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Send Us Your FeedbackSymposium MM: Biomolecular and Biologically Inspired Interfaces and Assemblies
SYMPOSIUM MM
November 25 - 29, 2007
Chairs
| Vincent M. Rotello Dept. of Chemistry University of Massachusetts 710 North Pleasant St. Amherst, MA 01003 413-545-2058 |
Jeffrey B. Tok Chemical Biology and Nuclear Science Division Lawrence Livermore National Laboratory BioSecurity and NanoSciences Laboratory, L-235 7000 East Ave. Livermore, CA 94551 925-423-1549 | |
| Molly M. Stevens Dept. of Materials Imperial College London Exhibition Rd. London, SW7 2AZ United Kingdom 44-20-7594-6804 |
Darrin J. Pochan Dept. of Materials Science and Engineering University of Delaware 201 DuPont Hall Newark, DE 19716 302-831-3569 | |
| Paula T. Hammond Massachusetts Institute of Technology Rm. 66-550 77 Massachusetts Ave. Cambridge, MA 02139 617-258-7577 |
Symposium Support
U.S. Army Research Office
(see Proceedings Library at www.mrs.org/publications_library)
as volume 1061E
of the Materials Research Society
Symposium Proceedings Series.
* Invited paper
TUTORIAL
MM/PP: Interfacing Quantum Dots, Metallic and Magnetic Nanoparticles with Biology
Sunday November 25, 2007
1:30 PM - 5:30 PM
Room 210 (Hynes)
Colloidal inorganic nanocrystals have several unique intrinsic photophysical properties that are not observed at the molecular level or shared by their bulk parent materials. These include size-dependent absorption and luminescence of semiconductor quantum dots (QDs), plasmonic absorption and Raleigh scattering from metallic nanoparticles, and size- and composition-dependent magnetic coercivity (and contrast) for magnetic nanocrystals. This has not only generated intense interest in understanding their fundamental properties but has also led to an explosion in applications ranging from electronic devices to lasing. Interfacing these materials with biology has, in particular, experienced a tremendous expansion in the past decade.
The most relevant issues in biology can be summarized in three main areas:
1. The development of surface functionalization techniques to render the nanocrystals hydrophilic, and the design of simple and reproducible conjugation methods that provide stable, compact, and biologically active hybrids
2. The development of targeted applications in biology that utilize the above physical properties to gain new understanding of complex biological phenomena, including: the design of specific assays based on fluorescence, magnetic contrast, and plasmonic absorption; the development of assays based on fluorescence energy transfer where both QDs and gold NPs can be combined; the delivery of NP-biomolecule cargos inside cells and tracking of intracellular protein movement and interactions
3. Usage of the information collected from these biological studies as feedback for chemists and physicists to help improve material design (new combinations and new properties) and to develop better understanding of their physical properties
The tutorial will provide an overview of the unique advantages of each type of nanocrystals (metallic, magnetic, and luminescence QDs) for use in biology. Instructors will address aspects such as: interfacing of NPs with biology (assembly of NP-bioconjugates with proteins, peptides, and DNA) and sensor design based on immuno-fluorescence, fluorescence energy transfer, colorimetric assays, magnetic contrast, and magnetic sensing. Issues such as intracellular delivery of NP cargos inside live cells (non-invasively) and protein tracking will also be addressed.
Instructors:
Jinwoo Cheon
Yonsei University, Republic of Korea
Hedi Mattoussi
Naval Research Laboratory
Vincent M. Rotello
University of Massachusetts
Chairs: Derek Woolfson and Michael Yu
Monday Morning, November 26, 2007
Room 210 (Hynes)
8:30 AM *MM1.1/NN1.1
Non-Canonical Amino Acids in Protein Engineering. David Tirrell, Caltech, Pasadena, California.
This lecture will describe several means by which non-canonical amino acids can be used to enhance the functionality of engineered proteins. Both translational and post-translational methods will be described. Examples will be chosen from biomaterials science, protein modification, protein evolution, and proteomic analysis.
9:00 AM *MM1.2/NN1.2
Stronger and Longer Synthetic Collagen. Ronald T. Raines, Department of Biochemistry, University of Wisconsin - Madison, Madison, Wisconsin; Department of Chemistry, University of Wisconsin - Madison, Madison, Wisconsin.
Collagen is the most abundant protein in the human proteome. The post-translational modification of collagen by the enzyme prolyl 4-hydroxylase increases markedly the conformational stability of the collagen triple helix. We have discovered that a previously unappreciated force—stereoelectronic effects—is responsible for this increased stability. By exploiting these stereoelectronic effects (e.g., the gauche effect and n→π* interaction) and reciprocal steric effects, we have created synthetic collagen of unprecedented stability. We have also used the molecular self-assembly of triple-helical fragments to create synthetic collagen of unprecedented length. These synthetic collagens have numerous applications in biotechnology and biomedicine. [This work is supported by NIH grant AR44276.]
9:30 AM MM1.3/NN1.3
Spatiotemporal Modification of Collagen Scaffolds Directed by Collagen Mimetic Peptide Derivatives. Allen Y. Wang1, Shirley Leong2, Catherine A. Foss3, Xiao Mo1, Martin G. Pomper3,4 and Seungju M. Yu1,4; 1Deptartment of Materials Science and Engineering, The Johns Hopkins University, Baltimore, Maryland; 2Department of Chemical and Biomolecular Engineering, The Johns Hopkins University, Baltimore, Maryland; 3Department of Radiology, The Johns Hopkins University, Baltimore, Maryland; 4Institute for NanoBiotechnology, The Johns Hopkins University, Baltimore, Maryland.
Functionalized collagen incorporating exogenous compounds may offer new and improved applications for collagen-based biomaterials especially in drug-delivery, multifunctional implants, and tissue engineering. We developed a specific and reversible collagen modification technique that utilizes associative chain interactions between synthetic collagen mimetic peptide (CMP), [(ProHypGly)x; Hyp:hydroxyproline] and natural type I collagen. Here we show temperature dependent collagen binding and subsequent release studies of a series of CMPs with varying chain lengths that indicate triple helical propensity driven binding mechanism similar to DNA strand invasion and exchange. The binding took place when melted, single strand CMPs were allowed to fold by cooling in contact with reconstituted natural collagens. The binding affinity is highly specific to collagen as CMP conjugated to gold nanoparticles revealed nanometer-scale repetitive binding locations along the length of type I collagen fibres and fluorescent CMPs could be used to selectively image collagens in ex vivo human liver tissue. When heated to physiological temperature, the bound CMPs discharged from the collagen at a sustained rate that correlated with CMP’s triple helical propensity suggesting that the sustainability is mediated by dynamic collagen-CMP interaction. We also report modification of collagen with linear and mutli-arm poly(ethylene glycol)-CMP conjugates. Due to the convenient nature of the modification procedure, pre-determined areas of collagen film were readily modified with PEG-CMP conjugates which exhibited temporary cell repelling activity at 37 degree C lasting up to 9 days. These results demonstrate new opportunities for targeting pathologic collagens for diagnostic or therapeutic application and for fabricating multifunctional collagen coatings and scaffolds that can temporally and spatially control the behavior of cells associated with the collagen matrices.
9:45 AM MM1.4/NN1.4
Building Tissue Engineering Scaffolds Directly from Extracellular Matrix Proteins with Microscale Spatial Control. Adam W Feinberg, Sean P Sheehy and Kevin Kit Parker; School of Engineering and Applied Sciences, Harvard University, Cambridge, Massachusetts.
We have developed a method for generating free-standing tissue engineering scaffolds that are spatially organized from the nanometer to millimeter length scales. These scaffolds are composed of extracellular matrix (ECM) proteins with the capability to create unique scaffold topologies that mimic in vivo structures. Integration of living cells into these ECM scaffolds should allow the generation of engineered tissues with a level of spatial control that exceeds what is possible with random mesh, sponge and gel scaffolds. Fabrication is based on microcontact printing of ECM proteins onto a transitional surface that serves as a temporary substrate during assembly. Multiple proteins are printed in a layer-by-layer process creating a microstructured, multi-component scaffold. The exact spatial structure and composition is controlled by altering the features of the polydimethylsiloxane (PDMS) stamp used for microcontact printing and/or by printing multiple proteins, multiple times at different angles. Upon dissolution of the transitional surface, the ECM scaffold is released into solution as a free-standing construct. Inherent protein-protein binding domains in the constituent ECM proteins hold the scaffold together providing structural integrity. In proof-of-concept experiments, we have fabricated “net-like” single component scaffolds composed of the ECM protein fibronectin (FN) and bi-component scaffolds composed of the ECM proteins laminin and FN. Composition and bioactivity of the ECM scaffolds has been verified by immunofluorescent staining with appropriate antibodies. This technology has potential use in a wide array tissue engineering applications. Initial cell seeding experiments have demonstrated the ability to generate highly anisotropic strands of muscle composed of neonatal rat ventricular cardiomyocytes. These myocardial fibers are typically ~20 μm wide and 100’s of μm long and demonstrate uniaxial, synchronized contraction similar to papillary muscle. Future work is aimed at expanding the types of ECM proteins that can be integrated into these scaffolds and building more complex tissue constructs.
10:00 AM MM1.5/NN1.5
Self-Assembling Hydrogels from Fibrin Coiled Coil Peptide-Polymers. Peng Jing and Joel H Collier; Biomedical Engineering, University of Cincinnati, Cincinnati, Ohio.
Fibrin-based gels are clinically useful as tissue sealants and are currently being explored as matrices for regenerative medicine. However, their utility is limited by incomplete compositional definition, heterogeneity, and a potential for pathogen transmission that is inherent in biologically sourced materials. As a novel approach to address these issues, we created fully synthetic analogs of fibrin gels that self-assemble via peptides inspired by fibrin’s coiled coil domains, which are critical in the oligomerization of the six chains that comprise the full protein. We started by synthesizing peptides between 35 and 37 amino acids long from the coiled coil domain of the gamma chain of human fibrin and investigated the effect of amino acid substitutions designed to stabilize multimerization through additional interhelical electrostatic pairings and improved packing of the hydrophobic core. Through these iterations we arrived at a 37-amino acid peptide with twelve substitutions that formed stable coiled coil dimers and tetramers, as shown by circular dichroism and analytical ultracentrifugation. Stable multimers were produced from both human fibrin sequences and mouse fibrin sequences (68% homology), and the peptides were non-cytotoxic in cultures of human endothelial cells. Conjugation of the modified human fibrin peptide to mono- and difunctionalized polyethylene glycol via maleimide-thiol chemistry produced self-assembling diblock and triblock molecules, the identity and purity of which were determined by ESI mass spectrometry and HPLC. PEG conjugation had negligible impact on the secondary structure of the peptide, both for the diblock and triblock. The triblock peptide-PEG-peptide, like the unconjugated peptide, formed mixtures of dimers and tetramers at concentrations above 0.6% w/v, and above 4% w/v it formed transparent hydrogels in neutral phosphate buffer. These gels had attractive mechanical properties, as the average plateau storage modulus of 8% w/v triblock gels was 570Pa and that of 12% w/v gels was 2500Pa. These values compare favorably to reported values for fibrin-based gels. Loss moduli were about one order of magnitude below storage moduli, indicating that the gels were elastic. In contrast, rheometry indicated that the diblock did not form gels at any concentration tested, up to 12% w/v. Additionally, triblock gels had attractive degradation properties, slowly dissolving in excess phosphate buffered saline by 50% after 4 days and entirely by 8 days. Collectively, these results indicate that the triblock peptide-PEG-peptide forms hydrogels that are promising candidates for further evaluation as fully synthetic analogs of fibrin gels, sealants, and matrices.
10:30 AM *MM1.6/NN1.6
Supramolecular Organization From Nanometers to Centimeters in Peptidic Materials. Samuel I. Stupp, Materials Science and Engineering, Chemistry and Medicine, Northwestern University, Evanston, Illinois; Institute for BioNanotechnology in Medicine, Northwestern University, Evanston, Illinois.
Over the past few decades, designed peptidic materials have been of great interest as biomaterials that can interface with cells in vitro and in vivo since they bear the potential to be both bioactive and biodegradable. Our laboratory has developed an extensive familiy of peptidic biomaterials in which the primary structural element is a cylindrical nanofiber that forms by self-assembly of molecules known as peptide amphiphiles. These amphiphiles contain both a peptide segment and a non-peptidic hydrophobic segment such as the tail of a fatty acid. Under appropriate conditions these molccules aggregate to form beta sheets which in turn collapse into cylindrical nanofibers with a hydrophobic core. Very recently we have discovered that these systems can be thermally and mechanically directed to create domains of co-aligned nanofibers that reach into macroscopic dimensions. Strings of peptidic material with lengths on the order of centimeters and containing aligned nanoscale fibers can be easily formed and even populated with cells. This lecture will describe the encapusulation and differentiation of human stem cells into these macroscopic strings. These self-assembling peptidic systems offer new opportunities to create cell assays and therapies in regenerative medicine.
11:00 AM MM1.7/NN1.7
Early Time β-Hairpin Peptide Self-Assembly into a Hydrogel Network. Tuna Yucel1,2, Joel P. Schneider3 and Darrin J. Pochan1,2; 1Materials Science and Engineering, University of Delaware, Newark, Delaware; 2Delaware Biotechnology Institute, Newark, Delaware; 3Chemistry and Biochemistry, University of Delaware, Newark, Delaware.
In dilute aqueous solution at pH=7.0 and T=22oC, MAX 1 peptide (NH2-(VK)4-VDPPT-(KV)4-CONH2) is unfolded and freely soluble. The peptide intramolecularly folds into a β-hairpin when the electrostatic interactions between charged lysine (K) amino acids are screened through an increase in the solution ionic strength. The β-hairpins consequently intermolecularly assemble via hydrophobic collapse and hydrogen bonding into a fibrillar hydrogel network. Here, we correlate a direct characterization of the temporal evolution of β-hairpin formation and intermolecular fibril formation with changes in viscoelastic properties. By combining the results of far-UV circular dichroism spectroscopy, cryogenic transmission electron microscopy, small angle neutron scattering, dynamic and static light scattering and dynamic oscillatory rheology, we observe that MAX 1 self-assembly proceeds by nucleation of semi-flexible β-sheet nanofibrils with monodisperse diameter (d~3 nm) that elongate and form branched fibril clusters. Under the assembly conditions studied here, these branched clusters had an apparent fractal dimension D~1.5 when they initially fill up the sample volume. This D value increases with peptide concentration, presumably due to increasing branching density. Clusters eventually interpenetrate and form a percolated network. Percolation leads to an ergodic to non-ergodic transition, as evidenced by a characteristic power law decay of the DLS autocorrelation function (g2(τ)~τ-0.45) followed by an increase in the frozen-in scattered intensity fluctuations due to gelation. Concurrently, the network rigidity increases significantly as observed by rheology. The self-assembly of MAX 1 was compared and contrasted with the self-assembly of biopolymer networks in literature. The potential biotechnological importance of the characterization of the early time β-hairpin self-assembly in the design of injectable hydrogels for in vivo tissue regeneration will be discussed. Ultimately, our goal is to understand possible biocompatibility-self-assembly-hydrogel material property relationships.
11:15 AM MM1.8/NN1.8
Modification of Liposomes using α-Helical Coiled-Coil Peptides. Hana Robson Marsden, Alexander Korobko and Alexander Kros; Chemistry, Leiden University, Leiden, Netherlands.
A system of active nanocapsules is investigated, using liposome capsules which are activated with interacting peptides. Lipopeptides are synthesized using a pair of peptides that form heterodimeric α-helical coiled-coils. The phospholipid tail of these hybrid molecules inserts into liposomes, resulting in vesicles with an outer surface studded with one of the two peptides. Reminiscent of cell membrane fusion, which requires the action of specific proteins, the interaction of these peptides facilitates liposome aggregation and probable fusion. The fusion of liposomes, accompanied by the mixing of liposome contents, would build up the complexity of the ‘lab in a vesicle’ concept.
11:30 AM *MM1.9/NN1.9
Nanofibers formed by Self-assembly of Multidomain Meptides: Applications for Bioengineering to Nanotechnology. Jeffrey D Hartgerink, Kerstin Galler, He Dong, Lorenzo Aulisa and Sergey E. Paramonov; Chemistry & Bioengineering, Rice University, Houston, Texas.
Control over the dimension of the assemblers has been a major challenge in the self-assembly of nanostructured materials. There are few effective approaches to confine the assembled objects in a defined dimension. In this paper we report on a series of multi-domain peptide molecules (MDPs), each of which consists of three functional domains that serve to control the organization and the extent of the self-assembly through a mechanism that is mediated by “molecular frustration”. We demonstrate that when forces favoring assembly are properly balanced with forces favoring disassembly, discrete nanofibers with controlled length result. In addition, we found that the ratio of domain size determines peptides’ secondary structure, which has a dramatic effect on their supramolecular nanostructure. This observation indicates a strong correlation between peptides’ molecular secondary structures and the self-assembled nano-structures. Due to the fact that the experiments were all performed under the physiological condition, we believe this architectural motif may be utilized for novel tissue regeneration strategies and other systems which require control over chemical organization at the nanoscale. Additionally, the high solubility (up to 1 wt%) of the nanofibers formed by at neutral pH allows for the use of a variety of spectroscopic measurements to help understand and further treat various diseases associated with protein aggregation.
Chairs: Paula Hammond and Seung-Wuk Lee
Monday Afternoon, November 26, 2007
Room 210 (Hynes)
1:30 PM *MM2.1/NN2.1
Genetic Control of the Synthesis and Assembly of Materials for Electronics and Energy. Angela Belcher, Ki Tae Nam, Yun Jung Lee, Dong-Soo Yun, Brian Neltner and Andrew Magyar; MIT, Cambridge, Massachusetts.
Organisms have been making exquisite inorganic materials for over 500 million years. Although these materials have many desired physical properties such as strength, regularity, and environmental benign processing, the types of materials that organisms have evolved to work with are limited. However, there are many properties of living systems that could be potentially harnessed by researchers to make advanced technologies that are smarter, more adaptable, and that are synthesized to be compatible with the environment. One approach to designing future technologies which have some of the properties that living organisms use so well, is to evolve organisms to work with a more diverse set of building blocks. These materials could be designed to address many scientific and technological problems in electronics, military, medicine, and energy applications. An example is a virus enabled lithium ion rechargeable battery we recently built that has many improved properties over conventional batteries. This talk will address scientific approaches to move beyond naturally evolved materials to genetically imprint advanced technologies including batteries, electochromic materials, and catalysis.
2:00 PM *MM2.2/NN2.2
Building from Bottom Up: Fabrication of Nanomaterials Using Peptide Motifs. Shugang Zhang, MIT, Cambridge, Massachusetts.
Materials science has generally been associated with metallurgy, alloy, ceramics, composites, polymer science, fiber spinning, coating, thin film, industrial surfactants and block copolymer development. That is about to change. Materials science will also expand to discovery and fabrication of biological and molecular materials with diverse structures, functionalities and utilities. The advent of nanobiotechnology and nanotechnology accelerated this trend. Similar as construction of an intricate architectural structure, diverse and numerous structural motifs are used to assemble a sophisticated complex. Nature has selected, produced and evolved numerous molecular architectural motifs over billions of years for particular functions. These molecular motifs can now be used to build materials from the bottom up. Materials science will begin to harness nature’s enormous power to benefit other disciplines and society. Zhang, S. (2002) Emerging biological materials through molecular self-assembly Biotechnology Advances 20, 321-339. Zhang, S. (2003) Fabrication of novel materials through molecular self-assembly. Nature Biotechnology 21, 1171-1178. Yokoi, H., Kinoshita, T. & Zhang, S. (2005) Dynamic reassembly of peptide RADA16 nanofiber scaffold. Proc. Natl. Acad. Sci.USA 102, 8414-8419. Zhao, X. & Zhang, S. (2006) Molecular designer self-assembling peptides. Royal Society of Chemistry 35, 1105-1110. Gelain, F., Bottai, D., Vescovi, A & Zhang, S. (2006) Designer self-assembling peptide nanofiber scaffolds for adult mouse neural stem cell 3-dimensional cultures. PloS ONE 1, e119, 1-11. Horii, A. Wang, X., Gelain, F. & Zhang, S. (2007) Biological designer self-assembling peptide scaffolds significantly enhance osteoblast proliferation, differentiation & 3-D migration. PloS ONE 2, e190, 1-9.
2:30 PM MM2.3/NN2.3
Development of Novel Hard-Tissue Regenerative Materials Through Directed and Natural Evolutionary Processes. Eddie Wang1 and Seung-Wuk Lee1,2; 1Bioengineering, University of California, Berkeley, Berkeley, California; 2Physical Biosciences Division, Lawerence Berkeley National Lab, Berkeley, California.
Bones are natural inorganic-organic nanocomposite materials with remarkable toughness and strength. Utilizing natural and artificial evolutionary processes, we have developed novel bone-mimetic nanocomposite materials to recapitulate bone’s unique properties. A functional biopolymer, composed of an elastin-like polypeptide (ELP) fused with a hydroxyapatite binding peptide (HBP), was synthesized using bacterial biosynthetic approaches. HBP is a twelve amino acid peptide previously identified through directed evolution by phage display that binds to and promotes nucleation of hydroxyapatite, the predominant mineral component of bones and teeth. The HBP sequence has been genetically engineered as an N and/or C-terminal addition to an ELP gene. The resulting composite protein (HBP-ELP-HBP) has been expressed and purified from E. Coli then chemically cross-linked through periodically spaced lysine residues to form a thermo-responsive gel. We believe this novel protein gel will retain the favorable properties of ELPs (elasticity, fatigue resistance, biocompatibility) and will also interface with hydroxyapatite through its HBP domains. We have combined the gel with hydroxyapatite crystals or precursor ions to characterize the construct’s hydroxyapatite binding and nucleating capabilities. We are now determining and optimizing the resulting composite’s mechanical properties. The resulting composite biomaterial may be useful for bone tissue engineering/repair or in dentistry as a treatment for dental caries.
2:45 PM MM2.4/NN2.4
Supramolecular Self-Assembly of a Metal-binding Polypeptide and Implications for Molecular Recognition. Christopher R So1, Emre E Oren1, Urartu Seker1,3, Brandon R Wilson1, John Kulp2, Candan Tamerler1,3, John S Evans2 and Sarikaya Mehmet1; 1Materials Science and Engineering, University of Washington, Seattle, Washington; 2Chemistry, New York University, New York, New York; 3Molecular Biology & Genetics, Istanbul Technical University, Istanbul, Turkey.
Recently, the utility of Genetically Engineered Peptides for Inorganics (GEPIs) has opened the prospect of achieving self-assembled hierarchical material systems due to their ability to recognize particular surfaces. One such polypeptide, 3x(MHGKTQATSGTIQS), has shown to possess properties of specificity towards gold, while being nonspecific to other noble metals (Pt), oxides (Quartz), or minerals/organics (mica, graphite), suggestive of such molecular recognition mechanisms. Here, we report novel atomic force microscopy (AFM) and molecular simulation studies that detail the formation of ordered assemblies of a gold binding protein (3r-GBP1). Simulated annealing molecular dynamics (SA/MD), based on nuclear magnetic resonance (NMR), indicates that the lowest energy structure of 3r-GBP1 features extended B-strand and random coil-like regions with repeating surface accessible side-chains identified as putative Au docking sites. Geometric models of lattice matching with the Au{111} reveal that the peptide aligns with both the <110> and <211> directions of the surface lattice. The AFM observation reveal a supramolecular assembly of the peptide forming crystallographically ordered six equivalent domains commensurate with the Au{111} surface lattice. To understand these supramolecular binding events, ex situ time-lapsed AFM experiments were carried out to quantitatively assess the kinetics of peptide assembly and to correlate the data to observed growth morphologies. Coverage trends from the concentration-varied experiment show high correlation to Langmuir fitting while approaching >90% coverage, in agreement with resulting surface plasmon resonance and quartz crystal microbalance analyses. These results provide initial insights into the molecular recognition mechanism(s) of peptide binding and self assembly on material surfaces.
3:30 PM *MM2.5/NN2.5
Targeted Protein Cage Architectures for Biofilm Imaging and Therapeutics. Trevor Douglas, Department of Chemistry and Biochemistry, Montana State University, Bozeman, Montana.
Diagnosis and treatment of infections can benefit from innovations that have substantially increased the variety of available multifunctional nanoplatforms. We have used an icosahedral viral nanoplatform to target a pathogenic, biofilm-forming bacterium, Staphylococcus aureus. Density of binding at the bacterial surface, mediated through specific protein ligand interactions, exceeded the density expected for a planar, hexagonally close-packed array. A multifunctionalized viral protein cage was used to load imaging agents (fluorophore and MRI contrast agent) onto these cells. The fluorescence- imaging capability allowed for direct observation of penetration of the nanoplatform into an S. aureus biofilm. Furthermore, the selectivity of antimicrobial photodynamic therapy (PDT) can be enhanced by coupling the photosensitizer (PS) to a targeting ligand. Nanoplatforms provide a medium for designing delivery vehicles that incorporate both functional attributes. We have used the photodynamic inactivation of Staphylococcus aureus, using targeted viral nanoplatforms conjugated to a photosensitizer (PS). Both electrostatic and targeted interactions were used to mediate PS nanoplatform delivery. Genetic constructs of a protein cage architecture allowed site specific chemical functionalization with the PS, and facilitated dual functionalization with the PS and the targeting ligand. These results demonstrate that multifunctional nanoplatforms based on protein cage architectures have significant potential as tools for both diagnosis and targeted treatment of recalcitrant bacterial infections.
4:00 PM MM2.6/NN2.6
Ferritin Cage Architecture with Superparamagnetic Iron Oxide Nanoparticle for Magnetic Resonance Contrast Agent. Masaki Uchida1,3, Masahiro Terashima4, Charles H. Cunningham5, Yoriyasu Suzuki4, Deborah A. Willits2,3, Ann F. Willis1,3, Philip S. Yang4, Michael V. McConnell4, Mark J. Young2,3 and Trevor Douglas1,3; 1Department of Chemistry and Biochemistry, Montana State University, Bozeman, Montana; 2Department of Plant Seicences, Montana State University, Bozeman, Montana; 3Center for Bio-Inspired Nanomaterials (CBIN), Montana State University, Bozeman, Montana; 4School of Medicine, Stanford University, Stanford, California; 5Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada.
Protein cage architectures are versatile nanoscale platforms amenable to both genetic and chemical modification, making them promising for cellular and molecular imaging. We have recently revealed that recombinant human H chain ferritin (rHFn), which has a 12 nm exterior diameter and an 8nm interior cavity, is a superior template for biomimetic mineralization and encapsulation of superparamagnetic iron oxide nanoparticles within its interior cavity. This nanoparticle is comparable in size to commercially available ultrasmall superparamagnetic iron oxide (USPIO) contrast agents for magnetic resonance imaging (MRI) but possess unique features not present in the commercially available USPIO contrast agents such as excellent homogeneity of particle size and demonstrated ability of further modification to impart cell-specific targeting. The aim of this research is to investigate the ability of a protein cage templated material to serve as a MRI contrast. By TEM and electron diffraction measurements, electron dense particles of magnetite (or maghemite) with narrow size distribution were formed within the rHFn after an iron oxide synthesis reaction. The average size of the particles increased form 3.6 to 5.9 nm with increasing theoretical Fe loading factor from 1000Fe to 5000Fe per cage. R2 relaxivity of the mineralized rHFn increased with increasing Fe loading factor and that of HFn5000Fe was comparable with that of a commercially available USPIO MR contrast agent. Cellular uptake of the mineralized protein cages was investigated in murine macrophage cells. The amount of Fe taken up by the cells cultured with the mineralized protein cage constructs was significantly more (9 to 39 fold) than that of the cells cultured with a commercially available USPIO, at equivalent Fe concentrations. The cells labeled with mineralized protein cages provided more intense dark MR images under a gradient echo sequence than those labeled with a commercially available USPIO. Since macrophage cells are involved in serious inflammatory diseases such as atherosclerosis, this composite magnetic cage material is expected to have great potential as a MRI contrast agent to assess inflammatory status in vivo.
4:15 PM MM2.7/NN2.7
Abstract Withdrawn
4:30 PM MM2.8/NN2.8
Protein-Mediated Assembly of Metal Nanostructures. Silke Behrens1, Wilhelm Habicht1 and Konrad Joachim Boehm2; 1Institute for Technical Chemistry, Forschungszentrum Karlsruhe, Karlsruhe, Germany; 2Leibnitz Institute for Age Research, Jena, Germany.
Self-assembled nanostructures represent interesting matrices to control the organization of inorganic matter at the nanometer scale. Beside other bio macromolecules, various proteins are known to form precisely defined nanostructures by self-assembly processes. Moreover, functional groups, e.g., SH-groups or imidazole heterocycles, exposed at the surface of proteins allow further chemical modification, including metal deposition. These attributes of proteins can be combined with appropriate chemical reactions to develop reliable bottom-up strategies for the formation of nanoscale hybrid materials with novel electronic, optical, or chemical properties. In this context, tubulin is a very interesting protein able to self-assemble in the presence of GTP into protofilaments, consisting of strictly alternating αβ-dimers. Under physiological conditions, the lateral linkage of these protofilaments results in the formation of microtubules. However, in cell-free environment, depending on co-factor and additive composition, the tubulin assembly and the arrangement of the protofilaments can be controlled to form a variety of other superstructures with defined nanometer-scaled geometry such as macrotubes, sheets, spirals, or rings. Zn2+ ions, e.g., direct the assembly of tubulin into sheets or macrotubes. In our approach, these protein assemblies were used as a functionalized scaffold where noble metal is generated in situ and deposited into particle arrays, reflecting the arrangement of tubulin subunits within the assembly. As a result particle arrays of different geometry and metal nanowires were obtained. The size and structure of the materials were examined using transmission electron microscopy and scanning force microscopy. Our study demonstrates a straightforward and rapid protein-based approach to obtain metal nanoparticle arrays and nanowires, not accessible via conventional synthetic methods.
4:45 PM MM2.9/NN2.9
Abstract Withdrawn
Chairs: Darrin Pochan and Vincent Rotello
Monday Evening, November 26, 2007
8:00 PM
Exhibition Hall D (Hynes)
MM3.1
Effects of Restriction Enzymes on DNA-mediated Assembly and Disassembly at a Fixed Temperature. Christopher K Tison and Valeria T Milam; Materials Science & Engineering, Georgia Institute of Technology, Atlanta, Georgia.
We use DNA as a tool to control the assembly and release of colloidal particles at a fixed temperature. By controlling the number and affinity of DNA duplexes between particle surfaces we induce weak, but complete, particle assembly through primary hybridization events. This particle assembly can then be redispersed without elevating the temperature through the addition of longer, competitive secondary targets with greater propensity for duplex formation than the primary target. We have investigated the effects of minimizing the number of DNA linkages between particles on particle assembly and redispersion by coupling controlled ratios of hybridizing probe strands and nonhybridizing diluent strands of equal base lengths. Diluent strands are then clipped by the restriction endonuclease AluI following surface immobilization to reduce the steric interference of these strands with hybridization events. Using flow cytometry, we find that the efficiency of AluI clipping on the surface of 1.04 µm polystyrene microspheres is initially low, but that over 75% of diluent strands can be cleaved after a five step digest procedure while leaving active probes intact for subsequent colloidal assembly. In addition, the location of the recognition sequence (to be clipped) with respect to the particle surface plays a significant role in optimizing the enzyme’s clipping activity. Microscopy studies indicate that particles conjugated with clipped diluent strands drive DNA-mediated assembly at lower probe concentrations than particles conjugated with unclipped diluent strands. Interestingly, however, this greater drive to assemble appears to compromise the ability to completely redisperse assemblies via competitive hybridization events. By finely controlling DNA-mediated assembly and disassembly at a fixed temperature, we continue to take steps towards designing model drug delivery vehicles with an intrinsic release mechanism.
MM3.2
Novel Protein/DNA/inorganic Biocatalytic Nanomaterials. Akhilesh Bhambhani1,2 and Challa V. Kumar2; 1Pharmaceutical Chemistry, The university of Kansas, Lawrence, Kansas; 2Department of Chemistry, University of Connecticut, Storrs, Connecticut.
Given the ubiquitous presence of heme proteins in biological systems, and their reactivity toward a number of substrates, we are interested in investing their catalytic properties in organized media. The lamellar solids α-Zr(IV) phosphate (α-Zr(HPO4)2.H2O, abbreviated as α-ZrP), for example, provided hydrophilic, negatively charged surfaces to entrap heme protein under benign condition. Upon binding to these anionic solids, bound proteins exhibited high temperature activities (>85 0C), while the corresponding free proteins denatured rapidly at this temperature. In an independent study, heme proteins were shown to bind to the double helical calf thymus DNA (DNA), and DNA inhibited the thermally-induced aggregation of Hb. This observation raised the interesting possibility of using DNA to improve the properties of intercalated Hb, and also test if Hb binding to DNA would assist DNA intercalation in the negatively charged galleries of α-ZrP. The first example of protein assisted DNA binding and DNA assisted protein activity enhancement at a layered inorganic solid, is reported here. Met-hemoglobin (Hb), for example, facilitated the co-intercalation of calf thymus DNA in the galleries of α-ZrP, and DNA did not bind to the solid in the absence of Hb. Exposure of a suspension of α-ZrP to a solution of Hb (100 µM, 6.45 mg/ml, 5 mM Tris 10 mM NaCl pH 7.2) and calf thymus DNA (262 µM in base pairs) resulted in the incorporation of the protein, and DNA in the galleries. Co-intercalation of DNA improved the structure and activity of intercalated Hb and also that of met-myoglobin (Mb). The circular dichroism (CD) spectra of the bound protein, for example, is almost super imposable with that of the free protein in solution. This result of Hb structure retention prompted the investigation of the activity of the bound Hb, in the presence of DNA. The peroxidase-like activity of bound Hb was enhanced five-fold when DNA was co-immobilized, much closer to that of the free Hb in solution. Similar results are indicated with Mb, and these enhancements in the properties of the bound protein are not limited to Hb. The strong role of DNA in enhancing bound protein properties is novel, and this is the first report of protein-assisted DNA binding to negatively charged, layered material. We suspect that the protein-DNA interactions play a major role in these nano-biocatalysts and these solids are promising for gene/RNA/drug delivery applications.
MM3.3
Asymmetric Functionalization of Gold Nanoparticles with Oligonucleotides. Xiaoyang Xu and Chad A Mirkin; Chemistry, International Institute for Nanotechnology, Evanston, Illinois.
Gold nanoparticles (AuNPs) were anisotropically functionalized with two different oligonucleotide sequences using magnetic microparticles (MMP) as geometric restriction templates for site-selective enzymatic extension of particle-bound oligonucleotides. The DNA-functionalized MMPs serve a dual-purpose: they allow site-specific modification of DNA-modified AuNPs, and they facilitate the separation and purification of the anisotropically functionalized AuNPs. The divalent linking capability of the resulting AuNPs allowed for the design and programmable assembly of discrete nanoparticle heterostructures. AuNPs functionalized in this way exhibit highly directional selectivity for hybridization with other nanoparticles, allowing the design and assembly of unique nanoparticle heterostructures, such as satellite, cat paw, and dendrimer-like structures.
MM3.4
Electronic Transport in DNA Segments with Diluted Base-pairing. Eudenilson L Albuquerque1, L. R. Da Silva1, F. F. De Moura2 and M. L. Lyra2; 1Fisica, UFRN, Natal, RN, Brazil; 2Fisica, UFAL, Maceió, AL, Brazil.
Due to their potential applications in nano-electronics, there has been a growing interest both in the synthesis and characterization of DNA-based molecules with periodic nucleotide sequences, as well as in the theoretical prediction of the electronic properties of model molecular structures [1]. Among these, molecules with binary periodic sequences have attracted a lot of attention due to their special electronic band structure composed of two main bands of allowed states separated by an energy gap. Such band structure is similar to those of solid-state semiconductors. At half filling, the presence of the energy gap gives to these molecules an intrinsic insulator character. The introduction of defects may generate states within the gap and substantially improve the conductance, specially of finite molecules. In single-strand molecules, defects may be originated within the own nucleotide sequence or by laterally attaching new structures at random. However, disorder modifies profoundly the nature of the electronic states in 1D systems. All states usually become exponentially localized for any amount of disorder. Such exponential localization competes with the above improvement on the conductance associated with the presence of states within the gap. Therefore, schemes for introducing defects that minimize the tendency of exponential localization of the electronic states are essential to tailor the electronic transport properties of DNA-based structures. Within the above context, we report in this work an analytical as well as numerical investigation of the one-electron states in single-strand binary DNA-based segments with diluted base pairing. Specifically, we will consider poly(CG) and poly(CT) segments at which guanine bases (G) are attached laterally at a fraction of the cytosine (C) bases. Within a tight-binding description, we will compute the density of states and eigenfunctions of the one-electron states. We will show that the model Hamiltonian for this system can be mapped onto that of the Anderson chain with diluted disorder [2]. We will explore the influence of the effective disorder on the nature of the one-electron states as well as on the wave-packet dynamics. In particular, we will show that in periodic segment formed with complementary units [as in poly(CG)], base pairing dilution indeed leads to a complete exponential localization of all one-electron states. On the other hand, in periodic segments with non-complementary units [as in poly(CT)] a resonant state is not affected by the disorder and remains extended. In the presence of such resonant state, the wave-packet develops a sub-diffusive dynamics. We would like to thank partial financial support from CNPq-Rede Nanobioestruturas (Brazilian Research Agency) [1] E.L. Albuquerque et al, Phys. Rev. E 71, 021910 (2005). [2] F.A.B.F. de Moura and M.L. Lyra, Phys. Rev. Lett. 81, 3735 (1998).
MM3.5
Conformation of DNA Oligos on Gold Nanoparticles. Katherine Alice Brown and Kimberly Hamad-Schifferli; Biological Engineering, Massachusettes Institute of Technology, Cambridge, Massachusetts.
DNA bases have been shown to respond differentially to the surface of gold nanoparticles (Au NPs). Surface affinities for individual nucleotides and homo-base oligonucleotides have been established. However, how the conformation of oligos varies with oligo sequence has not been studied systematically. We report a systematic study of the base dependent behavior of oligonucleotides covalently linked to Au NP surfaces. Comparison of conjugate size, coverage and ability to hybridize to compliments show the importance of sequence in selection of functional oligonucleotides. The effect of sequence motif location relative to the NP has also been studied. We show that the location of high affinity DNA sequences relative to the NP affects conjugate behavior markedly. Finally, the effects of NP size on DNA-surface interaction have been studied. Changing NP size shows that NP surface curvature affects DNA conformation and ability to hybridize to its target. These studies will allow effective selection of DNA sequences for numerous applications that require control of DNA behavior. An understanding of the behavior of such oligonucleotides is fundamental to fullfilling the promise of AuNP-DNA conjugates for numerous biological applications.
MM3.6
Selective DNA-Mediated Assembly of Gold Nanoparticles on Gold Patterned Substrates. Kim Elizabeth Sapsford1, Doe Park3, Ellen Goldman4, Edward Foos2, Arthur Snow2 and Mario Ancona3; 1CBMSE Code 6900, NRL, George Mason University and The Naval Research Laboratory, Washington, District of Columbia; 2Chemistry Division, Naval Research Laboratory, Washington, District of Columbia; 3Electronics Science and Technology Division, Naval Research Laboratory, Washington, District of Columbia; 4Center for Bio/Molecular Science and Engineering (CBMSE), Naval Research Laboratory, Washington, District of Columbia.
One requirement for the development of nanocluster-based electronics and sensors is the selective attachment of clusters to electronic substrates. In this paper we demonstrate protocols based on DNA hybridization for selectively and efficiently attaching 12nm gold (Au) nanoparticles to Cr/Au electrodes patterned onto SiO2 surfaces. The Cr/Au electrodes were defined using standard e-beam lithography, with initial test substrates having micron-scale features. Studies using optimized protocols were also carried out on substrates with nanometer-scale electrodes. To functionalize the electrodes with Au nanoparticles, single stranded (ss) DNA templates are first immobilized onto the Cr/Au electrodes, via a gold-thiol driven assembly. This is followed by hybridization of gold nanoparticles functionalized with complementary ssDNA [1,2]. The efficiency of the assembly was monitored by SEM. Various reaction conditions were investigated in order to maximize the density of Au nanoparticles immobilized on the electrode surfaces. These studies included varying the salt concentration, investigating the addition of surfactant and changing the DNA-to-thiol spacer used when depositing the ssDNA templates. Also studied were the effects of temperature, salt concentration and surfactant on the hybridization of the DNA functionalized Au nanoparticles to the templates. Optimized conditions gave a surface coverage of Au nanoparticles on the Au electrodes of about 40%. In all cases, gold nanoparticles functionalized with non-complementary DNA show no attachment. In addition, our protocol results in low-to-zero non-specific adsorption on all SiO2 surfaces. Finally, we investigated the DNA-driven assembly of multi-layers of gold nanoparticles. As expected, such depositions further increase the coverage, with 3 deposition cycles resulting in a surface coverage of approximately 60%. All of these experiments and the effects of the various parameters on the attachment of Au nanoparticles to electronic substrates will be discussed. 1) Jhaveri SD, Foos EE, Lowy DA, Chang EL, Snow AW, and Ancona MG. Nano Letters, 2004, 4, 737. 2) Hurst SJ, Lytton-Jean AKR, and Mirkin CA. Analytical Chemistry, 2006, 78, 8313.
MM3.7
Directed DNA-Metallization: Towards The Construction of Rationally Designed Conductive Nano Devices. Christian T. Wirges1, Katrin Gutsmiedl1, Johannes Gierlich1, Philipp M. E. Gramlich1, Glenn A. Burley1,2 and Thomas Carell1; 1Chemistry and Biochemistry, Chemistry and Pharmacy, Munich, Germany; 2Chemistry, Leicester, United Kingdom.
DNA represents an exceptional template for the preparation of nanowires since it is easily prepared and designed using a well established set of enzymatic reactions. Furthermore it has the intrinsic capacity of self-assembly according to the Watson-Crick rules which is useful for the predictable construction of nanoscale networks and assemblies.[1] Procedures have been developed to increase the conductivity of native DNA by coating it with a variety of different metals, among them silver, gold, copper and platinum.[2,3] These procedures however suffer from a lack of selectivity and typically high background metallization, as they only rely on electrostatic or coordinative interactions between the metal ions and the native DNA. Therefore, new innovative routes to DNA-templated metal nanowires are required if one wants to construct nanoscale electrical circuits based on DNA. Our group has developed a method to direct metal deposition to a particular sequence of DNA. To this end, modified nucleoside triphosphates have been synthesized and incorporated into DNA, leading to gene-specific metallization.[4,5] Metal deposition by the Tollens reaction of aldehyde-modified DNA with silver ions is confined exclusively to the modified DNA strands, leaving native DNA unmetallized.[5] AFM-measurements on 900mer aldehyde-modified strands have shown homogeneous metal deposition, yielding thin metal nanowires less than 10 nm in diameter.[6] Highly efficient silver reduction is achieved with novel dialdehyde-bearing nucleotides which can be set free from their protected form using a mild postsynthetic deprotection step. TEM and AFM measurements on metallized 2000mer dialdehyde-bearing DNA are currently underway. These experiments will give deeper insight into the morphology of the silver clusters which develop in the Tollens reaction on DNA. Complex DNA constructs consisting of unmodified and modified portions have been prepared by enzymatic ligation methods. These strands are currently used as templates for the preparation of patterned structures on DNA, e.g. metallized parts alternating with unmetallized or otherwise functionalized sections. These nanoscale devices will represent a significant step towards the long-term goal of constructing DNA-based electronic circuits. [1] N. C. Seeman, Nature 2003, 421, 427; P. W. Rothemund, Nature 2006, 440, 297. [2] E. Braun, Y. Eichen, U. Sivan, G. Ben-Yoseph, Nature 1998, 391, 775. [3] Review: Q. Gu, C. D. Cheng, R. Gonela, S. Suryanarayanan, S. Anabathula, K. Dai, D. T. Haynie, Nanotechnology 2006, 17, R14. [4] J. Gierlich, G.A. Burley, P. M. E. Gramlich, D. M. Hammond, T. Carell, Org. Lett. 2006, 8, 3639. [5] G. A. Burley, J. Gierlich, M. R. Mofid, H. Nir, S. Tal, Y. Eichen, T. Carell, J. Am. Chem. Soc. 2006, 128, 1398. [6] M. Fischler, U. Simon, H. Nir, Y. Eichen, G. A. Burley, J. Gierlich, P. M. E. Gramlich, T. Carell, Small 2007, 3, 1049-1055.
MM3.8
Evaluation of Hydrodynamic Size and Surface Charge Density of Surface Modified Au Nanoparticle-DNA Conjugates by Ferguson Analysis. Sunho Park1 and Kimberly Hamad-Schifferli1,2; 1Department of Mechanical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts; 2Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts.
Conjugates of gold nanoparticles and DNA (Au NP-DNA) have been extensively explored for their use in biological applications. However, DNA strands are known to adsorb onto the surfaces of Au NPs, which can limit the hybridization ability of Au NP-DNA conjugates. Therefore, chemical modification of Au NP surfaces was used to control the non-specific adsorption. DNA conformation was evaluated by Ferguson analysis and DNA function by hybridization studies. For particular conditions, DNA functionality was not diminished significantly. Ferguson analysis allows evaluation of conjugate hydrodynamic size and free mobility of molecules by gel electrophoresis at different gel polymer concentrations. Surface charge density of molecules is also evaluated by changing buffer concentration. Results show that Ferguson analysis is a reliable method to measure the size and surface charge density of Au NP-DNA and its derivatives when compared with actual DLS and zeta potential measurements.
MM3.9
DNA with Zip Codes: Addressable DNA Molecules and their Polymerization. Jong Bum Lee, Young Hoon Roh and Dan Luo; Biological Engineering, Cornell University, Ithaca, New York.
DNA is being used as an engineering material. Dendrimer-like DNA, a new type of DNA nanomaterial previously reported from our group, was used to develop a general synthesizing approach to create addressable molecules. The multivalent and anisotropic properties of branched DNA provide asymmetrical control over the placement of different functional moieties within one single molecule. Precise design, monodisperse synthesis, and controlled polymerization have been achieved. In particular, we have designed and synthesized two classes of such multifunctional hybrid molecules: 1) DNA-organic dye hybrid molecules and 2) DNA-gold nanoparticle-quantum dot hybrid molecules. We achieved in both cases very high purity anisotropic materials without any purification step. More importantly, these addressable, DNA-based anisotropic building blocks can be further polymerized via an enzyme (ligase) to create novel hybrid molecules, providing even more diversifications for future applications.
MM3.10
Nanofabrication Based on DNA Engineering and Microtemplated Dewetting. Wenlong Cheng, Nokyoung Park, Young Hoon Roh and Dan Luo; Biological and Environmental Engineering, Cornell University, Ithaca, New York.
The current challenge in applying chemically synthesized nanoscale building blocks to practical nanodevices is finding an efficient and economic way of precisely positioning them into designed microscale and nanoscale architectures. In particular, these applications will require not only an ordered nanoparticle (NP) assembly but also a precise control over geometry of the assemblies. For example, a NP-based plasmonic waveguide required a linear assembly of evenly-spaced NPs 1. Here, we show that the challenge can be met using our recently developed nanofabrication method, which is based on a combination of DNA ligand engineering and microtemplated dewetting. In addition to widely used alkyl ligands 2-10, we show that unique DNA engineering 11-13 allowed us not only to fabricate large-scale superlattices but also to tune inter-particle distance in broader size regime. We show that nanoparticle superlattices can be further shaped into various shaped nanoarchitectures with uniform features at a wafer-scale via a micro-templated dewetting (μTDW). μTDW is a new derivative soft-lithography technique, by which the minimum feature size is limited only by one single particle size. The methodology is not limited to spherical gold nanoparticles but provides general procedures for building nanoarchitectures from other types of nanoscale building blocks. References 1.Maier, S. A.; Kik, P. G.; Atwater, H. A.; Meltzer, S.; Harel, E.; Koel, B. E.; Requicha, A. A. G., Nature Materials 2003, 2, 229. 2.Murray, C. B.; Kagan, C. R.; Bawendi, M. G., Science 1995, 270, 1335. 3.Kiely, C. J.; Fink, J.; Brust, M.; Bethell, D.; Schiffrin, D. J. Nature 1998, 396, 444. 4.Shevchenko, E. V.; Talapin, D. V.; Kotov, N. A.; O'Brien, S.; Murray, C. B. Nature 2006, 439, 55. 5.Kalsin, A. M.; Fialkowski, M.; Paszewski, M.; Smoukov, S. K.; Bishop, K. J. M.; Grzybowski, B. A. Science 2006, 312, 420. 6.Bigioni, T. P.; Lin, X. M.; Nguyen, T. T.; Corwin, E. I.; Witten, T. A.; Jaeger, H. M. Nature Materials 2006, 5, 265. 7.Collier, C. P.; Saykally, R. J.; Shiang, J. J.; Henrichs, S. E.; Heath, J. R. Science 1997, 277,1978. 8.Korgel, B. A.; Fullam, S.; Connolly, S.; Fitzmaurice, D. Journal of Physical Chemistry B 1998, 102, 8379. 9.Zhang, J. P.; Liu, Y.; Ke, Y. G.; Yan, H. Nano Letters 2006, 6, 248. 10.Zheng, J. W.; Constantinou, P. E.; Micheel, C.; Alivisatos, A. P.; Kiehl, R. A.; Seeman, N. C. Nano Letters 2006, 6, 1502. 11.Li, Y. G.; Tseng, Y. D.; Kwon, S. Y.; D'Espaux, L.; Bunch, J. S.; Mceuen, P. L.; Luo, D. Nature Materials 2004, 3, 38. 12.Li, Y. G.; Cu, Y. T. H.; Luo, D. Nature Biotechnology 2005, 23, 885. 13.Luo, D. Materials Today 2003, 6, 38.
MM3.11
Single Walled Carbon Nanotube Fluorescence Detection of DNA Hybridization: Kinetics, Thermodynamics, and Applications. Esther Shu-Hsien Jeng, John D Nelson and Michael S Strano; Chemical Engineering, MIT, Cambridge, Massachusetts.
Single walled carbon nanotubes (SWNT) are excellent candidates for interfacing with, and detection of biomolecules. Individually dispersed semiconducting SWNT fluoresce at near-infrared wavelengths where interfering scattering from water and blood is low, and autofluorescence from cells is minimal. A two-step dialysis method is used to adsorb single stranded DNA onto the nanotube surface. Introduction of complementary strands results in DNA hybridization on the nanotube surface. This event is transduced through an energy change in the fluorescence of the SWNT. The kinetics and thermodynamics of this label free detection were studied to better understand the mechanism. Promising results also indicate that this mechanism has potential to be used in the detection of single point mismatches in the complementary strand.
MM3.12
Chitosan Biotinylation and Electrodeposition for Protein Assembly at Electrode Addresses. Gregory Payne1, Xiaowen Shi1, Yi Liu1, Angela T Lewandowski1,2, Hsuan-Chen Wu1,2, Li-Qun Wu1, Reza Ghodssi2, Gary W Rubloff2 and William E Bentley1,2; 1Center for Biosystems Research, University of Maryland Biotechnology Institute, College Park, Maryland; 2University of Maryland, College Park, Maryland.
The assembly of proteins at surfaces or within devices is important for a range of applications in medicine and biotechnology. Typically, spatial selectivity is conferred to protein assembly by printing or photolithographic fabrication methods. We are examining alternative, biofabrication methods that enlist the unique capabilities of biological materials for assembly. Specifically, we couple the stimuli-responsive properties of the aminopolysaccharide chitosan with the molecular recognition capabilities of biotin-streptavidin binding. We report that biotinylated chitosan retains stimuli-responsive properties and is capable of electrodepositing at specific electrode addresses. Once deposited, the biotinylated chitosan is capable of binding streptavidin which can mediate the subsequent assembly of biotinylated proteins. We demonstrate spatially-selective protein assembly using biotinylated Protein A and fluorescently-labeled antibodies. The capability of enlisting localized electrical signals to guide protein assembly is expected to offer considerable opportunities in microfluidic and lab-on-a-chip formats.
MM3.13
The Absorption of Polysaccharides and Polypeptides onto Calcite Surfaces. John Harding and Mingjun Yang; Engineering Materials, University of Sheffield, Sheffield, United Kingdom.
Organic molecules such as polysaccharides, polypeptides and proteins are believed to control the growth of minerals in biological systems. Examples of this phenomenon include coccoliths (calcite shields for many species of algae; for a review of the experimental data see [1]) and eggshells (see for example [2]). These molecules are believed to act both as templates for the overall crystal orientation and also as inhibitors of step growth, controlling the detailed shape. Simple structure-matching simulations have been performed to attempt to explain this behaviour , however they take no account of the relaxation of the molecules onto the surface or of the presence of water. We have performed a series of simulations on both stepped and flat surfaces typical of the ones observed with different polysaccharides and polypeptides to investigate the dependence of the absorption on both the calcite surface and the structure of the organic molecule. We have calculated the absorption energies of short chains of these units using a newly-developed set of consistent force-fields for the organic-mineral-water interface.[3] The calculations show that all units strongly bind to polar surfaces under acidic conditions (i.e. when organic carboxyl groups are expected to be ionised). This is simply explained as a consequence of electrostatic effects. More complex behaviour is observed for neutral and stepped surfaces. We discuss the behaviour in terms of the structures of the absorbed species in contact with various calcite surfaces and propose a set of rules for maximising the binding of more complex polysaccharides and polypeptides to calcite. [1] J.R. Young, S.A. Davis, P.R. Bown and S. Mann, J. Struct. Bio. 126 (1999) 195 [2] R. Lakshminarayanan, X.J. Loh, S. Gayathri, S. Sindhu, Y. Banerjee, R.M. Kini, and S. Valiyaveettil; Biomacromolecules 7 (2006), 3202 [3] C.L. Freeman, D.J. Cooke, J.H. Harding, J.A. Elliott, J. Lardge and D.M. Duffy; J. Phys. Chem. C in press.
MM3.14
Chitosan Films as Substrate to Line Patterning Technique of Graphite (LPTG). Application as Sensors. Clarice Steffens2, Douglas de Britto1 and Paulo Sergio de Paula Herrmann Jr1; 1Agricultural Instrumentation, Embrapa Agricultural Instrumentation, São Carlos, São Paulo, Brazil; 2Food Engineering, Universidade Regional Integrada (URI), Erechim, Rio Grande do Sul, Brazil.
Flexible and disposable films are important features to develop substrates to Line Patterning Technique of graphite (LPTG), with potential application to electronic devices. With this work has been proposing the use of a chitosan an edible film to be a substrate of the technique. LP is a method that takes advantage of differing rates of reaction for a material with the printed line on a substrate and the naked substrate surface itself. The technique was originally created on the basis of conductive polymer aqueous dispersions being more readily adhesive to hydrophilic surfaces, such as overhead transparencies, rather than to hydrophobic surfaces, such as a toner line printed by a laser printer onto an overhead transparency poly(ethylene teraphtale). In this experiment we are proposing to use a biopolymer from chitosan film as substrate of LPTG. Chitosan is a natural polymer, biodegradable and non-toxic linear polymer, commercially available by the deacetylation of chitin, an abundant polysaccharide extracted from the shells of shrimps and crabs. Commercial-grade chitosan and aqueous acetic acid solution (1%) were used to prepare chitosan and alkyl-chitosan derivative precursor solutions. Films were then prepared by solution casted onto acrylic an Petri dishes at room temperature. After drying the films were peeled from the dishes. The LPTG process consists in printing the mask on a conventional Laser printer. To make a conductive patterning on to a chitosan film it is necessary to dip it into the graphite dispersion for 1-2 seconds at room temperature. The aqueous dispersion of graphite (1:4 (weigth/weigth)) was used. After that it was then dipped into a toluene bath (eg. 200 mL toluene) at room temperature and ultrasonicated for 1 minute, leaving only the graphite on the film. A interdigitated electrode with four fingers in each side of the device was developed. The morphology of the chitosan film was studied with the atomic force microscopy (AFM). Images and roughness (RMS) were taken, using a Topometrix Discoverer TMX 2010 in standard contact mode. The influence of the organic vapor (methanol) in the variation of the electric response (Ω) was confirmed using a commercial multimeter. The RMS roughness of the sample, with area image of 400μm2, 100μm2, 25μm2 and 1.0μm2 were respectively 44.62nm, 18.99nm, 10.27nm and 5.75nm. An application of the chitosan film with graphite electrode like a disposable sensor to volatile organic compound (VOC) was investigated. The value of electric resistance (Ω) of the sensor with small short circuit in the end of electrode was 328,50KΩ ±0.76KΩ and the sensitivity (ΔR(%)) to methanol vapor was 19,3% and reversibility (η%) was 81,0%. A new substrate to LPTG was obtained, an investigation of the morphology (quantitative evaluation of the roughness and qualitative information with image) of the film with AFM was observed and an example of application was presented.
MM3.15
Preparation of Chitosan Sub-micron Beads by Phase Separation with Polyvalent Anion and their Evaluation as Bacteriostatic Materials. Shoji Nagaoka1, Kanako Saita1,2, Tetsuya Yamamoto3, Seitaro Kobayashi2, Ken Satoh4, Kenji Kurashiki5, Makoto Takafuji2 and Hirotaka Ihara2; 1Materials Development Department, Kumamoto Industrial Research Institute, Kumamoto, Kumamoto, Japan; 2Department of Applied Chemistry and Biochemistry, Faculty of Engineering, Kumamoto university, Kumamoto, Kumamoto, Japan; 3Daiichi Seimo Co. Ltd., Arao, Kumamoto, Japan; 4Nishinihon Nagase Co. Ltd., Fukuoka, Fukuoka, Japan; 5Muromachi Chemical Inc., Oomuta, Fukuoka, Japan.
In this report, we describe that the chitosan particles with series of sub-micron size or micron size could be prepared by “ionic exchange technique” using from inorganic polyvalent anion salt alone, without organic substance such as organic emulsifier and oil solvent. In addition, we also reported that the obtained particles showed antibacterial activity for Escherichia coli. Chitosan sub-micron particles were prepared by ion exchange phase separation method as follows: 1) chitosan was dissolved in lactic acid aqueous solution: 2) the obtained chitosan solution was dropped in polyvalent anion salt aqueous solution, i.e. dianion salt, trianion salt solution: 3) desalinating and deacidifying from aqueous dispersion of chitosan submicron particles was carried out by dialyzing tube method. The aqueous dispersion of chitosan particles, which was prepared using aqueous solution of polyvalent anion salt appeared cloudy. This phenomenon was attributed to cross-linking by ion interaction between sulfate anion and amino groups in glucosamine unit. The chitosan aggregates were confirmed to promote with increase of amount of Na2SO4 by dynamic light scattering method. This indicates that agglutination sites among chitosan particles increased as the number of the sulfate anion coupled with amino group increased. The chitosan particles could be prepared using polyvalent anion, i.e. Na2SO4. The formation mechanism of particle is attributed to crosslinking structure occurred by ion interaction between sulfate anion and amino groups. As results, the chitosan particles of sub-micron size were prepared by adding 1.0 equivalent of Na2SO4 toward an amount of amino groups to dispersion medium. In addition, we confirmed the antibacterial activity for Escherichia coli of obtained chitosan particles. Generally, it is well known that chitosan microparticle shows antibacterial activity only in an acidic medium. The antibacterial activity of chitosan is usually influenced by solubility of chitosan in solution under or above pH 6.5. On the other hand, the obtained chitosan particles were insoluble due to crosslinking by sulfate ion, even in acidic condition. And the above-mentioned chitosan sub-micron particles showed significantly antibacterial activity at concentration of 5.0 mg/ml for agar medium, in spite of incubation in neutrality condition (pH 6.5-7.0). These results were attributed to be microparticulated. Therefore, the antibacterial activity was considered to depend on relationship between the large surface area and amount of ammonium ion. In this report, we also discussed concerning the other evaluation, i.e. adsorption toward ammonium and acetic acid gas, hygroscopisity, porosity and surface area.
MM3.16
Electrochemical Measurement of Oligosaccharide Monolayers: Self Assembly and Lectin Binding. Joshua L Hertz1, David Lahr1, Ju-Hee Park2, Philip DeShong2 and Michael J Tarlov1; 1Chemical Science and Technology Laboratory, National Institute of Standards and Technology, Gaithersburg, Maryland; 2Department of Chemistry and Biochemistry, University of Maryland, College Park, Maryland.
Electrochemical sensors containing active biomolecules have the potential to be small, portable, and robust measurement devices for biomedical applications. The performance of these sensors can be greatly influenced by how the molecular ligands which selectively recognize the biological analyte are tethered to the electrode surface. Self-assembly provides an attractive method to fabricate this active sensing layer because of its ease and flexibility in tuning the sensor surface architecture. Here we report on the electrochemical measurement of monolayers of various thiol-modified oligosaccharides self-assembled on gold electrodes for use as biosensors. There is growing interest in carbohydrates as sensing films because they are known to be involved in a large variety of disease states and cell mediated processes. The capacitance of the gold-electrolyte interface, as determined by electrochemical impedance spectroscopy, is found to depend on the particular carbohydrate self-assembled on the gold surface. In addition, the binding of lectin proteins to specific saccharide monolayers can be detected by measuring changes in the electrode impedance. Results from chemical and structural characterization by x-ray photoelectron spectroscopy of the monolayers will also be presented. Finally, the practical utility of this measurement technique for biosensors will be discussed.
MM3.17
Alternative Substrate Materials for Supported Lipid Bilayers. Barbara Nellis1,2, Emel Goksu1, Marjorie Longo1, Alex Gash2, Joe Satcher2 and Subhash Risbud1; 1Chemical Engineering and Materials Science, University of California, Davis, Davis, California; 2Chemistry, Materials and Life Sciences Directorate, Lawrence Livermore National Labs, Livermore, California.
Supported biomembrane systems have become established tools for modeling the plasma membrane in cells. The current supports used suffer from several major drawbacks including a lack of separation between the substrate and the bilayer leading to one-sided bilayer access. Using porous materials to avoid this limitation is being explored but has been limited primarily to materials consisting of solid silica and alumina surfaces. In addition, chemical modification of surfaces has also been investigated. Here we explore bilayer formation on alternative porous materials using atomic force microscopy and epifluorescence microscopy. These porous materials are produced via a sol-gel synthesis technique where pore size and pore distribution can be controlled through reaction conditions. The chemistry and morphology of these different surfaces are believed to play a role in the stability of the lipid bilayer and on the ability to form domains in two-phase lipid systems.
MM3.18
Electrical Impedance Analysis of Phospholipid Bilayer Membranes for Enabling Engineering Design of Bio-based Devices. Stephen A. Sarles and Donald J. Leo; Center for Intelligent Material Systems and Structures (CIMSS), Virginia Tech, Blacksburg, Virginia.
Recent strides made at Virginia Tech have shown that the incorporation of ion-selection transporter proteins inserted into a supported BLM formed across an array of nano-pores can convert chemical energy available in adenosine triphosphate (ATP) into electricity. Experimental results from this work show that this system—called BioCell—is capable of 1.7μW of electrical power per square centimeter of BLM area and per 15μl of ATPase enzyme. Efforts to increase the power output and conversion efficiency of this process while simultaneously developing methods to effectively assembly and intelligently package this technology present unique engineering challenges. The phospholipid bilayer, as host to active biological proteins and channels, must be formed evenly across a supporting substrate, remain stable and yet fluid-like for protein folding and activation, and provide sufficient electrical insulation. This research commences a systems analysis (i.e. piece by piece) approach to develop an understanding of these materials, create design guidelines for producing engineering materials and devices using biological components, and to begin to unite the fundamentals of biology with the engineering process. We report first on the formation and characterization using electrical impedance spectroscopy (EIS) of a phospholipid bilayer formed across a porous substrate as a model and test-bed for bio-based designs. Preliminary results indicate a membrane resistance of 1.7MΩ-cm^2 at 0.01Hz for a SOPC lipid bilayer formed on track-etched polycarbonate substrates (400nm pores, %5-20 porosity). EIS measurements are performed to identify the electrical properties (membrane resistance and capacitance) of the lipid bilayer with respect to the phospholipid molecular structure, bilayer composition and supporting substrate. The results of this study will also be used to identify avenues for tailoring BLMs for specific applications and the BioCell will be used as a platform for demonstrating these relations in a modified BLM.
MM3.19
Failure Characteristics of Bilayer Lipid Membranes Formed over a Single Pore. David Hopkinson, M. Austin Creasy and Donald Leo; Mechanical Engineering, Virginia Tech, Blacksburg, Virginia.
Bilayer lipid membranes (BLMs) are formed from phospholipid molecules which self-assemble into a lipid bilayer with 4 − 8 nm thickness when submerged in an aqueous solution due to their amphiphilic nature. They are the primary structural component of cell membranes in living organisms and therefore are useful for modeling the properties of cells since they share many of the same chemical and physical properties. A new methodology has been developed to measure the mechanical integrity of a BLM formed over porous substrates. A custom test fixture was fabricated in which pressure is applied to a BLM in very fine increments. The pressure, monitored with a pressure transducer, is observed to increase until the BLM reaches its failure pressure, and then drops. In a previous study 1-Stearoyl-2-Oleoyl-sn-Glycero-3-Phosphocholine (SOPC) phospholipids as well as mixtures of SOPC and cholesterol were used to form BLMs over a track-etched porous polycarbonate substrate with pores ranging from 0.05 − 10 microns in diameter. Mixtures of SOPC/CHOL were used because studies have shown that cholesterol increases the strength of BLMs. The array of micro-BLMs was pressurized until failure, which resulted in a characteristic failure pressure curve. Failure pressures for the smallest pore size, 0.05 microns, were on the order of 400 kPa, and for the largest pore size, 10 microns, were on the order of 2 kPa. These pressure curves were successfully modeled according to the pressurization and flow of fluid throw the porous substrate. For this model the failure pressure of the micro-BLMs was assumed to follow a normal distribution, and the mean and standard deviation of the failure pressure were used as fitting parameters. For the current study, track-etched polycarbonate membranes have been modified by masking off and blocking all but a single pore with a range of 5 - 20 microns diameter. SOPC and SOPC/cholesterol BLMs were formed over the single pore and then pressurized using the previously described test apparatus. These single pore experiments are useful for determining the mean and standard deviation failure pressures of the multi-pore case which were previously found as fitting parameters. The failure pressure for a BLM formed over a single pore was found to be much higher than for an array of micro-BLMs formed over multiple pores. For SOPC BLMs formed over a 10 micron pore, for example, the failure pressure was on the order of 2 kPa for multiple pores, but 100 kPa for a single pore. This happens because when an array of many BLMs are pressurized simultaneously the weakest BLMs fail first, causing the overall failure pressure to be lower than that of a single BLM. Also, for some experiments the phospholipid mixtures have been marked using a di-8-ANEPPS fluorescent dye. Using a fluorescence equipped microscope, it was possible to image the BLM before and after pressurization.
MM3.20
Lipid-Enveloped Bioresorbable Nanoparticles as ``Synthetic Pathogens" for Vaccine Design. Anna Bershteyn, Tania R. Chan, José P. Chaparro, Richard S. Yau and Darrell J. Irvine; Materials Science and Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts.
The physicochemical context in which molecules are presented to immune cells has tremendous implications for the immune response to vaccination. For example, the spacing and organization of foreign proteins and polysaccharides displayed on the surface of a microbe may control its recognition by innate immune cells, such as dendritic cells (DCs), influencing pathogen uptake, DC activation, and subsequent immune responses. In addition, the conformation and chemical environment of antigens displayed at the microbe surface influences the specificity of the humoral immune response, because antibodies recognize antigen in its three-dimensional shape and context. To explore how the physical structure and organization of proteins expressed on the surface of pathogens influences the immune response, we have constructed “synthetic pathogens” consisting of a biodegradable poly(lactide-co-glycolide) core polymer coated by a phospholipid shell to mimic the surface of a lipid-enveloped pathogen. These particles, synthesized using an oil-in-water emulsion process, have an average diameter near either 100 nm, mimicking in size a lipid-enveloped viral pathogen, or 1 micron, mimicking a bacterial pathogen. Lipids dissolved in the oil phase act as emulsifiers in the particle synthesis, and self-assemble into monolayers or bilayers at the particle surface, as revealed by cryo-transmission electron microscopy (cryoEM) analysis of the particles. Functionalized lipid headgroups provide a means for conjugation of surface-bound molecules such as protein, polysaccharides, targeting moieties, or probes for detection. The bioresorbable core of the nanoparticles provides a physical support for the lipid surface layers, making these particles more stable than traditional liposomes, while eventual degradation of the polymer core allows for controlled release of encapsulated molecules. To increase the functionality of these lipid-enveloped particles, we encapsulated iron oxide nanoparticles within the biodegradable core, enabling magnetic capture of the particles and potentially providing contrast for in vivo tracking using magnetic resonance imaging (MRI). Analysis of the structural evolution of these particles over time by cryoEM revealed that, as the core polymer degrades by hydrolysis, a fluid-filled gap between the surface lipid bilayer and the particle core leads to gradual delamination of the lipid bilayers, potentially changing the surface display and mobility of ligands. In addition to providing a platform for systematic studies of pathogen recognition by the immune system, these biomimetic particles may be useful for implementing structural features of microbes in synthetic vaccines.
MM3.21
Reactive Multi-component Membranes: From Dynamic Shape Reconstruction to Self-Cleaning. Olga Kuksenok and Anna C. Balazs; University of Pittsburgh, Pittsburgh, Pennsylvania.
Using theory and computer simulations, we illustrate how external stimuli (i.e., light or flux of external reactants) can be utilized to effectively manipulate the dynamic functions of reactive, biomimetic membranes. The membrane consists of a ternary phase-separating mixture, in which an external stimulus initiates a chemical reaction that inter-converts A and B components. The third component in the ternary mixture, C, is non-reactive. We also assume that the A and B components have specified spontaneous curvatures. We focus on the dynamic response of this membrane to applied gradients and illustrate how these membranes can performs such specific functions as dynamic shape reconstruction, gradient sensing, and self-cleaning. For example, our results show that the relief of such a membrane can be dynamically reshaped by the external gradient. We also find that the non-reactive components within such membranes migrate along the external gradients; this effect can be used to separate non-reactive components within the reactive membrane, i.e., for an effective self-cleaning of the membrane.
MM3.22
Micro/Nano-patterning of Supported Phospholipid Membranes: From Sensor Design to Biophysical Studies. Jinjun Shi, Tinglu Yang and Paul S. Cremer; Department of Chemistry, Texas A&M University, College Station, Texas.
Micro/nano-patterning of supported phospholipid bilayers (SLBs) has shown considerable potential for exploring cell signaling, investigating cellular component organization, and the creation of a new generation of biosensors. In this talk, novel lithographic methods will be presented for the size-controlled patterning of SLBs from the microscale to the nanoscale. Using these methods, we can spatially address chemically distinct types of phospholipid bilayers on a single microchip. These arrays can, in turn, be employed in high throughput assay for biosensing, enzyme kinetics, and multivalent ligand-receptor isotherms. In fact, in conjunction with total internal reflection fluorescence microscopy (TIRFM), multiple equilibrium dissociation constants could be abstracted from one-shot binding experiments. Our studies on the effect of ligand density for multivalent CTB-GM1 interactions revealed that the CTB-GM1 binding weakened with increasing GM1 density from 0.02 mol% to 10.0 mol%. Such a result can be explained by the clustering of GM1 on the supported phospholipid membranes, which in turn inhibits the binding of CTB. Atomic force microscopy (AFM) directly verified GM1 clustering within glass-supported POPC bilayers. Understanding the underlying biophysical chemistry for multivalent interactions may provide insight into strategies for inhibitory drug design.
MM3.23
Preparation of Cell Rolling Surfaces by Controlled Covalent Immobilization Methods. Seungpyo Hong1, Allen Taylor2, Dooyoung Lee3, Michael King3, Shaoyi Jiang2, Robert Langer1 and Jeffrey Karp1; 1Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts; 2Chemical Engineering, University of Washington, Seattle, Washington; 3Chemical Engineering, University of Rochester, Rochester, New York.
Cell rolling is an important physiological and pathological process that is used to recruit specific cells in the bloodstream to a target tissue. The rolling behavior can be exploited for a variety of biomedical applications including separation of specific cells from blood. Although controlled immobilization methods of cell rolling mediating proteins such as P-selectin are required to effectively introduce cell specificity to engineered surfaces, most studies to date have relied on physisorption. Here we present chemical methods to covalently immobilize P-selectin to achieve better control over ligand density and orientation as well as longer functional stability. The SPR measurements revealed that P-selectin density was controlled by varying ratios between mono- and bi-functional polymer linkers on substrates. Immobilized P-selectin with proper orientation showed enhanced binding response against its antibody by ~2 folds as compared to randomly immobilized P-selectin of the same amount. To test the surface function stability, microsphere-Sialyl Lewis(x) conjugates and human primary neutrophils were flown into a flow chamber. Covalently immobilized P-selectin surfaces exhibited substantially longer functional stability than physisorbed controls, for the both cell mimics and live cells. This study represents chemical methods for enhanced P-selectin immobilization, which is essential for mimicking relevant complexities of the in vivo rolling response and for future development of devices for isolating specific cell types.
MM3.24
Layer-by-Layer Assembly of Biodegradable Block Copolymer Micelles for Applications in Drug Delivery. Byeong-Su Kim, Renee Smith and Paula T Hammond; Chemical Engineering, MIT, Cambridge, Massachusetts.
Layer-by-layer (LbL) assembly has been widely used as a versatile method for fabricating multilayer thin films with controlled structure and composition. Due to its facile, inexpensive, and environmentally friendly nature, LbL assembled multilayer thin films find their applications ranging from materials to biology. LbL assembly is typically based on sequential adsorption of materials with complementary functional groups employing electrostatic interaction, hydrogen bond, and coordination bond, which limits the incorporation of small, hydrophobic drugs into multilayer film. There is, therefore, widespread interest in finding ways to integrate therapeutic reagent into LbL film. Here, we describe the incorporation of amphiphilic block copolymer micelle as a nanometer-sized vehicle for hydrophobic drugs within the LbL multilayer films. In particular, we chose block copolymers containing biodegradable poly(ε-caprolactone) as a core block for controlled release, including poly(ethylene oxide)-block-poly(ε-caprolactone) (PEO-b-PCL), and poly(2-vinyl-N-ethylpyridinium bromide)-block-poly(ε-caprolactone) (P2VEP-b-PCL). We demonstrate the construction of polymer micelle containing films through different assembly condition employing either hydrogen-bonding for PEO-b-PCL micelle, or electrostatic interaction for P2VEP-b-PCL micelle. With these integrated nanostructures within LbL multilayer film, we have explored their potential uses as a platform for model drug incorporation and release under physiological condition.
Chair: Vincent Rotello
Tuesday Morning, November 27, 2007
Room 210 (Hynes)
8:30 AM *MM4.1
Large Synthetic Ion Channels from Nucleoside-Sterol Conjugates. Jeff Davis and Ling Ma; Chemistry and Biochemistry, University of Maryland, College Park, Maryland.
Lipophilic guanosine derivatives form G-quadruplexes in organic solvents. In this study, guanosine-sterol conjugates were used to build synthetic ion channels within phospholipid membranes. Our design involves using two hydrophilic guanosines to cap the ends of a hydrophobic bis-sterol. The ion transport properties of this compound were investigated by voltage-clamp experiments. Distinct conductances were observed, suggesting that the guanosine-sterol conjugate is able to form ion channels in phospholipid bilayer membranes. The important characteristics of these channels include large conductances and long-lasting open states. Reverse-potential measurements reveal selectivity for cations. Control experiments show that the guanosine moiety plays a key role in the self-assembly of the ion channels. Further evaluation of the structure-function relationships will be discussed.
9:00 AM *MM4.2
Mimicry of Protein Architectures and Assemblies with Unnatural Amino Acids. James S. Nowick, Department of Chemistry, Univ. of California, Irvine, Irvine, California.
Proteins achieve their roles as the primary actors in the processes of cellular life, through their structures, interactions, and size. This talk will describe my laboratory’s ongoing efforts to develop peptides that mimic the structures, interactions, and size scale of proteins. The first half of the talk will focus on peptides that mimic the structure and hydrogen-bonding interactions of protein β-sheets. The development of unnatural amino acids that impart β-sheet structure when incorporated into peptides has been central to these efforts. The second half of the talk will focus on the creation of peptides with well-defined shapes and nanometer-scale sizes. Structures prepared thus far include rods up to 10 nm in length and rings, triangles, and parallelograms up to 3 nm in size. The development of nanometer-scale rodlike and angular amino acids has been central to these efforts. Ongoing efforts to achieve more complex structures and interactions will be described.
9:30 AM MM4.3
Degradable Polymer Multilayers for Surface Delivery of Antibiotics. Helen F Chuang and Paula T Hammond; Chemical Engineering, MIT, Cambridge, Massachusetts.
Multilayered polymer thin films were constructed via the layer-by-layer (LbL) deposition technique using a unique water-hydrolyzable polycation, resulting in coatings that are stable in air but erode top-down in a layer-by-layer fashion when exposed to aqueous physiological environment. The multilayered nature of these films allows for the encapsulations and subsequent sequential releases of multiple drugs, with controls over the dosage and release kinetics of each encapsulated agent. We focus on the development of a coating for sequential delivery of pain killers with anti-inflammatories, antibiotics, and growth factors to be applied onto orthopedic implant surfaces. We have incorporated several classes of antibiotics into these films with a wide range of release rates and precise control over dosage. Preliminary in vivo studies using osteomyelitis models in rabbits will also be discussed. Aside from antimicrobial coatings, our findings can also be applied towards the coating-based delivery of other therapeutic agents, including proteins and nucleic acids, as well as other active ingredients in the non-medical industries.
10:15 AM *MM4.4
Crosslinked, Block-Copolymer Shields for Inorganic Nanoparticle Bioconjugates. T. Andrew Taton1, Chao Zuo1, Alexi J. Young1, Byeong-Su Kim1 and Yuji Shibasaki2; 1Chemistry, University of Minnesota, Minneapolis, Minnesota; 2Applied Chemistry, Iwate University, Morioka, Iwate, Japan.
We have recently reported that spontaneous self-assembly of block copolymer surfactants at the surface of inorganic nanoparticles, followed by chemical crosslinking to fix the polymer layer, yields composite nanostructures in water that exhibit the functionality, stability, and structural predictability of block-copolymer micelles as well as the unique optical and/or magnetic properties of nanoparticles. We find that, when the hydrophobic block of the copolymer shell is glassy (e.g., polystyrene or polymethylmethacrylate), the polymer provides an extremely effective barrier to Ostwald ripening and attack by competitive surface ligands. As a result, the morphology of Ag nanoparticles that have been encased in block copolymer shells do not change with time or light exposure. Likewise, CdSe nanoparticles inside copolymer shells do not leach ions into their surroundings or quench in the presence of oxidizers. We also find that crosslinking the copolymer shell plays an important role in enhancing particle stability against the high shear sometimes experienced in animal vasculature. We report that these features lead to enhanced in vivo performance, in zebrafish, for copolymer-encapsulated nanoparticles relative to those coated with non-crosslinked or small-molecule ligands and surfactants. Overall, we argue that attention to the chemical and physical properties of nanoparticle surface layers can improve the in vivo activity of nanobioconjugates.
10:45 AM MM4.5
Reversible Assembly of Polymer Main Chains at Interfaces. Michael J Serpe1,2, Farrell R. Kersey1,2 and Stephen L. Craig1,2; 1Department of Chemistry, Duke University, Durham, North Carolina; 2Center for Biologically Inspired Materials and Materials Systems, Durham, North Carolina.
The bridging of polymers between surfaces mediates a range of fundamental processes in the material and life sciences. Polymer bridging occurs when a polymer chain connects two separate surfaces via covalent or noncovalent interactions. Typically, the bridging polymer main chain is held together by covalent interactions, but small molecules can also assemble into polymer bridges via reversible, noncovalent interactions. These polymers, commonly referred to as main chain reversible polymers, differ from their covalent analogs in that they can dynamically adjust their size and shape in response to surface distance changes. In addition, reversible polymers equilibrate on a much shorter timescale than their covalent counterparts. Here, we will describe the process of main chain reversible polymer bridging created by DNA base pairing. The bridging process was studied by atomic force microscopy, and its dependence on the distance between surfaces and equilibration time was probed. The study revealed that the number of polymer bridges formed decreased as the gap width increased and the equilibration time decreased.
11:00 AM *MM4.6
Dithiocarbamate-Anchored Monolayers (DAMs): Surface Chemistry for Biological Interfaces. Alexander Wei, Chemistry Department, Purdue University, West Lafayette, Indiana; Birck Nanotechnology Center, Purdue University, West Lafayette, Indiana.
Dithiocarbamate-anchored monolayers (DAMs) are an appealing alternative to thiol-based SAMs for the functionalization of metal and inorganic substrates. Dithiocarbamate ligands can be formed under mild conditions by the simple condensation of amines with carbon disulfide, and adsorb onto substrates to form DAMs with high surface coverage. Current evidence suggests that DAMs have a much higher tolerance to environmentally harsh conditions than their alkanethiol-based counterparts; they can withstand a broad range of acidic and basic conditions, and resist desorption by competing surfactants or electrolytes. We illustrate the compatibility of DAMs at the interface of biological systems with several recent examples.
11:30 AM *MM4.7
Gold Nanoparticles as Scaffold for the Synthesis of Artificial Proteins. Lucia Pasquato, Dep. of Chemical Sciences, University of Trieste, Trieste, Italy; INSTM, Trieste, Italy.
Since the seminal work of Brust and Schiffrin in 1994 reporting a simple and reproducible synthesis of gold nanoparticles protected by a monolayer of alkanethiols (MPC)[1] these systems experienced an exponential growth of interest because of their unique properties as stability, solubility in organic or aqueous solvents modulated by the thiols properties, the easy tuning of the dimension determined by the reaction conditions and the facility to introduce a great variety of functional groups or biomolecules.[2,3] This is at the basis of the development of a multitude of applications of MPC in different fields as chemistry, biochemistry, material sciences, medicine, nanotechnologies. In this communication I will show as gold nanoparticles may be excellent scaffold to design self-assembled functional supramolecules able to mimic natural systems for recognition processes and catalysis. Recent results will be critically presented. For example, we have demonstrated that functional groups - small peptide-sequences - present in the monolayer of MPC can co-operate to undergo multivalent binding and to elicit catalytic activity.[4] One important feature is that the catalytic site is sensitive to the nature of the substrate and capable of regulation of its activity. [1] Brust, M.; Walker, M.; Betthell, D.; Schiffrin, D. J.; Whyman, R. J. Chem. Soc., Chem. Commun., 1994, 801-802. [2]Templeton, A. C.; Wuelfing, W. P.; Murray, R. W. Acc. Chem. Res., 2000, 33, 27-36. Badia, A.; Lennox, R. B.; Reven, L. Acc. Chem. Res., 2000, 33, 475-481. Daniel, M.-C.; Astruc, D. Chem. Rev., 2004, 104, 293-346. [3] Pasquato, L.; Pengo, P.; Scrimin, P. J. Mater. Chem. 2004, 14, 3481-3487. [4] Manea, F.; Bodar-Houillon, F.; Pasquato, L.; Scrimin, P. Angew. Chem. Int. Ed. 2004, 43, 6165-6169. Pengo, P. Polizzi, S.; Pasquato, L.; Scrimin, P. J. Am. Chem. Soc. 2005, 127, 1616-1617. Pengo, P.; Baltzer, L.; Pasquato, L.; Scrimin, P. Angew. Chem. Int. Ed. 2007, 45, 400-404.
Chair: Darrin Pochan
Tuesday Afternoon, November 27, 2007
Room 210 (Hynes)
1:30 PM *MM5.1
Nanoparticle-templated Protein Cage Assemblies. From Imaging Nanoprobes to Plasmonic Mmetamaterials. Bogdan Dragnea, Chemistry, Indiana University, Bloomington, Indiana.
Metamaterials have optical (or more general, electromagnetic) properties determined by their organized structure rather than inherited directly from the material properties of individual subunits. Metallodielectric metamaterials are composed of optically-resonant metal inclusions in a dielectric matrix and have subwavelength lattice periods. The optical response of metallodielectrics is intensely studied at present because of their freedom of design and promise for novel properties. Thus, predicted applications include better lenses, exotic coatings, new lasers, and miniaturization of photonic technologies beyond the diffraction limit. However, for metallodielectric metamaterials to be useful in the visible range of the electromagnetic spectrum, they require extended three-dimensional (3D) structures with lattice constants between 10 and 100 nm which are difficult to synthesize with current technologies. A solution to this problem is proposed based on a biological pathway to optical metamaterials. The building block for the new 3D metamaterial is a virus-like particle (VLP) which is a hybrid construct composed of a symmetric protein cage encapsulating an optically-active nanoparticle.
2:00 PM MM5.2
Viruses and DNA as Nanoscale Building Blocks: Using Biology to Control Interactions. Harry Bermudez, Polymer Science and Engineering, University of Massachusetts Amherst, Amherst, Massachusetts.
Controlling interactions between building blocks, in either self- or guided assemblies, is becoming increasingly important in creating functional materials. Viruses and DNA provide an unprecedented degree of flexibility by virtue of the >20 amino acids and the >4 nucleic acids. We first discuss our work using filamentous bacteriophage, where our strategy is to selectively alter surface features by focusing on spatially distinct proteins. Introduction of local reactivity in the form of single cysteines residues by site-directed mutagenesis allows for precise attachment of macromolecules such as polyethylene glycol (PEG), a known biocompatible and "stealthy" polymer. By varying the position of mutation in just one capsid protein we can achieve modulation of the virus (i.e., nanoparticle) reactivity. This ability to tune reactivity by both mutation type and position allows us to extend the traditional post-assembly surface modification. We will then briefly highlight our work using DNA to pattern surfaces and build nanoscale objects amenable to registration and subsequent assembly and functionalization. Extending and integrating the above types of modular systems is envisioned to lead to improved control over self-assembly in applications such as sensors and targeting. The approach described here is a general one and can be broadly utilized in other contexts (with other viruses or other polymers). Of obvious interest is broadening the scope of different shapes, sizes, and functionalities.
2:15 PM *MM5.3
Self-assembled Protein Cage Architectures - Size and Shape Constrained Templates for Magnetic and Catalytic Nanomaterials. Trevor Douglas, Department of Chemistry and Biochemistry, Montana State University, Bozeman, Montana.
The self-assembled architectures of viral capsids and ferritin-like proteins have been used as models for understanding processes of encapsulation of both hard and soft materials. We have explored modifications to the exterior and interior interfaces of viral (and other protein cage architectures) while maintaining assembly of stable icosahedral, cubic, or tetrahedral cage particles. This has allowed us to utilize the high symmetry of these architectures to engineer unique functionality for highly ordered multivalent presentation for controlled nucleation of hard inorganic materials and packaging of soft organic materials. We have probed the hard-soft interface using genetic and chemical modifications to direct the synthesis of magnetic and catalytic nanoparticles in a spatially controlled manner. The materials have been probed using high-resolution transmission electron microscopy, and cryo-electron microscopy and image reconstruction to elaborate key features of the protein-inorganic interface. The constrained protein environment plays an important role in tuning the properties of the encapsulated materials and results in unusual (and controllable) magnetic properties and enhanced catalytic properties. Magnetic exchange bias in constrained mixed metal oxide systems and catalytic hydrogen production with constrained inorganic nanoparticles will be discussed.
3:15 PM *MM5.4
Using Chemical Templates to Direct the Organization of Macromolecular Scaffolds. Jim J De Yoreo1, Sung Wook Chung1, Andrew D Presley2, George H Gilmer1, Selim Elhadj1, Raymond W Friddle1, Matthew B Francis2, Phillip L Geissler2, Ted M Tarasow1, Aleksandr Noy1, John E Johnson3 and Marianne E Manchester3; 1Lawrence Livermore National Laboratory, Livermore, California; 2University of California at Berkeley, Berkeley, California; 3Scripps Research Institute, La Jolla, California.
The use of macromolecular scaffolds for hierarchical organization of molecules and materials is a common strategy in living systems that leads to emergent behavior. One characteristic of this strategy is that it generates micron-scale structures from nm-scale building blocks. These in turn possess functionality defined at the Å-scale by active sites, typically on protein complexes. Here we describe efforts to mimic this strategy by creating nm-scale chemical templates to direct the organization of engineered macromolecules and complexes, such as RNA, proteins and viruses, into micron-scale patterns. These highly uniform building blocks then serve as scaffolds for the assembly of materials and molecules such as metallic and semiconductor nanocrystals or light absorbing centers. In the case of viruses, site-specific modification either through genetic manipulation or targeted chemical reactions define the sub-nm scale pattern of these functions. While these efforts are exploratory attempts to create a practical platform for device fabrication, they also provide well-controlled systems for investigating the factors that drive or limit macromolecular organization. Our approach is to use scanned probe lithography to fabricate patterns of self-assembled monolayers (SAMs) on Au or SiOx. The SAM monomers are designed for their ability to either bind to chemically or genetically modified sites on the target macromolecules or resist binding altogether. We then use in situ AFM to investigate either the dynamics of virus organization or nanocrystal growth at these templates. Virus organization is also modeled using kinetic Monte Carlo simulations, where adsorption energies for attachment depend on location on the template and number of nearest neighbor viruses. As a constraint on the models and a means of directly exploring the nature of the governing interactions, potentials and binding energies are determined from force spectroscopy (FS) where the viruses or RNA molecules are attached to the AFM tips using the same chemistry as for the template SAMs. Here we show results for three systems: RNA catalysts of Pd growth, 18nm disks formed from Tobacco Mosaic Virus (TMV) monomers, and 28nm icosahedral Cow Pea Mosaic Virus (CPMV) particles. For the aptamers, we show they can be reacted with SAM monomers to create patterns of surface-active catalysts and present results of kinetic studies on Pd nanocrystal formation. For CPMV and TMV we show that the morphological evolution predicted by the simulations resembles AFM observations for appropriate differences in adsorption energy between template and resist. FS measurements are compared to the predicted adsorption energies and are used to investigate the role of solvent interactions in inter-viral potentials. This work was performed under the auspices of the U. S. Department of Energy by the University of California, Lawrence Livermore National Laboratory under Contract No. W-7405-Eng-48
3:45 PM MM5.5
Synthesis and Characterization of Ferromagnetic Nanowires using Virus Particles. Gabriel George Baralia1, Anan Kadri2, Thomas Weitz1, Alexander M Bittner1, Sinan Balci1, Christina Wege2, Holger Jeske2 and Klaus Kern1; 1Nanoscale Science Department, Max Planck Institute for Solid State Research, Stuttgart, Germany; 2Department of Molecular Biology and Plant Virology, University of Stuttgart, Stuttgart, Germany.
Biological systems have intrinsic capabilities of molecular recognition and selectivity and therefore represent attractive templates for building nanostructures. An example is the Tobacco mosaic virus (TMV), a plant virus of tubular shape, 300 nm long with 18 nm outer and 4 nm inner diameters, which can be exploited to synthesize metallic nanowires. Here we show, by simple electrochemical deposition, synthesis of nickel (Ni) nanowires, 4 nm in diameter and micrometer long, within the central channel of the E50Q mutation (decreasing the overall negative charge) of the TMV. The electronic transport properties and the magnetoresistance of a single Ni nanowire are investigated after contacting (using e-beam lithography) on silicon substrates.
4:00 PM *MM5.6
Two Approaches for Directly Connecting Biological Events with Electronics. Gregory Alan Weiss1,2, Juan Diaz1, John Coroneus2, Jorge Lamboy1, Li-Mei Yang1, Phil Collins3 and Reg Penner1; 1Dept. of Chemistry, UC Irvine, Irvine, California; 2Dept. of Molecular Biology, UC Irvine, Irvine, California; 3Dept. of Biochemistry, UC Irvine, Irvine, California.
Our laboratory explores the intersection of biology and electronics in two systems. First, at the macroscopic scale, we collaborate with the electrochemist Prof. Reg Penner and co-workers to directly couple molecular recognition with an electronic signal. Our “virus electrodes” feature a dense virus layer, readily tailored for recognition of essentially any biomarker, covalently attached to a gold electrode surface through a self-assembled monolayer. The resistance of this virus electrode, ZRe, measured in the frequency range from 2 to 500 kHz in a salt-based pH 7.2 buffer, increased when the phage particles selectively bound either an antibody or prostate-specific membrane antigen (PSMA), a biomarker for prostate cancer. The second system leverages advances in microfabrication and the controlled synthesis of a single carbon nanotube contacting multiple electrodes. Together with our collaborator, physicist Prof. Phil Collins and co-workers, we have demonstrated conductance-controlled introduction of a single, carboxylate handle onto the sidewall of a nanotube connected into a nanocircuit. Using the electronic signature of the resultant nanocircuit, the single protein will be examined in real-time during protein unfolding, folding, binding, and, where applicable, catalysis.
4:30 PM MM5.7
Aptamer Incorporated Polyelectrolyte Multilayer Films Targeting Influenza Virus’s Hemagglutinin Binding Region. Srivatsan Kidambi1,2, Ilsoon Lee1 and Christina Chan1; 1Department of Chemical Engineering and Materials Science, Michigan State University, East Lansing, Michigan; 2Center for Engineering in Medicine, Harvard Medical School, Massachusetts General Hospital, Shriners Hospital, Boston, Massachusetts.
Rapid, accurate detection and prevention of influenza virus has important clinical and public health implications. In this work, we describe the engineering of highly customizable polyelectrolyte multilayer (PEM) films incorporated with hemagglutinin (HA) aptamer which binds and detects the virus’s HA receptor-binding region. Current techniques to detect the presence of virus involve expensive, multiple steps requiring several days to analyze and confirm the presence of the virus. We have engineered an aptamer based system targeting influenza virus using PEM films by capitalizing on electrostatic interaction between the polycations (PLL) and the negatively charged aptamer (HA aptamer). This system can be used both to detect and inhibit the virus. Aptamer arrays on PEMs were created by ionic interactions using microcontact printing (μCP) to detect for the HA antigens on the virus surface. The construction of the multilayer films of poly-L-lysine (PLL) and HA aptamer was also characterized. These films were deconstructed under physiological conditions (salt conc=0.25M) to release the HA aptamer, which can then target the HA region of the virus and passivate the virus. To our knowledge, this study represents the first report of incorporating aptamer within PEM films. This PEM and HA-aptamer based system provides a tool to 1) incorporate the HA aptamer within the PEM films which can be released with change in salt, pH or addition of enzyme providing it with the capability of targeting the HA and NA antigenic proteins on the virus surface and 2) detect and quantify the influenza related antigenic proteins (HA and NA).
4:45 PM MM5.8
Spontaneous Reduction of Silver Ions Mediated by Engineered M13 Virus, Yeast and Peptoid Oligomer. Ki Tae Nam1, Yun Jung Lee2, Eric M. Krauland3, Byoung-Chul Lee1, Angela M Belcher2,3 and Ron N Zuckermann1; 1Molecular Foundry, Lawrence Berkeley National Laboratory, Berkeley, California; 2Department of Materials Science and Engineering, MIT, Cambridge, Massachusetts; 3Bioengineering Division, MIT, Cambridge, Massachusetts.
Biological systems have developed biomineralization processes to nucleate, grow, and assemble inorganic materials. An important component of biomineralization is the protein or peptide template that controls the shape and crystal structure of biominerals as well as the assembly behavior. Peptides, biomolecules and organisms such as bacteria and fungi are reported to synthesize silver particles intra- or extracelluarly when they are exposed to silver salts. Although there is growing interest in the bioinspired synthesis of silver nanoparticles, a general understanding of growth is not yet known. The conformation, overall charge, and functional groups of biomolecules may all contribute to biological reduction in conjunction with solution pH, light, temperature, and other ions in solution. As an effort to contribute to the understanding of spontaneous reduction, we focused on the role of carboxylic acid containing peptides expressed on yeast surfaces and M13 virus scaffold. In this work, we report that the genetically engineered yeasts and viruses not only mediate the reduction of silver ions through expressed peptides, but also act as templates for controlled spatial growth of particles. The understanding obtained with peptides was extended to peptoid mediated synthesis of silver nanostructures. Peptoids are oligo-N-substituted glycine, designed to mimic peptides. Their side chains are appended to the amide nitrogen rather than to the alpha carbon like in a peptide. The designed peptoid can also reduce silver ion at room temperature without any reducing agents. This reduction process results in different silver nanostrucutres depending on the peptoid sequences. These insights into the spontaneous reduction of metal ions on biological scaffolds and peptoids will help further the formation of novel nanomaterials in biological systems.
Chairs: Darrin Pochan and Vincent Rotello
Tuesday Evening, November 27, 2007
8:00 PM
Exhibition Hall D (Hynes)
MM6.1
Templated Precipitation and Growth of Calcium Phosphate Nanocrystals on Self-Assembling Ionic Block Copolymers. Yusuf Yusufoglu1, Mathumai Kanapathipillai2, Aditya Rawal3, Yanyan Hu3, Eren Y Kalay1, Klaus Schmidt-Rohr3, Surya K Mallapragada2 and Mufit Akinc1; 1Materials Science and Engineering Department, Iowa State University, Ames, Iowa; 2Chemical & Biological Engineering, Iowa State University, Ames, Iowa; 3Chemistry, Iowa State University, Ames, Iowa.
In an effort to imitate the growth of natural bone, in which collagen (a hydrogel that acts as a template) provides sites for the nucleation and growth of inorganic dahlite (CO3HAp) phase, the polysulfobetaine-based zwitterionic and poly acrylic acid-based self-assembling ionic pentablock copolymers were employed as templates for the growth of calcium phosphate nanocrystals from aqueous solutions. Hierarchically assembled nanocomposite calcium phosphate-copolymer gels were prepared at pH ~5. Calcium phosphate nanoparticles were formed by the ionic interaction between the polymer and inorganic ions in the solution. XRD, TEM, NMR, TGA and SANS were used to characterize the self-assembled polymer-inorganic nanocomposite gels formed. XRD experiments revealed that the calcium phosphate in the zwitterionic copolymer gel was natural brushite, while the one in the PAA-based pentablock gel was synthetic brushite. This is consistent with the phases predicted by the calcium phosphate pH - stability diagram, that the most stable phase at pH ≤5 is brushite. Solid-state NMR experiments indicate that, in addition to crystalline brushite, significant amount of amorphous calcium phosphate is incorporated in to the nanocomposites. Moreover, TEM, solid-state NMR and SANS studies showed that the calcium phosphate precipitated on and interacted with the polymer micelles forming approximately 20 nm diameter nanospheres. The self-assembled nanocomposite calcium phosphate-copolymer hydrogels contained 15 weight percent calcium phosphate. Acknowledgment: This work was supported by the U.S. Department of Energy through Ames Laboratory. Ames Laboratory is operated through the U.S. Department of Energy by Iowa State University under contract number DE-AC02-07CH11358.
MM6.2
Bioinspired Synthesis of Cobalt Ferrite Nanocrystals. Tanya Prozorov1, Ruslan Prozorov1,2, Marit Nilsen-Hamilton4, Surya K. Mallapragada1,3, Balaji Narasimhan3 and Paul C. Canfield1,2; 1Materials Chemistry and Biomolecular Materials, Ames Laboratory, Ames, Iowa; 2Department of Physics and Astronomy, Iowa State University, Ames, Iowa; 3Chemical and Biological Engineering, Iowa State University, Ames, Iowa; 4Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, Iowa.
Magnetotactic bacteria produce ordered chains of uniform magnetite with various morphologies. Using a new bio-inspired route involving the novel acidic protein mms6 found in magnetotactic bacteria, we were able to synthesize a variety of nanostructured magnetic materials, some of which are not known to occur in Nature in living organisms. Mms6 protein was previously shown to facilitate shape-selective formation of magnetite. This bio-inspired route was used to obtain cobalt ferrite, CoFe2O4, which does not occur naturally in bacteria. We have covalently attached full-length mms6 and associated c-terminal mms6 protein to self-assembling polymers that form thermoreversible gels to control nanoparticle agglomeration and to enable templated hierarchical architectures resembling those observed in Nature. This new methodology enables facile room-temperature synthesis of complex magnetic nanomaterials with uniform and well-defined shapes, sizes and crystal structures, with nanocrystals in the range of 40-100 nm that are difficult to produce using conventional techniques. These nanocomposites exhibit unique magnetic and other properties not realizable via conventional synthesis methods. Overall, this bio-inspired strategy facilitates a bottom-up approach to design and synthesis of tailored nanostructured materials.
MM6.3
The Effect of Silk Fibroin Hydrogels, Poly-peptides, and Self Assembled Monolayers on Calcium Carbonate Crystallization. Ellen C Keene, Vijay Ravichandran and Lara A Estroff; Material Science & Engineering, Cornell University, Ithaca, New York.
A silk fibroin-like hydrogel, along with occluded glyco-proteins and a functionalized surface, is the current model in biomineralization for the layered organic matrix involved in the formation of the nacreous layer of mollusk shells (model developed by Falini et al.). Using this model from biology we created a biomimetic setup using silk fibroin hydrogels combined with self-assembled monolayers (SAMs) and acidic polypeptides to grow calcium carbonate crystals. Silk fibroin (from silkworm cocoons) hydrogel formation was characterized by circular dichroism (CD) spectroscopy, XRD, and SEM. Gelation was triggered by temperature (incubation at 60° C), and the conformational changes of the silk fibroin during gelation, from a random coil to a β-sheet, were assessed by CD spectroscopy and XRD. The gel samples were characterized by their rate of gelation and β-sheet character. The morphology of freeze dried gel samples were assessed by SEM. Based on these findings, a two step gelation mechanism is presented in which the protein first folds into β-sheets then later aggregate together to form an extended gel network. We studied the nucleation and growth of calcite in a matrix composed of silk fibroin hydrogels, with and without occluded polypeptides on top of self assembled monolayers (SAMs) on gold. Nucleation densities were found to depend both on the functionality of the SAM (carboxyl, hydroxyl, methyl terminated, or glass) and the silk structure (random coil versus β-sheet and aqueous versus gel). While previous studies have demonstrated the ability of SAMs to control the orientation of calcite crystals grown in solution and in agarose gels, the addition of silk to the crystallization assay suppresses the oriented nucleation. The SAM still retains the ability to increase the nucleation density. Grazing angle FTIR reveals that the silk adsorbs to the SAM surface thereby changing the surface functionality and eliminating the oriented nucleation. Finally, the addition of acidic polypeptides (e.g., poly(Asp) and poly(Glu)) to the silk fibroin hydrogel alters the crystal morphologies as compared to control crystals grown in solution or in the silk fibroin hydrogels alone.
MM6.4
Models for the Nucleation and Growth of Calcium Carbonate. John Harding1, Colin Freeman1, Mingjun Yang1, Dmytro Antypov2, David Cooke2, James Elliott2, Dorothy Duffy3, Michael Gillan3, Jennifer Lardge3, Michael Allen4, David Quigley4, David Quigley4, Mark Rodger4 and Tiffany Walsh4; 1Engineering Materials, University of Sheffield, Sheffield, United Kingdom; 2Materials, University of Cambridge, Cambridge, United Kingdom; 3Physics and Astronomy, University College London, London, United Kingdom; 4Chemistry, University of Warwick, Coventry, United Kingdom.
The nucleation and growth of calcium carbonate is important in fields from biomineralization through geology to industrial processing. Much work has been published in the area (a summary can be found in [1]), but there remains much to be understood. Simulation is a major tool in obtaining this understanding. We have developed a consistent set of potentials for carbonates (including a range of species in solution), organic functional groups and water based on well-known models for the carbonates, the AMBER forcefield for organic molecules and the TIP3P model for water. Interactions have been obtained by a variety of simple rules and validated by a range of ab initio calculations. This potential set has been used to investigate a range of nucleation and growth phenomena in calcium carbonate. We will also present results on how the growth and morphology of calcite can be controlled by simple ions, organic molecules such as polysaccharides or molecular arrays. Current and future work are addressing the problem of simulating the effects of peptides and proteins. Although it is usual to consider the nucleation of calcite on the basis of classical nucleation theory, other mechanisms, based on the crystallisation of amorphous calcium carbonate are also possible. Simulation of nano-particles is important because it allows size and shape-dependent properties to be studied directly. We have simulated nano-particles in vacuum and in water and shown the importance of size, the presence and structure of surface water and the effects of organic molecules and arrays. We shall also present initial results on the direct simulation of nucleation using enhanced molecular-dynamics methods. [1] J.H. Harding and D.M. Duffy; The challenge of biominerals to simulations; J. Mater. Chem. 16 (2006) 1105
MM6.5
Live-cell Cytoskeleton Dynamics using AFM in the Pico-newton Force Regime. Paul Campbell, University of Dundee, Dundee, United Kingdom.
Atomic force microscopy (AFM) has been used to image live PtK2 epithelial cells in vitro. By employing ultra low imaging forces (FL < 100pN) whilst operating in contact mode, it was possible to achieve spatial resolutions in the range of about 25nm, which was sufficient to easily resolve the constituent fibres of the cytoskeletal network and other sub-micron intra-cellular detail. Force distance curves were obtained which allowed a Hertzian analysis of the cellular elasticity and a measured value for the Young's modulus averaged over specific motility regimes. Time-lapse imaging with such low forces allowed the non-destructive observation of cytoskeletal reorganisation during motility over extended periods of up to 7 hours.
MM6.6
Abstract Withdrawn
MM6.7
Exploring Coiled-Coils Using Theoretical Methods and Applications to Bio-Inorganic Nanomaterials. Rachel D. Osborne1, Anna Laromaine1, Muthu Murugesan1, Sarah Harris2 and Molly M. Stevens1,3; 1Materials, Imperial College, London, United Kingdom; 2School for Physics and Astronomy, University of Leeds, Leeds, United Kingdom; 3Institute of Biomedical Engineering, Imperial College, London, United Kingdom.
Short peptides taking the tertiary structure of dimeric coiled-coils offer material scientists a versatile and reversible nanoswitch when coupled with inorganic nanoparticles. Careful engineering of the amino acid sequence allows the coiled-coil structure to be disrupted via a range of external stimuli including pH, temperature and light. Theoretical modelling of the coiled-coil systems can provide insights that result in improved properties and control over the peptide-inorganic nanomaterials. Utilizing the AMBER 9 suite of molecular modelling programmes to run 12ns simulations, the thermodynamics and kinetics of the yeast transcription factor, GCN4, and several derivates have been explored. The backbone RMSd values were < 1Å for all models showing good agreement with the starting structure. The dimeric coiled-coil models were analysed using the Lindemann’s principle, which confirmed a solid-liquid hybrid, similar to the larger proteins previously reported and also using principle component analysis to understand the conformational changes versus time of the coiled-coil. PC1 and PC3 showed perpendicular movements about the major axis of the superhelix, and PC2 showed an untwisting of the superhelix. We have utilised these findings to design and synthesise coiled-coil peptides which once immobilised onto gold nanoparticles generate reversible self-assembling materials with potential applications in drug delivery and biosensors.
MM6.8
Surface-induced Assembly of Fibrinogen Fibers on Clay Platelets. Jaseung Koo1, Tadanori Koga2, Dennis Galanakis3, Jonathan Sokolov1 and Miriam Rafailovich2,1; 1Materials Science, State University of New York at Stony Brook, Stony Brook, New York; 2Chemical and Molecular Engineering Program, State University of New York at Stony Brook, Stony Brook, New York; 3Department of Pathology, State University of New York at Stony Brook, Stony Brook, New York.
It is well known that fibrinogen molecules require thrombin in order to self-assemble into fibers. The role of the thrombin is to cleave two specific Arg-Gly bonds in the Aα and Bβ chains of the E domain, exposing active sites of the N-terminus on the central E domain which then interact complementary binding sites of the carboxy-terminus in the D domains from neighboring molecules. This successful completion of this process is a crucial step in building the fibrin gel that promotes to wound healing. Here we demonstrate that the formation of assembled fibers could also be induced by surface interactions alone without requiring thrombin cleavage. Hydrophoibic and hydrophilic surfaces were prepared by lifting functionalized clay films from the water surface using the Langmuir-Blodgett technique. The surfaces were then incubated with native human and bovine fibrinogens. We found that fibers, with the same dimensions as those observed in human fibrin gels, were formed on the hydrophobic surfaces in the absence of thrombin. Addition of thrombin did not significantly affect the dimensions. No fibers were found on hydrophilic surfaces, even though significant amount of proteins were adsorbed. In this case, scanning force microcopy revealed that the fibrinogen remained in its spherical form. The amount of proteins adsorbed to both surfaces was measured by an ELISA assay and found to be comparable. Fibrin molecules with only one arm also formed fibers on the surface, but the fibers were much thinner. No fibers were formed on molecules without any arms. We postulated that this difference would be ascribed to intermolecular interactions between αC domains which are become exposed due to the conformation of the molecules on the hydrophobic organoclay surface.
MM6.9
Novel Biopolymer-Clay Nanocomposites for Bone Tissue Engineering. Rajalaxmi Dash, Kalpana S Katti, Bedabibhas Mohanty and Dinesh R. Katti; Civil Engineering, North Dakota State University, Fargo, North Dakota.
Polymer-clay nanocomposites have received potential attraction because of their superior properties as compared to pure polymer but limited attention has given to biopolymer/ clay nanocomposites. Among biopolymers, chitosan-based materials are widely studied in the biomaterials field due to their biocompatibility, biodegradability, superior mechanical properties, gel forming property etc. In this work, we have developed a novel nanocomposite from chitosan, montmorillonite and a bioactive ceramic, hydroxyapatite (HAP). The composite was prepared by dispersing 10 wt% montmorillonite (MMT) in chitosan solution. To increase the bioactivity of the composite, hydroxyapatite was added to the chitosan-MMT system. The resulted chitosan/MMT/HAP composite was then characterized by X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), TGA, atomic force microscopy (AFM), DSC etc. XRD and AFM results indicate the formation of an intercalated nanostructure with an increase in d-spacing. Mechanical properties were evaluated using nanoindentation technique. The new composite exhibits significant improvement in mechanical property as compared to pure chitosan.
MM6.10
Electrospun Hydroxyapapatite-biocomposite Scaffolds for Tissue Engineering Applications. Koushik Ramachandran and P. Gouma; Materials Science and Engg, SUNY, Stony Brook, Stony Brook, New York.
This paper discusses the synthesis of natural polymer (cellulose acetate) and hydroxyapatite based polymer nanocomposite mats to mimic the extracellular matrix structure by electrospinning 3-D mats with hierarchical configuration. Solution of cellulose acetate in acetone and cellulose acetate and hydroxyapatite in acetone and acetic acid were used as precursors for the electrospinning process respectively. The morphology of the electrospun polymer composites and the effect of processing parameters on the production of the scaffolds are discussed. Human osteoblast cells have been cultured on these scaffolds to study cell differentiation and proliferation. The topology of the scaffolds and the effect of addition of nanoparticles like hydroxyapatite and their role in affecting the cell adhesion and growth have been studied. The mechanical properties of the bioscaffolds have been investigated in order to tailor the properties to meet the needs of tissue engineering applications.
MM6.11
Abstract Withdrawn
MM6.12
Functionalization of Micro- and Nanoparticles for Selective Attachment to Calcium Biomineral Surfaces. Stacey L. McLeroy1, Bruce Gnade1 and Jeffrey A. Cadeddu2; 1University of Texas at Dallas, Richardson, Texas; 2UT Southwestern Medical Center, Dallas, Texas.
Calcium-based biominerals are the primary component of most pathological biomineralization in humans, including atherosclerosis and kidney stones. The ability to deliver targeted therapeutic agents to a biomineralized surface opens a wide variety of treatment, therapy, and imaging options for doctors and surgeons. Inspired by the functionality of calcium binding proteins, we introduce a novel technique for selectively attaching both micro- and nanoparticles to calcium-rich surfaces such as hydroxyapatite and calcium oxalate. Preliminary results show selective attachment of iron oxide micro- and nano-particles to calcium-containing biomineral surfaces, although it is anticipated that a wide variety of particles or therapeutic agents could be functionalized using this technique. The resulting functionalized materials are characterized using Scanning and Transmission Electron Microscopy (SEM, TEM), Fourier Infrared Transform Spectroscopy (FTIR) and X-ray diffraction (XRD). In this work we also report optimization studies to increase the selectivity and specificity of the nano and micro particles for such biominerals. Toxicity and bio-compatibility tests are also reported.
MM6.13
Functional Bioplastics Made by Silk Fibroin Protein. Atsushi Kaneko1, Shinji Hirai1 and Yasushi Tamada2; 1Department of Materials Science and Engineering, Muroran Institute of Technology, Muroran, Japan; 2National Institute of Agrobiological Sciences, Tsukuba, Japan.
A resinified silk compact was formed from silk fibroin powder using a pulse current sintering apparatus under a temperature of more than 423 K and a uniaxially applied stress of 20 MPa. Addition of a small amount of water to the silk powder was required to form the resinified silk compact with good reproducibility. In a pulse electric current sintering apparatus, the heating is carried out by Joule heat inside the compacts induced by pulsed high DC current. The possibility of resinified silk compact as a new material was investigated by the evaluation of mechanical properties, thermal conductivity and dielectric properties at an ambient temperature. Compression tests and three-point bending tests using notched specimens were performed. The compression strength, Young’s modulus obtained by the compression test and rupture toughness gave 98 MPa, 5 GPa and 0.42 MPam1/2, respectively, and these values were superior to those of typical epoxy resin. The thermal conductivity by the steady method was 0.48 W/(mK) and this value was estimated as the same level with that of high-density polyethylene, which is known as a high thermal conductivity organic material. Furthermore, the dielectric constants and loss tangent of silk compacts were measured by employing the parallel plate method in the frequency range from 150 Hz to 1 MHz and by employing the open-ended reflection method in the frequency range from 200 MHz to 20 GHz. The dielectric constant was 4.5 at a frequency of 150 Hz and decreased gradually at higher frequency. The dielectric constant was 4 at a frequency of 20 GHz. The loss tangent was 0.01 at a frequency of 150 Hz and increased at higher frequency. However this value was not beyond 0.05 at a frequency of 20 GHz. Next, the EG-MS spectra were compared between the compacting temperatures of 343 K and 473 K. Consequently, moisture evaporation was observed near 343 K and 553 K. The former was considered to be due to moisture evaporation, and the latter was considered to be due to the cure or crosslinking reaction along with dehydration. Moreover, since a small amount of outgassing was observed near 573 K for M/Z in the range of 10-27 when the compacting temperature was 473 K, it was presumed that the silk compact starts to decompose. This decomposition affected the bending strength of the resinified compact, which decreased when the compacting temperature was 473 K, although the bending strength should increase with the compacting temperature. Although polylactic acid is currently used as a bioplastic, it has poor thermal resistance and mechanical strength since its glass transition temperature is 333 K, which is close to an ambient temperature. On the other hand, the resinified silk compact is expected to have excellent thermal resistance since the glass transition temperature of silk itself exceeds 443 K. Therefore, the resinified silk compact could be used as a novel environmentally friendly bioplastic, such as electrical material field.
MM6.14
Nuclear Morphology and Deformation in Micropatterned Cardiac Myocytes. Mark-Anthony Bray, Nicholas A. Geisse, Sean P. Sheehy and Kevin Kit Parker; School of Engineering and Applied Sciences, Harvard University, Cambridge, Massachusetts.
Cardiac tissue engineering requires finely-tuned manipulation of the extracellular matrix (ECM) microenvironment to optimize internal myocardial organization. In particular, the myocyte nucleus is mechanically connected to the cell membrane via cytoskeletal elements, making it a target for the cellular response to perturbation of the ECM. In this study, photolithographic methods were used to control ECM protein deposition, thereby altering the adherent geometry of cultured neonatal rat ventricular myocytes. Engineered two-dimensional tissue constructs and single-cell islands were assayed using live fluorescence imaging to examine nuclear position and morphology as a function of the imposed ECM geometry during diastolic relaxation as well as nuclear motion during systolic contraction. Image analysis showed that anisotropic tissue constructs cultured on microfabricated ECM lines possessed a high degree of nuclear alignment similar to that found in vivo; in contrast, isotropic tissues with no spatial guidance present are polymorphic in shape and the nuclei have an apparently random orientation. Similarly, nuclear eccentricity was also increased for the anisotropic tissues as compared to the isotropic tissue. Both of these observations suggest that intracellular cytoskeletal forces act to both realign and deform the nucleus as the cell boundaries are spatially confined. The cytoskeletal impact of ECM geometry upon nuclear positioning and morphology was further examined at the single cell level in myocytes cultured on ECM circles, rectangles and triangles. For all three shapes, the nuclear eccentricities were similar despite variation in cell and nuclear area; likewise, the nucleus was more likely to be restricted towards the middle of the cell than the periphery in all shapes. Examination of the cytoskeletal arrangement confirmed that the actin stress fibers followed consistent configurations as a function of cell shape, serving to limit the nuclear location and to impose unique patterns of force upon the nucleus. During systolic contraction, the nuclei were spatially displaced in a manner dependent on their intracellular position with respect to the axes of symmetry for each shape. Furthermore, the minor axis of the nucleus tended to deform more readily than the major axis during cellular contraction. Therefore, in both diastole and systole, forces transmitted to the nucleus through the cytoskeleton are not necessarily homogeneous across the nuclear surface area. Since chromatin is mechanically coupled to the nuclear lamina, differential perturbation of the genetic material due to nuclear deformation may occur at the single cell level or at the tissue level. Such findings have implications in understanding the genomic consequences and functional response of cardiac myocytes to their ECM surroundings under conditions of disease.
MM6.15
Cardiac Myocyte Cytoskeletal Architecture as a Function of Cell Shape. Mark-Anthony Bray, Sean P. Sheehy and Kevin Kit Parker; School of Engineering and Applied Sciences, Harvard University, Cambridge, Massachusetts.
The shape of a cardiac myocyte in vivo is a morphological indicator of myocyte health or pathophysiology. Proper cardiac function is dependent upon assembly of the cytoskeletal architecture and the organization of components of the contraction regulation system. In turn, cardiac cells organize both their contractile machinery and regulatory machinery based on geometrical cues from their environment. While the impact of cell-extracellular matrix (ECM) interactions upon cytoskeletal organization has been examined extensively on epithelium and fibroblasts, the same has not been performed for cardiac myocytes, especially in light of the additional contractile machinery they possess. For this study, photolithographic methods were utilized to control deposition of ECM proteins to produce rectangular islands of 2500 μm2 in area but with various aspect ratios ranging from 1:1 to 7:1. Cultured neonatal rat ventricular myocytes then adhered to the underlying ECM substrate via focal adhesions mediated through membrane receptors. The cytoskeletal arrangement was visualized with fluorescence imaging of fixed cells stained for the proteins actin (for the longitudinal contractile apparatus) and α-actinin (for the transverse Z-discs of the sarcomeric skeleton). Cells without the input of a geometric ECM pattern will assemble contractile sarcomere units but are unable to assign a spatial orientation amenable to organized force generation. In contrast, myocytes cultured on 2500 μm2 ECM patterns will align their sarcomeres in predictable and repeatable patterns. Furthermore, the actin cytoskeleton adapts to contractile stress fields generated at the cell corners by forming points of contact with the ECM through focal adhesions. Circular cells, which do not have corners to act as a geometric cue, do not have a well-organized sarcomeric alignment, which precludes contraction along an axis. Examination of the α-actinin shows a consistent pattern for cells of each rectangular aspect ratio. Since the sarcomere sub-units have a fixed spacing, this observation indicates that the cytoskeleton configuration is length-limited by the extracellular boundary conditions. These results suggest that modification of the extracellular microenvironment induces dynamic reconfiguring of the myocyte intracellular architecture, thereby optimizing contractility. Such knowledge is critical for the integration of regenerative cardiac tissue in vivo, as well as discovering the role of the extracellular environment on intracellular signaling pathways.
MM6.16
Spatiotemporal Dynamics of Cellular Traction Distribution During Fibroblast Migration. Zhi Pan1, Yajie Liu3, Kaustabh Ghosh2, Toshio Nakamura3, Richard Clark2 and Miriam Rafailovich1; 1Materials Science & Engineering, SUNY at Stony Brook, Stony Brook, New York; 2Biomedical Engineering, SUNY at Stony Brook, Stony Brook, New York; 3Mechanical Engineering, SUNY at Stony Brook, Stony Brook, New York.
Cellular traction forces are the physical interactions at cell-substrate interface. They are transmitted to the substrate through focal adhesions, which as they assemble and disassemble generate or release traction forces. This dynamic process directly determines where and how fast cells migrate. To better understand the spatiotemporal dynamics of cellular traction stresses (force per area), we measured their redistribution as a time sequence during fibroblast migration using optical digital image speckle correlation (DISC) technique in combination with finite element method (FEM) on a physiologically relevant substrate composed with cross-linked thiol-functionalized hyaluronan (HA-DTPH) and fibronectin functional domains (FNfds). We found that nuclear translocations, an indicator of cell migration, occurred in pulses, which was in concert with the cellular traction redistribution. Large pulsed-nuclear translocations only occur when rear tractions shuttled to a posterior nuclear location. Our results suggested that the reinforcing of tractions around the nucleus is a critical step in fibroblast migration. We also noticed that different substrate adhesiveness induced distinct distribution patterns of cellular traction stresses. Higher substrate adhesiveness with increasing FNfds density resulted in an increase in magnitude of cellular traction stresses across the whole cell, which hindered the relocation of rear tractions and made cell migrate slower. We anticipate these newly described spatiotemporal dynamics of cellular traction distribution will lead to further refinements of molecular dynamics during fibroblast migration.
MM6.17
Focal Adhesion Influence on Cell Stiffness and Cell Survival. Zhi Pan1, Fubao Lin2, Richard Clark2 and Miriam Rafailovich1; 1Materials Science & Engineering, SUNY at Stony Brook, Stony Brook, New York; 2Biomedical Engineering, SUNY at Stony Brook, Stony Brook, New York.
Focal adhesions are the physical connection between cell membrane and extracellular matrix (ECM). They are directly related to the arrangement of cytoskeleton and the transmission of cellular traction forces, which influence numerous cell behaviors and functions, such as cell adhesion, spreading, proliferation, and migration. In our previous study, we noticed that cell stiffness is a good mechanical indicator for cellular traction forces. When the cells generate greater traction forces, they become stiffer. Therefore, a correlation between cell stiffness and focal adhesion distribution is required to better understand how the physical connections to the surrounding environment influence cell behaviors such as cell survival. In this study, human dermal fibroblasts were cultured on 2D surfaces pre-coated by different concentration intact fibronectin or fibronectin functional domains. Distinct fibronectin functional domains (FNfds) were recognized by different adhesive receptors on cell membrane, which induced differential focal adhesion distribution and cell stiffness. We studied the focal adhesion distribution and cytoskeleton arrangement by staining vinculins and actin filaments, and measured the relative shear modulus of living fibroblasts by using AFM shear modulation force microscopy. Our data showed that the distribution of focal adhesion significantly influenced cell stiffness. Cells become stiffer with more focal adhesions distributed beneath the whole cell. These results were then correlated with auxiliary measurements of cell survival. We found that only cells with enough focal adhesion and stiffer cytoskeleton can survive well.
MM6.18
Thin Film Polymer Composites for Tissue Engineering of Neural Prostheses. Ning Han1, Craig Buckley1, Joseph F Rizzo3, Stuart F Cogan4 and Jessica O Winter1,2; 1Chemical Engineering, the Ohio State University, Columbus, Ohio; 2Biomedical Engineering, the Ohio State University, Columbus, Ohio; 3Center for Innovative Visual Rehabilitation, Boston VA/ Harvard MEEI, Boston, Massachusetts; 4EIC Laboratories, Norwood, Massachusetts.
Patients experiencing injury of the central nervous system have limited options for recovery. Some patients receive benefit from implanted neural prostheses, which use electrical stimulation to restore lost function. However, these devices do not repair damaged nerve; they merely take the place of affected tissue. Tissue engineering offers the promise of nerve regeneration and possible functional recovery, but there are several substantial impediments to use of this technique as a primary treatment. Our research examines the creation of hybrid devices that incorporate both technologies to produce a neural prosthesis with tissue engineering capabilities. These devices may bridge the gap between clinical implantation of tissue engineering strategies and existing neural stimulation technologies. Our approach consists of thin polymer films that may be applied to existing neural prostheses using standard lithography techniques. Films are comprised of a polymer hydrogel with encapsulated polymer microspheres and inorganic nanoparticles. These films possess many tissue engineering capabilities including potential for delivery of therapeutic or trophic compounds, modification of the electrode tissue interface through interaction with tethered cell adhesion molecules, and encapsulation of therapeutic stem cells. It is anticipated that neural prostheses modified with these films could be used to provide enhanced functional recovery by promoting neural regeneration and enhancing neuronal survival in the vicinity of neural prosthetic devices. We have examined the potential of polymeric films to enhance prosthetic devices using a retinal prosthesis platform. Our preliminary data shows that release of neurotrophins (i.e., brain derived neurotrophin, BDNF) enhances neurite extension near the device and that this effect is enhanced in the presence of cell adhesion molecules (i.e., laminin, collagen, and polylysine). We are currently examining the potential of these devices to support cell growth and delivery of biological agents, including the application of therapeutic stem cells.
MM6.19
Designer Functional Self-assembling Peptide Nanofiber Scaffolds for Angiogenesis Studies. Xiumei Wang1, Akihiro Horri1,2 and Shuguang Zhang1; 1Center for Biomedical Engineering, MIT, Cambridge, Massachusetts; 2Olympus America Inc., Center Valley, Pennsylvania.
A class of designer self-assembling peptide nanofiber scaffolds as a unique biological material has diverse and broad applications including for 3-D tissue cell culture, slow drug release and regenerative medicine. One of these peptide scaffolds, RADA16-I has been used in bone, cartilage, and neural regeneration and shown great promises. Here we report the development of several peptide nanofiber scaffolds designed specifically for angiogenesis study. Angiogenesis is a very important aspect in regenerative medicine. An adequate blood vessel supply to the newly formed tissue and within the transplanted scaffold is essential in determining the success of new tissue regeneration. We used designer functional self-assembling peptides through directly coupling RADA16-I with short biologically active motifs. In our study, the biologically active motifs include actin binding motif (ABM), fibronectin endothelial cells adhesion motif (FEA), RGD repetitive binding sequence (RRB) and angiogenesis growth factor derived peptide (AGF). We mixed 1% designer functional peptide solutions at a ratio of 1:1 with 1% RADA16-I solution to form functional self-assembling peptide nanofiber scaffolds. AFM examinations showed the molecular integrations of functional peptides into RADA16-I and formed well-ordered nanofibers. Compared with pure RADA16-I scaffold, designer peptide scaffolds AGF and RRB significantly increased human umbilical vein endothelial cells (HUVEC) viability and promoted cell proliferation. Furthermore, AGF and RRB motifs stimulated HUVECs migration from pure RADA16-I scaffold to functional peptide scaffolds. In addition, our results showed that AGF peptide scaffold provided an angiogenic environment and promoted HUVECs capillary-like network formation at both low and high cells seeding densities in short-term incubation. While on the RRB peptide scaffold, HUVECs formed capillary-like structure only after cells reached confluence. Our studies suggest that designer functional self-assembling peptide nanofiber scaffolds have promises for angiogenesis, which may be very useful for promoting diverse tissue regenerations.
MM6.20
Contact Guidance of Human Endothelial Progenitor Cells by Ordered Substrate Nanotopography. Chris John Bettinger1,2, Zhitong Zhang3, Sharon Gerecht4, Robert Langer3,5 and Jeffrey Borenstein2; 1Materials Science and Engineering, MIT, Cambridge, Massachusetts; 2MEMS Technology Group, Charles Stark Draper Laboratory, Cambridge, Massachusetts; 3Chemical Engineering, MIT, Cambridge, Massachusetts; 4Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, Maryland; 5Division of Health Science and Technology, Harvard-MIT, Cambridge, Massachusetts.
Mammalian cells respond to their substrates by complex changes in gene expression profiles, morphology, proliferation and migration. We report that substrate nanotopography leads to contact guidance effects in human endothelial progenitor cells (hEPCs). Fibronectin-coated poly(di-methyl siloxane) substrates with line-grating (600 nm ridges with 600 nm spacing and 600 +/- 150 nm feature height) induced alterations in gross cell morphology, proliferation rates, and migration velocity. Human EPCs cultured on nanotopographic substrates aligned and elongated parallel to the structures as demonstrated by cell alignment angle and a reduced circularity index. These morphological alterations were observed after 24 h and were maintained for up to 6 d. Human EPCs cultured on nanotopography also exhibited reduced proliferation as observed by reduced growth rates and reduced BrdU uptake. Migration velocity and total migration displacement was increased in hEPCs on nanotopographic substrates, as well. Human EPCs continue to express similar endothelial-specific markers, independently of nanotopography. Flow cytometry analysis showed that there was no significant difference in expression levels of CD31, von Willebrand Factor (vWF), or alpha-Smooth Muscle Actin (a-SMA). Similarly, hEPCs cultured on both flat and nanotopographic substrates expressed CD31, vascular endothelial cell adhesion molecule (VECAD), reactive to Ulex europaeus lectin type 1, and did not express a-SMA, as assessed by fluorescence microscopy. However, unlike flat substrates, culture of hEPCs on nanotopography resulted in their organization into supercellular band structures parallel to the grating axis after 6 days. An in vitro matrigel assay further revealed that these band structures ultimately led to ordered, enhanced sprouting (as determined by sprout length and orientation angle) of hEPCs grown on nanotopographic versus flat substrates. These results suggest that substrate nanotopography leads to morphological changes that can enhance endothelial tubular-like structure formation in vitro.
MM6.21
Biological Thin Films from Plasma Enhanced Chemical Vapor Deposition. A. Maruffo, J. Enlow, J. Slocik, Timothy Bunning and R. Naik; Air Force Research Laboratory, WpAFB, Ohio.
This study will discuss the novel fabrication of biopolymer films using plasma-enhanced chemical vapor deposition (PECVD) and amino acids. The introduction of tyrosine and tyrosine derivatives in the gas phase into an argon plasma stream resulted in controlled surface polymerization. These interfaces were explored for further specific biological binding events through the use of masks. Janus-like particles formed by polymerizing a thin polymer film on only one side of a spherical particle suspended on a surface is also discussed. The subsequent morphology and chemical composition of biopolymer films formed under different reaction conditions will be discussed.
MM6.22
Microcontact Printing of Melanin Thin Films for Neuronal Tissue Engineering Applications. Chris J. Bettinger1,2, Asish C Misra3, Robert Langer3,4 and Jeffrey T. Borenstein2; 1Material Science and Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts; 2MEMS Technology Group, Charles Stark Draper Laboratory, Cambridge, Massachusetts; 3Chemical Engineering, Massachusetts Insitute of Technology, Cambridge, Massachusetts; 4Harvard-MIT Division of Health Sciences and Technology, Cambridge, Massachusetts.
Melanins are a class of naturally occurring proteins that serves as a pigment for many organisms, and are rigid backbone, electrically conducting polymers with resistances on the order of 1 Ohm-cm. We hypothesized that the conductive nature of melanin could be an enabling property for the use of this material for neuronal tissue engineering applications. To test this hypothesis, we characterized the cell-biomaterial interaction of the PC12 neuronal cell line on melanin. Thin films of melanin were prepared by spin coating solvated synthetic melanin. Film morphology, thickness, and roughness were characterized by SEM, profilometry, and AFM, respectively. PC12 cells cultured on melanin films attached and exhibited neurite outgrowth in the presence of nerve growth factor (NGF). The micropatterning of proteins has proven to be an integral method for both elucidating fundamental mechanisms and engineering biological systems. We then hypothesized that melanin structures could be micropatterned as a method to support patterned neuron culture. We adapted soft lithography techniques to create a microcontact printing process for the controlled deposition of synthetic melanin with feature sizes ranging from 50 to 500 microns. Micropatterned melanin on silicon oxide substrates was used to control PC12 neuronal cell line morphology and placement via preferential adhesion to melanin structures relative to silicon oxide.
MM6.23
Synthesis and Characterization of Novel Biopolymer/Hydroxyapatite Fibers for Bone Tissue Engineering. Devendra Verma, Patrick Dunlap, Kalpana S Katti and Dinesh R. Katti; Civil Engineering, North Dakota State University, Fargo, North Dakota.
In the current work we have synthesized fibrous scaffolds of chitosan, polygalacturonic acid and hydroxyapatite for bone tissue engineering. The scaffolds were synthesized using freeze drying methodology. The thickness of fibers was observed to be in the range of 1-2 μm and the pores were in the range of 5-20 μm . Chitosan is a polyanionic biopolymer whereas polygalacturonic acid is polycationic biopolymer. The electrostatic interaction in these biopolymers leads to complex formation in solution. This polyelectrolyte complex scaffold maintains its structure under aqueous condition. Further analysis using atomic force microscopy suggests that the polyelectrolyte fibers are made of nanoparticles. These nanoparticles self assemble and form fibrous structures. The size of polyelectrolyte nanoparticles was in the range of 50-60 nm. Biocompatibility studies were also performed on these scaffolds using human osteoblasts cells. The osteoblasts cells did not attach on pure chitosan/polygalacturonic scaffolds. The osteoblast cells tend to form cell balls over these scaffolds. However, the chitosan/polygalacturonic acid/hydroxyapatite scaffolds exhibit excellent cell attachment, proliferation and differentiation. In this work, we also describe the results of synthesis and biocompatibility studies of polyelectrolyte fibers synthesized using electrospinning methods.
MM6.24
Biocompatibility Study of poly (ε-caprolactone)/Hydroxyapatite Nanocomposites. Rohit Khanna, Kalpana S Katti and Dinesh R. Katti; Civil Engineering, North Dakota State University, Fargo, North Dakota.
In recent years, polymer nanocomposites are being considered as ideal materials for making scaffolds for bone tissue engineering, due to combination of their excellent mechanical properties and biocompatibility. The primary objective of present work is to evaluate the effect of surface-modified hydroxyapatite (HAP) nanoparticles on biocompatibility of a nanocomposite consisting of poly(ε-caprolactone) (PCL) and HAP. Surface-modified HAP nanoparticles were prepared by mineralizing HAP in presence of PAAc (Polyacrylic acid). In exsitu HAP/PCL composite, the HAP nanoparticles are well-dispersed in the PCL matrix, where as, insitu HAP/PCL composite processed using surface-modified HAP, contains HAP agglomerates, which gives heterogeneity to the microstructure. In the prior work, we investigated the bioactivity of these composites in simulated body fluid, and insitu HAP/PCL composite showed better biocompatibility than exsitu HAP/PCL composite. Our on-going work attempts to evaluate the influence of different surfaces (PCL, PCL/HAP composites) on adhesion and proliferation of osteoblast cells. Our experimental results indicate that insitu HAP/PCL composites provide more favorable environment for protein adsorption as compared with unmodified HAP (exsitu HAP/PCL nanocomposite).
MM6.25
Enzymatic Synthesis of Amorphous Calcium Phosphate-Chitosan Nanocomposites and its Processing into Hierarchical Structures. Francisco delMonte, Maria C. Gutierrez and Maria L. Ferrer; Institute of Materials Science at Madrid, Spanish Research Council, Madrid, Spain.
Biomineralization offers the opportunity to produce highly organized nanocomposite structures, controlling specific architectures over extended length scales for a wide range of inorganic materials. Enzymatically assisted routes also offer the possibility to synthesize a number of materials with excellent control on the structural organization. In particular, HA precursors and different calcium carbonate precipitates could be obtained in solutions by enzyme-catalyzed decomposition of urea by urease. Furthermore, the gradual generation of base provided by urea hydrolysis has recently been used for the preparation of monolithic and homogeneous chitosan hydrogels. The homogeneous pH modulation besides the low temperature used for urea hydrolysis allow for the achievement of CHI hydrogels with a homogeneous 3D network structure with superior biotechnological performance than chitosan solutions gelled by neutralization with alkaline solutions, gaseous NH3 or dialysis. Here in, we applied the urease assisted hydrolysis of urea for the preparation of nanocomposites (of turbid appearance) based on calcium phosphate precipitates and CHI hydrogel (see Scheme I in downloaded file). The base generated by urea hydrolysis promoted both CHI gelation and calcium phosphate precipitation at biological temperatures (~37 degrees C). Otherwise (e.g., urea hydrolysis by thermal decomposition at 90 degrees C), CHI would undergo partial decomposition. Macroporous scaffolds (e.g.;, hierarchically organized) were obtained by a cryogenic process (named ISISA, ice segregation induced self-assembly) that simply consist on the unidirectional freezing (at -196 degrees C) of the hydrogel nanocomposites. Upon freezing, the ice formation (hexagonal form) causes every solute originally dispersed in the hydrogel to be segregated from the ice phase. After freeze-drying, the resulting hierarchical structures consists on well aligned micrometer-sized pores in the freezing direction corresponding to the empty areas where ice crystals originally resided, being the macrostructure supported by the matter (e.g., calcium phosphate nanoparticles dispersed within CHI matrix) accumulated between adjacent ice crystals. The excellent control on ice crystals formation, ice segregation matter and matter self-assembly between adjacent ice crystals allows ISISA for unique tailoring of the final macrostructural features of the resulting scaffolds. Thus, figure 1 (see at downloaded file) shows the porous channels of up to 90 micrometers that can be simply obtained by using different freezing rates in the application of the ISISA process to the hybrid hydrogel. The calcium phosphate nanoparticles entrapped within the CHI scaffold are identified as amorphous calcium phosphate as revealed by TEM, XRD and NMR experiments (see figure 1, right, at downloaded file).
MM6.26
Multilayer Composite Scaffolds: Combining Natural and Synthetic Polymers for Tissue Regeneration. Benjamin J. Lawrence1, Eric L. Maase1, Hsueh-Kung Lin2,3 and Sundararajan V. Madihally1; 1Chemical Engineering, Oklahoma State University, Stillwater, Oklahoma; 2Department of Urology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma; 3Department of Veterans Affairs Medical Center, Oklahoma City, Oklahoma.
Small intestinal submucosa (SIS) has generated immense interest in various tissue engineering applications due to its diverse favorable properties. However, as a natural matrix SIS has sample to sample heterogeneity issues which cause problems for industrial preparation [1]. As such, this study explored creation and characteristics of a composite polymer matrix, composed of both natural and synthetic polymers, that mimics the physical properties of SIS. Natural polymers (gelatin/chitosan, etc) have superior biological properties but they degrade slowly and lack physical strength. Synthetic polymers (PLA, PGA, PLGA, etc) have strength, elasticity, and tunable degradation properties but they lack biological activity. Therefore, three dimensional composite structures were developed by sandwiching an amorphous 50:50 PLGA film between two porous chitosan matrices. The outer chitosan layers provide biological activity, and the inner PLGA layer provides mechanical strength. PLGA films were mechanically perforated and porous chitosan matrix was formed sequentially on each side by controlled rate freezing and lyophilization. Scanning electron microscopy (SEM) and permeability studies confirmed that chitosan fills the perforations of PLGA membrane. Scaffolds showed a layered microarchitecture and remain largely impermeable to urea over an eight hour period. PLGA (19, 75, and 160 kDa) and composite matrices were analyzed for tensile strength which showed that composite matrices formed using 160 kDa PLGA had break stress greater then SIS (~4.5MPa). The composite material’s degradation characteristics over eight weeks in phosphate buffered saline solution containing 10 mg/L lysozyme were analyzed for weight loss. Additionally the molecular weight change of PLGA within the scaffold was analyzed by gel permeation chromatography. These results showed a 50% decrease in total weight and an 80% decrease in PLGA molecular weight. Cellular adhesion and actin distribution of two different cell types, canine smooth muscle cells and mouse embryonic fibroblasts, were evaluated by scanning electron microscopy and actin staining. Cells showed their typical spindle shape of the 3-D composite structures. Redistribution of actin fibers was also observed on 3-D chitosan matrices. In summary, the multilayer composite structure accentuates strengths of each compartment while minimizing their weaknesses and shows promise as a tissue engineering material. 1.Raghavan D, Kropp BP, Lin H-K, Zhang Y, Cowan R, Madihally SV. Physical Characteristics Of Small Intestinal Submucosa Scaffolds Are Location-Dependent. J. Biomedical Materials Research-Part A. 73A(1):90-9, 2005
MM6.27
In vitro Biomineralization Induced by Self-assembled Extracellular Matrix Proteins. Xiaolan Ba1, Yizhi Meng2, Yishu Huang3, Seo Young Kwak4, Shouren Ge1, Yixian Qin2, Elaine DiMasi4, Helga Fueredi-Milhofer5, Nadine Pernodet1 and Miriam Rafailovich1; 1Materials Sciences and Engineering, SUNY-Stony Brook, Stony Brook, New York; 2Biomedical Engineering, SUNY-Stony Brook, Stony Brook, New York; 3Ward Melville Highschool, Setauket, New York; 4National Synchrotron Light Source, Brookhaven National Laboratory, upton, New York; 5Chemistry, The Hebrew University, Jerusalem, Israel.
Extracellular matrix (ECM) proteins play an essential role during biomineralization in bone and engineered tissues. In a previous study [1], we showed that calcite preferentially nucleated on pure elastin fibers. However, the actual cellular ECM fibers are composed of a combination of proteins, primarily collagen, fibronectin and some elastin. Here we follow the calcium carbonate- and calcium phosphate- mineralization process in vitro when these ECM proteins are combined and determine the differences between these proteins in the biomineralization process. The surface morphology and mechanical properties of the protein fibers during the early stages were probed by atomic force microscopy (AFM) and shear modulation force microscopy (SMFM). The nucleation of the mineral crystals on the protein matrices was investigated by scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray diffraction (XRD) and optical microscopy. Preliminary data showed that the moduli of all protein fibers increased at the early stages, with collagen having the largest increase in supersaturated calcium bicarbonate solution. In metastable calcium phosphate solutions the modulus of the mixed elastin-fibronectin fibres increased to a greater extent than the moduli of the fibers composed of the single proteins. Longer exposure in the mineral solutions led to the formation of crystals templated along the self-assembled fiber structures.
Chair: Molly Stevens
Wednesday Morning, November 28, 2007
Room 210 (Hynes)
8:30 AM *MM7.1
Assembling Nanocomponents One by One with DNA. Hanadi Sleiman and Faisal Aldaye; Chemistry, McGill University, Montreal, Quebec, Canada.
A central challenge in nanoscience is the organization of functional components according to a deliberately designed pattern, and the ability to modify this pattern at will. Our research group has been examining the use of branched DNA molecules containing organic and inorganic vertices, as programmed templates to address the above goal. Specifically, we show a new method to label gold nanoparticles with DNA molecules, which serve to direct their organization into a discrete, cyclic hexagon, in a sequential and selective manner (Angew. Chem., 2006, 45, 2204). We also report a straightforward method, which uses a small number of dynamic DNA templates, to selectively organize gold nanoparticles into libraries of discrete and well-defined structures. This approach not only provides the ability to finely control the geometry of the assembly, and the precise position of each nanoparticle, but it also allows the modification and tuning of these structural features after the assembly. As such, the resulting nanoparticle groupings can undergo structural switching and write/erase functions with specific external agents. Access to libraries of precisely positioned particle groupings will allow for the systematic examination of their optical, electronic and catalytic properties as a function of structure, and will lead to advances in the use of these particles as components of nanoelectronic and nanophotonic circuitry, as plasmonic tools, and SERS substrates (J. Am. Chem. Soc., 2007, 129, 4130). Finally, we describe the first example of the use of a small molecule template to access a single DNA nanostructure from a library of multiple assemblies. By adding a guest molecule, an equilibrating library containing a DNA dimer, square, hexagon, etc. converges quantitatively into a DNA square. We apply this approach to predictably generate periodic 1-D DNA fibers extending over tens of microns, using two symmetrical DNA building blocks that otherwise assemble into ill-defined oligomeric networks. This opens the door the development of new, highly simplified methods which use a small number of building blocks and guest templates to access DNA nanostructures of high complexity.
9:00 AM MM7.2
Manipulating Assembly Kinetics and Interparticle Interactions in DNA-Nanoparticle Systems. Mathew M. Maye1, Dmytro Nykypanchuk1, Daniel van der Lelie2 and Oleg Gang1; 1Center for Functional Nanomaterials, Brookhaven National Laboratory, Upton, New York; 2Biology Department, Brookhaven National Laboratory, Upton, New York.
The development of DNA-based methodologies for the biomimetic assembly of nanomaterials has proven to be an attractive protocol due to the addressable nature of DNA controlled interactions. Using DNA-capped nanoparticles, we have recently been exploring approaches which utilize the tunable structural properties of DNA as a method towards tailoring nanoparticle self-assembly. By manipulating the rigidity of the nanoparticles DNA-capping by hybridization, we are able to selectively extend linker segments away from nanoparticle interfaces, where they are known to coil and coordinate. As a result, we have observed tunable reactivity and assembly kinetics. In addition, by controlling the composition and quantity of the DNA-capping we are able to regulate interparticle interaction energies, which allows for further regulation of assembly kinetics, relative aggregate size, and interparticle microstructure. Using an array of characterization techniques including; UV-vis, DLS, and SAXS with synchrotron radiation, valuable insights relating assembly morphology and interparticle spatial properties to DNA structure and reactivity have been obtained. These findings may aid in the design and construction of increasingly complex bio-inspired systems with desirable plasmonic and optoelectronic properties.
9:15 AM *MM7.3
Using DNA to Organize Nanoparticles and Functional DNA Units. Nadrian C. Seeman, Chemistry, New York University, New York, New York.
Structural DNA nanotechnology combines branched DNA motifs with cohesive ends to form objects, lattices and devices. It has been possible for many years to produce 2D arrays from a variety of DNA motifs that tile the plane. One of the chief goals of this approach is not merely to produce DNA arrangements, but to organize functional species. Here we report three examples of using DNA tiles to organize other materials. In the first case, we have developed a cassette that incorporates the robust sequence-dependent PX-JX2 nanomechanical device. We have used eight different three-domain motifs (TX) connected 1-3 to form a lattice containing gaps that are flanked by sticky ends. The cassette contains sticky ends complementary to those in the lattice, enabling us to insert the cassette into the lattice. The cassette also contains a robot arm whose position switches with the state of the device. We demonstrate by atomic force microscopy (AFM) that the device is functional following insertion. We are able to observe the switching of the arm between states within the context of the lattice. This system opens the door to a variety of multi-state nanomechanical systems. In the second case, we have attached a DNAzyme to a two-domain (DX) tile. This motif is then incorporated into a four-tile 2D DNA lattice, creating a pattern with stripes separated by 32 nm; every other stripe is a consequence of the presence of the DNAzyme. The DNAzyme cleaves itself in the presence of cupric cations, and we demonstrate this activity by the removal of the stripes due to the DNAzyme, leaving a pattern with stripes separated by 64 nm; the striped patterns are visualized by AFM. In principle, this system can lead to a 'developmental' pathway, by employing a variety of different DNAzymes that can have sequential impact on the pattern. In a third case, we have developed a robust 3-space-spanning motif, the 3D-DX triangle, held together by sticky-ended double cohesion. We use two of the propagation directions to form the 2D array, and then use the third direction as the site to attach a metallic nanoparticle. One strand of DNA is attached to the nanoparticle, and this strand is a strand that is an inherent part of the 3D-DX motif. By using two different DNA tiles, and two differently-sized nanoparticles, we can make uniform, gapped or checkerboard patterns, which are visualized by transmission electron microscopy. There is no obvious limit to the complexity of the nanoparticle pattern that can be formed. This research supported by NIGMS, NSF, ARO and the W.M. Keck Foundation.
10:15 AM *MM7.4
DNA Based Self-assembly of Nanostructures. Hao Yan, Arizona State University, Tempe, Arizona.
In recent years, structural DNA nanotechnology has fulfilled its promise for self-assembling both periodic and complex nanostructures. One of the original goals of this technology was to use self-assembled DNA nanostructures as scaffolds for directed molecular assembly. Here we present our recent progress toward this goal and discuss our recent efforts in DNA directed self-assembly of protein and metallic nanoparticle nanoarrarys.
10:45 AM MM7.5
Kinetics and Microscopic Structure of DNA Guided 2D and 3D Assembly of Nanoparticles. Oleg Gang1, Dmytro Nykypanchuk1, Mathew Maye1 and Daniel van der Lelie2; 1Center for Functional Nanomaterials, Brookhaven National Laboratory, Upton, New York; 2Biology Department, Brookhaven National Laboratory, Upton, New York.
An incorporation of DNA in nano-object design provides a unique opportunity to establish highly selective interactions between the components of nanosystems. In addition, the behavior of DNA in unusual geometries, cooperative phenomena related to their “crowding” on particles, and the interplay of diverse interactions can lead to new interesting phenomena. For 2D systems, the use of DNA’s hybridization furnishes a reversible, chemically weak and a highly selective way to assemble nanoparticles on surfaces. We studied in-situ the assembly kinetics of DNA-capped nanoparticles on surfaces as well as the structural changes with temperature in the resulting 2D DNA/nanoparticle system. Using high energy x-ray reflectivity, the microscopic structure of the 2D particle assembly was probed directly, while AFM and SEM were used to confirm a local particle’s arrangement. The observed changes in the DNA/nanoparticle array layer, both below and above the DNA melting temperature, reveal an evolution of particle-surface distances and surface coverages. These results provide insights into oligonucleotide chain conformations and energetics. For 3D systems, using small angle x-ray scattering, we investigated the correlation between internal structure of particle assemblies and DNA design. The recent results on pathways towards ordered phase formation will be discussed.
11:00 AM *MM7.6
Self-Assembly of DNA Nanostructures. Chengde Mao, chemistry, Purdue University, West Lafayette, Indiana.
Structural control at the nanometer scale is the key to the development of nanoelectronic devices. Self-assembly guided by information-containing-molecules is one promising approach to achieve this goal. Among many molecular systems, DNA stands out as one of the best choices. Because DNA is the universal genetic materials, its structures and physical/chemical properties have been extensively studied, and a rich array of manipulation tools have been developed. DNA has excellent molecular recognition capability and its structures can be precisely predicted. Branched DNA motifs have also been constructed. Combining all these factors together, DNA-based nanostructures have been rapidly developed. Here, the discussion will focus on the recent development of DNA nanostructures in my group. We believe that this work will help the integration of individual nano-components into large, functional architectures.
11:30 AM *MM7.7
Multilayered Films for the Delivery of DNA: Surface-Mediated Cell Transfection and New Approaches to Tunable Control over Film Erosion. David M. Lynn, Department of Chemical and Biological Engineering, University of Wisconsin - Madison, Madison, Wisconsin.
Layer-by-layer fabrication provides nanometer-scale control over the thicknesses and compositions of multilayered polymer films and has been investigated broadly in the context of potential biomedical applications. The ability to incorporate layers of biological polyelectrolytes into these materials renders these materials attractive as platforms for the controlled release of DNA, proteins, or other macromolecular therapeutics. We have added functionality to this class of thin films by designing and incorporating synthetic polyamines that can be degraded in aqueous media and promote the disruption of these films in physiologically relevant environments. We have demonstrated, for example, that films 100 nm thick can be fabricated from alternating layers of degradable polyamines and plasmid DNA and that these assemblies can be used to direct the surface-mediated transfection of cells. Layer-by-layer methods offer several potential practical advantages relative to conventional methods for the encapsulation and release of DNA from surfaces. First, these methods are entirely aqueous and permit precise control over the amount of DNA presented at a surface (by control over the numbers of layers deposited). Second, the ability to define the relative locations of multiple different layers in these assemblies presents opportunities to design films that provide control the timing and the sequence of the release of multiple different genes. Finally, the commingling of the DNA in these materials with alternating layers of polyamines (a class of materials used broadly to transfect cells) forms the basis of a systematic approach to the design of thin films that promote the internalization and processing of DNA by cells. This presentation will describe recent work on the fabrication of ultrathin films that provide spatial and temporal control over the release of DNA and other macromolecular therapeutics from surfaces. The influence and importance of polymer structure and film architecture on physical erosion and controlled release profiles will be discussed with an emphasis on methods and approaches that can be used to 1) provide broad and tunable control over release, 2) fabricate films that provide sophisticated control over the release of different multiple DNA constructs, and 3) enhance levels of surface-mediated cell transfection. The ‘toolkit’ of different polymers identified by these investigations makes possible the fabrication of ultrathin films that release transcriptionally active DNA for periods of several hours, several days, several weeks, or even several months depending on the structure of the polymer used. We will also describe the results of recent experiments that demonstrate the feasibility of using intravascular stents coated with these materials to promote localized transfection of vascular tissue in vivo. Finally, new approaches to the controlled disruption of these ionically crosslinked materials in aqueous media will be discussed.
Chair: Jeff Hartgerink
Wednesday Afternoon, November 28, 2007
Room 210 (Hynes)
1:30 PM *MM8.1
Self-Assembled Quantum Dot-Bioconjugates: Characterization and Use for Sensing and Probing Cellular Processes. Hedi Mattoussi1, Thomas Pons1, Kim E. Sapsford2 and Igor L. Medintz2; 1Optical Sciences Division, Code 5611, Naval Research Laboratory, Washington, District of Columbia; 2Center for Bio/Molecular Science and Engineering, U.S. Naval Research Laboratory, Washington, District of Columbia.
Colloidal quantum dots (QDs) have several unique optical and spectroscopic properties that are appealing for use in a variety of biological applications, ranging from immunoassay development to in vivo imaging. We have developed a conjugation technique based on non-covalent self-assembly to couple polyhistidine-appended biomolecules with hydrophilic CdSe-ZnS core-shell QDs. In this presentation, two particular aspects of such self-assembled conjugates will be discussed. In the first, we outline the advantages offered by QDs as exciton donors in developing Förster resonance energy transfer (FRET)-based sensing assemblies for the detection of specific targets. We will also investigate the use of our self-assembled QD conjugates to probe interactions at live cell membranes. In the second, we present a molecular characterization of the interactions driving the self-assembly between hydrophilic QDs and a series of proteins and peptides appended with various length polyhistidine tags, using either surface-immobilized QDs or freely diffusing QDs and proteins/peptides in solution. In particular, we discuss the derivation of a few key parameters, including the association and dissociation rates (kon and koff), and the dependence of those parameters on the polyhistidine size and the lateral extension of the hydrophilic ligands on the QD surface.
2:00 PM MM8.2
Hierarchical Assemblies of Weak Hydrogen Bonds form Ultra-strong Filaments by Nano-confinement. Sinan Keten and Markus J. Buehler; Department of Civil and Environmental Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts.
Protein materials with high beta-sheet content exhibit superior mechanical properties, as they are highly elastic, yet strong, despite predominantly weak interstrand hydrogen bonding. Based on theoretical analyses and molecular dynamics simulations, we report a novel, universally valid structure-property relationship used here to explain that the origin of the high strength and large elasticity of these super-fibres lies within hierarchical structures formed by weak hydrogen bonds. Our study reveals that in beta-sheets, at most five hydrogen bonds can break simultaneously, regardless of the strand length, limiting its strength to several hundred pN. This intrinsic strength limitation can be overcome by addition of entropic domains between short beta-strands with each five hydrogen bonds, thus enabling the formation of a template for large beta-sheet crystals with strengths many times larger than those of a single beta-strand. We further show that beta-sheet crystals consisting of stacks of strands must be confined to several nanometers to achieve robust resistance to shear deformation. The characteristic length scales predicted here correspond to those observed in spider silk and other structural proteins, and thus explain how these materials have the capacity for such extreme strengths. Our discovery elucidates a counter-intuitive concept of how strong materials can be created by utilizing a hierarchical composite of weak molecular interactions and entropic elasticity confined to characteristic dimensions.
2:15 PM MM8.3
Exploiting the Material Dependence of Magnetic Field Heating of Nanoparticles. Kimberly Hamad-Schifferli, Biological and Mechanical Engineering, MIT, Cambridge, Massachusetts.
Heating of magnetic nanoparticles by magnetic has been utilized for many applications, including cancer therapy, tumor killing, hyperthermia. Its use in drug delivery applications could be particularly useful because administering of the field is possible in a time-controllable manner We describe here use of nanoparticles in heating thermosensitive polymers that undergo dramatic volume changes upon heating. 12 nm Fe3O4 nanoparticles are incorporated into poly N-isopropyl acrylamide along with a molecule to be released. We apply alternating magnetic fields and can observe a ~20% volume decrease of the polymer. We also demonstrate release of the molecule into the surroundings. Heating characteristics of the nanoparticles in the polymer are quantified, in particular the specific absorption rate (SAR) of the nanoparticles. Effects of varying nanoparticle material and loading are also explored for independent release of two different molecules. These results could have significant ramifications on drug delivery in addition to the development of externally controlled polymer actuators.
2:30 PM MM8.4
Multimodal Functionalized Carbon Nanotubes for Targeted Self-assembly of Therapeutic Constructs. Carlos Hipolito Villa1, Michael R. McDevitt1, Diego A. Rey2, Juana Mendenhall3, Ian Ahearn4, Freddy E. Escorcia1, Carl A. Batt3,2, Mark R. Philips4 and David A. Scheinberg1; 1Molecular Pharmacology and Chemistry, Memorial Sloan Kettering Cancer Center, New York, New York; 2Biomedical Engineering, Cornell University, Ithaca, New York; 3Food Science, Cornell University, Ithaca, New York; 4Cell Biology and Pharmacology, NYU School of Medicine, New York, New York.
Carbon nanotubes have generated significant interest for their potential in novel therapeutic constructs because of their unique properties. The nanoscale of these materials opens the opportunity to target their site-specific assembly into more complex and multifunctional structures in a biological setting. In addition, because of the nanotubes’ large available surface area, a high degree of multivalency can be achieved, affording flexibility in assembly design. In this study, we chemically modified single wall carbon nanotubes (SWNT) to bear biological moieties that enable their targeted self-assembly. Fluorescein (FAM) labeled, backbone-modified single-stranded DNA oligonucleotides (ODN) are used as templates for subsequent annealing of complimentary strands that can be bound to a variety of substrates such as therapeutic isotope generators. These ODN are covalently bound to SWNT via carboxylic acid functionalities that are generated via nitric acid oxidation in the normal purification procedure. Using carbodiimide chemistry, this approach yields 10-20 ODN functionalities per 200 nm length of SWNT material. To enable specific targeting of the constructs, multiple copies of an αVβ3 integrin-targeting RGD peptide were bound to nanotube sidewalls through primary amines that are generated via azomethine ylide chemistry. The αVβ3 integrin is a well-known target for anticancer therapies and is primarly exposed on neovasculature, a particulary interesting site for in vivo assembly. The amine functionalization also serves to fully solubilize the nanotube materials for their use with biologics. The DNA-modified targeted nanotube construct, SWNT(RGD)(ODN-FAM), was characterized via atomic force microscopy, transmission electron microscopy, and a variety of spectroscopic techniques. The results show that we have successfully synthesized a multivalent, multifunctional nanotube platform capable of serving as an anchor for self-assembly. This anchor construct was demonstrated to specifically target αVβ3 expressing endothetial cells (HUVEC) in comparison to a non-specific peptide control, SWNT(RAD)(ODN-FAM), via confocal fluorescence microscopy. The ability to serve as a template for self-assembly was shown through spectroscopic demonstration of DNA annealing of complimentary ODN (cODN) onto the SWNT(RGD)(ODN-FAM). Further, through the use of Cy3 chromophore-labeled cODN, we demonstrated assembly through fluorescence resonance energy transfer (FRET) between the chromophores on complimentary sequences. The findings suggest that these nanoconstructs have potential as prototypes for in vivo self-assembly.
3:15 PM MM8.5
Abstract Withdrawn
3:30 PM MM8.6
Bio-Inspired Growth of Crystals: Hydrogels and Self-Assembled Monolayers. Lara Ann Estroff and Hanying Li; Dept. Materials Science and Engineering, Cornell University, Ithaca, New York.
The design of artificial models of biomineralization has resulted in the union of inorganic materials research and supramolecular organic chemistry. To begin unraveling the biological mechanisms of mineralization, and, in doing so, create new types of materials, there is interest in the design of supramolecular, organic assemblies to alter the growth of inorganic crystals. I will describe the application of a matrix composed of an agarose hydrogel on top of a carboxylate-terminated self-assembled monolayer (SAM) to control the nucleation and growth of calcite crystals. The design of this matrix is based upon examples from biomineralization in which hydrogels are coupled with functionalized, organic surfaces to control, simultaneously, crystal morphology and orientation. In the synthetic system, calcite crystals nucleate from the (012) plane (the same plane that is observed in solution growth). The aspect ratio (length/width) of the crystals decreases from 2.1 ± 0.22 in solution to 1.2 ± 0.04 in a 3 wt% agarose gel. One possible explanation for the change in morphology is the occlusion of gel fibers inside of the crystals during the growth process. In this work, we characterize the internal structure of calcite crystals grown in an agarose hydrogel and show that the gel-grown calcite crystals, like biogenic calcite crystals, incorporate the organic matrix. The gel fibers are uniformly distributed within the crystals, without changing the regular rhombohedral morphology of calcite crystals. Etching of the gel-grown crystals with distilled water reveals an interpenetrating network of gel fibers and crystalline material. TEM examination of microtomed slices shows directly the porous internal structures of the crystals with pores of 60-364 nm. Both electron-back scattered diffraction (EBSD) and selected area electron diffraction (SAED) demonstrate that the structures are single crystals of calcite. We are currently investigating the mechanism of occlusion and the structure and mechanical properties of the composite material. We suggest that in order to observe gel incorporation, a sufficient supersaturation is required to compensate for the surface energy of the internal pores and an adequate gel robustness is needed to balance the crystallization pressure. This work provides an in vitro platform to study the biomineralization of calcite and a potential approach to fabricate single crystals coupled with a large surface area.
3:45 PM MM8.7
Geckel: A Dry/Wet Adhesive Inspired by Mussel and Gecko Adhesion. Haeshin Lee1 and Phillip B Messersmith1,2,3; 1Biomedical Engineering, Northwestern University, Evanston, Illinois; 2Material Science and Engineering, Northwestern University, Evanston, Illinois; 3Institute for BioNanotechnology in Medicine, Northwestern University, Evanston, Illinois.
The adhesive strategy of the gecko relies on foot pads composed of specialized keratinous foot-hairs called setae, which are subdivided into terminal spatulae of dimensions approximately 200nm(1). The gecko adhesion has been explained by contributions of van der Waals and capillary forces(1,2). For this reason, the magnitude of adhesion force varies as a function of humidity but is decreased dramatically under water. A celebrated biological model for wet adhesion is a mussel, which is well recognized for its ability to adhere to wet surfaces. Mussels secrete byssal threads with adhesion pads at termini, in which unusual amino acid 3,4-dihydroxy-L-phenylalanine (DOPA) is found. DOPA is believed to be responsible for wet adhesion on solid substrates(3,4). In this abstract, we describe a new class of hybrid adhesive called ‘Geckel’, inspired by adhesive strategies of geckos and mussels(5) as well as recent progress. Geckel consists of an array of nanofabricated polymer pillars as a functional mimic of gecko’s feet onto which a mussel-mimetic polymer, p(DMA-co-MEA) e.g. poly(dopaminemethacrylamide-co-methoxyethylacrylate), was coated. Geckel showed several interesting adhesive properties which have never been demonstrated by other existing adhesives. First, Geckel showed a strong adhesion under both dry and wet conditions. In particular, it exhibited superior wet adhesion to current adhesives due to the mussel-mimetic polymer coating. Second, the adhesion is completely reversible both in air and under water, maintaining its adhesiveness over 1,000 contact cycles. Third, the reversible wet adhesion was effective to a variety of substrates (Si3N4, Au, and TiO2). To increase dry/wet adhesion power of Geckel, techniques that allow one to fabricate high density organic nanopillars are necessary. However, it is difficult to fabricate high- density patterns using electron-beam lithography because of so-called ‘proximity effect’. It is caused by electron scattering in resists and substrates, influencing the regions adjacent to those exposed by e-beam(6). Thus, we fabricated ‘mushroom’ like PDMS pillar to avoid the proximity effect with maximizing contact areas. References 1.K. Autumn et al., Proc. Nat. Acad. Sci. USA 99, 12252 (2002). 2.G. Huber et al., Proc. Nat. Acad. Sci. USA 102, 16293 (2005). 3.H. Lee, N. F. Scherer, P. B. Messersmith., Proc. Natl. Acad. Sci. USA 103, 12999 (2006). 4.J. H. Waite, N. H. Andersen, S. Jewhurst, C. Sun, J. Adhesion 81, 1 (2005). 5.H. Lee, B. P. Lee, P. B. Messersmith., Nature In press (2007). 6.T. H. P. Chang, J. Vac. Sci. Tech. 12, 1271 (1975).
4:00 PM MM8.8
Interfaces Involving Biomolecules and Inorganic Materials: The Solid State NMR Point of View. Christian Bonhomme1, Christel Gervais1, Florence Babonneau1, Michel Wong Chi Man2, Joel Moreau2, Bruno Alonso2, Satoshi Hayakawa3 and Akiyoshi Osaka3; 1universite P et M Curie, Paris, France; 2ENSCM, Montpellier, France; 3Okayama university, okayama, Japan.
The synthesis of structured hybrid and bioinspired materials is crucial. It is mainly based on molecular assemblies including organic or biological molecules [1]. Generally, H-bonding plays a prominent role in bridging organic entities. The inorganic component is based mainly on silica derivatives obtained by sol-gel hydrolysis and condensation reactions. Obviously, the properties of the obtained materials are strongly related to the interface between the organic and inorganic components. As a matter of fact, the interface remains difficult to characterize from a spectroscopic point of view and solid state NMR appears as a valuable tool of investigation. Materials based on ureidopyrimidinone (UPY) dimmers and Adenine (A) / Thymine (T) derivatives were synthesized and characterized by advanced solid state NMR techniques. Silylated UPY molecules were used as model compounds leading to structured organic-inorganic materials. High resolution 1H solid state NMR has been extensively used for the in-depth description of the H-bond networks, including very fast MAS experiments at very high field (for dramatic increase in resolution) and DQ (double quantum) recoupling experiments [2]. The chemical nature of the organic-inorganic interface has been illuminated by such techniques. In particular, it had been demonstrated that H-bond networks were preserved during sol-gel reactions and were comparable to those observed in the UPY crystalline precursors. Solid state NMR appears also as a valuable tool of investigation for the fine description of substituted hydroxyapatite (HAp) structures. Such substitutions are of prime importance for bioactivity. Dipolar (D) and scalar (J) techniques were implemented for the study of the spatial and chemical connectivities involving the 31P and 29Si/13C nuclei within the HAp structure [3]. By using fully enriched 13CO32- groups, 2D 13C/31P experiments were performed and the complete assignment of the 13C resonances (A, B and A/B sites) were obtained by using DFT calculations and ab initio estimation of all NMR parameters [4]. [1] a) J. J. E. Moreau et al., Angew. Chem., 43, 203, 2004. b) J. J. E. Moreau et al. New. J. Chem., 29, 653, 2005. [2] C. Bonhomme et al., Account Chem. Res., 2007, available online. [3] a) C. Lejeune, C. Bonhomme et al., Solid State NMR, 27, 242, 2005. b) C. Coelho, C. Bonhomme et al., J. Magn. Reson., 179, 114, 2006. [4] C. Gervais, C. Bonhomme et al., J. Phys. Chem. A, 109, 6960, 2005.
4:15 PM MM8.9
Protein Detection with Oscillating DNA Interfaces. Ulrich Rant1, Kenji Arinaga2,1, Erika Pringsheim1, Jelena Knezevic1, Shozo Fujita2, Naoki Yokoyama2, Marc Tornow3 and Gerhard Abstreiter1; 1Walter Schottky Institute, Technical University Munich, Garching, Germany; 2Fujitsu Laboratories Ltd., Atsugi, Japan; 3Technical University Braunschweig, Braunschweig, Germany.
Conventional protein assays using affinity probes which are immobilized on solid supports yield mere information whether target proteins (e.g. antibodies or enzymes) have been captured from solution, i.e., if target-probe recognition occurred. On the other hand, methods like electrophoresis, fluorescence-correlation-spectroscopy, or light scattering are used to characterize the size, shape, and charge of proteins based on the molecules’ mobility in the liquid/gel phase. We present a pioneering detection scheme that combines the advantages of both approaches. It yields binding affinity data and permits the differentiation of proteins according to their hydrodynamic friction and effective charge. For the first time, probe-target complexes are identified by their “kinetic fingerprints” using surface-bound probes. Moreover, the technique features a simple architecture of the bio-interface, a chip-compatible format, and does not require the labeling of target proteins. An electrically switchable DNA layer constitutes the sensor’s central functional element. DNA oligomers are chemically tethered to a gold electrode at one end. Their distal ends are modified with fluorescent- and affinity-labels. The former are used to monitor the conformation/orientation of the DNA relative to gold substrate in real-time, taking advantage of energy transfer that quenches the fluorophore emission as it approaches the surface. The affinity label serves as a specific capture probe for target proteins. The DNA molecules are driven to oscillate between “upright” and “lying” conformations by applying AC potentials (0.2 Hz - 200 kHz) to the supporting gold electrode. We describe the underlying principles of the DNA oscillation and explain how the dynamics of the switching process can be inferred from frequency response measurements. We demonstrate that the binding of proteins to the affinity-labels on top of the DNA oligomers (i) substantially alters the switching amplitude and (ii) slows the oscillation significantly. Both parameters are used to monitor target - probe recognition and provide complementary data. We present binding experiments using several antigen-antibody couples and evaluate quantitative binding data (affinity constants). Further, we discuss the influence of protein charge and size exemplarily for the biotin-avidin system. The different shifts in switching dynamics observed for various target-proteins are compared to the molecules’ hydrodynamic friction, which is determined by dynamic light scattering.
4:30 PM MM8.10
Real-Time Continuous Monitoring of Cocaine in Undiluted Blood Serum using Electrochemical Aptamer Sensors. James S Swensen1, Arica A. Lubin2, Rebecca Y. Lai2, Kevin W. Plaxco1,2 and H. Tom Soh1,3; 1Institute of Collaborative Biotechnology, University of California at Santa Barbara, Santa Barbara, California; 2Department of Mechanical Engineering, University of California at Santa Barbara, Santa Barbara, California; 3Department of Chemistry and Biochemistry, University of California at Santa Barbara, Santa Barbara, California.
The usefulness of a biosensor is determined by such factors as its sensitivity, specificity, speed and its robustness. RNA or DNA aptamers provide an important advantage in that, unlike antibodies, they can be chemically synthesized, are stable in long-term storage, and undergo reversible denaturation, which make them potentially field-portable. Previously, we have demonstrated the electrochemical aptamer-based (E-AB) sensor platform which is comprised of a redox-reporter attached to the sensing aptamers. Upon target binding, a conformational change is induced in the aptamer, altering the efficiency of electron transfer to the electrode, causing a measurable difference in the current to the electrodes to which the aptamers is immobilized. This platform is sensitive, specific, reagentless, reusable, and selective so that it can be used directly in serum, soil, and other complex samples. Typically, the integration of high performance electrochemical biosensors with microfluidic devices is challenging because of the inherent incompatibilities among biomolecular stability, signal transduction/amplification/detection schemes with microfabrication processes. Through the use of modular fabrication architecture, we have fully integrated the E-AB sensor into a continuous -flow, electrochemical detection device, in which we are able to perform in situ cleaning and preparation of the electrodes, as well as the in situ immobilization of probe aptamers. With this microfluidic device, we demonstrate the first, reagentless, continuous, real-time measurement of small molecules (Cocaine) directly in complex solutions. We report the continuous detection of clinically relevant cocaine concentrations ( < 25 µM) in undiluted blood serum, which, using traditional analytical methods, would have required the collection and chromatographic analysis of over 100 individual samples.
4:45 PM MM8.11
Charge Transfer of Locally Addressed Redox Proteins. Dirk Mayer, Florian Schroeper, Oliver Salomon, Daniel Schwaab and Andreas Offenhaeusser; IBN 2, FZ-Juelich GmbH, Juelich, Germany.
Recent work by various groups has pointed out the emerging interest of integrating biomolecules like proteins into electronic junctions. In this regard proteins and in particular redox proteins are of special interest, since they are involved in many redox processes for maintaining vital functions of organisms. These functions are often associated with exciting abilities regarding selective recognition and charge exchange. In this work cytochrome c (cyt c), a heme containing redox protein, was used as model system for studying general aspects of the integratation of proteins into conceptual metal -biomolecule - metal junctions. One basic aspects in bioelectronics (BE) as well as in molecular electronics (ME) is lithography, namely the local addressing of functional components. ME demands precise patterning capabilities with very high resolution, due to the small size of the molecules. In addition, BE demands gentle patterning processes, since biomolecules are fragile and require very specific conditions to keep their functionality, namely physiologic conditions. A very versatile technique attributed to Soft Lithography is Microcontact Printing (µCP). It allows the local addressing of biomolecular inks by a stamping process and thus can be used as lithographic method. In this presentation we will demonstrate the in situ patterning of proteins beyond the limits of photolithography and discuss the influence of the transfer process on the redox activity of cyt c by means of cyclic voltammetry. Furthermore, we investiated the immobilization of the proteins to conjugated and unconjugated linker molecules as well as the electronic properties of the resulting bio-inorganic interface by electrochemical methods. Finally, we report on the fabrication of conceptual metal - protein - metal junctions. We have pursued two general strategies for probing the electrical properties of biomolecules, namely techniques based on scanning probe microscopy and lithographically fabricated pairs of metallic electrodes in a crossbar configuration. The electrical properties of these junctions were studied by performing IV measurements. First information about the mechanism of the electron transport in this junctions could be derived on the basis of the determined tunneling parameter.
Chairs: Darrin Pochan and Vincent Rotello
Wednesday Evening, November 28, 2007
8:00 PM
Exhibition Hall D (Hynes)
MM9.1
Spontaneous Self-Assembly, Functionalization, and Meso-Scale Host-Guest Science of Organic Nanotubes. Naohiro Kameta1, Mitsutoshi Masuda1,2 and Toshimi Shimizu1,2; 1SORST, Japan Science and Technology Agency, Tsukuba, Japan; 2NARC-AIST, Tsukuba, Japan.
Tubular nanoarchitectures composed of carbon, metal, silica, polymers, and amphiphilic molecules have attracted much attention in nanotechnology and biotechnology fields. The organic nanotubes self-assembled from amphiphilic molecules should be not less useful than those mentioned above in terms of their characteristics including mild synthetic process, thermoreversible formation and deformation, tunable inner and outer surfaces, and controllable inner diameters [1]. Especially, the organic nanotubes have high biodegradability and compatibility, because the hydrophilic hollow cylinder functions as a suitable cavity for biomacromolecules, and plays important roles in acting as nano-containers and nano-channels [2]. Herein we report the exclusive self-assembly of the organic nanotubes with controlled inner diameters and surface properties, which were developed as meso-scaled hosts to encapsulate and transport biomacromolecules such as proteins and DNAs. Self-assembly of the bolaamphiphile, N-(2-aminoethyl)-N’-(beta-D-glucopyranosyl)-alkanediamide (1), gave two types of nanotubes with different inner diameters (20 and 80 nm) by optimizing pH conditions in aqueous solutions, the initial molecular packing, and the oligomethylene-spacer length of 1 [3,4]. Molecular packing analysis indicated that both nanotubes have different inner and outer surfaces covered with amino and glucose groups, respectively. To construct optical sensing system for recognition of biomacromolecular guest labeled with fluorescence acceptor, we modified the amino groups on the inner surface of the nanotubes covalently with a fluorescence donor molecule. Monitoring of the fluorescence resonance energy transfer (FRET) using time-laps fluorescence microscope allowed us to detect the encapsulation and nanofluidic behavior of spherical protein ferritin (12 nm wide) in the organic nanochannel (i.d. 80 nm) [5]. The analysis of the time-laps images enabled us to estimate the diffusion constant (D) of the ferritin in the nanotube channel. The D value is remarkably smaller than that of the ferritin in a bulk solution. We also clarified that the surface charge and diameters of the nanotube channels strongly affect the encapsulation behavior toward proteins and DNAs. References [1] T. Shimizu et al., Chem. Rev. 2005, 105, 1401-1443. [2] T. Shimizu, J. Polym. Sci. Part A: Polym. Chem. 2006, 44, 5137-5152. [3] N. Kameta et al., Adv. Mater. 2005, 17, 2732-2736. [4] N. Kameta et al., Langmuir 2007, 23, 4634-4641. [5] N. Kameta et al., Chem. Mater. 2007, ASAP Web release, DOI: 101021/cm070626p.
MM9.2
Fabrication of Nano-scaled Structures Using Genetically Engineered Tobacco Mosaic Virus. Mime Kobayashi1,2, Rikako Tsukamoto1,2, Kazutaka Ishikawa1, Hiromichi Nakagawa1,2, Shigeo Yoshii1,3, Hitoshi Tabata4, Yuichiro Watanabe5 and Ichiro Yamashita1,2,3; 1Materials Science, Nara Institute of Science and Technology, Nara, Japan; 2CREST, Japan Science & Technology Agency, Saitama, Japan; 3ATRL, Matsushita Electric Ind. Co., Ltd., Kyoto, Japan; 4Engineering, The University of Tokyo, Tokyo, Japan; 5Arts & Sciences, The University of Tokyo, Tokyo, Japan.
Tobacco Mosaic Virus (TMV) is a tube-shaped ribonucleoprotein with an inner diameter of 4 nm and 300 nm in length. We have succeeded in making CoPt, CoPt3 and FePt3 nano-scaled wires using TMV as a biotemplate (Table 1; Tsukamoto et al.Chem. Mater. (2007) 19, 2389-2391). The surface of TMV can also be genetically modified by introducing mutations in its coat protein. Electronic potential of inner and outer surfaces of TMV can be easily modified by mutation of amino acids. The technique makes it possible to explore optimal conditions for synthesizing metallic or semi-conducting nano-scaled wires inside TMV. Other Modification includes exposing substrate-specific binding peptide on its surface. Selective arrangement of TMV on a given surface has been tried using those recombinant TMVs. Our method is also useful for bridging the gap between biological and materials scientific approaches. Although pin-point assembly might not be feasible with current technique, our ultimate goal is to supply a nano-scaled structure for possible applications in constructing electronic devices by “biomineralization”. We are currently attempting to make wire-dot-wire structures using TMV and a cage-shaped Ferritin protein as a building block of nanoelectronic devices. This study is partially supported by Leading Project of MEXT, Japan.
MM9.3
Metal Nanoparticles Via Plant Protein Templates. Silke Behrens1, Oded Shoseyov2, Aron Heyman2 and Or Dgany2; 1Institute for Technical Chemistry, Forschungszentrum Karlsruhe, Karlsruhe, Germany; 2The Robert H. Smith Institute of Plant Science and Genetics, The Faculty of Agriculture, The Hebrew University, Rehovot, Israel.
Biological architectures have recently attracted much attention as spacially defined template for fabricating monodisperse nanoparticles. For example, the empty protein capsids of viruses, enzymes, or ferritin have been applied as nanobioreactors to fabricate monodisperse metal and metal oxide particles at the nanometer scale, e.g., paratungstate or decavanadate, iron oxide, Pd, or Co as well as metal nanoshells. Here, we address the specific nucleation of Pd particles by SP1 protein. SP1 is a plant protein (populus tremula) composed of 12 identical monomers self-assembling into an extremely stable ring-shaped structure, 11 nm in diameter, 3 nm heigh and with a central cavity of 3-4 nm. The protein complex displays a remarkable resistance to a variety of extreme conditions, including heat (melting point Tm ~ 107°C), proteases, detergents, chaotropic agents, and organic solvents and, thus, represents an ideal template for further chemical modification such as the manufacturing metal nanostructures. Following a bottom-up approach, we obtain Pd particles in the nanometer size range by in situ reduction of metal salts in the presence of histidine modified SP1. A colloidal solution is formed immediately which is stable over months. Transmission electron microscopy analysis reveals that the particle diameter corresponds to the size of the inner protein cavity. The particles are catalytically active as shown for the hydrogenation of nitrophenol and, thus, could be potentially applied as catalytic bio bar codes. The presented SP1 templating approach provides a great potential for functionalizing nanoparticles with affinity reagents for biomedical applications.
MM9.4
Novel Photoelectrochemical Cell Using Bacterial Light Antenna Structures. Arati Sridharan, Jit Muthuswamy and Vincent B. Pizziconi; Bioengineering, Arizona State University, Tempe, Arizona.
An emerging trend for next generation biophotonic devices is to merge the sensitive and selective nature of biological structures with conventional engineering platforms to attain higher device performance. While naturally-derived materials, such as the photosynthetic apparatus of a plant or the retinal photoreceptor, are known to sustain high phototransduction efficiencies even under low light intensity conditions, efforts to exploit or mimic its biophotonic functionality in an artificial setting have not yet been realized. In order to further investigate the above biohybrid approach, we present here preliminary work on the development of a novel photoelectrochemical cell that utilizes naturally derived bacterial light antenna structures, known as chlorosomes, as photoelectrical transducers. Chlorosomes, derived from the Chloroflexus aurantiacus bacterium, are highly efficient (69%-92%), supramolecular aggregates of bacteriochlorophyll-c molecules that were previously shown to be potential candidates for use in hybrid biophotonic devices. More recently, we have also demonstrated for the first time that chlorosomes are capable of photoelectron transfer in an aqueous environment. In this current study, the main focus of our photoelectrochemical cell is on the biohybrid interfaces, i.e., the chlorosome-electrode interface and the chlorosome-electrolyte interface which were characterized using electrochemical impedance spectroscopy, chronoamperometry, and relevant optical characterization methods. Preliminary results indicate that control electrochemical cells, consisting of indium tin oxide (ITO) substrates immersed in a phosphate buffered saline (PBS) solution, showed intrinsic photoactivity on the order ~20-30 nA at zero bias at a light intensity of ~50 mW/cm2. Similar studies run with ITO-based electrochemical cells immersed in an electrolyte composed of chlorosomes suspended in PBS showed decreased photocurrent changes (~Δ5 nA) under similar conditions. Yet, hybrid electrochemical cells comprised of chlorosomes immobilized onto the ITO substrate yielded enhanced photocurrents with an average increase of ~Δ5 nA upon light excitation, suggesting the chlorosome orientation at the interface in conjunction with other factors may play a critical role in the electron transfer process. In addition, initial results with chlorosome and alternative electrolytes, such as ferricyanide, suggest that electrolytes with low reduction potential may be needed to sustain a regenerative photoresponse. Further studies are needed to elucidate the mechanisms that govern electron transfer at the bioelectronic interface. Once achieved, it will potentially have significant impact on a wide range of practical applications ranging from fuel cell technologies to retinal prosthetics.
MM9.5
Synthesis and Characterization of Multifunctional Self-assembling Dendrimer Construct. Freddy E Escorcia, Molecular Pharmacology and Chemistry, Memorial Sloan-Kettering Cancer Center, New York, New York.
Dendrimers are novel, globular, synthetic nanoparticles whose geometry and readily modifiable surface moieties have spawned considerable interest in their potential as medicinal carriers. These properties could be exploited to create a new class of multivalent, multifunctional devices to achieve tumor targeting and site-specific assembly. Here, we describe the synthesis and characterization of a generation six polyamidoamine dendrimer (DEN) construct containing single-stranded modified-backbone oligonucleotides (ODN) to serve as a scaffold for subsequent specific assembly via complementary oligonucleotide (cODN) annealing. In order to obtain a more robust spectral handle during construct synthesis, DEN were first conjugated to cascade blue chromophore (CB) on their primary surface amines, resulting in approximately one CB per DEN. Subsequently, single stranded Cy3-labeled phosphothiorate oligonucleotide (ODN-Cy3) were covalently coupled to DEN using the sulfo-SMCC biological linker, yielding approximately five ODN per DEN. This new construct, DEN(CB)(ODN-Cy3) was characterized by UV-Vis spectrophotometry and HPLC. DEN(CB)(ODN-Cy3) was incubated with single-stranded fluorescein labeled complementary ODN and DNA annealing was observed spectrophometrically. Additionally, fluorescence resonance energy transfer (FRET) between FRET pairs fluorescein and Cy3 on complementary ODNs provided further evidence of assembly. Because these cODNs could be coupled to a variety of agents such a radioisotope chelates or generators, our results hold promise for self-assembly of therapeutic dendrimer-based devices.
MM9.6
AFM Studies Reveal Multi-stage Pathway of Prion Aggregation Involving Stable Oligomeric Intermediates. Kang Rae Cho1,2, Yu Huang2, Shuiliang Yu3, Man-Sun Sy3 and James J. De Yoreo1; 1Chemistry, Materials and Life Sciences Directorate, Lawrence Livermore National Laboratory, Livermore, California; 2Department of Materials Science and Engineering, University of California, Los Angeles, Los Angeles, California; 3Department of Pathology, Case Western Reserve University, Cleveland, Ohio.
Aggregation of individual proteins into larger scale structures, from which functional properties emerge, is a common architectural element in living tissues. But for some proteins, uncontrolled aggregation is triggered by a misfolding reaction that transforms the secondary structure from mainly alpha-helical to beta-sheet, leading to devastating diseases. Studying the aggregation of these proteins may help to define strategies both for synthetic formation of functional protein aggregates and therapeutic approaches to halting pathogenesis. The prion protein (PrP) is the sole causative agent of a set of neurodegenerative disorders, such as bovine spongiform encephalopathy and Creutzfeld-Jakob disease. The key event in prion diseases is the oligomerization of cellular prion protein (PrPc) into high beta-sheet content scrapie prion protein (PrPsc) and subsequent aggregation of PrPsc into amorphous and fibrillar deposits in the brain. It is believed that familial prion disease is caused by a mutation of the normal amino acid sequence, leading to spontaneous misfolding of mutant prion proteins into PrPsc. Within this framework, we used Atomic Force Microscopy to study the aggregation pathways of recombinant human prion proteins, and specifically compared the difference in oligomerization and aggregation behavior between normal cellular prion protein (PrPcwt) and a mutant protein (PrPc10or), which contains five extra copies of the octapeptide repeat region believed responsible for the misfolding reaction. Experiments were carried out in partly denaturing acid solution condition (20uM protein, 50mM NaAc, 150mM NaCl, pH 4) that triggers the oligomerization and aggregation. Our experiments show that both PrPcwt and PrPc10or have the same aggregation pathway to the formation of nonfibrillar aggregates, but do not follow a conventional nucleation and growth mechanism. First, stable trimers or tetramers form from monomers in solution. These then aggregate together to form larger oligomers(such as octamers and 9mers). These are then the fundamental units that form all larger aggregates. The difference between PrPcwt and PrPc10or is in the rates of oligomerization and oligomer aggregation, leading to different size aggregates. The mutant protein (PrPc10or) shows higher oligomerization and aggregation rates with the same solution conditions, resulting in larger aggregates. The ultimate source of these rate differences appears to be the differences in solubility. The results give a clear picture of the steps that lead from protein monomers to large-scale protein aggregates. They support the hypothesis that mutations in the octapeptide repeat region induce protein instability leading to spontaneous formation of PrPsc, which slowly accumulates over time, eventually leading to disease. This work was performed under the auspices of the U. S. Department of Energy by the University of California, Lawrence Livermore National Laboratory under Contract No. W-7405-Eng-48.
MM9.7
Adsorption Behavior of Linear and Cyclic Genetically Engineered Platinum Binding Peptides. Brandon Ruf Wilson1, Urartu Seker1,2, Candan Tamerler2 and Mehmet Sarikaya1,2; 1Material Science, University of Washington, Seattle, Washington; 2Istanbul Technical University, Istanbul, Turkey.
Recently, phage and cell-surface display libraries have been adapted for selecting short peptides for a variety of inorganic materials. Despite the enormous number of inorganic-binding peptides reported, there is still a limited understanding of molecular mechanisms of peptide recognition of and binding to solid materials. As part of our goal of designing these peptides, understanding their binding behavior, and applying peptides in molecular assembly; we discuss the effect of molecular structural constraints on the quantitative binding characteristics of peptides selected to bind to noble metal platinum. We studied peptide sequences in two confirgurations: one, a Cys-Cys constrained “loop” sequence that mimics the domain used in the pIII tail sequence of the phage library construction, and the second is the linear form, without the loop. Both sequences were analyzed for their adsorption behavior on Pt thin films by surface plasmon resonance (SPR) spectroscopy and for their conformational properties by circular dichroism (CD). We find that the cyclic peptide of the integral Pt-binding sequence possesses single or 1:1 Langmuir adsorption behavior and displays equilibrium and adsorption rate constants that are significantly larger than those obtained for the linear form. Conversely, the linear form exhibits biexponential Langmuir isotherm behavior with slower and weaker binding. Furthermore, the structure of the cyclic version was found to adopt a random coil molecular conformation, whereas the linear version adopts a polyproline type II conformation in equilibrium with the random coil. The 2,2,2- trifluoroethanol titration experiments indicate that TFE has a different effect on the secondary structures of the linear and cyclic versions of the Pt binding sequence. We conclude that the presence of the Cys-Cys restraint affects both the conformation and binding behavior of the Pt-binding sequence and that the presence or absence of constraints can be used to tune the adsorption and structural features of inorganic binding peptide sequences. Mainly supported by NSF-UW/MRSEC, and also by AFOSR-Bioinspired Materials, NSF-BioMat, and SPO/Turkey (CT).
MM9.8
Synthesis and Self-assembly of DMPC-conjugated Gold Nanoparticles. Subhasish Chatterjee1, Markrete Krikorian2 and Bonnie Gersten1; 1Chemistry, The Graduate Center, CUNY and Queens College, Flushing, New York; 2Towsend Haris High School, Flushing, New York.
Bioconjugated nanomaterials play a promising role in the development of novel supramolecular structures, molecular machines, and biosensing devices. In this study, lipid-capped gold nanoparticles were synthesized and allowed to form a self-assembled monolayer structure. The nanoparticles were prepared by a phase transfer method, which involved the reduction of potassium tetrachloroaurate(III) by sodium citrate in an aqueous solution and the simultaneous transfer of the reduced species to an organic medium containing DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine). The resulting nanoparticles were characterized using Uv-vis spectroscopy and dynamic light scattering particle-size analysis. In addition, the gold nanoparticles were examined using Transmission Electron Microscopy (TEM). The diameters of the nanoparticles ranged from 25 to 30 nm. The Langmuir-Blodgett technique was used to assemble the DMPC-capped nanoparticles onto a water subphase at room temperature. The measurement of the compression isotherm confirmed the assemblage of lipid capped gold nanoparticles. This method of synthesis of ordered structures utilizing biomolecular interactions would be useful in manufacturing novel metamaterials and nanocircuits.
MM9.9
Laser Photothermal Heating of Different Gold Nanorods: Selectivity and Application in Gene Regulation. Andy Wijaya1, Joshua Alper2, Lauren DeFlores4, Andrei Tokmakoff4 and Kimberly Hamad-Schifferli2,3; 1Department of Chemical Engineering, MIT, Cambridge, Massachusetts; 2Department of Mechanical Engineering, MIT, Cambridge, Massachusetts; 3Biological Engineering Division, MIT, Cambridge, Massachusetts; 4Department of Chemistry, MIT, Cambridge, Massachusetts.
Gold nanorods can be heated to extremely high temperatures using laser pulses. We explored the application of this technique for controlling biological function. The seed-mediated synthetic method has been shown to be capable of producing gold nanorods with various aspect ratios. This level of control is achieved by varying the amount ratio of the spherical gold nanoparticle seeds to the other chemical precursors. These gold nanorods are unique due to its shape-dependent optical properties, especially their absorption bands. Increasing the aspect ratio red-shifts the longitudinal plasmon band. Numerous studies have also shown that irradiation of gold nanorods by ultrafast pulsed laser at their longitudinal plasmon bands increased the local temperature and even induced the shape transformation of these nanorods. We investigated the localized heating of two different gold nanorods which have two different longitudinal plasmon bands by utilizing femtosecond laser pulses. We studied their heating selectivity when they are excited at different wavelengths which correspond to their longitudinal plasmon bands. Thiol dsDNA was conjugated to these nanorods. Its complement is functionalized with different fluorophores for different nanorods. Their de-hybridizations were studied to characterize the localized laser-induced heating of these nanorods. Our study indicated that relatively high selectivity can be achieved. Application of this technique towards selective control of gene expression will be discussed.
MM9.10
Electrostatic Force Microscopy of Protein Arrays and Biofilms for Bio-sensor Applications. You Jin Oh and William Jo; Physics, Ewha Womans University, Seoul, South Korea.
Microcontact printing (μCP) is a useful tool to pattern a wide range of molecules and nanoscale dots into microscale features on different types of substrates. We report a straightforward approach to the 2-dimensional immobilization of bovine serum albumin (BSA) and poly-ethylene glycol (PEG) by the μCP method. After immobilization of the proteins with a line and space shaped PDMS mold, the same regions were printed with another stamp. Sequentially, we obtained immobilization of the 2-dimensional BSA and PEG lattices on a glass surface. It is found that the lattice deposited by the μCP method, which has 2~4 nm height is an effective chemical barrier for bacteria behavior. In addition, the formation of O157:H7 biofilm was observed by atomic force microscopy (AFM) and force characteristics to examine surface charges of the bacteria by electrostatic force microscopy (EFM). We explored these bacterial electrical properties with more detail and accuracy than available by more traditional zeta potential measurement. The EFM measures the variation and potential energy difference between tip and cell surface arising from non-uniform charge distributions and local variations in surface work function. We show high resolution images of the biofilm formation and local charge distribution simultaneously. The EFM images depict directly how the surface charge is distributed on the bacterial surface. Moreover, EFM images indicated that electrostatic force is a sign of extra-cellular matrix of the bacteria and formation of biofilm.
MM9.11
Multifunctional Magnetic Gold Nanocomposites: Breast Cancer Detection via Magnetic Resonance Imaging and Localized Synchronous Therapy. Jaewon Lee1, Jaemoon Yang1, Jinyoung Kang3, Yong-Min Huh2,3 and Seungjoo Haam1,3; 1Department of Chemical Engineering, Yonsei University, Seoul, South Korea; 2Department of Radiology, Yonsei University, Seoul, South Korea; 3Graduate Program for Nanomedical Science, Yonsei University, Seoul, South Korea.
Nanomaterials are increasingly used in biomedical fields due to their remarkable physicochemical properties on the nanoscale. For example, the local temperature around a nanocomposite composed of a dielectric core and an outer thin metal shell can be sharply increased by near infrared (NIR) laser illumination, due to the surface plasmon resonance (SPR) effect of the metal shell. The hyperthermia induced by SPR, in turn, leads to a non-invasive thermocidal method for treating cancer without any side effects or operations. Magnetic nanocrystals (MNCs) have been used for MR imaging, cell labelling, and magnetic cell separation. In particular, we recently demonstrated that MnFe2O4 MNCs serve as excellent magnetic resonance imaging (MRI) probes for non-invasive in vivo monitoring of molecular and cellular events. By hybridizing MnFe2O4 MNCs and a gold nanoshell, the dual tasks of cancer detection by MRI and cancer treatment by hyperthermia might be accomplished simultaneously. Thus, we report a novel multifunctional magnetic particles-gold nanocomposite (MGNC) system for targeted cancer detection by MRI and a synchronous cancer therapy via therapeutic antibody and hyperthermia effects. The quadruple stratigraphical structure of MGNC is depicted in Scheme 1. For a MR contrast agent, we used magnetic kernels (MKs), which are composed of a number of ultrasensitive magnetic nanocrystals (MNCs). MKs were then embedded in the dielectric silica layer by a slightly modified Stöber method. The synthesized core shell type of magnetic-silica nanoparticles (MSNPs) were then coated with a gold nanolayer. To increase the colloidal stability and antibody conjugation efficacy, the gold nanolayer surface was enveloped with polyethylene glycol (PEG). A therapeutic antibody, Erbitux (ERB), was further conjugated on the outer ends of PEG for EGFR specific tumor cell targeting, both to localize NIR laser beam and to image their events through MRI. Furthermore, growth inhibition effect of cancer cells is expected via the blocking of epidermal growth factor receptor (EGFR) signalling pathway. The superb MR contrast agent property of our MGNC system stems from the intrinsic high emu/g of MnFe2O4 nanoparticles as well as the clustering of magnetic nanocrystals aided by polymer. Furthermore, for effective thermal therapy using NIR laser, 820 nm of λmax was achieved by separating a gold nanoshell from embedded MKs by dielectric silica layer. In summary, ERB conjugated MGNCs selectively recognized the target cancer cell lines and were effectively taken up by the cells. We clearly demonstrated that ERB-conjugated MGNCs had an excellent synchronous therapeutic efficacy stemming from therapeutic antibody and NIR laser-induced SPR, which implies the great therapeutic potentials of MGNCs for simultaneous diagnosis and treatment of cancer.
MM9.12
The Effect of Single Molecule Fluctuations on Multi-Molecular Enzymatic Assemblies for Sensor Applications. Nily Dan, Chemical and Biological, Drexel University, Philadelphia, Pennsylvania.
Recent studies show large fluctuations in the instantaneous reaction rate of single enzyme molecules. In large systems (containing many enzyme copies) and/or after a long period of time, the observed reaction rate converges to the expected, Michaelis-Menten (MM) value. However, in cells and enzyme-based nanosensors that contain few enzymes (10-106), fluctuations in the reaction rate of individual molecules may significantly affect performance and response. Here we develop a model for reaction kinetics in systems containing a finite number of fluctuating enzyme molecules. Using β-galactosidase as a test case, we find that a single enzyme molecule displays significant deviations from the expected MM reaction rate over time scales of 10-30 s, orders of magnitude longer than the characteristic kinetic-state fluctuation time. In multi-molecular systems, the time required to approach the steady-state rate depends on the acceptable deviation (error): For a fixed deviation value, the time to convergence decreases inversely with the number of enzyme molecules. For a fixed system size, the time required decreases with the square of the acceptable deviation. These results can be applied to the design of reliable enzyme-based nano-scale sensors.
MM9.13
Nanoparticle-based Sensing of Bacterial Toxins. James E. Ghadiali, Anna Laromaine and Molly M. Stevens; Department of Materials, Imperial College, London, United Kingdom.
Nosocomial bacterial infection is a prevalent and costly problem faced routinely in modern Western medicine. The pathologies associated with these infections, often fatal to the young, elderly or those with compromised immunity, are largely mediated by the secretion of a variety of protein toxins from the bacteria, which assist in its ability to colonise its host, promote reinfection and subsequent transmittance to other individuals. There is, understandably, great incentive to develop new techniques to aid in drug discovery, preventative detection and point of care diagnostics in order to minimise the financial burden and human cost of these bacterial infections in healthcare. To this end, we have developed a simple, label-free activity assay for the detection of a model bacterial toxin. We have employed the optical properties of metallic nanoparticles, functionalized with a range of peptides and nucleic acids, together with principles of biological self-assembly, to design a convenient colorimetric biosensor amenable to high-throughput screening. We demonstrate how simple hydrogen bonding motifs can be used to drive nanoparticle aggregation in response to toxin activity and further characterise the system using a variety of spectroscopic techniques including UV-visible absorbance spectroscopy, TEM, ATR/FTIR and surface enhanced Raman scattering.
MM9.14
Immobilization of RNase A to Si-g-PAA Brush: Study of Binding Kinetics and Relative Activity. Padma Gopalan1, Franz Himpsel2, Sean Cullen1, Xiaosong Liu2 and Ian Mandel1; 1Materials Science and Engineering, University of Wisconsin-Madison, Madison, Wisconsin; 2Department of Physics, University of Wisconsin-Madison, Madison, Wisconsin.
Immobilization of biomolecules on substrates is of great interest for applications in biosensors and biotechnology. In the last few years’ research in this area has examined a range of binding chemistries, substrates, and techniques for immobilizing mainly small molecules such as biotin and proteins such as enzymes and antibodies. Immobilized enzymes are particularly of interest due to their wide scale use in numerous biotechnology processes as they can be easily removed from the reaction mixture preventing cross-contamination. Ribonuclease A (RNaseA) is a single subunit enzyme used in the removal of RNA from DNA preparations. This is a critical step in the downstream processing of biopharmaceutical plasmid DNA that is used as an active pharmaceutical ingredient in gene therapy and DNA vaccination. Immobilization of RNaseA on a solid support allows for easy separation of the enzyme from the plasmid DNA mixture. Polymer brushes offer certain advantages over other materials such as hydrogels and polymer coatings as they are covalently anchored to the substrate providing excellent mechanical stability and provide a three dimensional template with functionality controllable by monomer type and brush length. Here, we examine the feasibility of using polymeric brushes as three dimensional scaffolds to covalently immobilize RNase A. Poly(acrylic acid) brushes synthesized using atom transfer radical polymerization (ATRP) were used to immobilize RNaseA by conventional EDC/NHS coupling and high-capacity NTA-Cu2+ complexes. The amount bound by each method and further activity of the enzyme after immobilization was examined. The immobilization was investigated by ellipsometry, XPS, TGA, and NEXAFS. The activity of the immobilized RNAse A was determined using UV absorbance. The polymer brushes immobilized twenty times as much enzyme when compared with monolayer coverage’s. As the thickness of the brush increases, the surface density of RNase A increases with the high capacity binding showing a marked improvement from conventional binding at all brush lengths. With PAA brushes, we were able to bind 11.0 micro g/cm2 with NTA-Cu2+ and 5.8 micro g/cm2 with standard coupling agents. Immobilization kinetics for both methods was characterized by ellipsometry. The maximum relative activity of 0.80 was achieved by optimizing the brush length and binding chemistry. While the temperature dependence of activity for PAA-RNase A was similar to the free enzyme, the RNase A bound by the NTA-Cu2+ showed no temperature dependence. This is probably due to binding of Cu2+ predominantly to histidine residues located in the active site of the enzyme, which drastically, reduced the activity of the enzyme.
MM9.15
Nanomechanical Switching in Lateral Force of Stimulus-Responsive Biomimetic Macromolecular Layers. Miao Ye and Christine Ortiz; Department of Materials Science and Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts.
Inspired by the structure of a major compressive, shear and tensile load bearing extracellular matrix proteoglycan of articular cartilage, aggrecan, which consists of a core protein with covalently bound, closely spaced (~2-4 nm) and negatively charged chondroitin sulfate glycosaminoglycan side chains, we prepared stimulus responsive polymer layers via chemically end-attached "brush-brushes" formed by chemisorption of mono(end)-functional thiol-terminated poly(methacrylic acid-g-ethylene glycol) (HS-poly(MAA-g-EG)) with two different macromolecular architectures (Mn=27K, PEG graft density, PEG(%)=7.7%, backbone contour length, Lcontour=41.1 nm; Mn=17K, PEG(%)=1.9%, Lcontour=39.8 nm). The lateral force interaction between the end-grafted polymer layers and a probe tip (nominal radius ~50nm) functionalized with a OH-terminated SAM (HS(CH2)11OH) was studied in buffered aqueous solutions at pH9-pH4 and an ionic strength of 0.005M via contact angle AFM on micro-contact printed samples at a scan angle of 90°. Using a modified wedge method for nanosized probe tips, the lateral sensitivity of the probe tip was calibrated to be 124.2 nN/V, which was used to convert the lateral cantilever signal (volt) into later force (nN). As pH decreased, both polymer layers exhibit an abrupt change in lateral proportionality coefficient (ratio of lateral force over normal force), μ, between pH7.1 and pH6. At pH4-5, the lateral force increased almost linearly as the normal force for both the 17K and 27K polymer layers, which coefficients μ were ~0.63-0.65 and ~0.78-0.79 respectively. At pH7.1-9, first, the μ dropped significantly down to ~0.20-0.34 for the 17K polymer and ~0.12-0.20 for the 27K polymer; second, there were two regimes for the correlation between lateral force and normal force. At smaller normal load (<~6nN for 17K, <~8nN for 27K), the lateral force was small (~1nN for 17K and ~2nN for 27K) and independent of the normal force. When higher normal forces were applied (>~8nN) to the probe tip, lateral force increased approximately linearly as normal load. A similar two-regime behaviour has also been found in the lateral force microscopy of cartilage aggrecan macromolecules using an OH-functionalized nanosized probe tip, which was attributed to the intersurface interaction between probe tip and the substrate when full aggrecan compression and tip penetration occur at high normal force. As pH decreases, the conformational transition of the polymer brushes will lead to a collapsed conformation and a hydrophobic surface, the adhesion between the OH-functionalized probe tip and the hydrophobic surface along with the contribution between the probe tip-substrate interactions make μ much higher than at high pH. The 27K polymer had higher μ values at pH<6 (0.89±0.19) but smaller μ at pH>7.1 (0.21±0.04), indicating that a more dramatic change in lateral force coefficient is expected for stimulus-responsive graft copolymers with higher side chain grafting density.
MM9.16
Silicon-Based Nanoscale Field Effect Transistor for Chemical and Biological Sensing. Yu Chen1, Xihua Wang1,2, Mi K Hong1,2, Shyamsunder Erramilli1,2,3, Carol Rosenberg4 and Pritiraj Mohanty1; 1Department of Physics, Boston University, Boston, Massachusetts; 2The Photonics Center, Boston University, Boston, Massachusetts; 3Department of Biomedical Engineering, Boston University, Boston, Massachusetts; 4School of Medicine, Boston University, Boston, Massachusetts.
Ultrasensitive detection of chemical and biological species is fundamental to biomolecular analysis. Nanotechnology, by using nanoscale material, such as nanowires, nanotubes, nanocrystals, nanocantilevers, and quantum dots, makes it possible to greatly enhance the detection sensitivity as the signal can be effectively transduced because of large surface-to-volume ratio. Here, we report successful development of a silicon-based nanoscale gated biological field effect transistor (BIOFET) for label-free detection. The silicon nanowire fabricated using top-down approach can not only enhance performance but also allow on-chip integration and scalable manufacturing. We validate our device function on calibrated pH solutions and the amplified detection of model proteins in solution. We also demonstrate ultrasensitive detection of breast cancer serum biomarker protein CA15.3 down to levels of concentration less than 40 Units/milliliter, relevant for clinical use.
MM9.17
Abstract Withdrawn
MM9.18
Toxicity of BSA-stabilized Silver Nanoparticles on Immune Circulating Cells. Imani Hayman1, Patrick Mehl2, Veena Kapoor4 and Otto Wilson3; 1Pharmaceutical Science, Howard University, Washington, District of Columbia; 2Biomagnetics Group, Vitreous State Laboratory, Catholic University, Washington, District of Columbia; 3Biomedical Engineering, Catholic University, Washington, District of Columbia; 4Flow Core Facility, Experimental Transplantation and Immunology Branch, National Institutes of Health, Bethesda, Maryland.
Silver nanoparticles have shown immense potential in many biomedical applications, specifically wound healing. These nanoparticles reduce the degree of inflammation in wounds and increase the rate of wound healing overall in a dose-dependent manner. Moreover, silver nanoparticles exhibit antibacterial and antimicrobial properties. While the mechanism of action for silver nanoparticles is not clear, current studies focus on the effect of silver nanoparticles on recipient cells and tissues. It is shown that silver nanoparticles are more toxic to these recipient cells in comparison to other metal nanoparticles. This suggests that the bactericidal properties of the silver nanoparticles are size dependent. Our present work investigates the toxicity level of silver nanoparticles on specific immune circulating cells. The approach is to report the LD50 level as a function of the ratio of the nanoparticles concentration (ppm) to the cell concentration (cell number/ml) used in the assays. This method allows a normalization of the LD50 capable to compare the toxicity of the nanoparticle on different types of cells. Next, the localization of the silver nanoparticles within the cells will be determined, and the toxic mode of action of the nanoparticles will be modulated by the modification of the synthesis method.
MM9.19
Micropatterning Cardiac Myocytes On Self Assembled Polyelectrolyte Multilayers Vipra Dhir1,2, Anupama Natrajan4,2, Anindarupa Chunder3,2, Lei Zhai3,2 and Peter Molnar4,2; 1Mechanical, Material & Aerospace Engineering, University Of Central Florida, Orlando, Florida; 2Nano Science Technology Center, University Of central Florida, Orlando, Florida; 3Department Of Chemistry, University Of Central Florida, Orlando, Florida; 4Department of Biomolecular Science, University Of Central Florida, Orlando, Florida.
Self assembly of weak polyelectrolytes based on Hydrogen bonding were studied for their cell resistance. Coatings based on dilute solutions (0.01 M) of Poly(acrylic acid) (PAA)/ Poly(acryl amide) (PAAm) were deposited by layer by layer technique. The coatings were crosslinked thermally in order to make them stable. The coatings were patterned by Laser ablation using a photomask to create adjacent cytophobic and cytophillic surfaces. Neonatal Cardiac myocytes were plated on the patterned substrates. Cell growth was observed in patterns obtained by ablation. The patterns were observed for their long term stability, it was observed that the patterns were stable more than a period of 40 days. Beating cardiac patterns were obtained. These patterned systems can find applications in biosensors and cell based drug screening devices.
MM9.20
Abstract Withdrawn
MM9.21
Cardiac Myocyte Size and Cytoskeletal Architecture are Independently Regulated by Cell Shape. Nicholas Andrew Geisse, Sean P. Sheehy and Kevin Kit Parker; School of Engineering and Applied Sciences, Harvard University, Cambridge, Massachusetts.
The cardiac myocyte has an extraordinarily complex structure which dictates its function and interaction with other cells in the myocardium. In vivo cardiac myocytes have a shape that approximates a rectangular box with a ~7:1 aspect ratio. During hypertrophy, the structure of these cells changes such that the aspect ratio falls, and this maladaptive structural change is accompanied by dysfunction in both contractility and Calcium handling. We hypothesized that the boundary conditions of these cells affects the structure of the cytoskeleton and the cytoplasm. Using microcontact printing, we have engineered silicone substrates in order to make them amenable to the culture of individual cardiac myocytes with precisely controlled boundary conditions by printing micron-sized islands of extracellular matrix protein (ECM). Using this technique, we fabricated cardiac myocytes with shapes similar to those seen in the healthy and hypertrophic phenotypes. We show that for cells grown with imposed boundary conditions, cytoskeletal architecture is dependent on the shape of the cell. Further, the cytoplasm is organized such that the contractile machinery lies in a plane at the cell-substrate interface. Using atomic force microscopy (AFM), we show that the cell size (as measured by volume per unit area) remains constant regardless of the shape of the ECM boundary conditions imposed upon it, while myofibrillar architecture changes drastically. This relationship between cell-substrate contact area and cell volume also holds true for cells that are grown in the absence of substrate-mediated 2D boundary conditions. We postulate that the extracellular shape of the cardiac myocyte affect self assembly and organization of the cytoplasm and organelles, but that cell volume control is regulated by a different mechanism. Our results suggest that the size of the engineered cardiac myocyte may be independent global cytoskeletal architecture of the cytoskeleton in vitro. Further studies will reveal if 3D boundary conditions are required to mediate the kinds of cytoskeletal architectures that directly affect cell volume.
MM9.22
Phase Transition Behaviors and Lipid Interactions in Myelin Sheath. Younjin Min, Kai Kristiansen, Joe A Zasadzinski and Jacob N Israelachvili; Chemical Engineering, UCSB, Santa Barbara, California.
Myelin is a stacked membrane structure that allows for fast, efficient conduction of nerve impulses. It has 8 kinds of lipid molecules on two alternating bilayers and proteins such as Myelin Basic Protein (MBP) which has an important role in maintaining myelin structure. The compact bilayer organization of healthy myelin is believed to require a well-defined range of lipid and protein composition, and bilayer-bilayer interaction. Even though we know that multiple sclerosis (MS) is a morphological transformation involving loss of adhesion between myelin lamellae and sometimes formation of myelin vesicle, its mechanism and causes for demyelination are still under investigation. We have used fluorescence microscopy, Langmuir isotherm, and Langmuir-Blodgett techniques to investigate how lipid composition of myelin lipid system affects the phase transition behaviors of myelin monolayers and bilayers depending on lateral pressure, temperature, and pH conditions. We have also been studying the topographic changes and the interactions of two symmetrical myelin bilayers in the absence and presence of MBP using Atomic Force Microscopy (AFM), and Surface Forces Apparatus (SFA) techniques. Our findings clearly show how lipid compositions are related to their interactions and strongly support our hypothesis about which there is a reduced adhesion in diseased myelin membranes. The acute experimental allergic encephalomyelitis (EAE) in the common marmoset which is a highly relevant model of MS was used as a comparison.
MM9.23
Protein Fingerprinting Using Flat-Surface Electrophoresis Perumal Ramasamy1, Raafat M Elmaghrabi2, Gary Halada1 and Miriam Rafailovich1; 1Materials science and Engineering, SUNY Stony Brook, Stony Brook, New York; 2Physiology and Biophysics, SUNY Stony brook University, New York, New York.
Developments in the field of proteomics are highly encouraging for medical researchers. While gel electrophoresis offers a successful means for protein recognition, it requires the use of a relatively large system and a rather long period of time. Here, a protocol is developed for electrophoresis of proteins using flat surfaces based on the principles of electrophoresis of DNA on flat surfaces. By further adapting this system, it is hoped to create a portable device for protein electrophoresis. Droplets of fluorescently tagged proteins such as albumin, casein, poly-L-lysine and their mixtures were placed on glass surfaces in an electrophoretic cell and allowed to dry. TBE buffer was added to the cell and the migration of the salt complexes was monitored using confocal microscopy. We show that different protein - salt complexes have different mobilities on a flat surface. The shape and size distributions of the protein-salt complexes and their mixtures on surfaces were also studied using atomic force microscopy and were found to be dependent upon the proteins. It is observed that the native charge of the proteins play a dominant role in the migration of the protein-salt complexes in the electrophoretic cell. From the morphology of the protein droplets it is observed that large aggregates are formed when oppositely charged proteins are mixed. Light scattering measurements and zeta potential measurements confirm the difference in the size and shape of the aggregates in solution leading to different nobilities of the protein-salt aggregates during electrophoresis.
MM9.24
A Light Scattering Study of Interactions of Oppositely Charged Proteins in Solution. Perumal Ramasamy1, Raafat M Elmaghrabi2, Gary Halada1 and Miriam Rafailovich1; 1Materials science and Engineering, SUNY Stony Brook, Stony Brook, New York; 2Physiology and Biophysics, SUNY Stony brook University, New york, New York.
In experiments involving electrophoresis of proteins in gels, it was observed that the mobility of FITC tagged albumin (FITC albumin) was greater than that of TRITC tagged albumin (TRITC albumin). To further understand the effects of tagging proteins with fluorescent dyes, interactions of cationic proteins FITC albumin, TRITC albumin, untagged bovine serum albumin (BSA), FITC casein and casein with anionic proteins ploy-L-lysine and FITC tagged poly-L-lysine (FITC poly-L-lysine) were studied using dynamical light scattering. It was found that aggregates formed by the interaction of FITC albumin with poly-L-lysine were larger than those formed by the interaction between poly-L-lysine and BSA or poly-L-lysine and TRITC albumin. Using zeta potential measurements it was observed that irrespective of the fluorescent tags attached to them, the zeta potential values of cationic proteins changed from negative to positive with increasing amounts of poly-L-lysine. It was also observed that addition of small amounts of poly-L-lysine to solutions containing FITC albumin decreased the zeta potential drastically. To explain this data, we are proposing a model that suggests that low concentrations of poly-L-lysine serve as scaffold - like structures on which several FITC albumin molecules anchor. Using UV spectrophotometry it was observed that when FITC albumin was added to FITC poly-L-lysine, there was a shift in the absorption spectra due to conjugation. However no shift was observed when TRITC albumin was added to FITC poly-L-lysine. We conclude that FITC appears to change the surface charge of albumin significantly and thereby influencing its behavior in solution and its interaction with anionic poly-L-lysine.
MM9.25
Electrostatic Field Calculations for Electrophoresis Using Surfaces. Perumal Ramasamy1, Raafat M Elmaghrabi2 and Gary Halada1; 1Materials science and Engineering, SUNY Stony Brook, Stony Brook, New York; 2Physiology and Biophysics, SUNY Stony brook, New York, New York.
Conventionally, separation of DNA and proteins is done electrophoretically on various types of gels. However, these techniques are frequently time consuming. Also, it is difficult to retrieve the samples from gels after the electrophoresis. One way to overcome this problem is by using solid surfaces as possible separation tools for electrophoresis. During electrophoresis, the migration of DNA/protein close to the surface depends upon the frictional forces and the chemical interactions between the surface used for separation and the DNA/protein molecules. Surface electrophoresis can be relatively rapid, and it is easy to retrieve the samples from the electrolytic buffer following separation. The first step towards surface electrophoresis is to understand the electric field distribution in the electrophoretic cell using the surface as the separation medium. The distribution of electric field in and near the surface of the electrophoretic cell determines the motion of proteins in the buffer and along the surface. This is a complicated problem, influenced by buffer ion concentration, electrode configuration, and surface and substrate conductivities. Steady state calculations approximating the experimental geometry were made for different arrangements of electrodes using MAFIA (Computer Simulation Technologies) program and Electro static field (ESTAT) programs. Electric field distributions in both conducting surfaces like ITO (Indium Tin Oxide) (Kevley Technologies), gold and aluminum and non-conducting surfaces were studied. In order to measure the EOF (define - what is EOF?) of the buffer neutrally charged fluorescent Poly-Styrene (PS) beads of 1 µm diameter (Fluo spheres F-13083; red fluorescent (580 / 605nm); 1x1010 beads/ml from Molecular probe) were included in the buffer and imaged using confocal microscope. It was observed that the electric filed was highly modified by various factors including the conducting nature of the surface, position of the electrodes, salt concentration in the buffer and distance from the separation surface.
MM9.26
Prostate-specific Antigen Detection Using Optical Resonant Reflection Biosensor. Kyung-Hyun Kim, Chul Huh, Jongchul Hong, Hyun sung Ko, Wanjung Kim and Gun yong Sung; ETRI, Daejeon, South Korea.
As the need for rapid, ultrasensitive, and economical methods for the detection of biomedical entities, various label-free biosensor technologies are increasingly studied or developed. We fabricate an optical resonant reflection biosensor, which is consist of a nanoscale resonate grating patterns on a glass substrate via nanoimprint. The resonantly reflected wavelength is modified by the attachment of biomolecules to the resonant filter, so that small changes in surface optical density, which is resulted antigen-antibody interactions of biomolecule. We describe the design of an optical resonant reflection biosensor and experimental results for antigen-antibody interactions upon resonant filter surface. For the sensitivity and efficiency improvement of our biosensor, we introduce protein G, promoting the biding and enhancing orientation of the antibody, and various surface chemical treatment methods. As a result of prostate-specific antigen (PSA) detection, We successfully measure the shifts of resonantly reflected wavelengths as function of antigen concentration and time on the biosensor surface. And we also report the various immobilization data are affected by surface chemical treatment conditions. Currently, the limits of detection of prostate-specific antigen (PSA) using our biosensor is an about of 1 ng/ml.
MM9.27
Electrochemical Detection of Biomolecules Patterned on Electrode Arrays by Biocompatible Photolithography. Mònica Mir2, Srujan Kumar Dondapati2, Maria Viviana Duarte2, Ioanis Katakis2, Margarita Chatzichristidi1, Konstantinos Misiakos1, Panagiota S. Petrou1, Sotirios E. Kakabakos1 and Panagiotis Argitis1; 1Inst. of Microelectronics and Inst. of Radioisotopes and Radiodiagnostic Products, NCSR "Demokritos", Athens, Greece; 2Bioengineering and Bioelectrochemistry Group, Departament d'Enginyeria Química, Universitat Rovira i Virgili, Tarragona, Spain.
The problem of fabricating closely spaced electrochemical detectors that can be modified selectively so as to create multianalyte arrays, especially biosensor arrays, suitable for incorporation in microsystems and small volume diagnostic devices is treated in this work. 20 micron-spaced gold electrodes were fabricated by photolithography on silicon wafers. All the electrodes were covered by a photoresist that could be washed off by treatment under conditions that allowed retention of biomolecule activity. It was therefore possible to expose and modify sequentially the electrodes of the array with different biomolecules achieving multianalyte devices. In the cases where direct electron transfer from the biomolecules (especially redox enzymes) was expected, a blocking step preceded subsequent exposures in order to avoid “stray” currents from subsequent biomolecules non specifically adsorbed on previously modified electrodes. Such “blocking” was made with insulating polyelectrolytes in such a way that allowed efficient electron transfer from the redox enzymes but insulating electrically subsequent layers. This concept was demonstrated with selective detection of oligonucleotides, antigens, and enzyme substrates in two-analyte systems in order to assure that the method is compatible with all three types of biorecognition elements. The photoresist that can be processed under conditions allowing the retention of biological activity photoresist was based on a co-polymer of different methacrylate monomers conferring useful functionalities to the resulting material and a photoacid generator, as it has been reported in previous publications by the NCSR Demokritos group. Electrochemical transduction was achieved by redox polymers and electrochemical “blocking” through poly(vinylsulfonate) and poly(vinylpyridine) derivatives. Electrode pairs modified with capture probes could differentiate clearly target oligonucleotides with five mutations in the gene region relevant to breast cancer mutations. A pair of electrodes modified with glucose and sarcosine oxidase respectively could simultaneously detect both analytes with less than 5% interference from electrode crosstalk. Finally, a pair of electrodes modified with thyroxine could differentiate the presence of thyroxine through a competition assay and result to significantly different response as that form interfering proteins. Although the methods of detection used were rudimentary, each “array” was composed of only two electrodes, and the electrode spacing was of 20 microns, the results demonstrate that the method proposed is generic and could be used for patterning of electrochemical multianalyte biosensors at even higher resolution.
Chair: Jeffrey Tok
Thursday Morning, November 29, 2007
Room 210 (Hynes)
8:00 AM *MM10.1
Low-Temperature Kinetically Controlled Nanofabrication of Semiconductor Thin Films and Nanoparticles. Daniel E. Morse, Richard Brutchey, Birgit Schwenzer and John Gomm; Institute for Collaborative Biotechnologies and the California NanoSystems Institute, University of California, Santa Barbara, California.
We developed a generic new, biologically inspired low-temperature route for the kinetically controlled synthesis of a wide range of nanostructured metal oxide, -hydroxide, -phosphate and bimetallic perovskite semiconductor thin films and nanoparticles without the use of organic templates. Post-synthesis conversion to the nitrides, sulfides and phosphides has been demonstrated. Because no organics are used, this new biologically inspired synthesis method yields exceptionally pure inorganic semiconductors, and thus is potentially integrable with MOCVD, CMOS and other conventional manufacturing methods. Employing gentle catalysis at low temperature, this method can preserve the intermetallic organization of bimetallic precursors that are thus incorporated into crystalline solids as bimetallic molecular units without phase segregation. This has led to the first low-temperature synthesis of 6 nm barium titanate nanoparticles with low polydisperisty, good electronic properties and no organic contaminants. We also have used this process for the low-temperature synthesis of a wide range of supported (substrate-grown) and unsupported (free-standing) nanostructured thin films. One such material is strongly photoconductive cobalt hydroxide-based thin film material that exhibits high dopant density, high surface area of single-crystal domains, long minority carrier lifetime and strong absorption in the visible, making it potentially attractive for photovoltaic applications. Conversion to the oxide yields a nanostructured material now under investigation for its advantages in high power-density 3-dimensional batteries. A wide range of other materials made by this low-temperature process offers unique combinations of structures and properties not readily attainable by conventional high-temperature process; these exhibit potential advantages now under investigation for improved energy conversion and storage, ferroelectric random access memory (FeRAM), infrared detectors and flexible displays.
8:30 AM MM10.2
Biomimetic Assembly of Functional Nanomaterials using Synthetic Polymer Substrates and Biological Polymers. Song Jin, Stephen A. Morin and Fairland Amos; University of Wisconsin-Madison, Madison, Wisconsin.
We apply the principles inspired by the biomineralization processes to the assembly of nanoscale functional materials into nanoscale systems. In biomineralization, “matrix” macromolecules can efficiently induce the nucleation of inorganic crystals at specific locations with controlled size and morphology, and defined growth orientation. By carefully controlling the surface organic molecules, heterogeneous nucleation at designated regions can be promoted while homogeneous nucleation elsewhere can be completely suppressed, therefore enabling the controlled bottom-up assembly of inorganic nanomaterials directly from solution. Surface carboxylic groups on selective regions were generated on commodity engineering polymers such as polycarbonate (PC) and poly(ethylene teraphthalate) (PET) by direct patterned UV irradiation through a photomask and then were successfully employed to selectively nucleate nanoscale crystalline inorganic materials, such as semiconductors ZnO and CdS, directly from aqueous solutions to form patterned arrays. We have demonstrated the application of such arrays of functional materials in, for example, arrays of photodetctors that have high performance yet retain the low cost roll-to-roll solution processing on flexible polymer substrates. We have extended this biologically inspired approach to truly bottom-up nanoscale assembly using self-assembled nanostructured block copolymers and synthetic collagen protein fibers, which can have applications in information storage, quantum electronics and biosensors. The preliminary unpublished results will be discussed.
8:45 AM MM10.3
The Mechanism of Chitosan Enhanced Lung Surfactant Adsorption. Patrick C Stenger, Omer A Palazoglu and Joseph A Zasadzinski; Chemical Engineering, University of California, Santa Barbara, Santa Barbara, California.
Chitosan is a biocompatible polysaccharide derived from deacetylated chitin and this paper will demonstrate how its unique material properties as a cationic polyelectrolyte enhance surfactant adsorption to the air-liquid interface. These properties are of therapeutic interest in Acute Respiratory Distress Syndrome (ARDS) where the lung surfactant (LS),,which modulates surface tension in the alveoli, is inhibited by the competitive adsorption of surface active serum proteins. Utilizing a cycling Langmuir trough as an in vitro model for LS/serum protein competitive adsorption, we show that LS adsorption to the interface is restored by chitosan addition suggesting a promising therapy for ARDS. Fluorescence microscopy images of the interface show distinct morphological changes between serum protein and LS-covered regions offering a visual confirmation of chitosan enhanced LS adsorption. LS is ~70 wt % dipalmitoylphosphatidylcholine (DPPC) and freeze fracture transmission electron microscopy images of DPPC vesicles treated with chitosan show distinct morphological changes. While untreated DPPC vesicles are ~50 nm in diameter and uniformly distributed through the solution, images of chitosan exposed vesicles show fusion and numerous ~50 nm vesicles entrapped inside larger vesicles, similar to the "vesosomes" used for drug delivery. This competitive adsorption of serum proteins to the alveolar air-liquid interface can be modeled as an energy barrier to LS adsorption and can be analyzed using a variation of the classical Smolukowski description of colloidal stability. The serum proteins generate both a steric and electrostatic barrier to lung surfactant adsorption due to a net repulsion between the negatively charged surfactant aggregates and serum proteins. Previous work has shown that mono and divalent salts restore serum protein inhibited LS to the interface by lowering the electrostatic barrier to adsorption via charge screening and charge neutralization. Here we show that the chitosan concentration necessary to restore LS adsorption is three orders of magnitude lower than divalent salt due to a greater number of positive charges per molecule. Additionally, increasing the chitosan concentration beyond the optimum value results in significantly less LS adsorption suggesting a charge neutralization and reversal mechanism similar to multivalent ions.
9:00 AM MM10.4
Molecular Mechanics of Stutter Defects in Vimentin Intermediate Filaments. Theodor Ackbarow and Markus J. Buehler; Laboratory for Atomistic and Molecular Mechanics, Civil and Environmental Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts.
Biological materials such as bone, skin, spider silk or cells consist of proteins, the building blocks of life. In cells, the mechanical properties are mainly determined by the cytoskeleton, a network composed of actin filaments, microtubules and intermediate filaments. Vimentin coiled-coil alpha-helical dimers are elementary protein building blocks of intermediate filaments, which determine the large deformation behavior of cells. Here we focus on a detailed analysis of the structure-property relationship of coiled-coiled dimers under presence of molecular defects. Different coiled-coil ‘defects’ were found in nature, whereas skips, stutters and stammers are the three major irregularities. These defects are characterized by the insertion of one, three and four additional amino acids in the coiled-coil characteristic seven residue repeat pattern, leading to a change in the coiled-coil geometry. Notably, all intermediate filament dimers feature a highly conserved ‘stutter’ region, a sequence of amino acids that interrupts the superhelical coiled-coil arrangement of the two alpha-helices, leading to a parallel arrangement of the alpha-helices in this region. Earlier studies have suggested that the stutter plays an important role in filament assembly. Here we show that the stutter also has a strong influence on the mechanical behavior of vimentin dimers. We develop an Extended Bell Model to provide a theoretical description of the unfolding behavior of coiled-coil structures, capable of capturing different molecular geometries and loading rates. The Extended Bell Model predicts that the stutter represents a molecular weakening defect at which unfolding occurs at lower forces than in the rest of the protein. Our studies suggest that the presence of the stutter leads to a softer structure with more homogeneous plastic strain distribution under deformation. The predictions by the Extended Bell Model are confirmed by large-scale MD simulations of three model systems: Two parallel alpha-helices, a coiled-coil dimer, as well as a coiled-coil dimer with a stutter, which is the combination of the first two model geometries. The simulations prove that in agreement with the prediction based on our Extended Bell Model, the stutter represents the locations at which the protein structure has the least resistance to unfolding. We discuss the implications of this molecular architecture in terms of its biological function and possible applications of this nano-structural design for problems in engineering.
9:15 AM MM10.5
Electrical Characterization of Bio-templated Nanostructured Photovoltaic Material. John H Joo1, Elaine D Haberer1, Jennifer C Hsieh2, Chung-Yi Chiang2, Angela M Belcher2 and Evelyn L Hu1; 1Materials, University of California- Santa Barbara, Santa Barbara, California; 2Massachusetts Institute of Technology, Cambridge, Massachusetts.
In polymer solar cells, interpenetrating networks of the different polymers can improve carrier collection efficiency, thus improving the overall efficiency of the device. This nanostructuring should have similar benefits for inorganic solar cell materials, but seems more challenging to realize. We have used selective biotemplating of M13 bacteriophage to form a composite material composed of an interpenetrating network of metal and semiconductor nanowires. The goal is the formation of an efficient photon-absorbing material that at the same time allows for efficient collection of the photo-generated charge. Semiconducting CdSe nanoparticles are functionalized with positively charged ligands which attach to the negatively charged capsid of the M13 bacteriophage through electrostatic interactions. These nanowire building blocks act as the photo-active component. Metallic Au nanoparticles are attached to the M13 bacteriophage through a material-specific peptide displayed along the major coat. The mixture of the different nanowires creates a composite composed of an interpenetrating network of Au nanowires and CdSe nanowires. We present electrical characteristics of the bio-templated CdSe-Au composites. M13 films infiltrated with CdSe nanoparticles using circular TLM patterns were measured with a resistivity of ~107 Ohm-cm, which may be an underestimate due to current spreading. Photoluminescence measurements on the films did not show a red shift in nanoparticle emission, suggesting the nanoparticles are not in close proximity to one another. Difficulties in obtaining densely packed nanoparticles in the composite by infiltration of the M13 films with CdSe nanoparticles required a new assembly and measurement technique. To ensure densely packed composites, phage-nanoparticle complexes were assembled in solution, centrifuged, and deposited on test patterns with 210 μm wide Au stripes. A 500 μm Au line is deposited on top of the agglomerate through a shadow mask. Resistance is measured across the intersection of these lines. Both M13 only films and non-templated nanocrystalline solids are used as controls for electrical measurements to evaluate the effects of the bio-templating technique.
9:30 AM MM10.6
Colloidal Satellites as Optical Diagnostic Tools for Mutation Analysis. Valeria Tohver Milam1,2, Sonya Parpart2 and Chris K. Tison1; 1School of Materials Science & Engineering, GA Tech, Atlanta, Georgia; 2Dept of Biomedical Engineering, GA Tech, Atlanta, Georgia.
DNA is a versatile tool for colloidal assembly. Past work has focused on 1) employing perfectly matched sequences to form duplexes between particle surfaces and drive colloidal assembly; and 2) thermal melting or dissociation of duplexes to break assemblies apart. Here, we take an alternative approach to both the assembly and disassembly process. We employ target sequences with a single point mutation to assemble colloidal satellites. Each colloidal satellite is comprised of a large, nonfluorescent core particle surrounded by a single layer of fluorescent nanoparticles. In order to break apart or disassemble the colloidal satellites, perfectly matched target sequences are added to the suspension. Flow cytometry results indicate that these secondary targets can competitively displace the mismatched targets to, in principle, cause disassembly. Fluorescence and confocal microscopy studies, however, indicated that the extent of disassembly was largely dependent on the nanoparticle size as well as the difference in hybridization activity between the mismatched and matched targets. These results indicate that DNA-linked colloidal satellites present exciting potential as optical diagnostic tools for monitoring sequence-specific mutations identified in many diseases such as breast cancer.
9:45 AM MM10.7
Precursor to Beta-Sheet Crystal Formation in the Polymer-Water System. Xiao Hu1,2, David Kaplan2 and Peggy Cebe1; 1Physics and Astronomy, Tufts University, Medford, Massachusetts; 2Biomedical Engineering and Chemical and Biological Engineering, Tufts University, Medford, Massachusetts.
We investigated the polymer-water interaction in a model fibrous protein. Biodegradable Bombyx mori silk fibroin film with bound water molecules inside was analyzed in this study. Time-resolved techniques of Fourier Transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), and its temperature-modulated variant (TMDSC), were used to monitor the detailed structural changes of silk fibroin during heating and during isothermal crystallization above the glass transition temperature, Tg (~451K). Results provide a clearer picture of how silk fibroin chains can self-assemble from the non-crystalline, water-soluble structure to the beta-sheet-crystal rich insoluble final spun structure. The intermolecular bound water molecules, acting as a plasticizer, will strongly affect the secondary structure of silk fibroin. DSC study shows that silk fibroin displays a water-induced glass transition around 353K during quick heating, resulting from a formation and “melting” of temporary bound water-silk structure. Real-time FTIR study shows the tyrosine (Tyr) side chains spectrum shift to a lower frequency during heating below Tg, indicating change of the microenvironment in the silk fibroin chains. A dense “precursor” protein structure will be formed with evaporation of the water molecules, promoting the folding of the fibroin chains into beta-pleated-sheet crystals. During isothermal crystallization above Tg, silk fibroin has a clear phase transition from the secondary structures of non-crystalline random coil and alpha helix to the beta pleated sheet crystals, while the tertiary structure remains stable in this process. This study provides a deeper understanding of the formation of beta pleated sheets during the crystallization process in proteins, with implications for the crystallization of naturally occurring silk fibers from animals such as the silkworm and spider.
10:30 AM MM10.8
On The Nature of Electrical Transport in Bacterial Nanowires. Mohamed Y El-Naggar1, Yuri A Gorby2 and Kenneth H Nealson1; 1University of Southern California, Los Angeles, California; 2J. Craig Venter Institute, La Jolla, California.
Bacterial nanowires are conductive pilus-like appendages produced by bacteria, most notably some metal-reducers, in direct response to electron acceptor limitation. These recently discovered biomolecular wires represent a new paradigm in extracellular electron transfer, but the mechanism of electron transport remains unclear. We here report quantitative measurements of transport across bacterial nanowires produced by Shewanella oneidensis MR-1, whose electron transport system is currently being investigated for bioremediation and renewable energy recovery in microbial fuel cells. Oxygen-limited chemostat cultures of Shewanella oneidensis strain MR-1 (wild-type) produced a high density of electrically conductive bacterial nanowires. Using scanning electron microscopy (SEM) and atomic force microscopy (AFM), we observe wire bundles ranging from 50 to 150 nm in diameter and extending well beyond a cell’s length. These assemblies appear to form coordinated junctions between cells, and the conductive nature of these appendages and junctions thereof was confirmed by scanning tunneling microscopy (STM) and conductive atomic force microscopy (CAFM). Current-voltage spectroscopy reveals interesting non-linearities in the current response to applied bias, and provides insight into the electronic structure of the nanowires. We will qualitatively deduce this structure and discuss the implications of this finding, while taking into account the role and properties of the redox-active c-type cytochromes that are thought to be important constituents in these unique supramolecular assemblies. Finally, this combination of long-range electrical transport and redox proteins in one ready-made system motivates us to consider the integration with inorganic systems and the applicability as building blocks and connectors for biosensors and bioelectronic devices.
10:45 AM MM10.9
Bioselective Assembly of Dynamic Nanoarchitectures. Erik David Spoerke1, Judy Hendricks1, Adrienne Greene1, George Bachand1, Bruce Bunker1, Robert Haddon2 and Elena Bekyarova2; 1Sandia National Laboratories, Albuquerque, New Mexico; 2University of California - Riverside, Riverside, California.
Microtubules and motor proteins are critical components in the dynamic assembly and organization of nanomaterials within living cells. Our work aims to specifically utilize these "smart" biological tools to direct nanostructure assembly. We have developed an integrated approach to materials assembly that uses selectively functionalized lithographic substrates as platforms for bioassembly. Building from these substrates, we take advantage of the bioselective interactions between microtubules and motor proteins to first organize microtubule networks on the lithographic platforms and second template the assembly of nanocargo onto these organized microtubules. Engineering into the system control over the bioactive nature of the microtubule/motor protein interactions affords subsequent control of the assembly and disassembly of the organized nanostructures. This approach represents a promising new method for future nanoscale materials manipulation and organization.
11:00 AM MM10.10
Intercalation and Mechanical Behavior of Dye Doped Electrospun DNA Nanofibers. Yogesh Ner1, Gregory A. Sotzing1,3, Jeffrey A. Stuart2,3 and James G. Grote4; 1Polymer Program, Univ. of Connecticut, Storrs, Connecticut; 2Center for Nanobioelectronics, University of Connecticut, Storrs, Connecticut; 3Dept. of Chemistry, University of Connecticut, Storrs, Connecticut; 4Materials and Manufacturing Directorate, AFRL/MLPJ,, US Air Force Research Laboratory, Wright-Patterson Air Force Base, Ohio.
Nanostructures and nanoassemblies based on DNA are of special interest due to their potential for application in the areas of molecular electronics, nanoscale robotics, DNA-based computation, and advanced functional biomaterials. Herein we present bio-functional nanofibers derived from DNA complexed with a cationic surfactant. The surface modified material exhibits greatly improved processability, and can be easily electrospun to nanofibers of DNA. A fluorescent intercalating hemi-cyanine dye was incorporated into the nanofiber in order to examine intercalation behavior and DNA accessibility in the nanofibers. From this, we determined that DNA double helix structure is preserved and is available for small molecule intercalation. Furthermore, the combination of high fiber aspect ratio, confined geometry, and chromophore intercalation served to enhance the resulting fluorescence intensity as compared to that produced by cast films of the dye incorporated into DNA. Mechanically stretching non-woven, free-standing DNA nanofiber meshes resulted in alignment of DNA molecules along the stretch axis, representing direct observation of stress-induced anisotropy and DNA’s nano-assembly in the bulk scale.
11:15 AM MM10.11
Silica Nano-architectures Templated by Genetically Engineered Biomolecular Nanofibers. Chuanbin Mao1 and Fuke Wang1; 1Chemistry & Biochemistry, University of Oklahoma, Norman, Oklahoma; 2Chemistry & Biochemistry, University of Oklahoma, Norman, Oklahoma.
Filamentous phage (~1µm long and ~7 nm wide) is a biomolecular nanofiber made of a protein shell encasing DNA. Foreign peptides can be genetically displayed on the side wall to modify its surface chemistry. The peptide display enables us to control the materials synthesis on the surface of phage. We found that the engineered phage could direct the formation of phage-silica composite nanofibers with controlled nano-architectures. When positively charged peptides were displayed on phage, silica formation was templated by individual phage nanofibers. However, when negatively charged peptides were displayed on phage, the phage nanofibers self-assembled into a higher-order structure while templating the silica formation, leading to the formation of hierarchically structured nanofibers. Non-engineered phage formed a very long bundle and templated the formation of long silica fibers. Silica formation was found to be site-specific and catalyzed by the amino acids of the coat protein of the phage. The diameter and length of fibers can be tuned by the concentration of the phage and silica precursors. We envision that upon removal of the biological templates, mesoporous nanofibers can be synthesized. The pore sizes will be uniform as they are defined by the monodisperse viruses. They can be further used as templates to grow nanowires of different properties (e.g., metallic, semiconducting, and magnetic) with controlled organization.
11:30 AM MM10.12
Heterotropic Multivalency: a New Paradigm for Protein Assembly at Interfaces. Jurriaan Huskens, Molecular Nanofabrication group, University of Twente, MESA+, Enschede, Netherlands.
Multivalency is the phenomenon that describes the interaction between multivalent receptors and multivalent ligands. It is well known to play a pivotal role in biochemistry, particularly in protein-carbohydrate interactions, both in solution and at interfaces. In particular in the latter case, multivalency is often poorly understood in a quantitative sense. Supramolecular host-guest chemistry has been well established in solution, but its use at interfaces remains limited to for example sensor development for specific guest compounds. In order to build assemblies at surfaces through supramolecular interactions for nanotechnological applications, this inevitably leads to the use of multivalent interactions [1]. After an introduction into multivalent binding, we introduce the concept of molecular printboards, which are self-assembled monolayers functionalized with receptor groups suitable for nanofabrication [2]. The design of guest molecules allows precise control over the number of interacting sites and, therefore, over their (un)binding strength and kinetics. The newest results deal with heterotropic multivalency, which is the use of multiple interaction motifs. This has been applied to the controlled, selective, and specific binding of metal-ligand coordination complexes [3] and proteins [4]. The current paper will focus on the newest results dealing with the assembly of histidine-tagged proteins [5], antibodies, and cells. [1] J. Huskens, A. Mulder, T. Auletta, C. A. Nijhuis, M. J. W. Ludden, D. N. Reinhoudt, J. Am. Chem. Soc. 2004, 126, 6784; A. Mulder, T. Auletta, A. Sartori, S. Del Ciotto, A. Casnati, R. Ungaro, J. Huskens, D. N. Reinhoudt, J. Am. Chem. Soc. 2004, 126, 6627. [2] M. J. W. Ludden, D. N. Reinhoudt, J. Huskens, Chem. Soc. Rev. 2006, 35, 1122; J. Huskens, Curr. Opin. Chem. Biol. 2006, 10, 537; O. Crespo-Biel, B. J. Ravoo, D. N. Reinhoudt, J. Huskens, J. Mater. Chem. 2006, 16, 3997. [3] O. Crespo-Biel, C. W. Lim, B. J. Ravoo, D. N. Reinhoudt, J. Huskens, J. Am. Chem. Soc. 2006, 128, 17024; C. W. Lim, O. Crespo-Biel, M. C. A. Stuart, D. N. Reinhoudt, J. Huskens, B. J. Ravoo, Proc. Natl. Acad. Sci. USA 2007, 104, in press. [4] M. J. W. Ludden, M. Péter, D. N. Reinhoudt, J. Huskens, Small 2006, 2, 1192. [5] M. J. W. Ludden, A. Mulder, R. Tampé, D. N. Reinhoudt, J. Huskens, Angew. Chem. Int. Ed. 2007, 46, 4104.
11:45 AM MM10.13
Evolutionary Screening of Selective Biomimetic Receptors for Chemical Detection. Justyn Jaworski1,4, Digvijay Raorane2, Jin Huh1,3, Arunava Majumdar2,4,5 and Seung-Wuk Lee1,3; 1Bioengineering, UC Berkeley & UCSF, Berkeley, California; 2Mechanical Engineering, UC Berkeley, Berkeley, California; 3Physical Biosciences Division, Lawrence Berkeley National Labs, Berkeley, California; 4Materials Sciences Division, Lawrence Berkeley National Labs, Berkeley, California; 5Materials Science and Engineering, UC Berkeley, Berkeley, California.
Current commercially available chemical sensors, while very sensitive, face the common challenge of selectivity. The selectivity of a receptor to its respective target molecule is of incredible importance, since various interfering agents exists which are capable of creating false positive signals. By mimicking biology, we have demonstrated the use of sequence-specific biopolymers to generate highly selective receptors for a given target. This form of receptor based chemical interactions has the advantage over current sensor coatings materials in terms of selectivity and chemical/structural diversity. Additionally, our approach exploits a peptide based receptor embedded in a hydrogel for use in gas phase sensing. We have created highly selective peptide based receptors for targets of interest, trinitrotoluene (TNT) and 2,4-dinitrotoluene (DNT), in which we have shown extraordinary selectivity against these two very similar molecules. Furthermore, our identified DNT receptor has revealed the ability to selectively bind gas phase target molecules. These experiments exhibit the possibility to translate receptor screening in liquid phase to identifying a receptor coating capable of gas phase binding. The potential of such biomolecular recognition elements may create a new class of chemically specific sensor coatings based on evolutionary screened receptors.
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