovine serum albumin (BSA)) was evaluated by using electrophoretic. Phosphoproteins (Phosphorylase a, β-casein) sepalated from complex proteins mixtures at the following condition: Buffer solution (5 mL) is 0.1 M acetic acid/NaOH, pH 3.1. The concentration of model phosphoproteins is 10 mg/L. We also discussed about phosphopeptide enrichment and MALDI-TOF MS analysis.
9:00 PM - SS8.38
Formation of Collagen Type I Fibrils in a Microfluidic Device for Cell Culture Applications.
Tighe Spurlin 1 , Sam Forry 1 , Gregory Cooksey 1 , Anne Plant 1
1 Biochemical Sciences Division, National Institute of Standards and Technology, Gaithersburg, Maryland, United States
Show AbstractCell culture in microfluidic devices potentially provides several advantages over traditional cell culture, including: improved temporal and spatial control of the soluble environment, ability to integrate multiple analytical techniques on chip, automated fluid manipulation, reduced reagent consumption, and the ability to perform many experiments in parallel. Optimizing cell behavior inside the devices is an area of ongoing study; thus well controlled and reproducible techniques that lead to physiologically relevant extracellular matrix coatings in devices are needed. Collagen type I is the most prevalent extracellular matrix protein found in vivo. We have previously shown that films of collagen fibrils formed at alkanethiol self-assembed monolayers provoke cell response similar to those seen in cells grown on collagen gels, and provide unique advantages over gels in terms of handling, imaging with optical microscopy, and characterization with surface analysis techniques. Here, we provide details concerning the formation of collagen fibrillar films in PDMS microfluidic devices. These films are similar in ability to invoke reproducible cell response when compared to collagen fibrillar thin films formed on self assembled monolayer coated surfaces. AFM data shows that the structures and coverage of collagen fibrils formed on chip appear to be identical to those at self assembled monolayer coated surfaces.
9:00 PM - SS8.39
Covalent Immobilization of Type I Collagen onto Titanium Implants with Polydopamine Coating.
Xiaohua Yu 1 , Yong Wang 1 , Mei Wei 1
1 Department of Chemical, Materials, and Biomolecular Engineering, University of Connecticut, Storrs, Connecticut, United States
Show AbstractThe osteointegration property of titanium implants is closely related to their surface properties such as surface chemistry, composition and topography. Being the main component in extracelluar matrix (ECM) protein, collagen has been successfully immobilized on titanium surface using various methods. It has been proved that collagen immobilized on titanium induced positive effects of cell adhesion, proliferation, and differentiation on titanium. However, passive adsorption of collagen onto titanium surface could have its own disadvantages. The resulting collagen coating is normally too unstable to be used for long-term applications. To avoid this problem, collagen can be covalently bond to the titanium surface to achieve the desired biocompatibility as well as the long-term stability. Dopamine, which is the main component of the adhesive protein secreted by mussel, has been proved to be an efficient binding agent which forms strong covalent and noncovalent bonds with various substrates. In this study, the surface of the titanium implants was modified by a polydopamine coating followed by covalent coupling of fibrillar type I collagen to improve the surface biocompatibility of titanium implants. The biocompatibility of the collagen surface was evaluated by seeding with MC3T3-E1 cells.Our X-ray photoelectron spectroscopy (XPS) results show that a homogeneous polydopamine coating was successfully formed on the surface of titanium substrate. It was also confirmed that the type I collagen coating formed on polydopamine is much stronger than that formed through a simple adsorption method. The immunostaing and SEM results also demonstrated that MC3T3-E1 cells had faster adhesion and better spread on the type I collagen coating immobilized with dopamine compared to the collagen coating adsorbed onto titanium. In summary, a simple and efficient method has been developed to covalently immobilize type I collagen onto titanium implants which can effectively improve the bonding strength as well as the biocompatibilty of the coating.
9:00 PM - SS8.40
Evolution of Surface Morphology, Elasticity and Adhesion Properties of Protein Layers on Dental Enamel
Sandip Basu 1 , Jennifer Gronlund 2 , Guofeng Xu 2 , Adrian Mann 1
1 Materials Science and Engineering, Rutgers University, Piscataway, New Jersey, United States, 2 , Colgate Palmolive Company, Piscataway, New Jersey, United States
Show AbstractThe present work discusses the evolution of surface morphology, elasticity and adhesion properties of protein-rich bio-layers grown on inorganic substrates. As an example, salivary pellicle on dental enamel has been characterized using atomic force microscopy (AFM). Salivary pellicle is a nanoscale protein-rich film formed by adsorption of saliva on dental enamel. It plays an important role in lubrication and protecting the teeth from chemical attack. Characterizing these thin pellicle layers is important not only for dental research, but also to understand the growth process and physical nature of protein-rich bio-layers on inorganic substrates. The composition and structure of these protein-rich films is known to vary with thickness and it is expected that their properties will show similar variations. All these changes in morphology, elasticity and adhesion can impact the colonization of the films by bacteria. We carried out AFM imaging and nanoscale mechanical characterization on pellicle samples grown on dental enamel for three different periods of time – 30 minutes, 2 hours and 8 hours. Statistical analyses of surface features show significant changes in surface morphology of the protein layers during the growth process. While the mean width of surface features decreases, the adhesion strength of the protein layers increases with time of growth. We also determined the elastic properties of these pellicle layers by AFM force measurements. Herein, we demonstrate the correlation between the nanomechanical and morphological properties during growth of protein layer on inorganic substrate, and in particular salivary pellicle on dental enamel.
9:00 PM - SS8.41
Molecular Dynamics Simulation on Adsorption of Ribosomal Protein L2 onto Silica Surface.
Ryo Tosaka 1 , Hideaki Yamamoto 1 , Iwao Ohdomari 1 2 , Takanobu Watanabe 1 2
1 , WASEDA Univ., Shinjyuku-ku,Tokyo Japan, 2 , WASEDA INN, Shinjyuku-ku,Tokyo Japan
Show Abstract Immobilizing proteins on a solid surface is an essential technique for bio-sensors and bio-chips. Recently, experimental reports showed that the ribosomal protein L2 (RPL2) adsorbed tightly on a silica surface [1], and RPL2 is expected to be used as a new cross-linker between a protein and a silicon material. Here we adopted the RPL2 as a model protein to analyze the interaction between bio-molecules and solid surfaces. We modeled the RPL2 and silica substrates and investigated the adsorption mechanism and the origin of high affinity of RPL2 for silica surfaces by molecular dynamics simulation. The substrate was modeled by cristobalite SiO2 structures. The (100) surface of the model was terminated with OH groups. Here we modeled three types of silica surfaces supposing at different pH values of 5, 7, and 9. Since the dissociation constant of the silanol groups on the top of silica surfaces is 7.2, the model surface at pH 5 was fully caped with OH groups. For pH 7 and 9, 50% and 100% of hydrogen atoms in OH groups were dissociated. As the initial structure of RPL2, the crystal structure of PDB ID: 2Z4L [2] was adopted. The RPL2 is composed of 273 amino acids, and amino acid sequences 61-202 are known as a RNA binding domain (we define it as domain 2). We define amino acid sequences 1-60 and 203-273 as domain 1 and domain 3, respectively. The RPL2 model was placed above the silica surface with a separation of 4 nm. A rectangular parallelepiped solvent box was created so as to surround the RPL2 and the substrate by means of the LEAP module in the MD simulator AMBER 9. A three-dimensional periodic boundary condition was imposed. The dimension of the bounding box is 10.9 nm × 10.9 nm × 11.5 nm. We performed MD simulations with the AMBER potential for several nanoseconds. We analyzed pH value dependence of the affinity, b-factor of the RPL2 before and after the adsorption and initial orientation dependence of adsorption configuration of the RPL2. After MD calculation for 2 ns, we found that the affinity of the RPL2 to the silica surface was stronger at higher pH value and that the domains 1 and 3 adsorbed more tightly than the domain 2. The domains 1 and 3 charge positively at 11e C and 16e C, respectively, at pH 7. Moreover, the atomic vibration in the domains 1 and 3 was restrained after the adsorption although the domains 1 and 3 showed a high b-factor and the structural flexibility. Hence, driving force of the adsorption of the RPL2 onto the silica surface is attributed to the Coulombic interaction between them, and the structural flexibility of the domains 1 and 3 may farther contribute to the high affinity. We verified that these trends were independent of an initial orientation of the RPL2 in the calculated models. We will report on the quantitative analysis about the affinity of the RPL2 for the silica surface.[1] K. Taniguchi et al , Biotech. Bioeng, 96, 1023 (2007). [2] Borovinskaya, M. A. et al , Nat. Struct. Mol. Bio. 14, 727 (2007).
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Amphiphilic PEO-Silanes: Effect of Siloxane Tether and PEO Architecture on Blood Protein Resistance.
Brennan Bailey 1 , Ranjini Murthy 1 , Melissa Grunlan 1
1 Biomedical Engineering, Texas A&M University, College Station, Texas, United States
Show AbstractDuring surface induced thrombosis, adsorbed blood proteins mediate cell adhesion and activation leading to clot formation. Thus, it is desirable for blood-contacting materials to repel the adsorption of blood proteins. Among the polymeric materials which have desirable bulk properties but suffer from poor resistance to blood proteins are silicones (e.g. PMDS), poly(ethylene terephthalate) (PET), polypropylene (PP) and polyethylene (PE). Their poor protein resistance is attributed to their hydrophobicity since proteins prefer to adsorb onto hydrophobic, non-polar surfaces. In contrast, poly(ethylene oxide) (PEO) is a neutral, hydrophilic polymer which exhibits unusually high protein resistance because of its hydrophilicity and configurational mobility. A new strategy is presented to prepare both crosslinked PEO-modified silicone coatings as well as surface-grafted coatings with enhanced protein resistance using novel amphiphilic PEO-silanes containing flexible siloxane tethers. Amphiphilic PEO-silanes were prepared with systematic variations to key structural features, including: siloxane tether length, PEO segment length, and PEO architecture. The PEO-silanes had the general formula: α-(EtO)3Si(CH)2)2-oligodimethylsiloxanen-block-[PEO8-OCH3] (n = 0, 4, and 13; linear architecture) (a-c) and α-(EtO)3Si(CH2)2-oligodimethylsiloxanen-block-[PEOm-OCH3]2 (n = 0, 4, and 13; m = 6 and 12; branched architecture) (1a-3a and 1b-3b). Thus, the PEO segment is distanced from the crosslinkable (EtO)3Si- group by an oligodimethylsiloxane tether that is highly flexible. PEO-modified crosslinked silicones were prepared by phosphoric acid (H3PO4)-catalyzed sol-gel condensation of α,ω-bis(Si-OH)PDMS (P, Mn= 3000 g/mol) in a 2:3 molar ratio (a-c) or (1a-3a and 1b-3b) to P. In addition, PEO-silanes (a-c) were grafted onto silicon wafers which served as a model biomaterial surface. Protein resistance was enhanced with increased siloxane tether length which may be due to the enhanced configurational mobility of the PEO segments as well as the amphiphilic nature of the surface.
9:00 PM - SS8.44
Fluorescence Recovery after Photobleaching for Quantitative Characterizing Protein-nanostructured Surface Interaction.
Pasquale Scopelliti 1 , Borgonovo Antonio 1 , Luca Giorgetti 1 2 , Paolo Milani 1
1 CIMAINA and Department of Physics, Università degli Studi di Milano, Milan Italy, 2 European School of Molecular Medicine and Department of Experimental Oncology, European Institute of Oncology, Campus IFOM-IEO, Milan Italy
Show AbstractHere we present fluorescence recovery after Photobleaching (FRAP) as a novel tool for quantitatively studying protein surface interaction [1]. FRAP is a confocal microscopy-based technique used for different applications such as studying the mobility of proteins and receptors on the cell membrane or measuring in vivo molecules diffusion coefficients. We have developed a protocol based on the combination of confocal microscopy and FRAP for characterizing protein surface interaction and we have shown that this technique has several advantages compared to existing ones. We demonstrate that confocal microscope can be used for imaging in real time the protein layer adsorbed onto a surface in presence of protein solution in a wide range of concentrations (from 10 nM up to 10 uM). Thanks to the possibility to image the protein layer and proteins in solution, this technique allows reliable background estimation and easy quantification of the fluorescence signal. Moreover we have developed a FRAP protocol in order to obtain fluorescence recovery curve of proteins adsorbed on a surface. We demonstrated that with this technique, using a simple set-up (no protein solution flux is needed), it is possible to measure with a single experiment the amount of adsorbed proteins, the interaction kinetic parameters and the mobile/immobile part ratio. FRAP has been applied for characterizing the interaction between Bovine Serum Albumin and nanostructured titanium dioxide produced with Supersonic Cluster Beam Deposition (SCBD) [2, 3, 4]. Since these films have a nanoscale roughness that can be tailored and regulated from 1 nm up to 30 nm, without changing surface chemistry, they are particularly suitable for the investigation of the role of the nanoscale roughness in the process of protein-surface interaction [5]. The fully FRAP characterization was performed for different surface roughness and different protein concentrations. Results show that nanoscale roughness influences the amount of adsorbed proteins, the kinetics parameters and the values of the mobile/immobile part ratio. These results, together with results obtained with another technique developed in our laboratory (Protein Surface Interaction Microarrays), give important indications about the interaction mechanism and highlight the central role played by nanoscale roughness in this process [1].[1] Scopelliti P., et al., to be submitted.[2] Wegner K. et al., J Phys D: Appl Phys 2006;39:R439.[3] Carbone R. et al., Biomaterials 2006;27(17):3221(9).[4] Carbone R. et al., Biomaterials 2007;28(13):2244(53).[5] Giorgetti L. et al., Langmuir 2008;24(20):11637(44).
9:00 PM - SS8.45
Effect of Metallic Nanoparticle Morphology on the Structure and Function of Adsorbed Proteins.
Jennifer Gagner 1 2 , Sudipa Panigrahi 3 , Jonathan Dordick 1 3 , Richard Siegel 1 2
1 Rensselaer Nanotechnology Center, Rensselaer Polytechnic Institute, Troy, New York, United States, 2 Materials Science & Engineering, Rensselaer Polytechnic Institute, Troy, New York, United States, 3 Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy, New York, United States
Show AbstractMetallic nanoparticles of varying size and morphology were conjugated with the biological molecules lysozyme and horseradish peroxidase in order to understand the factors influencing the structure and function of these proteins upon adsorption. Prior literature has reported the effect of nanoparticle size and curvature on the structure and activity of adsorbed enzymes, indicating that immobilized proteins may experience a greater loss of secondary structure on larger nanoparticles due to increased interaction with the particle surface. The present study extends that work to include metallic nanoparticles, initially gold, accounting for the effect of material, size, morphology, aspect ratio, surface chemistry, and crystal structure on protein adsorption. Gold nanoparticles of similar size, but in the shape of spheres, rods, and cubes have been synthesized through seeded growth methods. Each particle type was carefully controlled for similar surface chemistries in order to normalize the effect of the stabilizing ligand on protein adsorption; ligand replacement was confirmed through Fourier transform infrared analysis. Monodisperse size distributions and crystal structure have been determined through transmission electron microscopy. Initial circular dichroism studies reveal that adsorbed proteins show secondary structure loss upon conjugation, with adsorption patterns correlated to particle curvature and structure shown through ultraviolet-visible spectroscopy. Elucidation of factors influencing particle adsorption, surface interaction, and structure when immobilized on metallic nanoparticles can improve our understanding and control of nanoparticle interactions in vivo, with broad implications for biosensing, drug delivery and targeted therapy.
9:00 PM - SS8.47
Molecular Failure Mechanisms of Cross-linked Type I Collagen.
Sebastien Uzel 1 , Markus Buehler 2
1 Mechanical Engineering, MIT, Cambridge, Massachusetts, United States, 2 Civil Engineering, MIT, Cambridge, Massachusetts, United States
Show AbstractType I collagen is the most abundant form of collagen, responsible for the strength and integrity of connective tissues such as bone, tendon, cornea or cartilage. Type I tropocollagen molecules self-assemble into highly organized fibrillar structures, held together via intermolecular covalent bonds. In this study we present an atomistic-level investigation of the chemomechanical properties of intermolecular cross-links in collagen fibrils. These cross-links play a fundamental role in collagenous tissues as they directly relate to the stability, the stiffness and the strength of the material. Recent experimental studies suggest that the cross-link formation occurs between telopeptidic regions and a portion of the triple helical domain of a neighboring molecule, specifically between lysine and hydroxylysine rich sites. One of these cross-links was observed to be located between LYS(17) of the C-terminal telopeptidic region the HLY(87) in the helical domain of the adjacent molecule, albeit the detailed molecular structure of the cross-link is not known. Here we report an atomistic representation of this cross-link structure obtained based on a bottom-up computational approach, and subject it to different loading conditions using steered molecular dynamics. We carry out large-scale atomistic simulations with the first principles ReaxFF reactive force field in explicit solvent, and report a detailed analysis of the mechanical response of the cross-link structure, associated deformation mechanisms, and provide theoretical predictions of its stiffness and its strength. Loading conditions considered for the analysis include shear loading, tensile loading and twisting. The effect of amino acid mutations on cross-link mechanics and stability is investigated. The results of full-atomistic simulations are implemented in a coarse-grained model to evaluate the mechanical behavior at the larger fibril level.
9:00 PM - SS8.48
Multibranched Amorphous Graphitic Carbon Nanostructure for Protein Immobilization.
Marilin Perez 1 , Luis Fonseca 1 , Francisco Sola 1 , Oscar Resto 1 , Vincent Rotello 2 , Oscar Miranda 2 , Kai Griebenow 1
1 Institute for Functional Nanomaterials, University of Puerto Rico, San Juan, Puerto Rico, United States, 2 Department of Chemistry, University of Massachusetts, Amherst, Massachusetts, United States
Show AbstractMultibranched amorphous graphitic carbon nanostructures were grown on porous alumina substrates by the electron beam induced deposition method in a TEM (or TEM-EBID method). These nanostructures have intrinsic geometrical aspects that can be exploited in protein immobilization applications. On one hand, the fractal array of the branches in each nanostructure minimizes steric effects and offers a larger number of active sites per nanostructures than related systems like carbon nanotube bundles. At the other hand, their morphological structure makes them mechanically stable to withstand chemical manipulations required by the immobilization protocols. In this work we report brief information about the synthesis and the characterization of the nanostructures and introduce the procedures used to activate the surface. The surface carboxylation of the branches is demonstrated by the study of selective surface bonding of gold nanoparticles with amine groups and carboxylic groups surface termination, respectively. The structural stability of the nanostructures during the chemical procedures is investigated and explained. The first experiments on the immobilization of glucose oxidase are discussed.
9:00 PM - SS8.49
Peptide-directed Co-assembly of Organic and Inorganic Nanoentities on Solid Surfaces.
Marketa Hnilova 1 , Turgay Kacar 1 2 , Christopher So 1 , Ersin Oren 1 , Candan Tamerler 1 2 , Mehmet Sarikaya 1 2
1 Department of Material Science and Engineering and GEMSEC, University of Washington, Seattle, Washington, United States, 2 Department of Molecular Biology and Genetics and MOBGAM, Istanbul Technical University, Istanbul Turkey
Show AbstractImmobilization and patterning of biomolecules onto patterned solid surfaces with high affinity and specificity is a major challenge in building nanosensing devices and proteomic arrays. Currently, immobilization and patterning of biomolecules mostly require covalent attachments of target molecule to solid substrates through chemical reagents, e.g. thiols and silanes. Although used in many successful applications, these linkers, however, have certain disadvantages, due to their limited material specificity and restricted control over their molecular organization on substrates, immobilization via random orientation. Biocombinatorially selected inorganic-binding peptides with specific material affinity offer opportunities for assembling nanoscale organic and inorganic entities on various solid substrates, including metals, oxides, semiconductors, and solid polymer. Here, we demonstrate the utility of a bi-functional peptide construct in the assembly of nanoentities, e.g., metal and oxide nanoparticles, and fluorescent molecules. Specifically, we use biocombinatorially selected gold-binding and quartz-binding peptides, AuBPs and QBPs, respectively, and make several bi-functional peptide constructs functionalized with biotin, fluorescent-molecule, and also AuBP-(linker)-QBP bi-functional peptide constructs. Using AuBP-bio or QBP-bio, for example, one can immobilize streptavidin or any streptavidin functionalized nano-entity (such as quantum dots, QDs) on either patterned or nanoparticle gold or silica, respectively. Here, we demonstrate the utility of combinatorial gold-binding and quartz-binding peptide linkers in the directed co-assembly of biomolecules and quantum dots on gold/silica surfaces using their material-specificity characteristics and soft lithography technique. These molecular biomimetics approaches of peptide-directed immobilization have significant implications in a wide range of potential applications, particularly in controlled bottom-up assembly schemes utilizing hybrid nanostructures towards building novel nanophotonic and nanosensing devices. This research is supported by GEMSEC, an NSF-MRSEC at the University of Washington, and NSF-IRES/TUBITAK programs.
9:00 PM - SS8.51
Nanoengineered Calcium Phosphate Substrates for Decoupling the Effects of Nanotopopraphy and Biochemical Cues in Integrin-Mediated Cell Adhesion.
Rebecca Kraft 1 , Parimal Bapat 1 , Marco Bottino 1 , Brad Eaton 1 , Renato Camata 1
1 Dept. of Physics, University of Alabama at Birmingham, Birmingham, Alabama, United States
Show AbstractCell adhesion processes are mediated by the integrin family of transmembrane glycoproteins. Over two dozen different integrins are known to be expressed in humans and contribute to the specific binding of cells to proteins of the extracellular matrix (ECM). Emulating the ECM microenvironment of natural tissue and understanding how such an environment affects integrin function is a major goal of regenerative medicine and tissue engineering. Recent studies of cell behavior on surfaces patterned with gold nanoparticles and conjugated to RGD peptides show that integrin function and thereby cell migration and orientation can be controlled by how adhesive ligands are patterned and spaced on a surface. The elucidation and exploitation of the basic mechanisms through which such chemical and topographical features determine cell behavior represent one of the most important challenges in biomaterials research. In this work we have combined laser and aerosol techniques to create nanoengineered substrates comprising calcium phosphate nanoparticles of well controlled size on atomically flat SiO2 layers. In our process, gas-suspended calcium phosphate nanoparticles are generated by the ablation of a solid hydroxyapatite (HA) target in an Ar/H2O ambient using the focused beam of a KrF excimer laser. The nanoparticle aerosol formed in the laser ablation plume undergoes in-flight annealing and passes through an ionization zone where it acquires an equilibrium charge distribution. The charged calcium nanoparticles are then sorted according to size based on their different migration velocities in an electric field across a particle-free laminar gas stream. Size-selected nanoparticles are deposited by electrophoresis on SiO2, leading to a surface of bioactive nanoislands on an otherwise nonfouling surface. Atomic force microscopy shows that this technique can create uniformly dispersed nanoparticle deposits with diameter tunable in the 3–100 nm range with a size dispersion of ~15%. Control of the excimer laser fluence, Ar:H2O ratio in the ambient and in-flight annealing temperature allows tailoring the aerosol to nanoparticles of the desired calcium phosphate phase and crystallinity, including HA, tricalcium phosphate, and amorphous HA. For example, X-ray diffraction from samples obtained at 950°C (in-flight annealed for 5 seconds) show that the nanoparticle phase changes from HA to tricalcium phosphate as the laser fluence varies from 1.7 to 5.4 J/cm2. These nanoengineered substrates provide sufficient spatial specificity to allow direct observation of peptide adsorption on the calcium phosphate nanoislands. This is particularly the case for RGD peptides conjugated to polyacidic aminoacid domains, which have high affinity for calcium phosphate surfaces. Such substrates can enable studies that decouple the effect of nanotopography and biochemical motifs on cell adhesion and motility, with relevance for applications in bone tissue engineering and regeneration.
9:00 PM - SS8.52
Investigation of Long-Term Protein Adsorption on Antifouling Polypeptoid Brushes.
Aaron Lau 1 , Andrea Statz 1 , Jinghao Kuang 1 , Chunlai Ren 2 , Igal Szleifer 1 , Phillip Messersmith 1
1 Biomedical Engineering, Northwestern University, Evanston, Illinois, United States, 2 National Laboratory of Solid State Microstructures, Nanjing University, Nanjing 210093 China
Show AbstractLong term prevention of non-specific protein adsorption and cell adhesion on surfaces of medical devices is critical to their proper function. Since adsorbed proteins mediate cell-surface interactions, the development of surfaces resistant to protein adsorption is of major interest. However, experimental methods for measuring protein fouling typically probe time-scales (minutes to hours) that are much shorter than medical device lifetimes. In this study, we evaluate, over several weeks, the resistance to protein adsorption of novel N-substituted polypeptide (polypeptoid) brushes developed in our laboratory, and compare the results with predictions obtained from a molecular theory of polymer and protein adsorption. The polypeptoids are resistant to enzymatic degradation and have a precisely defined molecular weight. Long term protein adsorption is characterized in two ways: i) AFM measurements of the protein surface coverage and layer thickness; and ii) quantification of fibroblast adhesion as a proxy for resistance to protein adsorption. Useful information for the rational design of antifouling polymer brush architectures, especially in terms of brush density and brush chain length, is obtained.
9:00 PM - SS8.6
Nanoengineered Biofunctionalized Glass Fibers with Defined Curvature to Investigate Neural Cell Migration.
Sebastian Kruss 1 , Joachim Spatz 1 , Tobias Wolfram 2
1 New Materials and Biosystems, MPI for Metals Research, Stuttgart Germany, 2 Research Group for Biomedical Engineered Surfaces, MPI for Metals Research, Stuttgart Germany
Show AbstractA critical question in cortical histogenesis is the control of neuronal cell migration in the developing brain. A system of radial glial fibers has been shown to provide the primary pathway for CNS neuronal migrations. Although great research effort has been invested there is still a considerable lack of knowledge of important external cues on neuronal guidance. Glial fiber diameter R (and curvature κ=1/R) and the nanoscopic arrangement of adhesion molecules on the interaction area with the neuronal cell are estimated to play a very important role.We developed a glass fiber based device with defined and tunable physical properties (diameter and curvature). In addition nanostructured and controlled biofunctionalized surfaces on fibers were generated. Finally, we investigated how the engineered devices influenced neural cell migration and adhesion. Commercially available glass fibers were etched with hydrofluoric acid (HF) to yield fibers with defined diameters of 20 to 300 µm (κ=50/mm-3,3/mm). Diblock copolymer micelle nanolithography was used to decorate the glass fibers with gold-nanoparticles with different interparticle distances ranging from 40 to 200 nm. Distances were changed on single gradient fibers to further investigate the impact of biomolecule density on cell migration. The Au-decorated glass fibers were treated with poly-l-lysine-grafted-polyethylene glycol (PLL-g-PEG) or silanePEG to prevent unspecific adsorption of proteins or peptides. Au-nanoparticles were decorated with different histidine-tagged proteins (N-Cadherin, L1, and Agrin) via a mono-thiol NTA linker or decorated with peptides (RGD from fibronectin or IKVAV from Laminin1) via direct immobilization with a sulfhydryl group. The Ni-NTA system facilitates the immobilization of single proteins in a site directed fashion, which resembles the original orientation of the protein at the cell membrane. Glass fibers were analyzed with scanning electron microscopy for evaluation of the diameter and curvature and biofunctionalization was analyzed by fluorescence microscopy. Different neural cell lines (mouse neuroblastoma N2a, rat neuroblastoma B35, and human neuroblastoma SHSY-5Y) were plated on glass fibers. Binding capacity of cells and cell morphology were evaluated by phase contrast microscopy and correlated with different fiber diameters (curvatures), interparticle distances and biomolecules. Live-cell imaging was performed on different glass fibers to investigate their effects on the cellular migration behavior of single cells. Specifically designed glass fibers proved to be valuable tools for the study of complex biological problems like cell migration and adhesion. With the defined environment of tunable nanostructured glass fibers, differences in those biological functions could be correlated with quantifiable changes on the engineered material. The system at hand might be applicable as a research tool for first steps towards specifically engineered implants.
9:00 PM - SS8.8
Free Energy Adsorption at Mineral Surfaces - When Does Entropy Matter?
Colin Freeman 1 , John Harding 1
1 Engineering Materials, University of Sheffield, Sheffield United Kingdom
Show AbstractThe binding of biomolecules to materials requires careful consideration of the complex interface [1]. There are the roles of different functional groups within the molecule, the surface chemistry and ordering of the ions within the material and in most cases the impact of water [2]. Analysing all these interactions involves both enthalpic and entropic changes that must be defined if a free energy of adsorption is to be calculated [3].Direct calculation of the free energy with molecular dynamics is not possible since entropy is not an average property of the system. Thermodynamic integration allows us to calculate the free energy change between two systems along a pathway. For the purposes of adsorption we can analyse the energy change as the molecule approaches the surface.We examine the adsorption of several molecules onto calcite surfaces varying in size from a methanoic acid to the protein ovocledin-17 associated with the production of eggshell. In each case we analyse the methods of binding and the effect on the molecule, surface and solvent. We conclude this analysis with estimates of the free energy of adsorption and the role of entropy in molecular binding.[1] CL Freeman, JH Harding, DJ Cooke, JA Elliott, DM Duffy, JS lardge, J. Phys. Chem. C 111 (2007) 11943-11951[2] CL Freeman, I Asteriadis, M. Yang, J. Phys. Chem. C 113 (2009) 3666-3673[3] S Kerisit, SC Parker J. Am. Chem. Soc. 126 (2004) 10152
9:00 PM - SS8.9
Fabrication of Biomimetic Apatite/collagen Composite Coating on Ti6Al4V Discs.
Zengmin Xia 1 , Mei Wei 1
1 , University of Connecticut, Storrs, Connecticut, United States
Show AbstractHydroxyapatite and type I Collagen, the major components of bone tissues, are broadly utilized to coat biomaterial surfaces for enhanced bioactivity. Apatite coating techniques are well realized and developed by many researchers. Using a biomimetic coating technique, bone-like apatite coating with a fast-forming rate, tailored surface morphologies has been achieved in our previous work. The apatite layer demonstrates a high bonding strength and good affinity to bone tissue. However, few works were found in literature on the biomimetic formation of apatite/collagen composite coating onto metallic implants. To the best of our knowledge, there has been no previous study on the adhesive strength test between the hybrid coating and the substrate. The challenge in obtaining high quality apatite/collagen coating lies in the difficulty to control the highly sensitive processes of apatite/collagen co-deposition and collagen fibrillization.In the present work, apatite/collagen coating was prepared by soaking the UV irradiated Ti6Al4V discs in a collagen-containing modified simulated body fluid (m-SBF). UV irradiation was applied to create a hydrophilic surface, which induced the composite coating formation on Ti6Al4V discs. The surface morphology and composition of the hybrid coating at different coating stages were characterized by FESEM, XRD and FTIR. Moreover, the coating conditions, such as pH and collagen concentration of the m-SBF, were varied to achieve the optimal composite coating. A homogenous apatite/collagen biomimetic coating was successfully prepared using an accelerated biomimetic process in our current work. The adhesive strength test suggested that a bonding strength as high as ~18MPa was attained for the apatite/collagen coating on UV irradiated Ti6Al4V substrate. The coating composition and morphology could be tailored by carefully adjusting the initial pH and collagen concentration of the m-SBF solution. The mechanism of the coating formation was investigated based on the results from SEM, XRD and FTIR examinations. In summary, the composite coating can be potentially applied to different implant surfaces to enhance osteointegration of the implants to surrounding bone.
Symposium Organizers
Jose A. Garrido Technische Universitaet München
Erika Johnston Genzyme
Carsten Werner Leibniz Institute of Polymer Research
Thomas Boland The University of Texas-El Paso
SS9: Diamond and Bioinspired Materials
Session Chairs
Thursday AM, December 03, 2009
Ballroom A (Hynes)
9:30 AM - **SS9.1
Ultrastable Biomolecular Interfaces to Nanostructures and Nanostructured Materials.
Robert Hamers 1
1 Department of Chemistry, University of Wisconsin-Madison, Madison, Wisconsin, United States
Show AbstractThe covalent modification of nanomaterials with biological molecules of interest can confer excellent biomolecular recognition properties and control their interactions with the environment. Our work has been addressing the fundamental chemistry in formation of bio-nano hybrid materials and structures, and the application of these in novel sensing applications.The need for high chemical stability drives a particular interest in nanostructured carbon including thin-film diamond and carbon nanofibers, and in nanostructured metal oxides. Photochemical grafting of alkenes has provided one attractive route to formation of ultrastable interfaces that serve as a starting point for making bio-nano hybrids. Mixed layers in which ethylene glycol oligomers are mixed with linker molecules exposing reactive amine or carboxylic acid groups can be used to reduce nonspecific binding and optimize overall selectivity on diamond and other forms of carbon. One novel approach to forming nano-bio sensing systems has been the use of biologically functionalized carbon nanofibers as the basis for a nanoscale biologically selective ‘fuse’ that provides direct digltal bio-to-electronic signal transduction. The high stability of carbon, and especially the mechanical properties of diamond, can also also be used to enhance the properties of nanomechanical approaches to real-time biosensing. Finally, while carbon provides the highest stability, metal oxides such as TiO2 and ZrO2 are also of interest as hard, oxidatively stable materials that can be easily applied to a wide range of materials at moderate temperatures to form thin films with controlled porosity that are transparent to visible light and stable over a wide range of pH. We will present resent recent results on the formation of biological interfaces to metal oxide nanoparticles and thin films and discuss the factors that control stability and selectivity of their interactions.
10:00 AM - SS9.2
Detecting of ATP via Aptamer on Diamond Surface.
Yuichiro Ishiyama 1 , Shinya Tajima 1 , Hirofumi Arai 1 , Yoko Ishii 1 , Hiroshi Kawarada 1
1 Science and engineering, WasedaUniversity, Shinjyuku-ku, Tokyo, Japan
Show AbstractHigh reusability of covalent immobilization on sp3 carbon based biointerface is one of the most attractive characteristics for biosensor application of diamond. In our previous work, DNA hybridization was detected by fluorescence observation and by electrolyte solution gate field-effect transistors (SGFETs) for potentiometric observation. During a number of hybridization and denaturation cycles on the diamond surface, both reversible changes in fluorescence and gate potential were very reproducible throughout a series of measurements [1,2]. This point is advantageous for the development of regenerable biochips based on diamond substrate. Additionally, diamond has many other attractive characteristics for application in sensor devices, such as wide potential window, physicochemical stability, and potential for simple surface modification. In this study, we developed the adenosine triphosphate (ATP) biosensor by using ATP binding DNA aptamer for the first time on diamond. Aptamers are useful oligonucleotide (Short DNA or RNA) for preferentially detecting particular biomolecules such as protein or smaller molecules by its specific binding characteristic to these molecules. [3] The ATP binding DNA aptamer is a single strand deoxynucleotide binding specifically to ATP. [4] The presence of ATP was detected by fluorescence disappearance as described in the following. First, the complementary DNA to the ATP aptamers were immobilized directly on the diamond surface without linker molecules, and a duplex was formed by the hybridization between the immobilized complementary DNA and the ATP aptamers with fluorescent dye Cy5.After the hybridization, the duplex fluorescence was observed. Then, by the introduction of ATPs, the aptamer changed its structure from the duplex to the aptamer/ATP complex and was released from the immobilized DNA, due to the preferential binding of the aptamer to the ATP. The disappearance of luminescent source after the aptamer/ATP complex was washed out from the diamond surface indicates that the ATP reaction with the ATP aptamers took place. The regeneration (or initialization) of the duplex of ATP aptamer labeled with Cy5 can be carried out and checked by the luminescence. This sequential observation indicates the potential application of ATP aptmter on diamond as a reusable biosensor. For label-free detection using SGFET , ATP binding can be detected as decrease of channel surface hole density influenced by the negative charge decrease due to the release of negative charged aptamer. This study is in progress.[1] J.H.Yang, H.Kawarada et.al. Appl. Phys. Express: 1, (2008) 118001[2] S.Kuga, H.Kawarada et.al. J. am. Chem. Soc. 130, (2008) 13251[3] A.D.Ellington, J.W.Szostak Nature 346 , (1990) 818-822[4] D.E.Huizenga, J.W.Szostak Biochemistry 34 (1995) 656–665
10:15 AM - SS9.3
A Platform Approach to Gene Delivery Via Surface Modified Nanodiamonds.
Xueqing Zhang 1 , Mark Chen 1 , Robert Lam 1 , Xiaoyang Xu 1 , Eiji Osawa 2 , Dean Ho 1
1 , Northwestern University, Evanston, Illinois, United States, 2 NanoCarbon Research Institute, Shinshu University, Nagano Japan
Show AbstractGene therapy holds great promise for treating diseases ranging from inherited disorders to acquired conditions and cancers, and has been used with some success since the first gene therapy clinical trials began. Nonetheless, because a method of gene delivery that is both effective and safe has remained elusive, these successes were limited. Functional nanodiamonds (NDs) are rapidly emerging as promising carriers for next-generation therapeutics with demonstrated potential. Here we introduce NDs as vectors for in vitro gene delivery via surface-immobilization with 800 Da polyethyleneimine (PEI800) and covalent conjugation with amine groups. It is interesting to find that both low molecular weight (LMW) PEI coated nanodiamonds (ND-PEI800) and amino-conjugated nanodiamonds (ND-NH2) showed stronger DNA binding capacity, better biocompatibility and cellular internalization in comparison with pristine NDs. Transfection experiments were performed in HeLa cells with all three ND carriers. The transfection efficiencies varied as a function of the ND/DNA weight ratio and declined in the following order: ND-PEI800 > PEI800 > ND-NH2 > ND > Naked DNA. Specifically, ND-PEI800 showed the most efficient transfection in HeLa cells when using different plasmids, and was 400, 70 and 800 times more efficient than ND-NH2, PEI800 alone and unmodified ND, respectively. Because the PEI800 densely coats the ND surface with amino groups, the ND-PEI800 may possess the properties of a proton sponge, which may aid in rupture of and escape from endosomes into the cytoplasm. Therefore, we designed PEI800-modified NDs possessing both the biocompatibility of a LMW PEI, and the high transfection efficiency of a high molecular weight (HMW) PEI. Additionally, we demonstrated that the enhanced delivery properties were exclusively mediated by the hybrid ND-PEI800 material, and not exhibited by any of the materials alone. This represents a simple and efficient platform approach towards gene delivery via DNA-functionalized NDs, and serves as a rapid, scalable, and broadly applicable gene therapy strategy.
10:30 AM - SS9.4
Para-Xylylene-Encapsulation of Ultrananocrystalline Diamond-Based Microchip Platforms for Controlled Multi-therapeutic Delivery.
Erik Robinson 1 , Paola Bruno 2 , Dieter Gruen 2 , Dean Ho 1 3 4
1 Mechanical Engineering, Northwestern University, Evanston, Illinois, United States, 2 Materials Science Division, Argonne National Laboratory, Argonne, Illinois, United States, 3 Biomedical Engineering, Northwestern University, Evanston, Illinois, United States, 4 Robert H. Lurie Comprehensive Cancer Center, Northwestern University, Evanston, Illinois, United States
Show AbstractUltrananocrystalline diamond (UNCD) thin films serve as promising platforms for medical applications due to their biocompatibility, scalable chemical vapor deposition parameters (CVD), and capacity for interface with a broad array of biologically active compounds ranging from small molecules, to proteins and DNA/RNA. Furthermore, the unique UNCD material properties result in its resistance to oxidation and serve as optimal foundations for generating highly stable UNCD surfaces that have been derivatized with biological molecules [1]. Current requirements for the optimization of implantable drug delivery devices include the ability to controllably release therapeutic compounds while minimizing burst release and supporting their sustained elution. Furthermore, material selection for device as well as drug packaging plays a key role in mediating long term device stability/bioamenability, preservation of drug structure and activity, and overall system function. As such, para-xylylene serves as a favorable route towards the encapsulation of UNCD-based devices as it is FDA-approved for the coating of approved implants due to its inert properties and perpetual stability following insertion, among other advantages. We have previously shown that therapeutically-active collagen networks interfaced with UNCD films were capable of mediating localized/sustained drug release [2]. Towards the construction of implantable devices that harness UNCD-mediated slow release/biostability properties, this work demonstrates the fabrication of para-xylylene A (dixA)-encapsulated UNCD microchips for the controlled elution of multiple agents including insulin (protein) and ibuprofen (small molecule anti-inflammatory) through the dixA structure with modulated porosity. Due to the unique amine group presentation of the dixA material, arginine-glycine-aspartic acid (RGD) peptides were further conjugated to the device surface to mediate enhanced biocompatibility. Following CVD deposition UNCD nanofilms onto silicon substrates, devices were functionalized with therapeutic agents and subsequently coated with dixA. Coating porosity was controlled via variations to the amount of dixA deposited which was utilized to dictate drug release. Sustained therapeutic delivery as well as reductions in burst release was observed via UV-vis spectrophotometry while confirmation of RGD functionalization was performed via atomic force microscopy and bicinchoninic acid (BCA)assays. These metrics serve as indicators for the potential of the para-xylylene-UNCD device as a biostable implant for sustained multi-agent treatment.[1] Gruen, D.M. Annu. Rev. Mater. Sci. 1999, 29, 211-259.[2] Huang, H.; Chen, M.; Lam, R.; Bruno, P.; Robinson, E.; Gruen, D.;, Ho, D. J. Phys. Chem. B, 2009, 113, 2966-2971.D. Ho acknowledges support from NSF grants CMMI-0846323 and CMMI-0856492. Work at Argonne was supported by the U.S. Department of Energy, BES-Materials Sciences, under Contract DE-AC02-06CH11357.
10:45 AM - SS9.5
Modified Nanodiamonds for Specified Adsorption of Fluorescent Dyes and Toxins.
Natalie Gibson 1 , Olga Shenderova 1 2 , Tzy-Jiun Mark Luo 1 , Zachary Fitzgerald 1 , Donald Brenner 1
1 Materials Science and Engineering, North Carolina State University, Raleigh, North Carolina, United States, 2 , International Technology Center, Research Triangle Park, North Carolina, United States
Show AbstractNanodiamonds (NDs) have gained heightened attention due to their desirable chemical, physical and biological properties. NDs’ biocompatibility, biological stability and absence of cytotoxicity lends themselves for use in a wide range of applications, including drug delivery agents, carriers for active molecules, bioloabels and enterosorbents for toxin neutralization. Their specific surface areas (300 to 400 m2/g by detonation synthesis) and abundant surface chemistries have shown high affinities for adsorbing various biological molecules such as proteins and enzymes. However, NDs, commercially available under numerous vendors, show inconsistencies with surface functional groups, aggregate sizes, and impurity contents that may hinder adsorption characteristics. Research explores NDs modified under various treatments to enhance binding selectivity toward target chemicals. A variety of target chemicals were chosen to simulate possible applications as biolabels and enterosorbents. Fluorescent dyes, propidium iodide, pyranine and ABTS were used in the development of a ND biolabeling complex. Binding mechanisms and controllable release of the dyes were observed using UV-Vis spectroscopy and cyclic voltammetry measurements. Adsorption capacities and strengths of the dyes were determined through use of the Langmuir isotherm and related transform calculations. Potential enterosorbent applications for aflatoxin B1 (AfB1) adsorption by ND substrates were also explored. AfB1, produced by certain strains of Aspergillus flavus and Aspergillus parasiticus, is a potent carcinogen and mutagen. If consumed by humans or animals aflatoxicosis, suppression of the immune system and death have all been observed. Deactivation of these toxins requires an effective substrate that is biocompatible, chemically inert and can remain highly dispersed under severe pH changes in the gastrointestical tract. NDs have been shown to bind strongly to various biological molecules and resist agglomeration in a wide range of pH, making them a candidate to adsorb AfB1. Furthermore, ND functionalization may be used to gain high selectively for the toxin and prevent uptake of essential nutrients and vitamins. Initial studies examined the toxin adsorbing behaviors of AfB1 on modified ND substrates using UV-Vis spectroscopy, chromatography and cyclic voltammetry. Characteristics of NDs influencing adsorption will be reported including effects of various surface groups, electrostatic interactions, aggregate sizes and porosities.
11:30 AM - **SS9.6
Hierarchically Structured Conjugated Polymers.
Holger Frauenrath 1
1 Institute of Materials, Ecole Polytechnique Federale de Lausanne (EPFL), Lausanne Switzerland
Show AbstractBiomaterials derive their often extraordinary properties from the hierarchical structure formation of typical biopolymers. New materials with interesting properties may be obtained if nature’s strategy to prepare such materials can be mimicked and transferred to synthetic conjugated polymers.For this purpose, we utilized diacetylene macromonomers based on β-sheet-forming oligopeptide-polymer conjugates as supramolecular building blocks. The monomers gave rise to supramolecular polymers with a finite number of strands, a uniform diameter of a few nanometers, and defined superstructures, depending on the exact pattern of amide hydrogen bonding sites. These were then converted into conjugated polymers under retention of their hierarchical structures, leading to soluble poly(diacetylene)s with single-stranded, double-helical, or quadruple-helical quaternary structures. The multiple-helical polymers exhibited a rich dynamic folding behavior similar to biopolymers upon the addition of hydrogen-bond-breaking cosolvents.The diacetylene macromonomers served as a model system to improve our understanding of how to use amide hydrogen-bonding sites in order to control the placement and reactivity of molecular precursors for hierarchically structured organic materials. Related conjugated oligomers (oligothiophenes, oligophenylenes, oligoethynylenes) have been prepared and investigated as molecular optoelectronic materials themselves, or as molecular precursors for the preparation of functional carbon nanostructures as well as carbon materials.
12:00 PM - SS9.7
Biomimetic Nanostructured Surfaces with Designer Mechanics and Geometry for Broad Biological Applications.
Alexander Epstein 1 , Boaz Pokroy 1 , Joanna Aizenberg 1 2
1 School of Engineering and Applied Sciences, Harvard University, Cambridge, Massachusetts, United States, 2 Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts, United States
Show AbstractBiology abounds with examples of functional structures, whose properties are unmatched in today’s smart synthetic materials. There is a growing body of information describing natural structures with sophisticated design strategies that lend the organisms superior mechanical, optical, adhesive, self-cleaning, actuation and sensing capabilities. Notably, the common feature of these largely unrelated designs is the use of fibers and high-aspect-ratio nano- and micro-structures. While nanostructured superhydrophobic surfaces inspired by the lotus flower and the adhesive properties of gecko feet have been mimicked with success, actuation/sensing at the sub-micron scale is more challenging. In the current study we use a truly “materials” approach to develop a low-cost procedure for producing a bio-inspired arbitrarily-designed actuated surface with high-aspect-ratio nanostructures that are themselves responsive to a variety of stimuli and have a finely-tuned geometry and stiffness. [1] In our approach, we adapt and significantly extend the soft lithography technique as a low cost alternative to conventional lithography for the high resolution replication of nanostructured substrates in polydimethylsiloxane (PDMS). However, PDMS is not the final nanostructured material; it is used as an intermediate elastomeric mold to cast a replica in the material of choice, including metals, ceramics, or polymers. This enables the fabrication of a biomimetic array of stable, high-aspect-ratio features whose stiffness and sensitivity can be varied by five orders of magnitude by tailoring the final material—as we demonstrate by varying ratios of two epoxy resins—and the geometry of the nanostructures can be tuned by applying a specific deformation to the PDMS mold. Our soft lithographic method therefore transcends one-to-one replication and allows us to produce nanostructures with nontrivial geometries that would be expensive or infeasible via conventional lithography: arbitrary cross-sectional shapes, tilt orientations, and 2D lattices different from the master. The resulting bio-inspired surfaces offer multifunctional characteristics that include superhydrophobic character, actuation under e-beam and numerous other stimuli, as well as field-sensing capabilities. We have also developed complementary techniques via reactive ion etching to directly tune the micro/nanopost sensitivity for specific applications. The use of these structures in biomedical and antifouling applications will be discussed.[1] B. Pokroy, A.K Epstein, M.C.M. Persson-Gulda, and J. Aizenberg. Fabrication of Bioinspired Actuated Nanostructures with Arbitrary Geometry and Stiffness. Advanced Materials 2009, 21, 463-469.
12:15 PM - SS9.8
Self-assembling Biomimetic Surface Coatings from Modular Proteins.
Stephen Fischer 1 2 , Fan Wan 1 , Zeinab al-Rekabi 1 , Scott Dick 1 , James Harden 1 3
1 Physics, University of Ottawa, Ottawa, Ontario, Canada, 2 Materials Science and Engineering, Johns Hopkins University, Baltimore, Maryland, United States, 3 Ottawa Institute of Systems Biology, University of Ottawa, Ottawa, Ontario, Canada
Show AbstractDe novo protein design and recombinant DNA methods have been used to develop a library of proteins for creating designer biofunctional interfaces. These are diblock protein polymers composed of a surface-active, amphiphilic block joined to a disordered, water soluble block with an end terminal bioactive domain. The amphiphilic block has a strong affinity for many synthetic polymer surfaces, providing a facile means of imparting biological functionality to otherwise bio-neutral materials through physical self-assembly. We have incorporated a series of bioactive end domains into this diblock motif, including sequences that encode specific cell binding and signaling functions of extracellular matrix constituents (e.g. RGD and YIGSR), and ligands that facilitate the association of biomacromolecules such as growth factors and cytokines. In this talk, we show that these diblock constructs self-assemble into robust biofunctional surface coatings on several model synthetic polymer materials. We demonstrate that surface adsorption of the proteins has minimal impacts on the presentation of the bioactive domains in the soluble block, enabling us to tune surface ligand density. And, through the use of microscopic and cell proliferation assays, we show that the resulting biofunctional interfaces are capable of inducing appropriate cellular responses in a variety of human cell types. Such a combinatorial approach to creating tailored biointerfaces, in which the end user can choose from a library of bioactive modules, mixing and matching as needed, is a promising strategy for biomedical applications.
12:30 PM - SS9.9
Bio-inspired materials: a new solid state NMR characterization – MACS, Magic Angle Coil Spinning.
Christian Bonhomme 1 , Florence Babonneau 1 , Dimitris Sakellariou 2 , Pedro Aguiar 2
1 , universite P et M Curie, Paris France, 2 , CEA Iramis, Saclay France
Show AbstractSolid state Nuclear Magnetic Resonance (NMR) – and its latest experimental, instrumental and theoretical developments – appears as a remarkable tool of investigation for hybrid materials and interfaces [1-2]. Very recently, we have proposed new syntheses of hybrid silicas based on molecular recognition through H-bonding. Homo-association of silylated ureidopyrimidinone (UPY) entities, followed by standard hydrolysis and condensation sol-gel reactions, led to nanostructured silica derivatives [3]. The key characterization of the H-bond networks was based on advanced 1H double quantum dipolar recoupling techniques allowing for the detailed description of proton connectivities in the crystalline precursors (silyl-UPY), as well as in the amorphous final materials. The approach was successfully extended to Adenine (A) and Thymine (T) silylated derivatives. Homo-associations (A/A and T/T), as well as hetero-association (A/T), were clearly evidenced by high resolution 1H NMR [4]. By using such silylA and silylT precursors, ways are open to "real" nanostructured bioinspired materials, exhibiting H-bond driven interfaces. As a matter of fact, the main drawback of NMR remains sensitivity, excluding the study of small amounts of matter (roughly one hundred of micrograms). Recently, Sakellariou and coworkers [5] proposed the so-called MACS (Magic Angle Coil Spinning) technique, combining the fast rotation at the magic angle of the sample and of a micro-coil (located inside the main rotor). A gain of 10 in sensitivity (and 100 in time) was expected. In this communication, we show the first 1D and 2D 1H MACS NMR spectra of hybrid derivatives related to a single nanostructured film (TEOS/CTAB/PhenylSi(OEt)3), corresponding to 100 micrograms of matter. The sensitivity is high, as "noiseless" spectra were obtained in less than 2 min. The technique can be extended to other important nuclei, such as 13C, 29Si … In our opinion, the NanoMACS approach should be of great help in a very near future for the multinuclear and multidimensional NMR characterization of a large variety of bio-inspired materials, including the precise description of the bio-inorganic interfaces. The selectivity of NMR combined with much enhanced sensitivity opens obviously new perspectives in the field of bio-inspired materials. [1] C. Bonhomme et al. Accounts Chem. Res., 40 (2007) 738 [2] N. Baccile et al. Chem. Mater., 19 (2007) 1343 [3] G. Arrachart et al., Chem. Eur. J., 15 (2009) 5002 [4] G. Arrachart et al., J. Mater. Chem., 18 (2008) 392 [5] D. Sakellariou et al., Nature, 447 (2007) 694.
12:45 PM - SS9.10
A Bio-inspired Approach to Tip-based Nanofabrication at Peptide-derivatized Templates.
Sungwook Chung 1 2 , Michael Nielsen 1 , Byoung-Chul Lee 1 , James DeYoreo 1 , Jonathan Felts 3 , William King 3
1 Molecular Foundry, Lawrence Berkeley National Laboratory, Berkeley, California, United States, 2 Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California, United States, 3 Department of Mechanical Science and Engineering, University of Illinois Urbana-Champaign, Urbana, Illinois, United States
Show AbstractDeveloping generic platforms to create deterministic patterns of nanostructures at surfaces is one of the central challenges of nanotechnology. To address this challenge, we are developing a tip-based approach to fabrication of nanowires and nanoparticles that relies on patterning of functional biotemplating molecules (BTMs) selected for their ability to mediate material-specific formation of inorganic solids under mild reaction conditions. Our strategy is to pattern maleimide terminated self-assembled monolayers (SAMs), which either specifically react to linker molecules for functional BTMs or directly conjugate to BTMs. By coupling these linkers through DNA primers to distinct BTMs presenting complimentary sequences, multiple materials can be incorporated into a single pattern. These patterns are then backfilled with a PEG-like SAM that acts as a resist against unwanted formation of the target materials. Here we demonstrate this approach through both micro-stamping and scanned-probe patterning using a peptide molecule, one of our BTM candidates, that code for nucleation of gold. We show that both the dimension and morphology of gold wires can be controlled by optimizing the pattern geometry and growth conditions. Finally, we show how the use of heated cantilevers enables control over the dimensions of the BTM patterns and present a thermally activated linker-BTM reaction scheme that can, in principle, create templates with feature sizes of ~ 10 nm.
This work was performed under the auspices of the U.S. Department of Energy by the University of California, Lawrence Berkeley National Laboratory (LBNL) under the U.S. Department of Energy Contract No. DE-AC02-05CH11231.
SS10: Patterning and Topography
Session Chairs
Thursday PM, December 03, 2009
Ballroom A (Hynes)
2:30 PM - **SS10.1
Massively Parallel Polymer Pen Lithography.
Zijian Zheng 1 , Weston Daniel 1 , Louise Giam 1 , Fengwei Huo 1 , Andrew Senesi 1 , Gengfeng Zheng 1 , Chad Mirkin 1
1 , Northwestern University, Evanston, Illinois, United States
Show AbstractFabricating protein micro and nano arrays in a low-cost and high throughput manner is important for many applications ranging from drug screening, medical diagnostics, biosensors, to fundamental biological studies.[1] Conventional approaches for making protein microarrays include photolithography and inkjet printing while recent work, however, has focused on the miniaturization of protein patterns towards the nanoscale. In this regime, high density arrays provide increased detection sensitivity and, in principle, would allow one to screen millions of biomarkers in one chip. Such protein nanopatterns also can provide insight into important fundamental biological processes, such as cell adhesion and differentiation. Moreover, the ability to place an array of proteins or even multiple protein structures underneath a single cell opens up the opportunity to study multivalent interactions between a cell and a surface, and points to a major capability of nanoarray technology not afforded by analogous microscale structures. Dip-pen nanolithography (DPN)[2] and Polymer Pen Lithography (PPL)[3] are versatile “direct write” methods which allow one to generate structures in parallel over large areas with sub-micrometer resolution. In particular, PPL uses soft polymeric tip arrays which allow for novel force-dependent patterning reminiscent of contact printing while preserving time-dependent nanoscale patterning characteristic of DPN. We have demonstrated a novel way of using a PPL array mould to localize different inks on the pens of an array. This strategy for localizing inks of interest on the nanoscale tips of a two-dimensional PPL array allows for the multiplexed patterning of protein nano and micro arrays in a high throughput and low-cost manner. The resulting protein structures are not only functional, but also can be prepared with no evidence of cross-contamination over large areas. This novel method is materials-general and can be applied to large scale, multiplexed nano- and micropatterning of many biomolecules.[1] I. Balboni, et al. Annu. Rev. Immuno. 2006, 24, 391.[2] R. D. Piner, et al. Science. 1999, 283, 661.[3] F. Huo, et al. Science. 2008, 321, 1658.
3:00 PM - SS10.2
Biomimetic Nanopatterns Achieved by Combining Molecularly Imprinted Polymers with Soft Lithography.
Helene Lalo 1 , Etienne Dague 1 , Christophe Vieu 1 , Karsten Haupt 3 , Cedric Ayela 2
1 , LAAS-CNRS; University of Toulouse; 7, avenue du Colonel Roche F-31077, Toulouse France, 3 , Université de Technologie de Compiègne UMR 6022, Compiègne France, 2 , Laboratoire de l'Intégration du Matériau au Système UMR 5218; University of Bordeaux, Talence France
Show AbstractNanobiochips are of main interest since high level of integration for high-throughput molecular screening and testing can be achieved. Biomacromolecules patterned at nanoscale showed enhanced biological recognition capabilities when compared to standard formats. However, natural biomolecules still suffer of poor stability when processed out of their native conditions, reducing their potential when used in severe environments. To close the gap, biomimetic polymers based on molecular imprinting (MIPs) are being evaluated as a powerful alternative. But creating 3D nanopatterns of active MIPs is hard to achieve.Among available nanopatterning tools, soft lithography was considered for its simplicity, low-cost and showed good compatibility when combined with polymers as active layer. Indeed, we describe the simultaneous use of three structuring techniques, soft lithography, nanomolding on a sacrificial soluble polymer, and molecular imprinting, to nanopattern polymers with specific molecular recognition properties. PDMS Stamps were composed of lines of 400µm long, 500nm wide with a pitch of 1µm and 160nm deep to form networks of 400x400µm2. Before processing, stamps surface was made hydrophilic by oxygen plasma treatment, to allow spreading of prepolymers. After deposition on silanized glass surfaces and photopolymerization, MIPs nanolines of 400µm long, 660nm wide with a pitch of 1µm and 140nm high were obtained. From those results, the transfer process was studied since MIPs nanopatterns are wider than features defined on the PDMS mold. The details of the nanopatterning principles of MIP materials will be thus presented together with the interpretation of the effect of the plasma treatment of the PDMS mold.But, beyond the nanopatterning of MIPs with high resolution by soft lithography, their molecular recognition capability has to be demonstrated. Printed MIPs were based on amino acid derivative Boc-L-Phenylalanine (Boc-L-Phe) as template, while they were able to rebind a fluorescent analogue Dansyl-L-Phenylalanine (Dansyl-L-Phe). Boc-L-Phe was chosen as template since it avoids residual fluorescence of entrapped molecules that could not be removed from the polymer matrix. Rebinding of Dansyl-L-Phe was clearly demonstrated using fluorescence microscopy since a significant and reproducible fluorescence signal was measured on MIP while a weak signal was found on a non-imprinted polymer, acting as a control. Also, when the analyte was extracted from the polymer, the fluorescence level was close to the background noise. Finally, through AFM imaging of the patterns before and after recognition of the target molecules, we will discuss the dimensional changes occurring in the MIP material after analyte rebinding.
3:15 PM - SS10.3
Addressable Lipid Rafts Nanoarrays Toward Advanced Nanomedicine.
Tomoji Kawai 1 , BongKuk Lee 1 , HeaYeon Lee 1
1 , Osaka university , Osaka Japan
Show AbstractBiocompatible integrated nanopattern requires the fabrication of appropriately designed nanomatrix for high sensitivity homogenous assays, which are capable of ultimately mimic the physiological environment. We constructed a simple method for the construction of nano(submicro) arrays of tethered lipid bilayer raft membranes (tLBRMs) in a nanopatterned Poly(vinyl alcohol)(PVA) hydrogel on a gold substrate comprising a biosensing platform, using ultraviolet-nanoimprint lithography (UV-NIL), as an inert barrier to prevent biofouling. Lipid rafts are cholesterol- and sphingolipid-rich domains that function as platforms for signal transduction and other cellular processes. Tethered lipid bilayers have been proposed as a promising model to describe the structure and function of cell membranes. The robust structures of the nanopatterned PVA hydrogel were stable for up to three weeks in phosphate-buffered saline solution despite significant swelling (100% in height) by hydration. The PVA hydrogel strongly restricted the adhesion of vesicles, resulting in an array of highly selective hydrogel nanowell. The tethered lipid bilayer raft membranes were not formed by direct vesicle fusion, although raft vesicles containing poly(ethylene glycol) lipopolymer were selectively immobilized on gold substrates patterned with PVA hydrogel. These results suggest that the fabrication of inert nanostructures and the site-selective modification of solid surfaces to induce vesicle rupture may be essential in the construction of tethered lipid bilayer raft membranes nanoarrays using stepwise self-assembly. It is envisioned that the miniaturized integrated nanowell array-chip system has excellent advantages over conventional instrumental systems for analysis of biomaterials such as compactness, economical, rapid, and multiplex capability. Furthermore, the nanowell array-chip system should be compatible with a variety of nanodevices that aim at high throughput analysis.
3:30 PM - SS10.4
Versatile Approach to High-Throughput Cellular Microarrays Using Thiol-ene Click Chemistry.
Nalini Gupta 1 , Craig Hawker 1
1 Materials Research Lab, University of California at Santa Barbara, Santa Barbara, California, United States
Show AbstractMicroarray technology has garnered much attention as a simple and efficient method to screen large libraries of proteins, peptides, DNA, among other biological molecules for a variety of applications. Cellular microarrays in particular have been used to screen the interactions between cells and variety of (bio)molecules such as proteins, peptides and synthetic polymers. Inkjet printing has been widely investigated in forming well-defined microarrays due to the low-cost and automated methodology, however, most are printed on stiff glass substrates which may affect cellular responses. We sought to form microarrays on soft hydrogel substrates with covalently bound peptides in order to effectively screen integrin-receptor interactions with various cell types in a high-throughput manner. Through a novel strategy, the facile fabrication of functionalized high-throughput microarrays embedded at the surface of a hydrogel matrix was developed using thiol-ene click chemistry. This user-friendly strategy provides a platform for the immobilization of a combination of bioactive and diagnostic molecules, such as peptides and dyes, at the surface of poly(ethylene glycol) based hydrogels. The robust and orthogonal nature of the thiol-ene chemistry allows for a range of covalent attachment strategies in a fast and reliable manner. In this work, two strategies for the attachment of bioactive molecules were examined: a) the direct attachment of the biomolecules of interest to form the microarray, and b) the post-modification of a pre-formed microarray with orthogonal, click-type reactive functional groups. The accessibility of these cellular microarrays to screen peptide-ligand activity was demonstrated with the RGD peptide sequence and fibroblast cells with selective adhesion only being observed when the active RGD peptide was covalently immobilized on the surface of the array. The orthogonality of the printed arrays was further analyzed with functional dyes through azide-alkyne, amine-active ester, hydrazide-aldehyde and thioester-cysteine (native chemical ligation) coupling chemistries. In all cases, selective conjugation was observed in the printed arrays without any non-specific binding to the hydrogel substrate.
3:45 PM - SS10.5
Pixelated Conducting Polymer Surfaces for Control of Cell Adhesion.
Esma Ismailova 1 , Sang Yang 1 , Alwin M.D. Wan 1 , George G. Malliaras 1 2 , Claudia Fischbach 3 , Roisin Owens 2
1 MSE, Cornell University, Ithaca, New York, United States, 2 , Centre Microelectronique de Provence, Gardanne France, 3 Biomedical Engineering, Cornell University, Ithaca, New York, United States
Show AbstractConducting polymers offer unique opportunities as electrically “active” substrates for cell growth. We report pixelated surfaces in which individual pixels can be electrically switched between two states – one that promotes cell adhesion and one that does not. We investigated the effectiveness of such surfaces in the deterministic placement of various cells, including neurons. We discuss the dynamics of cell hoping among different pixels and correlated it with device geometry.
4:30 PM - **SS10.6
Nanopatterned Interfaces to Control Stem Cell Fate.
Kevin Healy 1
1 MSE, Univ. of California at Berkeley, Berkeley, California, United States
Show AbstractBiomimetic materials have been designed to emulate the regulatory processes involved in cell fate determination by employing ligands inspired by the natural extracellular matrix, cell–cell contacts, proteolytic remodeling, and growth factors with precisely engineered density, nanoscale arrangement, and presentation. While biochemical signals that modulate cell function have been extensively studied, only recently have the mechanical properties of a cell’s microenvironment been shown to regulate its behavior. We propose that altering the physical state of a stem cell, via spatial clustering of its adhesions with a surface, will influence self-renewal and differentiation to a specific phenotype. Therefore, we have developed nanopatterned cell culture substrata where the size, peptide ligand density, number/cell body, and spatial arrangement of integrin-engaging domains can be varied to control cell and colony morphology. We wish to use these materials to explore the nanoscale spatial arrangement of cell adhesion domains and assess their effect on the self-renewal and directed differentiation of stem cells.
5:00 PM - SS10.7
Controlling Cell Rolling by Patterning of Receptors Using Microcontact Printing.
Chia-Hua Lee 1 , Suman Bose 2 , Jeffrey Karp 3 4 5 , Rohit Karnik 2
1 Materials Science and Engineering, MIT, Cambridge, Massachusetts, United States, 2 Mechanical Engineering, MIT, Cambridge, Massachusetts, United States, 3 , Harvard-MIT Division of Health Science and Technology, Cambridge, Massachusetts, United States, 4 Department of Medicine, Brigham and Women’s Hospital, Cambridge, Massachusetts, United States, 5 , Harvard Medical School, Boston, Massachusetts, United States
Show AbstractSeparation and isolation of cells from a heterogeneous population is important for diagnostic and therapeutic applications and for biomedical research. We recently discovered that the trajectories of cells rolling in a microfluidic device can be controlled using edges of receptor patterns at an angle to the direction of fluid flow. This discovery opens the possibility of tuning cell rolling interactions and deflecting and collecting cells in a simple, flow-though device for label-free separation or analysis of cells. Cell rolling is a physiological phenomenon involving transient receptor-ligand interactions that is involved in homing and extravasation of leukocytes, hematopoietic and mesenchymal stem and progenitor cells, and metastatic cancer cells.In this work, we demonstrate the feasibility of microcontact printing as a versatile method to pattern P-selectin edges for controlling cell rolling. Microcontact printing stamps were fabricated in PDMS by SU-8 molding process. The stamp with multiple straight bands was first inked with a solution of PEG molecules, dried, and pressed onto a gold surface to be patterned. After selective deposition of PEG molecules, the bare areas were then filled with P-selectin receptors. AFM and SEM characterization revealed sharp and straight edges that could direct the trajectories of rolling HL60 cells and neutrophils. HL60 myeloid cell suspension (~105 cells/mL) was flowed over the surface in flow chamber at shear rates ranging from 0.1 dyn/cm2 to 0.6 dyn/cm2 and edge angles ranging from 5° to 25°. Cells were clearly seen to roll on plain P-selectin regions, encounter an edge, and then roll along the edge at an angle to the direction of fluid flow. Matlab image analysis revealed that the distance traveled by the HL60 cells along the edges decreased with increasing angle of the edge with respect to the direction of fluid flow. Cell rolling velocities were higher on the edges as compared to plain P-selectin regions, and exhibited a maximum at an edge angle of 20°, suggesting that only cells with the strongest adhesive interactions could roll along the edges angles larger than 20°. Our results show that patterning of receptor edges can be used to tune cell rolling adhesion and control the transport of cells. Such substrates could find applications in separation and sensing of leukocytes for point-of-care diagnostics or isolation of viable stem cells and cancer cells for therapeutics or personalized medicine.
5:15 PM - SS10.8
The Impact of Material Nanotopography on Cell Functions and Filopodia Extension: Experiments and Modeling.
Lei Yang 1 , Qunyang Li 1 3 , Viswanath Chinthapenta 1 , Brian Sheldon 1 , Thomas Webster 1 2
1 Engineering, Brown University, Providence, Rhode Island, United States, 3 Department of Mechanical Engineering, University of Pennsylvania, Philadelphia, Pennsylvania, United States, 2 Department of Orthopaedics, Brown University, Providence, Rhode Island, United States
Show AbstractExploring the cell-material interface is an emerging area of great interest in biomaterial science. Specifically, creating nanostructured surface interfaces to improve biomaterial efficacy is one of these key focus topics. As an example, an increasing number of studies have demonstrated the positive role nanostructured surfaces can have towards promoting various cell functions. However, the relevant mechanism behind this improvement in biological interactions at the cell-implant interface is not well understood. For this reason, here, osteoblast (bone forming cells) and fibroblast (fibrous, soft tissue forming cells) functions (including adhesion, proliferation, and differentiation) on two carefully fabricated diamond films with dramatically different topographies were tested. The results over all the time periods tested revealed greater cell responses on nanocrystalline diamond (grain sizes <100nm) compared to microcrystalline diamond (grain sizes 200~1000nm). In order to understand this positive impact of diamond nanotopography on cell responses, fibronectin absorption and subsequent cell spreading were studied. More importantly, cell filopodia extensions were also studied through computational mechanical modeling and live cell imaging. A deflection-diffusion model of cell filopodia extension was established and clearly suggested that increasing the lateral dimension or height of nanometer surface features could inhibit cell filopodia extension and decrease cell spreading. This modeling result was supported by real time live cell imaging of osteoblast and fibroblast filopodia extension on nanocrystalline and microcrystalline diamond. Both the experiments and modeling from this study indicated that a nanometer surface topography can control cell responses to promote implant efficacy.
5:30 PM - SS10.9
Investigation of Cellular Adhesion by Nanopillar Array and Super-resolution Microscopy.
Jau-Ye Shiu 1 , ChiungWen Kuo 1 , Peilin Chen 1
1 Research Center for Applied Sciences, Academia Sinica, Taipei Taiwan
Show AbstractCellular adhesion plays a very important role in many biological processes such as embryonic development, tissue function, inflammation and wound healing. Most tissue cells are not viable when suspended in a fluid. These cells need to adhere to solid surfaces, which can resist to the pushing and the pulling forces exerted by cells. The adhesion of cells on the soft materials is known to be characteristic of important phenotypes, which leads to the development of cancer identification by measuring the cell growth on soft agar gels. Recently, it has been shown that the differentiation of mesencymal stem cells (MSCs) depended on the matrix elasticity, which indicated that it is necessary to optimize the stiffness of matrix to promote regeneration. Therefore, it is important to investigate the mechanical force of the cell adhesion. In the past few years, our group has developed two techniques, polymeric nanopillar arrays and super-resolution microscopy, to investigate the cellular adhesion process. By a combination of nanosphere lithography and nanoimprint lithography, the polymeric nanopillar arrays have been fabricated routinely with various aspect ratios. Upon the cell adhesion on the polymeric nanopillars, the cells exert traction forces, which deflect the tip of nanopillars. By calculating the displacements of the tips using the Young’s modulus of the polymer, the evolution of the traction force during the cell adhesion process can be monitored as a function of time. This technique allows us to measure the adhesion force in the range of uN to pN and to map the cellular adhesion force with sub-micrometer resolution. The formation of focal adhesion on the nanostructures was also investigated by the super-resolution microscopy, which is cable of localizing the position of individual focal adhesion proteins inside living cells with 10 nm lateral resolution.
5:45 PM - SS10.10
Engineering the Interface of Nanoparticles to Biology.
Kimberly Hamad-Schifferli 1 , Marie-Eve Aubin-Tam 2 , Wonmuk Hwang 3
1 Biological Engineering and Mechanical Engineering, MIT, Cambridge, Massachusetts, United States, 2 Mechanical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States, 3 Biomedical Engineering, Texas A & M University, College Station, Texas, United States
Show AbstractMany have been inspired by Nature to utilize biology for applications in computation, self-assembly, and mechanics. Because biomolecules are inherently on the nanoscale, nanotechnology has emerged as an appropriate means for controlling biology. This requires both understanding the inorganic properties of the nanoparticle as well as creating an interface that is compatible with the complex and highly disordered environments of real biological systems. We will discuss the use of nanoparticles composed of Au, Fe3O4, Fe2O3, CoFe2O4, and similar materials in biological applications by engineering both the inorganic properties of the nanoparticles along with creating optimal biological interfaces. We study the interface between the nanoparticle and covalently linked proteins and DNA. Labeling proteins with nanoparticles has been utilized for many applications but often the structure of the protein in the conjugate is not characterized. In addition, site-specific labeling of the protein with a nanoparticle has been achieved for only a limited set of proteins and nanoparticles. We present work in which we study the interface between nanoparticles and the protein cytochrome c. We vary nanoparticle ligand and composition, as well as labeling site on the protein. Biophysical techniques such as quantitative gel electrophoresis, circular dichroism, and optical spectroscopy are used to characterize the structure of the protein in the conjugate. These experiments allow us to understand some of the chemical interactions involved in non-specific adsorption, and come up with general design rules for optimal conjugation.