Shashi Murthy Northeastern University
Henry Zeringue University of Pittsburgh
Saif Khan National University of Singapore
Victor Ugaz Texas A&M University
Dept of Chemical Engineering, Northeastern University
Microfluidic ChipShop GmbH
OO1: Frontiers in Lab-on-a-Chip Research I
Tuesday AM, April 14, 2009
Room 3016 (Moscone West)
9:00 AM - **OO1.1
Precision Measurement in Biology.
Stephen Quake 1 Show Abstract
1 Bioengineering and Applied Physics, Stanford University and Howard Hughes Medical Institute, Stanford, California, United States
Is biology a quantitative science like physics? I will discuss the role of precision measurement in both physics and biology, and argue that in fact both fields can be tied together by the use and consequences of precision measurement.The elementary quanta of biology are twofold: the macromolecule and the cell. Cells are the fundamental unit of life, and macromolecules are the fundamental elements of the cell. I will describe how precision measurements have been used to explore the basic properties of these quanta, and more generally how the quest for higher precisionalmost inevitably leads to the development of new technologies, which in turn catalyze further scientific discovery. In the 21st century, there are no remaining experimental barriers to biology becoming a truly quantitative and mathematical science.
9:30 AM - **OO1.2
Nanoplasmonics and Integrated Biological Microprocessor for Molecular Medicine.
Luke Lee 1 2 3 Show Abstract
1 Department of Bioengineering, University of California, Berkeley, Berkeley, California, United States, 2 Berkeley Sensor and Actuator Center, University of California, Berkeley, Berkeley, California, United States, 3 Biomolecular Nanotechnology Center, University of California, Berkeley, Berkeley, California, United States
In this talk, I will discuss nanoplasmonics for molecular spectroscopic imaging and optogenetics. Using new paradigms of biological inspiration and understanding of electron transfer mechanism in biological systems, we have developed quantized Plasmon Resonance Energy Transfer (PRET) nanospectroscopy for molecular imaging of living cells. For the remote optical control of gene regulation and protein expression, we have accomplished Oligonucleotides on a Nanoplasmonic Carrier Optical Switch (ONCOS). ONCOS allows on-demand gene silencing with nanometer-scale spatial resolution and localized temperature controls in living cells. The ONCOS and PRET are being applied for molecular/cellular diagnostics, therapeutic applications, and experimental system biology since it will provide us precise spatial and temporal information of living cellular mechanism. Bionanophotonic molecular ruler is also accomplished to measure the dynamics of DNA and protein interactions and understanding cellular dynamics. In-vivo Surface Enhanced Raman Spectroscopy (SERS) probes, in-vitro integrated nanofluidic SERS, and optofluidic ICs are developed for label-free molecular diagnostics and drug discovery. In order to accomplish physiologically relevant cell culture platforms, we have developed Biological Application Specific Integrated Circuits (BioASICs) for single cell biophysics, quantitative cell biology, and cell-based diagnostics by connecting novel microfluidics and nanofluidic circuits, which can impact on high-speed and high-content quantitative biology in new ways. We are creating a library of these “building blocks" to develop innovative single cell array, dynamic cell culture array, and cellular microprocessors, which can help us to develop an innovative personalized medicine.
10:00 AM - **OO1.3
Polymer-based Micro- and Nanofluidic Chips: Simple Strategies for Their Surface Modifications to Accommodate Applications in Biology.
Steven Soper 1 3 , Robin McCarley 1 3 , Michael Murphy 3 2 , Sunggook Park 3 2 , Dimitris Nikitopoulos 3 2 Show Abstract
1 Chemistry, Louisiana State University, Baton Rouge, Louisiana, United States, 3 Department of Mechanical Engineering, Louisiana State University, Baton Rouge, Louisiana, United States, 2 Center for BioModular Multi-Scale Systems, Louisiana State University, Baton Rouge, Louisiana, United States
We are developing polymer-based micro- and nanofluidic chips that can be produced in a high production mode and at low cost using replication technologies. Due to the low cost of producing these chips, they can be configured into a disposable format, appropriate for many sensitive clinical applications such as in vitro diagnostics (personalized medicine, detection of infectious diseases in 3rd world countries), bio-security (point-of-use monitoring of food and water supplies), and forensics (assist in reducing large backlog of DNA testing). Due to the high surface-to-volume ratio associated with any microfluidic and especially nanofluidic chip, engineering bio-compatible surfaces to minimize deleterious effects it may play on the assay performance must be realized. While techniques for modifying glass and quartz surfaces using siloxane-based chemistry are well established, surface modification chemistries for polymers are not. We have developed simple and robust UV-induced modification protocols that can be invoked on a variety of polymeric materials to create functional scaffolds from which bio-compatible surfaces can be grafted to. In this presentation, we will demonstrate the utility of these modification protocols for several application areas: (1) DNA probes can be grafted to embedded PMMA waveguides to create low density arrays with sampling accomplished via evanescent excitation. The utility of this strategy will be demonstrated for the creation of universal DNA microarrays that can be used for screening clinical samples for multi-drug resistant strains of mycobacterium tuberculosis. (2) Creating nano-textured surfaces using a variety of polymer supports via lost mold techniques (nanoporous alumina), which consist of ultra-high aspect ratio (>600) polymer nanopillars. These nano-textured surfaces can be UV-modified and functional entities subsequently grafted to them without losing their structural integrity. Applications of this technology for solid-phase bioreactors to perform proteolytic digestions of intact proteins will be discussed. (3) Two-phase flows (droplet microfluidics) provides the ability to significantly enhance the throughput of sample processing, appropriate for such applications as drug discovery where combinatorial libraries must be screened against a therapeutic target. Unfortunately, the carrier fluids typically used for droplet microfluidics, such as perfluorocarbon solvents, do not support stable two-phase flows in many polymeric materials. UV-activated surfaces can be functionalized with siloxane-containing perfluorocarbon chains to create highly wetable surfaces for the perfluorinated carrier fluids. We will show the generation of stable two-phase flows in PMMA surface modified with perfluorinated polymers and the use of this technology for high throughput drug screening.
10:30 AM - **OO1.4
Molecular Gates for Attoliter Manipulation, Analysis, and Deposition of Biofluidic Compounds.
Mark Shannon 1 2 Show Abstract
1 Mechanical Science and Engineering, University of Illinois, Urbana, Illinois, United States, 2 Beckman Institute, University of Illinois, Urbana, Illinois, United States
There have been tremendous advancements and discoveries in biofluidic sciences that can enhance pharmaceutical research, medical diagnosis, and combinatorial biochemistry. Recently, in the field of micro-nanofluidics, it has become possible to selectively add and subtract attoliter volumes (e-21 m^3) of different solutes from fluid streams. The key discovery that enables this performance is the molecular gate: a deceptively simple construct that interconnects microfluidic channels with nanopore membranes. The molecular gate can digitally pass and control sub-attomoles of materials with applied electric potentials, much like diodes and electronic switches pass and control electrons, but with more complexity since molecules can undergo chemical reactions, changing both the composition and behavior of the fluid. Applications of these molecular gates include the fields of micro-total analytical systems (μTAS) for bio-chemical analysis of solutions, water purification, and combinatorial chemistry. A fluidic chip that employs molecular gates has been developed by several investigators to separate, manipulate, and analyze minute amounts of specified molecular compounds, such as toxins and proteins, from biological samples such as blood, saliva, and natural water, and to print sub-picoliters of compounds to substrates for submicron spotting. The ultimate objective of this work is to develop a system built on this chip that can be computer controlled to rapidly analyze a wide number of low-concentration proteins and metabolites in serum for diagnosis and detection of trace compounds, as well as to analyze huge numbers of combinations of compounds. In this talk, I will focus on the design and fabrication of the molecular gate, and present some initial results in the ability to separate, mix, react, and deposit compounds using molecular gates. While the results are promising, molecular gates are still in their infancy, and many of the observations are not fully understood. Much is not yet known in the fluid and molecular transport and reaction properties of these gates, and the coupling between the mechanical, electrical, and chemical fields within nanopores. I will close the talk with some open questions and challenges to utilizing molecular gates in microdevices, which if solved will advance biological analysis.
11:30 AM - **OO1.5
Microfluidics and nanofluidics for Labs-on-a-Chip.
Albert van den Berg 1 Show Abstract
1 Faculty of Electrical Engineering, Mathematics and Computer Science, University of Twente, Enschede Netherlands
Recent developments in microfluidics have enabled precise manipulation of fluids on the nanoliter scale, leading to realization of Labs-on-a-Chip for a variety of applications. Some examples of this will be presented like field-effect flow control, induced field electrokinetics, and flow independent droplet generation. New nanofluidics phenomena will be discussed, such as electrical field dependent mobility of DNA in nanoconfinements may lead to new methods for rapid DNA analysis. Finally, some examples of applications of Labs-on-a-Chip like lithium analysis for manic depressive patients, a million well Petri dish and genetic modification using single cell electroporation will be demonstrated and the future potential of Labs-on-a-Chip for medical applications will be shown.
12:00 PM - **OO1.6
Droplets & Microfluidics: Novel Tools in Nanomaterial Synthesis.
Andrew deMello 1 Show Abstract
1 Department of Chemistry, Imperial College London, South Kensington United Kingdom
Recent years have seen considerable progress in the development of microfabricated systems for use in the chemical and biological sciences. At a primary level, interest in miniaturized analytical systems has been stimulated by the fact that physical processes can be more easily controlled and harnessed when instrumental dimensions are reduced to the micron scale. For example, it is well recognized that when compared to macroscale instruments, microfluidic systems engender a number of distinct advantages with respect to speed, analytical throughput, reagent usage, process control, automation and operational and configurational flexibility. In general terms, such systems define new operational paradigms and provide predictions about how molecular synthesis and analysis might be revolutionized in the coming years.
Nanomaterials exhibit optical and electronic properties that depend on their size and shape, and are seen as tailored precursors for functional materials in biological sensing and optoelectronics. These critical dependencies indicate that ‘bottom-up’ approaches for nanomaterial synthesis must provide for fine control of the physical dimensions of the final product. Synthetic routes have attracted interest owing to their versatility and ease of use, but for many applications deviations about the mean particle diameter must be <1% to achieve the desired selectivity. This is beyond the tolerance of standard macroscale syntheses, and it is almost always necessary to use some form of post-treatment to extract the desired particle size. Accordingly, nanoparticles with narrow size distributions can only be extracted, through complex, costly and low-yielding routes. Microfluidic systems provide an ideal medium for nanoparticle production. Since both mass and thermal transfer are rapid, temperatures may be defined with precision or varied on short timescales. Additionally, reagents can be rapidly and efficiently mixed to ensure homogeneous reaction environments, while allowing for additional reagents to be added at predefined times. My lecture will describe how we have utilized microfluidic reactors to perform highly efficient nanomaterial synthesis. The system incorporates a microfluidic reactor to perform synthesis and an in-line optical spectrometer to monitor the emission spectra of the emergent particles. Acquired data are assayed using a control algorithm which reduces each spectrum to a scalar ‘dissatisfaction coefficient’ and then intelligently updates the reaction conditions in an effort to minimise this coefficient and so drive the system towards a desired goal. In this way ‘intelligent’ synthesis of nanoparticles of varying size, shape and size-distribution becomes possible.
Furthermore, I will discuss how droplets formed spontaneously when multiple laminar streams of aqueous reagents are injected into an immiscible carrier fluid, can be used for nanomaterial synthesis.
12:30 PM - **OO1.7
Stop Flow Lithography to Create Functional Microparticles.
Patrick Doyle 1 Show Abstract
1 Chemical Engineering, MIT, Cambridge, Massachusetts, United States
Microfluidic devices offer the ability to finely control physical and chemical conditions which is advantageous for materials synthesis. Several groups have used multi-phase microflows to produce microparticles and capsules. Surface tension limits these particles to be spheroids. In this talk we will introduce a new technique entitled Stop Flow Lithography (SFL) which couples microfluidics and projection lithography to create microparticles with unprecedented chemical and geometric complexity. We will first demonstrate the versatility of SFL by showing how it can be used to create materials ranging from soft cell-laden microgel blocks for applications in tissue engineering to ceramic microcomponents for MEMs to TMV virus-patterned particles. Next we will discuss a specific application of SFL to create barcoded microparticles for highly multiplexed bioassays. Our new barcoding approach not only outperforms existing technologies in terms of multiplexing capability, but has better sensitivity, specificity and is much more versatile.
Shashi Murthy Northeastern University
Henry Zeringue University of Pittsburgh
Saif Khan National University of Singapore
Victor Ugaz Texas A&M University
OO4: Materials Synthesis on Chip
Wednesday AM, April 15, 2009
Room 3016 (Moscone West)
9:30 AM - OO4.1
Biofabrication and Enzymatic Functionalization of Free-standing Chitosan Membranes in Microfluidic Network.
Xiaolong Luo 1 3 , Dean Larios Berlin 1 3 , Jeffrey Gair 1 , Gary Rubloff 2 3 Show Abstract
1 Bioengineering, University of Maryland, College Park, College Park, Maryland, United States, 3 Institute for Systems Research, University of Maryland, College Park, College Park, Maryland, United States, 2 Materials Science, University of Maryland, College Park, College Park, Maryland, United States
We demonstrate formation of enzymatically active semi-permeable membranes in bioMEMS by exploiting the pH-responsive solubility of the amino-polysaccharide chitosan. A membrane of insoluble chitosan is formed in a microfluidic network at the fluidic interface between a near-neutral (pH 4.8) chitosan solution and an alkaline (pH 10) buffer solution, yielding a free-standing porous chitosan membrane. The abundant primary amine groups on the chitosan were then used for conjugation to active biomolecules.The microfluidic network was fabricated with PDMS by soft lithography and packaged to a glass slide. A gas pressure-driven external manifold is used to maintain a balanced mass flow delivery rate for both the denser chitosan solution and the less viscous base solution. The chitosan membrane grows at the interface of an acidic chitosan solution and a basic buffer solution in the direction of fluid flow until channel geometry separates the two streams. The membranes are stable in mild pH and can be dried for dissection and characterization. The thickness of chitosan membranes was determined to be ~30µm thick and 50µm high (microchannel height), and was grown across a 1.0mm opening. Importantly, the chitosan membrane is removable by acidic solution, demonstrating the free-standing membrane to be a versatile structure that enables in situ operations such as closing, opening and gating in microfluidic network.We exploit tyrosinase conjugation to biofunctionalize the free-standing membrane in the microfluidic network. Specifically, we investigated enzymatic conversion of SAH (S-andenosylhomocysteine) to SRH (S-ribosylhomocysteine) intermediary by the enzyme Pfs (S-adenosylhomocysteine nucleosidase), a primary step in bacterial synthesis of the cell signaling molecule Autoinducer-2 (AI-2). The enzyme Pfs, with an engineered pentatyrosine tag on the C-terminus, was immobilized on the chitosan membrane by permeating a Pfs solution with activation enzyme tyrosinase through the membrane to efficiently bind Pfs to the chitosan primary amines.Enzyme activity was then measured by flowing SAH substrate through the membrane, collecting the downstream reaction products and evaluating by high performance liquid chromatography (HPLC). The HPLC analysis results show that the enzyme immobilized on the chitosan-membrane efficiently convert 93.7% of substrate into products at 1µL/min flow rate. This conversion efficiency is high compared to our previous studies of enzyme immobilized on electrode surfaces in bioMEMS, underscoring the benefits of enzyme-decorated free-standing chitosan membranes. These membranes expand the functionality of chitosan as a versatile biointerface to enable site-specific, electrode-free, enzyme immobilization for metabolic engineering applications in bioMEMS. We are currently pursuing more complex microfluidic networks, flow recipes, and control systems that are promising for various applications of the free-standing chitosan membrane.
9:45 AM - OO4.2
Ultra-Soft Microfluidics: Photo-Induced Structures from Lipid Bilayers.
Linda Hirst 1 , Jing Yuan 2 Show Abstract
1 Natural Sciences (physics), UC Merced, Merced, California, United States, 2 Physics, Florida State University, Tallahassee, Florida, United States
The self-assembly of biological amphiphiles has proved a fascinating topic in recent years, and phaseseparation phenomenon in the cell membrane have attracted a great deal of attention. The hollow cylindrical lipid tubule is of particular interest due to its potential relevance to intercellular transporting channels and applicability to controlled-release systems, chemical micro-reactors and nano-conduits. Phase co-existence in the lipid bilayer has recently been observed in biologically-relevant three- component giant unilamellar vesicles. We have generated stable, photo-induced micron-scale phaseseparation in lipid tubules formed from ternary lipid mixtures, inducing a new bilayer disc structure. This investigation not only aids in our understanding of lipid sorting phenomena in cell membranes, but is also a fascinating route to the generation of new functional structures.J.Yuan and L.S. Hirst, J. Am. Chem. Soc. 130 (6), 2067 -2072 (2008).
10:00 AM - OO4.3
Tunable Synthesis of Metallic Nanoparticles in Three-phase Microfluidic Segmented Flows.
Saif Khan 1 , Suhanya Duraiswamy 1 Show Abstract
1 Chemical and Biomolecular Engineering, National University of Singapore, Singapore Singapore
We present a continuous flow method to synthesize metallic nanoparticles employing three-phase microfluidic segmented flow to effectively isolate growing nanocrystals from the microchannel walls while ensuring facile product recovery. We demonstrate the applicability of this method in the preparation of spherical and rod-shaped gold nanocrystal dispersions of varying aspect ratios. Continuous flow microfluidic synthesis methods operate at steady state and offer excellent control over reaction conditions such as reagent addition, mixing and temperature. Isolation of growing particles from the microchannel walls is a critical requirement in the implementation of such methods to prepare colloidal metal dispersions. We employ three-phase microfluidic segmented flow comprising of a train of alternating gas and aqueous segments riding on a thin annular wetting film of silicone oil to isolate the reagents and growing particles from the microchannel walls. We demonstrate the applicability of this method in the preparation of rod-shaped gold nanocrystal dispersions. In our method, nitrogen gas, gold nanoparticle seed suspension and aqueous reagent solutions are separately delivered into one arm of a microfluidic T-junction, and a cross-flowing silicone oil is delivered into the other arm. At low volumetric oil flow rates relative to the aqueous streams, bubbles and drops are alternately pinched off at the T-junction and assemble downstream into an alternating bubble-drop train flowing through the oil. Chaotic advection within the drops rapidly mixes the aqueous reagents. The oil only flows as a thin wetting film encapsulating the bubble-drop ensemble and isolates growing particles from the microchannel walls, while the injected gas functions as the carrier fluid. This method uses far less oil than purely drop-based approaches, and therefore circumvents the formation of stable water-in-oil emulsions at the device outlet which make product recovery a challenging task.
10:15 AM - OO4.4
Microscale Bioactive Patterning of Silicone Rubber for Incorporation in Microfluidic Channels
Jessica McLachlan 1 , Natasha Patrito 1 , Sarvesh Varma 1 , Jayna Chan 1 , Peter Norton 1 Show Abstract
1 Chemistry, University of Western Ontario, London, Ontario, Canada
In the fields of medical diagnostics and bioanalysis, a recent trend towards the use of whole cells as biosensors makes the ability to pattern cells critical to the development of new high throughput screening devices. Selective deposition of molecules which influence cell adhesion leads to the spatial control of cell growth. Fabrication of these substrates often relies on photolithography or microcontact printing to locate extracellular matrix components, other adhesive and inhibitory molecules. Many of these techniques are not robust enough to withstand biological conditions or microfluidic environments for extended periods of time.Our research is focused on the patterned functionalization of poly(dimethylsiloxane) (PDMS), a hydrophobic elastomer frequently used for the rapid prototyping of microdevices. Recently, we have reported a novel surface treatment, which renders PDMS hydrophilic and, in turn, promotes cell adhesion. Thin metal films are deposited onto PDMS in the presence of a gaseous plasma. Removal of the deposited metal exposes roughened PDMS regions enriched with hydrophilic oxygen-containing species. The use of a metal has an advantage over typical plasma treatment of PDMS in that the metal prevents hydrophobic recovery until it is removed, making the substrates “shelfable”. Two different routes to the micropatterning of this surface modification have been explored: stencil masking and photolithography. These patterning methods allow us design hydrophilic features in practically any geometric design or size within the resolution limitsof photolithography. Optical microscopy has been used to visualize cell growth on the patterned substrates. Further, the cell-substrate interactions have been studied using confocal and atomic force microscopy. Distinct differences between cell growth on stencil mask and photolithographically patterned substrates have been observed. To explain these differences contact angle measurement and cell growth has been performed on modified PDMS which has recovered its hydrophobicity. We have utilized this modification to create a new PDMS bonding protocol as the silanol-enriched polymer surface is amenable to irreversible bonding with glass, PDMS or silicon substrates. Immediately prior to bonding, the protective metal layer is removed by immersion in an aqueous etchant, exposing the adhesive surface. Employing this technology, PDMS-glass and PDMS-PDMS microfluidic devices were fabricated and the adhesive strength was quantified by tensile and leakage testing. Combining this new protocol with micropatterning has allowed for the fabrication of patterned and irreversibly sealed PDMS microchannels.The patterned channels can be integrated into more complex microfluidic devices allowing for precise control over cell location inside the device.
10:30 AM - OO4.5
Nanoparticle Synthesis on a Programmable Microfluidic Droplet System.
Woon Seob Lee 1 , Duckjong Kim 1 , Se-kwon Kim 2 , Jong Wook Hong 1 Show Abstract
1 Materials Research and Education Center, Department of Mechanical Engineering, Auburn University, Auburn, Alabama, United States, 2 Marine BioProcess Research Center, Pukyong National University, Busan Korea (the Republic of)
The use of droplet-based microfluidics offers a number of advantages over conventional flow control technology. The micro- or nanometer sized droplets have high surface area to volume ratios, so a fast reaction is possible because their heat and mass transfer times and diffusion distances are very short. Various microfluidic droplet formation methods have been proposed to achieve control the size, shape, and monodispersity of micro droplets. However, they need complicated structures and/or accurate flow rate control. In addition, it is hard to change the concentration of reagents every time. Therefore, it is difficult to parametric study according to the changing the concentration of mixture. Here, we report on the digital microfluidic droplet system for synthesis of nanoparticles, which is capable of independent control over the droplet formation timing and the droplet size. In addition, this system can be applied to a parametric study to extend the number of reaction chemicals. By using the proposed system, we can control the concentration ratio of the mixtures. We have analyzed and compared the property change of nanoparticles according to changing the concentration ratio of the mixture. CdCl2 and Na2S were used to make CdS nanoparticles. The concentration ratio of the mixture was changed by changing the volume ratio of the mixture in the microfluidic chip. The spectrophotometer showed different peak according to changing the Na2S volume ratio. This system has a great potential for developing materials because it can control the concentration ratio of the mixture each time, and there are no limitations to the number of reaction chemicals and combinations of the mixture.
11:15 AM - OO4.6
Patterning Colloidal Gold Nanoparticles on a Device Surface through Chemical Self Assembly.
Sarah Adams 1 , Regina Ragan 1 Show Abstract
1 Chemical Engineering and Materials Science, University of California, Irvine, Irvine, California, United States
Fabrication of ordered nanoscale metallic structures as lab-on-a-chip devices provides the opportunity for cost-efficient development of single-molecule level detection limits for fluidic analysis. Metal nanostructures enable field-enhanced chemical and biological detection due to the strong near field coupling between closely spaced noble metal nanostructures. Yet challenges still exist for low-cost fabrication of metal nanostructure arrays with features sizes and/or inter-particle spacing on the sub-10 nm length scale. Through a series of chemical self assembly techniques, we have developed a simple and cost-efficient design for assembling colloidal gold nanoparticles on a nanoscale ordered surface array. Self-organized polymer templates, consisting of poly(methyl methacrylate) domains in a phase-separated polystyrene-b-poly(methyl methacrylate) diblock copolymer thin film template, were chemically modified for controlled placement of monodisperse Au nanoparticles. Chemically synthesized colloidal gold nanoparticles, measured at 5, 10, and 20 nm diameter using dynamic light scattering techniques and scanning electron microscopy (SEM), were attached to these surface amine regions using 1-Ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride linking chemistry and N-hydroxy sulfosuccinimide stabilizer with an organic ligand, thioctic acid, on the nanoparticle surface. Optimization of thioctic acid functionalized particles in aqueous solution was analyzed to increase the electrostatic stability from zeta potential measurements as well as to reduce the presence of aggregate formation as observed in solution with dynamic light scattering spectroscopic analysis and on the attached surfaces with SEM analysis. Atomic force microscopy and SEM images demonstrate that Au nanoparticles are preferentially immobilized on poly(methyl methacrylate) domains in polystyrene-b-poly(methyl methacrylate) templates using this method. The fabrication method of Au nanoparticle array assembly described here can be generalized to fabricate a variety of materials, structure and patterns for these lab-on-a-chip devices. By altering the volume fraction of the PS and PMMA polymer domains, we have produced both hexagonal arrays and lamellar distributions of PMMA blocks within a PS matrix. With controlled placement of gold nanoparticles using chemical assembly on a variety of substrates, including silicon, glass, and gold, ordered arrays of metal nanoparticle clusters can be fabricated for devices.
11:30 AM - OO4.7
Directed Fluidic Assembly of Nanotube Networks for Transistors and Aqueous Chemical Sensors
Melburne LeMieux 1 , Nishant Patil 1 , Mark Roberts 1 , Stefan Mannsfeld 1 , Justin Opatkiewicz 1 , Zhenan Bao 1 Show Abstract
1 Chemical Engineering, Stanford, Stanford, California, United States
The directed placement and controlled assembly of single-walled carbon nanotubes (SWNTs) represents the biggest obstacle preventing the widespread application of these 1D materials with superior electronic properties. For such devices to be integrated onto disposable plastic chips, the SWNT networks should be fabricated from solution rather than the more commonly used approach of high temperature chemical vapor deposition. Here, we present methods to fabricate and pattern SWNT network thin film transistors (TFTs) via directed fluidic flow patterns, using this dynamic interface to control the SWNT alignment and density. We demonstrate that SWNT TFT performance is affected by various surface treatments and the characteristics of the fluidic flow during fabrication. Finally, the fluidic assembly at room temperature allows for deposition onto plastic surfaces enabling low-voltage operation of the SWNT TFTs and hence operation in aqueous environments. Protecting our water supply has become increasingly important due to higher contaminant levels and the threat of terrorism. We show that these carefully assembled SWNT TFTs are suitable for threat monitoring and diagnostics by detecting analytes in water at ppb concentration.
11:45 AM - OO4.8
The On-chip, Directional Growth of Metallic and Polymeric Nanowires.
Bret Flanders 1 , Prem Thapa 1 Show Abstract
1 Physics, Kansas State University, Manhattan, Kansas, United States
We present an electrochemical approach to the on-chip, template-free growth of conducting metallic and polymeric nanowires along predictable, inter-electrode paths up to 100 μm in length. This approach is called directed electrochemical nanowire assembly (DENA). In this technique, an alternating voltage is applied to an electrode immersed in a simple salt solution in order to induce nanowire growth. The nanowire is grown from the electrode to within ± 2 μm of a user-specified target lying within a ~140 ° angular range and a ~100 μm radius of the electrode tip. The target may be a second electrode or a submicron object such as a biological cell cultured onto the substrate in the inter-electrode gap. A long range component of the applied voltage-signal defines the growth-path. This component originates in the balance that is established between the electrode flux, which describes the finite amount of material that can be deposited per unit time, and the flux due to the steady drift of cations towards the electrode. As an application of this methodology, we demonstrate the use of DENA-grown polymeric nanowires for measuring the forces exerted by individual Dictyostelium cells. A electrotactical approach is employed to induce an individual Dictyostelium cell to attach a pseudopod to the tip of a polythiophene wire that is grown part way across the electrode-gap. The flexibility of the wire allows cell-induced deflections from the equilibrium position of the wire to be observed through an optical microscope. In turn, the deflection-amplitudes are directly proportional to the forces exerted by the cell. The (preliminary) force-determinations that we have made using this technique range from 5 nN to 20 nN. Recent work on the application of this technique to articulating the migratory mechanisms of various cell-types will be reported. Ozturk B, Talukdar I and Flanders B N 2007 Directed growth of diameter-tunable nanowires Nanotechnology 18 365302Thapa P S, Barisci J N, Yu D J, Wicksted J P, Baughman R and Flanders B N 2008 Directional growth of conducting polypyrrole and polythiophene nanowires Appl. Phys. Lett. Submitted
12:00 PM - OO4.9
Gradient Lithography of Engineered Proteins to Fabricate Cell Culture Microenvironments.
Sheng Wang 1 2 , Cheryl Wong Po Foo 3 , Mu-ming Poo 4 , Sarah Heilshorn 3 , Xiang Zhang 1 5 Show Abstract
1 NSF Nanoscale Science and Engineering Center (NSEC), University of California Berkeley, Berkeley, California, United States, 2 Applied Science and Technology Graduate Program, University of California Berkeley, Berkeley, California, United States, 3 Department of Materials Science and Engineering, Stanford University, Stanford , California, United States, 4 Department of Molecular and Cell Biology, University of California Berkeley, Berkeley, California, United States, 5 Materials Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California, United States
Protein patterning is a key technique for various applications including biosensors, drug screening, developmental biology research, and tissue engineering. Many techniques have been developed to pattern proteins including contact imprinting, optical lithography, microfluidics, and diffusion processes. However, it is still challenging to achieve protein patterns with complex geometries, gradient distributions, or three-dimensional structures, as well as to produce mechanical and topographic properties comparable to native microenvironments that can meet the requirements of many developmental biology research interests, such as stem cell differentiation, directed cell migration, neuronal axon guidance, and tissue engineering etc. Here, we demonstrate a simple dynamic mask lithography method which is capable of fabricating any geometric topography or concentration gradient from an artificial extracellular matrix (aECM) protein that is compatible for cell culture.
12:15 PM - OO4.10
Fabrication of Microfluidic Devices for Droplet Generation based on Dry Film Resist
Patrick Leech 1 , Nan Wu 2 , Yonggang Zhu 2 Show Abstract
1 CSIRO, Materials Science and Engineering, Clayton, Victoria, Australia, 2 CSIRO, Materials Science and Engineering, Highett, Victoria, Australia
The generation of droplets in a microfluidic system has become a preferred method of performing highly reproducable experiments . The definition of the master pattern in these devices has typically been based on SU8 resist. In this paper, we present an alternative method of fabrication of the master pattern based on dry film resist. Dry laminar resist requires fewer process steps than SU8 resist although with some restrictions on the attainable aspect ratio due to resolution limits. In the first part of this paper, the resolution limits and aspect ratio of test features in Shipley 5038 laminar resist were established as a function of exposure dose, the thicknesses of resist (using multiple layers up to 140 µm) and mask parameters (with comparison of film transparency and Cr masks). The optimized process was then applied in the fabrication of flow focusing microdroplet devices incorporating channel widths of 50 µm - 200 µm. The channels formed in dry laminar resist were characterised by smooth, straight sidewalls with an aspect ratio of 2:1. Replication of the pattern as a Ni shim has allowed hot embossing in PMMA and the sealing of micro-channels. Experimental results using these flow focusing devices have shown a controlled variation in size and distribution of oil droplets in water by altering the ratio of liquid volumes and flow rates. The average size of droplets decreased monotonically from 40 to 100 µm with increase in oil flow rate. The use of dry film resist provides a method of rapid fabrication of prototype microfluidic channels for experiments in droplet formation.  A.H. Huebner, S. Sharma, M. Srisa-Art, F. Hollfelder, J.B. Edelm and A.J. deMello, LabChip 8, 1244 (2008).
OO5: Cell Manipulation & Biomimetics on Chip
Wednesday PM, April 15, 2009
Room 3016 (Moscone West)
2:30 PM - OO5.1
Capture and Release of Cardiac Fibroblasts in Microfluidic Devices using Peptide-Functionalized Alginate Gels
Brian Plouffe 1 , Melissa Brown 2 3 , Milica Radisic 2 3 , Shashi Murthy 1 Show Abstract
1 Chemical Engineering, Northeastern University, Boston, Massachusetts, United States, 2 Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario, Canada, 3 Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Ontario, Canada
Microfluidics offers an economical platform for selective cell capture from small sample volumes in the order of microliters while maintaining high throughput and purity comparable to conventional fluorescent- and magnetic-activated cell sorting. Adhesion based micro-scale devices that possess surface-immobilized ligands have recently emerged as a tool for selective cell isolation by positive selection. The ability to handle small volumes and point-of-care operation makes this technology highly desirable for applications in tissue engineering and regenerative medicine. An important design objective that must be met for these applications, however, is the ability to detach the captured cells non-destructively. We describe the application of peptide functionalized alginic acid via carbodiimide chemistry as a methodology for cell capture and release in microfluidic channels. An alginate-peptide complex is adsorbed on the surface of the channels and rinsing with Ca2+ ionic solutions results in a thin hydrogel layer on the channel surface. Rat cardiac fibroblasts were injected into the device and subsequently captured from the flow stream by the adsorbed ligand. Following capture, the hydrogel can be dissolved using ethylene diamine tetraacetic acid (EDTA), a strong chelator of divalent ions. The conjugation of Arg-Gly-Asp-Ser (RGDS), a tetrapeptide to the alginate backbone showed a two-fold and four-fold increase in fibroblast adhesion compared to unconjugated alginate and bare glass controls, respectively. Rinsing with EDTA solution resulted in over 95% cell release within the device. This suggests that the RGDS-alginate complex can be utilized as an effective method for selective cell capture and subsequent release. This method is simple and amendable to all micro-scale devices that operate in a low fluid shear stress regime. In addition, the conjugation chemistry utilized in this work can be applied to a wide range of molecules containing primary amines and cellular release requires no application of external forces such as heat, electrical potential, or photo-activation.
2:45 PM - OO5.2
Electronically Modulated Mixing of Neurotransmitters for Spatio-temporal Control of Nerve Cell Signalling Using an Organic Electrophoretic Delivery Device.
Karin Larsson 1 3 , Klas Tybrandt 2 3 , Sindhulakshmi Kurup 1 3 , Daniel Simon 2 3 , Peter Kjall 1 3 , Joakim Isaksson 2 , Edwin Jager 2 3 , Mats Sandberg 4 , Magnus Berggren 2 3 , Agneta Richter-Dahlfors 1 3 Show Abstract
1 Department of Neuroscience, Karolinska Institutet, Stockholm Sweden, 3 , Strategic Research Center for Organic Bioelectronics (OBOE), Stockholm Sweden, 2 Department of Science and Technology, Linköping University, Norrköping Sweden, 4 , Acreo AB, Norrköping Sweden
Taking advantage of the combined electronic and ionic conductivity of conjugated polymers, we have established a novel technology platform to achieve precise, spatio-temporal control of cell signaling. With the organic electrophoretic device, a miniaturized version of the previously reported organic electronic ion pump (OEIP, Nature Materials 2007), we demonstrate the ability to deliver multiple bio-substances at high spatio-temporal resolution. This device is designed to fulfill several important requirements of cell biologist’s, e.g. individual cell addressing and the possibility of obtaining strict control of dynamic parameters without convective disturbances. Electronically controlled multiplexing offers simultaneous or sequential delivery of bio-substances with independently modulated delivery rates. Using the organic electrophoretic device we demonstrate that multiplex delivery of the neurotransmitters acetylcholine and nicotine can reliably induce robust Ca2+ responses, as assessed by microscopy-based real-time single cell Ca2+ imaging, in human SH-SY5Y neuroblastoma cells. The device can be fabricated using photolithography, which enables integration with other organic or inorganic solid state systems. Our findings highlight the potential of further development of solid state systems to create an interface where complex, high-resolution, signal patterns are generated to control cell physiology at the sub-cellular level.
3:00 PM - OO5.3
Peptide-Functionalized Surfaces in Microfluidic Devices for Cell Separation by Negative Selection
James Green 1 , Milica Radisic 2 3 , Shashi Murthy 1 Show Abstract
1 Chemical Engineering, Northeastern University, Boston, Massachusetts, United States, 2 Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario, Canada, 3 Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Ontario, Canada
Microfluidic cell separation systems have emerged as attractive alternatives to traditional techniques in recent years. These systems offer the advantages of being able to handle small sample volumes and at the same time achieve highly selective separation at low cost. Microfluidic devices with surface-immobilized adhesion molecules can achieve separation by exploiting ligand-receptor interactions. When ligands are immobilized on the microfluidic channel surfaces, the resulting cell capture devices offer the typical advantages associated with microfluidic systems, with the added benefit of not requiring complex fabrication schemes or pre-processing incubation. This presentation will describe how a microfluidic system of devices coated with peptides can be utilized to deplete a cell suspension of endothelial cells, smooth muscle cells, and fibroblasts in order to isolate a fourth cell type, adipose-derived stem cells, by negative selection. The peptides utilized are arg-glu-asp-val (REDV), val-ala-pro-gly (VAPG), and arg-gly-asp-ser (RGDS). The significance of this approach is that it can be utilized to isolate stem and progenitor cell populations from digested tissue as a precursor to conventional tissue engineering on scaffolds or cell-based regenerative therapeutics. Furthermore, this approach could be an effective way to isolate stem/progenitor cells whose markers are not fully characterized.
3:15 PM - OO5.4
Rapid One-step Fabrication of 3D Branched Microvascular Flow Networks in Plastic Substrates.
Jen-Huang Huang 1 , Jeongyun Kim 1 , Arul Jayaraman 1 , Victor Ugaz 1 Show Abstract
1 Chemical Engineering, Texas A&M University, College Station, Texas, United States
Standard photolithography-based micromachining techniques are widely used to construct 2D microchannel networks, but the inherently planar nature of these processes limits their usefulness in the creation of 3D structures. More recent developments have enabled fully 3D flow networks to be produced using processes including solid freeform fabrication, stereolithography, and 3D printing. But many of these methods involve serial ‘direct writing’ processes that require timescales on the order of hours to days and are not practical for mass production. In addition, no single technique has proven ideal to construct microchannel networks that incorporate a wide range of size scales (µm to mm). Here, we describe a new process that enables 3D branched microvascular networks to be constructed in a single step. This technique employs an electrostatic discharge phenomenon that occurs when a dielectric medium is energized by a strong electric field and subsequently discharged to form branched “tree-like” channels in polymer substrates. Suitable space charge distributions are generated by irradiating the sample with an electron beam so that the energy released upon discharge is sufficient to locally vaporize and fracture the material, leaving behind a network of branched channels in a tree-like fractal structure. Beam intensity and spatial irradiation profile (width and penetration depth) can be adjusted to control the location and morphology of the discharge structures. The embedded patterns exhibit a self-similar fractal network structure, with channel characteristic dimensions ranging from approximately 10 µm to 1 mm in diameter. Interconnected networks with multiple fluidic access points can be straightforwardly constructed using a multi-step process whereby the substrate is re-irradiated by the electron beam in order to nucleate additional discharges. Flow and interconnectivity are characterized by direct visualization of tracer dye solutions, both in 2D by recording images of a subset of the network occupying a specific focal plane, and in 3D by using confocal laser scanning microscopy.
3:30 PM - OO5.5
A Chip Based Patch Clamp Device.
Piyush Verma 1 , Nick Melosh 1 Show Abstract
1 Materials Science and Engineering, Stanford University, Stanford, California, United States
Patch clamping is the foremost technique for measuring the activity of ion channels within cells, and is vitally important for drug screening and electrophysiology. Patch clamp devices rely upon formation of a tight membrane-material seal in order to prevent ion leakage, with the dominant architectures based on cell suction into cylindrical holes in glass, PDMS, or silicon. However, large numbers of simultaneous experiments, cell toxicity, and low-resistivity seals are still issues with these platforms. Here we report an alternative approach by functionalizing a 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 electrical patch-clamp measurements with these post-electrodes on lipid vesicles and red blood cells to determine sealing resistance. Due to their small size and very low access resistance, these probes may provide high resolution, low-noise patch clamp measurements in an array format.
3:45 PM - OO5.6
Integrated On-Chip Blood Separation and Cancer Marker Detection
Ophir Vermesh 1 , Rong Fan 1 Show Abstract
1 , California Institute of Technology, Pasadena, California, United States
Blood comprises the largest and deepest version of the human proteome and is thus the most important fluid for clinical disease diagnostics. Nevertheless, only a handful of plasma proteins are utilized in routine clinical tests. This is due to a host of reasons, including the intrinsic complexity of the plasma proteome, the heterogeneity of human diseases and the fast kinetics associated with protein degradation in sampled blood. Simple technologies that can sensitively sample large numbers of proteins over broad concentration ranges, from small amounts of blood, and within minutes of sample collection, would assist in solving these problems. Herein, we report on the Integrated Blood Barcode Chip (IBBC) which is designed for on-chip separation of plasma from a fingerprick of whole blood, followed by the rapid measurement of a panel of plasma proteins. This platform holds potential for inexpensive, minimally-invasive, and informative point-of-care clinical diagnoses.
4:00 PM - OO5.7
Critical Size, Dynamic Range, and Throughput Improvements in Sorting by Deterministic Lateral Displacement Enabled by Triangular Posts.
Kevin Loutherback 1 2 , Kevin Chou 2 , Jason Puchalla 3 , Robert Austin 1 3 , James Sturm 1 2 Show Abstract
1 Princeton Institute for the Science and Technology of Materials, Princeton University, Princeton, New Jersey, United States, 2 Electrical Engineering, Princeton University, Princeton, New Jersey, United States, 3 Physics, Princeton University, Princeton, New Jersey, United States
Deterministic Lateral Displacement (DLD) arrays have been shown to sort particles (100 nm - 30 micron) with high speed and resolution based on fluid flow through an array of circular posts tilted a small angle (< 10 degrees) with respect to the direction of the fluid flow [LR Huang, Science. 304 (2004)]. Particles suspended in the fluid smaller than the critical size follow the fluid direction while larger particles follow the array axis. The critical diameter is typically 40% of the gap between the posts, so sorting a heterogeneous mixture of particle sizes is often difficult because particles larger than the gap between posts will clog the device. As a result, multiple array stages are necessary to separate out large particles from the fluid containing target particles. In this work, we show by both experiment and modeling that by changing the post shape from circles to triangles, we are able to decrease the critical particle size for a fixed gap by bewteen 20% (11.3 degree tilt - e 1/5) and 40% (2.9 degree tilt - e 1/20). These improvements are fundamentally a result of the post geometry. Using triangular posts results an asymmetric velocity profile across the gaps that is biased towards the triangle vertex. This leads to a narrower stream width and a smaller critical particle size for the same gap. This phenomena enables three practical advantages. First, a larger gap size can be used for a given critical size, reducing the likelihood that a device will clog. Second, it also allows for faster separations for a fixed ratio of critical diameter to gap by allowing increased array tilt - the angle at which large particles travel compared to small particles is larger. Third, DLD arrays with triangular posts require a lower pressure drop for the same critical diameter and flow speed since the pressure drop is proportional to gap^-2.
4:30 PM - OO5.8
The Normal and Shear Strength of the Cell-Implant Interface: Accelerated Negative Buoyancy as a Method of Cell Adhesion Assessment.
Helen Griffiths 1 , Charles Collier 1 , Athina Markaki 1 2 , James Curran 1 3 , Trevor Clyne 1 Show Abstract
1 Materials Science and Metallurgy, University of Cambridge, Cambridge United Kingdom, 2 Department of Engineering, University of Cambridge, Cambridge United Kingdom, 3 , Keronite International Ltd, Cambridge United Kingdom
The strength of adhesion at the cell-substrate interface is an important parameter in the design of many prosthetic implant material surfaces, due to the desire to create and maintain a strong implant-tissue bond. This study focuses on the mechanical strength of the interface and the ease of cell removal from ceramic coatings using normal and shear forces, but also looks at cell proliferation rates on the same series of surfaces.This systematic study of cell proliferation and adhesion has been carried out on a series of oxide coated Ti6Al4V-based substrates with a range of surface morphologies and chemistries. Oxide coatings were formed using Plasma Electrolytic Oxidation (the PEO process). Cells were seeded at a low concentration onto substrates and proliferation monitored for up to three weeks. The same cell concentrations were seeded on samples for adhesion testing. These were cultured for a few days to ensure well established adhesion of viable cells. The normal and shear strength of osteoblasts (bone cells) and chondrocytes (cartilage cells) adhered to these substrates was measured using accelerated negative buoyancy within an ultracentrifuge. The variation in proliferation rates on, and adhesive strengths to, the range of coatings, is discussed and related to morphological and chemical differences in the coatings. A comparison is made between the normal and shear strengths of the cell-coating bonds and the differences between the behaviour of the two cell types discussed.
4:45 PM - OO5.9
Multiplexed Nanowire Nanoelectronics – Cardiomyocytes Interfaces
Tzahi Cohen-Karni 2 , Brian Timko 1 , Lucien Weiss 1 , Charles Lieber 1 2 Show Abstract
2 School of Engineering and Applied Sciences, Harvard University, Cambridge, Massachusetts, United States, 1 Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts, United States
Nanostructures and nanostructured substrates show enhanced coupling to artificial membranes, cells, and tissue. Such nano-bio interfaces exist at a length scale natural for biological systems and offer better sensitivity and spatial resolution as compared to conventional planar structures. In this work, we report the electrical properties of silicon nanowires (SiNWs) interfaced with cultured embryonic chicken cardiomyocytes. We developed a scheme that allows us to manipulate the interfaced cells while monitoring their electrical activity. In addition, we demonstrate for the first time multiplexing on the sub-cellular level, thereby exceeding the spatial and temporal resolution limits of other electrical recording techniques. The flexible assembly of arrays of SiNWs could prove useful for fundamental studies of ion channel biophysics, real-time drug assays and creation of semiconductor / muscle hybrids.
5:00 PM - OO5.10
Assessing Functional Heterogeneity at Single-cell Level using Integrated Microchips.
Rong Fan 1 2 , Ophir Vermesh 1 2 , Habib Ahmad 1 2 , Chao-Chao Liu 2 , Gabe Kwong 1 2 , James Heath 1 2 Show Abstract
1 Nanosystems Biology Cancer Center(NSBCC), California Institute of Technology, Pasadena, California, United States, 2 Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California, United States
It is now widely recognized the immune system plays an intricate and often paradoxical role in tumor development and metastasis. Persistent infection leads to chronic inflammation that can often increase the risk of malignant tumor progression. For example, IL-1b secreted from macrophages in an innate response, is found to mediate the transition of prostrate tumor cells from androgen dependent to the more malignant androgen independent form. However, both anti-tumor and pro-tumor effects of the immune system are subjected to the regulation via the same panel of cell-cell signaling molecules such as cytokines and chemokines that form a cell-cell communication network. In order to harness the anti-tumor capability of the immune system, it is crucial to unravel such regulatory network at the single and few cell levels, and compare that back to the observations that can be made at the tumor level. We developed a large scale microfluidic device that is integrated with a DNA-encoded antibody microarray (DEAL). This device enables the detection of a dozen proteins potentially secreted from a single cells or a small number of cells compartmentalized in a tiny microchamber. Using this technique, we observed the secretion of at least six proteins – TNF-a, IL-1b, IL-10, IL12, GM-CSF, and MCP-1 from single and/or small colonies of human monocyte cells. These cells undergoing directed differentiation into macrophage lineage exhibit remarkable functional heterogeneity in terms of cytokine secretion profile. This is attributed to not only the intrinsic stochastic gene expression but also cell-cell communications. The colony-size-dependent secretion profile can provide a functional phenotypic signature for the cells of different physiological origins, and thus be exploited for differential analysis of a heterotypical population of tumor-associated immunocytes and study of their co-evolution with tumors.
5:15 PM - OO5.11
Femtosecond Laser Ablation to Create Nanometer-scaled Cell Adhesion Ligand Patterns.
Ray Schmidt 1 , David Hwang 2 , Naomi Kohen 3 , Lara Gamble 4 , David Castner 4 5 , Costas Grigoropoulos 2 , Kevin Healy 3 1 Show Abstract
1 Bioengineering, University of California, Berkeley, Berkeley, California, United States, 2 Mechanical Engineering, University of California, Berkeley, Berkeley, California, United States, 3 Materials Science and Engineering, University of California, Berkeley, Berkeley, California, United States, 4 Department of Bioengineering, National ESCA & Surface Analysis Center for Biomedical Problems, University of Washington, Seattle, Washington, United States, 5 Department of Chemical Engineering, National ESCA & Surface Analysis Center for Biomedical Problems, University of Washington, Seattle, Washington, United States
The goal of our project is to fabricate interfaces for mammalian cell culture that control cell fate via the spatial distribution of individual focal adhesions that cells use to interrogate the interface. To create nano-scale cell adhesion sites on a surface, an ultrathin protein adsorption resistant polyethylene glycol (PEG) brush layer was synthesized via surface initiated atom transfer radical polymerization (SI-ATRP). The surface chemistry was verified with XPS and the growth kinetics of the brush were monitored in a quartz crystal microbalance with dissipation (QCMD). The film was selectively ablated using focused femtosecond laser pulses, exposing the underlying quartz substrate as centers for adsorption of cell-adhesive molecules. Currently, 250nm features can regularly be ablated with a processing wavelength of 400nm and a 50X objective, based on atomic force microscopy (AFM) scanning of the ablated features. Higher resolution features (~50-100nm) have also been observed to appear irregularly at lower pulse energies, and may require a multishot ablation process to ensure regularity. A wide range of feature arrangements can be generated on a single substrate, including gradients of feature size, feature spacing, or individual cell islands, which will allow us to decouple the effects of cell size and shape, focal adhesion placement, and ligand input to the cell. Each variable can be modulated independently to determine the effects on cellular function and fate for primary bone marrow stem cells. Surfaces with varying pitch, feature diameter, and overall projected adhesion ligand density were generated to study the effects of these variables on cell function. Mesenchymal stem cells attached to features down to 250nm, with significant changes in nuclear distention observed depending on the ligand surface density presented to the cell, suggesting these patterned surfaces will be invaluable in studies examining stem cell fate determination.
5:30 PM - OO5.12
Bio-inspired Microfluidic Propulsion Through Magnetically-actuated Cilia.
Syed Khaderi 1 , Michiel Baltussen 2 , Patrick Anderson 2 , Daniel Ioan 3 , Jaap Den Toonder 2 4 , Patrick Onck 1 Show Abstract
1 Zernike Institute for Advanced Materials, University of Groningen, Groningen Netherlands, 2 , Eindhoven University of Technology, Eindhoven Netherlands, 3 , University Politehnica of Bucharest , Bucharest Romania, 4 , Philips Research, Eindhoven Netherlands
A rapidly growing field in biotechnology is the use of lab-on-a-chip devices to analyse bio-fluids. Such fluids have to be preprocessed (for example, mixed with other fluids) and transported to and from one or many micro-chambers where the biochemical analyses are performed. The microfluid transport through these stages is usually performed by downscaling conventional methods such as syringe pumps, micropumps, or by exploiting electro-magnetic actuation, as in electro-osmotic and magnetohydrodynamic devices. However, the use of electric fields may induce heating, bubble formation and pH gradients from electrochemical reactions. In this work, we explore a new way to manipulate fluids in microfluidic systems, inspired by nature, through the magnetic actuation of artifical cilia.Fluid dynamics at the micrometer scale is dominated by viscosity rather than inertia. This has important consequences for fluid propulsion mechanisms. In particular, mechanical actuation will only be effective in propelling fluids if their motion is cyclic, but asymmetric in shape change. Nature has solved this problem by means of hair-like structures, called cilia, whose beating pattern is asymmetric and consists of an effective and a recovery stroke. While natural cilia use an internal forcing system based on motor proteins (dyneins), the key challenge for its artificial equivalent is the design of an externally-applied loading system that will generate a similar non-reciprocating motion. In this work we report on the identification of two different magneto-mechanical configurations that can do so. The first is based on a magnetic instability that develops when the applied magnetic field is opposite to the direction of the magnetization in a permanently magnetic film. In a second configuration we will demonstrate that asymmetry can be achieved in a super-paramagnetic film, based on the intricate inter-play between the geometry of the film, the externally-applied field and the internally-induced magnetization.By simultaneously solving the elasto-dynamic, magnetostatic and fluid mechanics equations, we show that the amount of fluid propelled is proportional to the area swept by the cilia. We delineate the functional response of the system in terms of three dimensionless parameters that capture the relative contribution of elastic, inertial, viscous and magnetic forces.
5:45 PM - OO5.13
Thermochemical Nanolithography of Multi-functional Templates for Selective Assembly of Bioactive Proteins.
Debin Wang 1 3 , Vamsi Kodali 1 4 , William Underwood 2 3 , Jonas Jarvholm 2 3 , Takashi Okada 2 3 , Simon Jones 2 3 , Mariacristina Rumi 2 3 , Zhenting Dai 5 , William King 5 , Seth Marder 2 3 , Jennifer Curtis 1 4 , Elisa Riedo 1 3 Show Abstract
1 School of Physics, Georgia Institute of Technology, Atlanta, Georgia, United States, 3 Center for Organic Photonics and Electronics, Georgia Institute of Technology, Atlanta, Georgia, United States, 4 Petit Institute for Bioengineering and Biosciences, Georgia Institute of Technology, Atlanta, Georgia, United States, 2 School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, Georgia, United States, 5 Department of Mechanical Science and Engineering, University of Illinois Urbana-Champaign, Urbana, Illinois, United States
Protein patterning at the molecular scale is crucial for fundamental studies in cell biology and for biomedical applications such as protein chips, drug screening, biological sensors, and tissue engineering. Atomic force microscopy (AFM) based techniques have been successful in generating protein nano-arrays on various substrates. However, several challenges still exist in terms of resolution, writing speed, cost, substrate choice, protein bioactivity, multi-component patterning, and surface passivation. Here, we report the use of thermochemical nanolithography (TCNL) [1, 2] combined with post covalent functionalization (PCF) and molecular recognition (MR) on a polymer surface to produce multiplexed nanopatterns at speeds of mm/s. These patterns can then be functionalized under native conditions to create tailored nano-assemblies of two different species of proteins coexisting on the same surface. The proteins attach selectively and strongly to the nanopatterns via covalent and/or specific biotin-streptavidin interactions, while retaining their ability to interact specifically with other proteins in buffered solution. At present, this method has produced nanopatterns of bio-active proteins with features as small as 40 nm on polymer films. The ability to produce high resolution nanopatterns of two distinctive species of proteins on the same polymer surface opens up new possibilities to study protein-protein interactions and cell biology. As a proof of concept, we have fabricated a two-protein nanopattern mimicking the surface of an antigen presenting cell (APC) for future controlled studies of the spatial organization of the T-cell immunological synapse . We foresee that the TCNL/PCF/MR can have a direct impact on the development of biosensors, nanoscale manipulation of biological macromolecules, and on many cell studies that require interaction with two or more proteins in tailor-made patterns. 1. R. Szoszkiewicz, T. Okada, S. C. Jones, T.-D. Li, W. P. King, S. R. Marder, and E. Riedo, Nano Lett. 7, 1064 (2007).2. D. B. Wang, R. Szoszkiewicz, T. Okada, S. C. Jones, M. Lucas, J. Lee, W. P. King, S. R. Marder, and E. Riedo, Appl. Phys. Lett. 91, 243104 (2007).3. D. Wang, V. Kodali, W. D. Underwood, J. E. Jarvholm, T. Odaka, S. J. Jones, C. Rumi, W. P. King, S. R. Marder, J.E. Curtis and E. Riedo, (2008) submitted
Shashi Murthy Northeastern University
Henry Zeringue University of Pittsburgh
Saif Khan National University of Singapore
Victor Ugaz Texas A&M University
OO9: Porous Materials in Labs on a Chip
Thursday PM, April 16, 2009
Room 3016 (Moscone West)
4:15 PM - OO9.1
Porous Silicon in Lab-on-a-Chip Technology: Main Features and Applications in Biosensing Platforms.
Luca De Stefano 1 , Edoardo De Tommasi 1 , Ilaria Rea 1 , Ivo Rendina 1 Show Abstract
1 , Institute for Microelectronics and Microsystems, National Council for Research, Naples Italy
The efficiency of porous silicon (PSi) as a transducing element in label-free biosensing has been widely demonstrated in recent years. Furthermore, PSi presents some characteristics, such as low cost in the fabrication process, compatibility with microelectronic technologies (and, thus, the possibility to be integrated in hybrid systems such MEMS and MOEMS), very high specific surface (200-500 m^2*cm^−3), which are fundamental features when a lab-on-a-chip is designed. PSi is obtained from electrochemical etching of crystalline silicon in a hydrofluoric solution, finally leading to a sponge-like, nanocrystalline structure. The morphology of the surface can be precisely controlled by varying the composition of the etching solution, the doping of the silicon substrate, and the current density used to etch the surface. Computer-controlled production can create silicon films with precise thickness and pore sizes that range from a few nanometers up to microns. Moreover, because the etching process is self-stopping, stacks of multiple layers, each one with different porosity, can be fabricated in a single run. Examples of multi-layered structures include Fabry-Perot interferometers, Bragg reflectors, micro-cavities, resonant mirrors and even complicated quasi-periodic sequences. The ability to perform optical sensing by means of PSi structures relies on changes in the photonic properties of the material, such as photoluminescence or reflectance, when exposed to gaseous or liquid samples. For sensing to be selective, these interactions must be made specific by chemical or physical modification of the surface, i.e. by means of its functionalization. Thus, by substituting the superficial Si-H bonds with Si-C or Si-O-C bonds, it is possible both to thermodynamically stabilize the sensor surface and to link the proper biological probes on it. We tested and successfully employed several functionalization techniques, from UV-stimulated photochemical functionalization to pure chemical ones. Very recently we made use of synthesized biocompatible multiblock copolymers based on Poly(e-caprolactone) (PCL), containing pendant functional groups regularly spaced along the chain, as coatings of PSi optical structures, in order to realize a new class of implantable biosensors. In present work, several examples of PSi-based biosensors are described and the perspective to obtain, starting from this technology, fast, simple, specific, sensitive and multiplexed sensor systems integrated in low cost labs-on-a-chip is deeply investigated.
4:30 PM - OO9.2
Efficient Nanoporous Silicon Membranes for Integrated Microfluidic Separation and Sensing Systems.
Nazar Ileri 1 2 , Pieter Stroeve 1 , Sonia Letant 2 , Jerald Britten 2 , Hoang Nguyen 2 , Cindy Larson 2 , Saleem Zaidi 3 , Ahmet Palazoglu 1 , Roland Faller 1 , Joseph Tringe 2 Show Abstract
1 , University of California Davis, Davis, California, United States, 2 , Lawrence Livermore National Laboratory, Livermore, California, United States, 3 , Gratings, Inc., Albuquerque, New Mexico, United States
Nanoporous devices constitute emerging platforms for selective molecule separation and sensing, with great potential for high throughput and economy in manufacturing and operation. Acting as mass transfer diodes similar to a solid-state device based on electron conduction, conical pores are shown to have superior performance characteristics compared to traditional cylindrical pores. Such phenomena, however, remain to be exploited for molecular separation. Here we present performance results from silicon membranes created by a new synthesis technique based on interferometric lithography. This method creates ~mm2 planar arrays of uniformly tapered nanopores in silicon with pore diameter 100 nm or smaller, ideally-suited for integration into a multi-scale microfluidic processing system. Molecular transport properties of the devices are compared against state-of-the-art polycarbonate track etched (PCTE) and anodic aluminum oxide (AAO) membranes. Mass transfer rates up to 15X greater than achievable with existing commercial sieve technology are shown to be highly controllable with surface functionalization and electric fields applied to pore surfaces. Experimental results on molecular separation efficiency are reported, together with complementary results from molecular dynamics simulations. This work was performed under the auspices of the U.S. Department of Energy by Lawrence Livermore National Laboratory under Contract DE-AC52-07NA27344 and by UC Davis. The work was supported by LLNL LDRD and by University of California Systemwide Biotechnology Research & Education Program GREAT Training grant 2007-03.
4:45 PM - OO9.3
Open-Pore Microfluidic Tissue Engineering Scaffolds.
George Engelmayr 1 , Jane Wang 2 3 , Jeffrey Borenstein 3 , Robert Langer 1 , Lisa Freed 1 Show Abstract
1 Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States, 2 Department of Materials Science and Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States, 3 Biomedical Engineering Center, Charles Stark Draper Laboratory, Cambridge, Massachusetts, United States
Microfabrication techniques have shown promise in generating tissue engineering scaffolds with structural and mechanical properties more closely resembling those of native tissues. Toward more biomimetic tissue engineered myocardium, we recently demonstrated that anisotropic scaffolds with unique accordion-like honeycomb pore structures can be fabricated by excimer laser microablation of the bioresorbable elastomer poly(glycerol sebacate) (PGS) (Engelmayr et al., Nature Materials, DOI: 10.1038/nmat2316). Indeed, thin (~ 250µm) accordion-like honeycomb scaffolds seeded with neonatal rat heart cells exhibited closely matched anisotropic mechanical properties compared with adult rat right ventricular myocardium, directionally-dependent electrical excitation thresholds (p<0.05), and greater heart cell alignment (p<0.0001) compared with isotropic controls. However, toward generating thicker, metabolically demanding engineered tissues, previous studies have demonstrated significant benefits of perfusion strategies in maintaining cell viability and function. Here we report on the development of open-pore microfluidic scaffolds incorporating through-thickness open pores for cell attachment and tissue formation with microfluidic flow channels integrated within the walls of the pores. Open-pore microfluidic scaffolds were fabricated from PGS by a combined excimer laser microablation-lamination technique previously used to fabricate multi-layered PGS scaffolds with 3D interconnected pore networks. Studies are underway to characterize the effects of microfluidic scaffold perfusion on cell viability, differentiation and function. Open-pore microfluidic scaffolds could potentially be useful in sustaining cell viability and function in thicker engineered tissues.
5:00 PM - OO9.4
Sub-5 nm FIB Direct Patterning of Nanopores.
Gierak Jacques 1 , Birgitta Schiedt 1 Show Abstract
1 , CNRS-LPN, Marcoussis France
Nanopores open the perspective of studying the confinement, dynamics and transport properties of single macromolecules at nanometre scales with a temporal resolution of some microseconds. Synthetic or artificial nanopores, nano-engraved within membranes as a template for nano-pore and nano-mask fabrication is an application field of growing interest. In contrast to lithographical approaches, where additional wet etching after the irradiation is used to develop pores, a FIB system can produce holes directly at specified locations with customised organisation and shape. One the other hand the main limitation with FIB is the achievable resolution that is limited in the range of 50 nm. Therefore FIB techniques are used in complement with sculpting methods to achieve smaller pore sizes. There is a considerable application potential for such nano-sized holes or nano-pores aiming at using such membranes for example as stencils or masks to grow or depose nanostructures, or to fabricate single molecule electronic detectors or sensors. In this presentation we propose to detail an innovative FIB instrument and advanced methodology we have carefully optimised in our Laboratory for achieving deep sub-10 nm nano-pores fabrication capability. We will describe and illustrate this potential in presenting a method capable to fabricate directly nano-pores as small as 3 nm in relatively large quantities. We will summarise the optimisation efforts we have conducted aiming at (i) fabricating thin (10 nm to 100 nm thick) and high quality membranes as a template for the nano-pores, and at (ii) performing efficient and controlled FIB nanoengraving of such a delicate media. We will describe the integration method we have use for integrating these artificial nanopores in an electrophoresis experiments and our preliminary measurements.
5:15 PM - OO9.5
Functionalized Porous Nanostructured Thin Films for Immobilization of Biomolecules.
Jonathan Kwan 1 , Jeremy Sit 1 Show Abstract
1 Electrical and Computer Engineering, University of Alberta, Edmonton, Alberta, Canada
Amine-modified solid surfaces are commonly used for applications such as protein and DNA immobilization. They function as a cross-linker to attach other molecules with specific functionality to the surface. However, due to the use of a planar surface, the functionality of these surfaces is inherently limited. For lab-on-a-chip or sensing applications, this is a crucial factor affecting sensitivity, which is directly related to the surface area. We present the use of high surface area, porous, nanostructured thin films fabricated by the glancing angle (GLAD) technique as the solid support for amine modification. GLAD affords direct control over the porosity and surface area of the films and can be used with a wide range of materials. Thus, by chemically modifying these films we may directly address the sensitivity factor affecting micro-scale devices.In this work, SiO2 vertical post nanostructures were fabricated on silicon substrates and used as the solid support for chemical modification. Vapour-phase deposition of an amine-terminated precursor, 3-aminopropyltrimethoxysilane, was employed to terminate the surface of the nanostructured film with amine moieties. To investigate the accessibility of the amine groups for a given density on the surface of the films, deposition time and precursor concentrations were varied to produce a range of amine densities. The modified films were characterized with x-ray photoelectron spectroscopy, Auger electron spectroscopy, and scanning electron microscopy, and compared to their planar counterparts. The results demonstrate that these nanostructured thin films are a viable platform for increasing sensitivity in micro-devices.