Symposium Organizers
Vincent M. Rotello University of Massachusetts
Jeffrey B.-H. Tok Lawrence Livermore National Laboratory
Molly M. Stevens Imperial College London
Darrin J. Pochan University of Delaware
Paula T. Hammond Massachusetts Institute of Technology
MM1/NN1: Joint Session
Session Chairs
Derek Woolfson
Michael Yu
Monday PM, November 26, 2007
Room 210 (Hynes)
9:30 AM - **MM1.1/NN1.1
Non-Canonical Amino Acids in Protein Engineering.
David Tirrell 1
1 , Caltech, Pasadena, California, United States
Show Abstract10:00 AM - **MM1.2/NN1.2
Stronger and Longer Synthetic Collagen.
Ronald Raines 1 2
1 Department of Biochemistry, University of Wisconsin - Madison, Madison, Wisconsin, United States, 2 Department of Chemistry, University of Wisconsin - Madison, Madison, Wisconsin, United States
Show AbstractCollagen 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.]
10:30 AM - MM1.3/NN1.3
Spatiotemporal Modification of Collagen Scaffolds Directed by Collagen Mimetic Peptide Derivatives.
Allen Wang 1 , Shirley Leong 2 , Catherine Foss 3 , Xiao Mo 1 , Martin Pomper 3 4 , Seungju Yu 1 4
1 Deptartment of Materials Science and Engineering, The Johns Hopkins University, Baltimore, Maryland, United States, 2 Department of Chemical and Biomolecular Engineering, The Johns Hopkins University, Baltimore, Maryland, United States, 3 Department of Radiology, The Johns Hopkins University, Baltimore, Maryland, United States, 4 Institute for NanoBiotechnology, The Johns Hopkins University, Baltimore, Maryland, United States
Show AbstractFunctionalized 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.
10:45 AM - MM1.4/NN1.4
Building Tissue Engineering Scaffolds Directly from Extracellular Matrix Proteins with Microscale Spatial Control.
Adam Feinberg 1 , Sean Sheehy 1 , Kevin Parker 1
1 School of Engineering and Applied Sciences, Harvard University, Cambridge, Massachusetts, United States
Show AbstractWe 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 ~20μm 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.
11:00 AM - MM1.5/NN1.5
Self-Assembling Hydrogels from Fibrin Coiled Coil Peptide-Polymers.
Peng Jing 1 , Joel Collier 1
1 Biomedical Engineering, University of Cincinnati, Cincinnati, Ohio, United States
Show AbstractFibrin-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.
11:30 AM - **MM1.6/NN1.6
Supramolecular Organization From Nanometers to Centimeters in Peptidic Materials.
Samuel Stupp 1 2
1 Materials Science and Engineering, Chemistry and Medicine, Northwestern University, Evanston, Illinois, United States, 2 Institute for BioNanotechnology in Medicine, Northwestern University, Evanston, Illinois, United States
Show AbstractOver 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.
12:00 PM - MM1.7/NN1.7
Early Time β-Hairpin Peptide Self-Assembly into a Hydrogel Network.
Tuna Yucel 1 2 , Joel Schneider 3 , Darrin Pochan 1 2
1 Materials Science and Engineering, University of Delaware, Newark, Delaware, United States, 2 , Delaware Biotechnology Institute, Newark, Delaware, United States, 3 Chemistry and Biochemistry, University of Delaware, Newark, Delaware, United States
Show AbstractIn 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.
12:15 PM - MM1.8/NN1.8
Modification of Liposomes using α-Helical Coiled-Coil Peptides.
Hana Robson Marsden 1 , Alexander Korobko 1 , Alexander Kros 1
1 Chemistry, Leiden University, Leiden Netherlands
Show AbstractA 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.
12:30 PM - **MM1.9/NN1.9
Nanofibers formed by Self-assembly of Multidomain Meptides: Applications for Bioengineering to Nanotechnology.
Jeffrey Hartgerink 1 , Kerstin Galler 1 , He Dong 1 , Lorenzo Aulisa 1 , Sergey Paramonov 1
1 Chemistry & Bioengineering, Rice University, Houston, Texas, United States
Show AbstractControl 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.
MM2/NN2: Joint Session
Session Chairs
Paula Hammond
Seung-Wuk Lee
Monday PM, November 26, 2007
Room 210 (Hynes)
2:30 PM - **MM2.1/NN2.1
Genetic Control of the Synthesis and Assembly of Materials for Electronics and Energy.
Angela Belcher 1 , Ki Tae Nam 1 , Yun Jung Lee 1 , Dong-Soo Yun 1 , Brian Neltner 1 , Andrew Magyar 1
1 , MIT, Cambridge, Massachusetts, United States
Show Abstract3:00 PM - **MM2.2/NN2.2
Building from Bottom Up: Fabrication of Nanomaterials Using Peptide Motifs.
Shugang Zhang 1
1 , MIT, Cambridge, Massachusetts, United States
Show AbstractMaterials 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.
3:30 PM - MM2.3/NN2.3
Development of Novel Hard-Tissue Regenerative Materials Through Directed and Natural Evolutionary Processes.
Eddie Wang 1 , Seung-Wuk Lee 1 2
1 Bioengineering, University of California, Berkeley, Berkeley, California, United States, 2 Physical Biosciences Division, Lawerence Berkeley National Lab, Berkeley, California, United States
Show AbstractBones 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.
3:45 PM - MM2.4/NN2.4
Supramolecular Self-Assembly of a Metal-binding Polypeptide and Implications for Molecular Recognition.
Christopher So 1 , Emre Oren 1 , Urartu Seker 1 3 , Brandon Wilson 1 , John Kulp 2 , Candan Tamerler 1 3 , John Evans 2 , Sarikaya Mehmet 1
1 Materials Science and Engineering, University of Washington, Seattle, Washington, United States, 3 Molecular Biology & Genetics, Istanbul Technical University, Istanbul Turkey, 2 Chemistry, New York University, New York, New York, United States
Show AbstractRecently, 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.
4:30 PM - **MM2.5/NN2.5
Targeted Protein Cage Architectures for Biofilm Imaging and Therapeutics.
Trevor Douglas 1
1 Department of Chemistry and Biochemistry, Montana State University, Bozeman, Montana, United States
Show AbstractDiagnosis 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.
5:00 PM - MM2.6/NN2.6
Ferritin Cage Architecture with Superparamagnetic Iron Oxide Nanoparticle for Magnetic Resonance Contrast Agent.
Masaki Uchida 1 3 , Masahiro Terashima 4 , Charles Cunningham 5 , Yoriyasu Suzuki 4 , Deborah Willits 2 3 , Ann Willis 1 3 , Philip Yang 4 , Michael McConnell 4 , Mark Young 2 3 , Trevor Douglas 1 3
1 Department of Chemistry and Biochemistry, Montana State University, Bozeman, Montana, United States, 3 Center for Bio-Inspired Nanomaterials (CBIN), Montana State University, Bozeman, Montana, United States, 4 School of Medicine, Stanford University, Stanford, California, United States, 5 Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada, 2 Department of Plant Seicences, Montana State University, Bozeman, Montana, United States
Show AbstractProtein 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.
5:30 PM - MM2.8/NN2.8
Protein-Mediated Assembly of Metal Nanostructures.
Silke Behrens 1 , Wilhelm Habicht 1 , Konrad Joachim Boehm 2
1 Institute for Technical Chemistry, Forschungszentrum Karlsruhe, Karlsruhe Germany, 2 , Leibnitz Institute for Age Research, Jena Germany
Show AbstractSelf-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.
MM3: Poster Session I
Session Chairs
Darrin Pochan
Vincent Rotello
Tuesday AM, November 27, 2007
Exhibition Hall D (Hynes)
9:00 PM - MM3.1
Effects of Restriction Enzymes on DNA-mediated Assembly and Disassembly at a Fixed Temperature.
Christopher Tison 1 , Valeria Milam 1
1 Materials Science & Engineering, Georgia Institute of Technology, Atlanta, Georgia, United States
Show AbstractWe 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.
9:00 PM - MM3.10
Nanofabrication Based on DNA Engineering and Microtemplated Dewetting.
Wenlong Cheng 1 , Nokyoung Park 1 , Young Roh 1 , Dan Luo 1
1 Biological and Environmental Engineering, Cornell University, Ithaca, New York, United States
Show AbstractThe 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.References1.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.
9:00 PM - MM3.11
Single Walled Carbon Nanotube Fluorescence Detection of DNA Hybridization: Kinetics, Thermodynamics, and Applications.
Esther Jeng 1 , John Nelson 1 , Michael Strano 1
1 Chemical Engineering, MIT, Cambridge, Massachusetts, United States
Show AbstractSingle 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.
9:00 PM - MM3.12
Chitosan Biotinylation and Electrodeposition for Protein Assembly at Electrode Addresses.
Gregory Payne 1 , Xiaowen Shi 1 , Yi Liu 1 , Angela Lewandowski 1 2 , Hsuan-Chen Wu 1 2 , Li-Qun Wu 1 , Reza Ghodssi 2 , Gary Rubloff 2 , William Bentley 1 2
1 Center for Biosystems Research, University of Maryland Biotechnology Institute, College Park, Maryland, United States, 2 , University of Maryland, College Park, Maryland, United States
Show Abstract9:00 PM - MM3.13
The Absorption of Polysaccharides and Polypeptides onto Calcite Surfaces.
John Harding 1 , Mingjun Yang 1
1 Engineering Materials, University of Sheffield, Sheffield United Kingdom
Show AbstractOrganic 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.
9:00 PM - MM3.14
Chitosan Films as Substrate to Line Patterning Technique of Graphite (LPTG). Application as Sensors.
Clarice Steffens 2 , Douglas de Britto 1 , Paulo Sergio Herrmann Jr 1
2 Food Engineering, Universidade Regional Integrada (URI), Erechim, Rio Grande do Sul, Brazil, 1 Agricultural Instrumentation, Embrapa Agricultural Instrumentation, São Carlos, São Paulo, Brazil
Show AbstractFlexible 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.
9:00 PM - MM3.15
Preparation of Chitosan Sub-micron Beads by Phase Separation with Polyvalent Anion and their Evaluation as Bacteriostatic Materials.
Shoji Nagaoka 1 , Kanako Saita 1 2 , Tetsuya Yamamoto 3 , Seitaro Kobayashi 2 , Ken Satoh 4 , Kenji Kurashiki 5 , Makoto Takafuji 2 , Hirotaka Ihara 2
1 Materials Development Department, Kumamoto Industrial Research Institute, Kumamoto, Kumamoto, Japan, 2 Department of Applied Chemistry and Biochemistry, Faculty of Engineering, Kumamoto university, Kumamoto, Kumamoto, Japan, 3 , Daiichi Seimo Co. Ltd., Arao, Kumamoto, Japan, 4 , Nishinihon Nagase Co. Ltd., Fukuoka, Fukuoka, Japan, 5 , Muromachi Chemical Inc., Oomuta, Fukuoka, Japan
Show AbstractIn 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.
9:00 PM - MM3.16
Electrochemical Measurement of Oligosaccharide Monolayers: Self Assembly and Lectin Binding.
Joshua Hertz 1 , David Lahr 1 , Ju-Hee Park 2 , Philip DeShong 2 , Michael Tarlov 1
1 Chemical Science and Technology Laboratory, National Institute of Standards and Technology, Gaithersburg, Maryland, United States, 2 Department of Chemistry and Biochemistry, University of Maryland, College Park, Maryland, United States
Show AbstractElectrochemical 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.
9:00 PM - MM3.17
Alternative Substrate Materials for Supported Lipid Bilayers.
Barbara Nellis 1 2 , Emel Goksu 1 , Marjorie Longo 1 , Alex Gash 2 , Joe Satcher 2 , Subhash Risbud 1
1 Chemical Engineering and Materials Science, University of California, Davis, Davis, California, United States, 2 Chemistry, Materials and Life Sciences Directorate, Lawrence Livermore National Labs, Livermore, California, United States
Show AbstractSupported 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.
9:00 PM - MM3.18
Electrical Impedance Analysis of Phospholipid Bilayer Membranes for Enabling Engineering Design of Bio-based Devices.
Stephen Sarles 1 , Donald Leo 1
1 Center for Intelligent Material Systems and Structures (CIMSS), Virginia Tech, Blacksburg, Virginia, United States
Show AbstractRecent 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.
9:00 PM - MM3.19
Failure Characteristics of Bilayer Lipid Membranes Formed over a Single Pore.
David Hopkinson 1 , M. Creasy 1 , Donald Leo 1
1 Mechanical Engineering, Virginia Tech, Blacksburg, Virginia, United States
Show AbstractBilayer 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.
9:00 PM - MM3.2
Novel Protein/DNA/inorganic Biocatalytic Nanomaterials.
Akhilesh Bhambhani 1 2 , Challa V. Kumar 2
1 Pharmaceutical Chemistry, The university of Kansas, Lawrence, Kansas, United States, 2 Department of Chemistry, University of Connecticut, Storrs, Connecticut, United States
Show AbstractGiven 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.
9:00 PM - MM3.20
Lipid-Enveloped Bioresorbable Nanoparticles as ``Synthetic Pathogens" for Vaccine Design.
Anna Bershteyn 1 , Tania Chan 1 , José Chaparro 1 , Richard Yau 1 , Darrell Irvine 1
1 Materials Science and Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States
Show AbstractThe 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.
9:00 PM - MM3.21
Reactive Multi-component Membranes: From Dynamic Shape Reconstruction to Self-Cleaning.
Olga Kuksenok 1 , Anna Balazs 1
1 , University of Pittsburgh, Pittsburgh, Pennsylvania, United States
Show Abstract9:00 PM - MM3.22
Micro/Nano-patterning of Supported Phospholipid Membranes: From Sensor Design to Biophysical Studies.
Jinjun Shi 1 , Tinglu Yang 1 , Paul S. Cremer 1
1 Department of Chemistry, Texas A&M University, College Station, Texas, United States
Show AbstractMicro/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.
9:00 PM - MM3.23
Preparation of Cell Rolling Surfaces by Controlled Covalent Immobilization Methods.
Seungpyo Hong 1 , Allen Taylor 2 , Dooyoung Lee 3 , Michael King 3 , Shaoyi Jiang 2 , Robert Langer 1 , Jeffrey Karp 1
1 Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States, 2 Chemical Engineering, University of Washington, Seattle, Washington, United States, 3 Chemical Engineering, University of Rochester, Rochester, New York, United States
Show AbstractCell 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.
9:00 PM - MM3.24
Layer-by-Layer Assembly of Biodegradable Block Copolymer Micelles for Applications in Drug Delivery.
Byeong-Su Kim 1 , Renee Smith 1 , Paula Hammond 1
1 Chemical Engineering, MIT, Cambridge, Massachusetts, United States
Show AbstractLayer-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.
9:00 PM - MM3.3
Asymmetric Functionalization of Gold Nanoparticles with Oligonucleotides.
Xiaoyang Xu 1 , Chad Mirkin 1
1 Chemistry, International Institute for Nanotechnology, Evanston, Illinois, United States
Show AbstractGold 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.
9:00 PM - MM3.4
Electronic Transport in DNA Segments with Diluted Base-pairing.
Eudenilson Albuquerque 1 , L. Da Silva 1 , F. De Moura 2 , M. Lyra 2
1 Fisica, UFRN, Natal, RN, Brazil, 2 Fisica, UFAL, Maceió, AL, Brazil
Show Abstract9:00 PM - MM3.5
Conformation of DNA Oligos on Gold Nanoparticles.
Katherine Brown 1 , Kimberly Hamad-Schifferli 1
1 Biological Engineering, Massachusettes Institute of Technology, Cambridge, Massachusetts, United States
Show AbstractDNA 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.
9:00 PM - MM3.6
Selective DNA-Mediated Assembly of Gold Nanoparticles on Gold Patterned Substrates.
Kim Sapsford 1 , Doe Park 3 , Ellen Goldman 4 , Edward Foos 2 , Arthur Snow 2 , Mario Ancona 3
1 CBMSE Code 6900, NRL, George Mason University and The Naval Research Laboratory, Washington, District of Columbia, United States, 3 Electronics Science and Technology Division, Naval Research Laboratory, Washington , District of Columbia, United States, 4 Center for Bio/Molecular Science and Engineering (CBMSE), Naval Research Laboratory, Washington, District of Columbia, United States, 2 Chemistry Division, Naval Research Laboratory, Washington , District of Columbia, United States
Show Abstract9:00 PM - MM3.7
Directed DNA-Metallization: Towards The Construction of Rationally Designed Conductive Nano Devices.
Christian Wirges 1 , Katrin Gutsmiedl 1 , Johannes Gierlich 1 , Philipp Gramlich 1 , Glenn Burley 1 2 , Thomas Carell 1
1 Chemistry and Biochemistry, Chemistry and Pharmacy, Munich Germany, 2 , Chemistry, Leicester United Kingdom
Show AbstractDNA 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.
9:00 PM - MM3.8
Evaluation of Hydrodynamic Size and Surface Charge Density of Surface Modified Au Nanoparticle-DNA Conjugates by Ferguson Analysis.
Sunho Park 1 , Kimberly Hamad-Schifferli 1 2
1 Department of Mechanical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States, 2 Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States
Show AbstractConjugates 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.
9:00 PM - MM3.9
DNA with Zip Codes: Addressable DNA Molecules and their Polymerization.
Jong Bum Lee 1 , Young Hoon Roh 1 , Dan Luo 1
1 Biological Engineering, Cornell University, Ithaca, New York, United States
Show AbstractDNA 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.