Bionanomaterials Lab - UC Berkeley and Lawrence Berkeley National Laboratory

Research Program at the Lee Lab

Biomimetic Interfacial Nanomaterials

The primary goals of our research program are to create precisely defined biomimetic nanomaterials and to use them to study complex interfacial phenomena and to develop novel, functional materials and therapeutics.

Jump to section:

A. Atomic Level Investigation of Biomolecular Interfaces on Bone
B. Molecular Level Interactions at Bone Biointerfaces
C. Microscopic Level Interactions at Bone Biointerfaces
D. Macroscopic Level Interactions with Proteins and Cells
E. Virus-based Piezoelectric Energy Generation
F. Molecular recognition based biomimetic biosensor development


An understanding of the complex interfaces that exist between proteins, minerals, cells and their surrounding solutions in biological systems is critically important in biomaterial development because the reactions and interactions occurring at these interfaces govern their properties and functionalities. In our research group, we used chemical and biological approaches to create precisely defined nanomaterials, to investigate complex phenomena at their interfaces, and to develop novel, biomimetic, functional materials. Specifically, we focused on bone and its basic building blocks to study the fundamental mechanisms of bone mineralization and resorption and to develop bioinspired functional materials (Fig. 1). Calcified mineral tissues, such as bones and teeth, are remarkable components of our bodies that provide both structural functions as well as a means of storing essential minerals. Bones are composed of organic proteins, mainly collagenous-matrices, and inorganic crystals, hydroxyapatite, that are combined in hierarchically organized structures. The resulting composite structures are strong and fracture-resistant but dynamic in their ability to exchange mineral ions for the regulation of many biological functions. In the last decade, significant progress has been made in understanding the factors that cause bone mineralization, resorption, disease, and fractures. However, an understanding of the molecular mechanism of bone mineralization and resorption remains elusive. Attaining this knowledge will allow for advancements in disease treatment and synthetic orthopedic material development and will provide new information for designing materials and devices to solve challenging science and engineering problems.

Figure 1. Schematic illustration of our biomimetic interfacial nanomaterials research program.

For the last five years, we have created tractable model systems related to bone biointerfaces and developed novel bone-protein mimetic macromolecules that can be used for hard and soft tissue engineering materials and therapeutics (Figure 1). A) We investigated the atomic level bone crystal-solution interface by synthesizing single crystal hydroxyapatite with (100) or (001)-dominant surfaces and characterized their properties using in situ atomic force microscopy. Our studies were performed in real time under precisely defined conditions. We used various inorganic and organic modifiers to regulate bone surfaces under static and dynamic conditions. We first demonstrated that the bone crystal surfaces changed in a crystal structure dependent manner, exhibiting step shapes and heights that dynamically evolved upon the application of external cues (defects, ionic concentration, fluoride ions, amino acids and etc). B) We investigated molecular level bone crystal-peptide interfacial interactions using high throughput phage peptide library screening. We mimicked the evolution processes of bone proteins and discovered new collagen-like and other bone protein-like peptides which bind our well-characterized (100) HAP surfaces. These peptides exhibited bone crystal nucleation and dissolution inhibition activities that were verified using various microscopy techniques. Based on our AFM and phage display work, we also proposed possible mechanisms by which bone proteins and crystals interact. C) We investigated microscopic protein-protein and protein-crystal interactions using specifically designed protein sequences. Through a recombinant synthesis approach, we created bone-protein like biomacromolecules and incorporated them into organic/inorganic bone-mimetic composite materials. By varying the protein sequences and analyzing the resulting composites' microstructures and mechanical properties we were able to investigate the effects of protein-protein, protein-crystal, and protein-ion interactions. This allowed us to determine mechanisms by which the mechanical properties of bones are modulated. We verified that protein-crystal interactions were critical for enhancing the mechanical properties of the nanocomposites. D) We investigated macroscopic cell-protein interfaces using collagen-mimetic nanofiber matrices. We developed novel phage-based nanofiber matrices that could easily be engineered to display various biochemical cues and form self-assembled nanostructures for regulating cellular behaviors. Using this phage-based matrix system, we investigated various protein and cellular interfaces and developed a topical therapeutic material.

Our efforts to characterize these complex biointerfacial interactions and to develop precisely defined biomimetic materials are critical for understanding not only bone- or tooth-related diseases but also for designing functional materials and therapeutics. We believe that the successful completion of our research will broadly impact many areas of science and engineering. The following is a brief description of our major achievements and ongoing efforts:

A. Atomic Level Investigation of Biomolecular Interfaces on Bone

Jump to section:

B. Molecular Level Interactions at Bone Biointerfaces
C. Microscopic Level Interactions at Bone Biointerfaces
D. Macroscopic Level Interactions with Proteins and Cells
E. Virus-based Piezoelectric Energy Generation
F. Molecular recognition based biomimetic biosensor development
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We developed bone model surfaces whose structures were defined at the atomic level and investigated their structure dependent characteristics using in situ atomic force microscopy techniques. Mature mammalian hard tissues are composed mainly of calcium phosphates in the form of mineralized biological apatites. Biological apatites vary in impurity content but are most closely related to hydroxyapatite (HAP), Ca10(PO4)6(OH)2. Tremendous effort has been made to determine how various molecules interact with biologically-derived and synthetic apatites in order to better understand their roles in hard tissue biomineralization and integrity. However, the interactions between bone crystal and other biological molecules are not understood at the molecular level due to the complexity of bone structures, the lack of a well-defined model system, and the lack of tools to investigate bone mineral's complex interfaces. Therefore, we used two crystal surfaces [(100) and (001) HAP] (Fig. 2A and B) as models to study events at the bone interface: The former was used to model the bone surface at which proteins interact, and the latter was used to model the surface at which bone crystal growth dominantly occurs, when interfaced with collagen. Using in situ atomic force microscopy techniques, we investigated these HAP crystals under precisely controlled fluid conditions and demonstrated the first detailed characterization of hydroxyapatite interface properties at the molecular level. We verified that the bone crystal surfaces possessed crystallographic structure dependent properties, exhibiting specific asymmetric step shapes, heights, and mirror symmetry that dynamically evolved upon the application of external cues. These cues included local structural defects (Langmuir 2008), ionic strength (JPC 2009), fluoride ions (Langmuir 2010 revision), amino acids (Langmuir 2011 submitted), and peptides (Langmuir 2011).

A.1. Bone crystal surfaces possess crystallographic structure dependent properties: Our recent publications (Kwon et al, JPC 2009, Langmuir 2008, Langmuir 2009) demonstrate novel bone surface properties that were discovered through in situ AFM analysis of (100) HAP surfaces:

  1. HAP bone crystal surfaces possess crystal structure dependent properties. The (100) HAP surfaces mainly exhibit steps parallel to the elongated axes of the HAP crystals or angled approximately 54o to the HAP crystal's long axis (HAP [001] direction).
  2. Model bone surfaces exhibit asymmetric elongated hexagonal etch pits during demineralization. Under acidic buffer conditions, HAP crystals exhibit stochastically positioned elongated hexagonal etch pits oriented parallel to the [001] direction (Fig. 2C).
  3. Asymmetric etch pits evolve on HAP surfaces due to its molecular symmetry. HAP (100) surfaces possess mirror axes across the [100] and [010] directions but not across the [001] direction. Therefore, the evolution of HAP surface structures is associated with this mirror symmetry.
  4. HAP crystal features are determined by the unit cell structure of Ca10(PO4)6(OH)2. Vertical sectional analysis shows that the height of HAP surface steps is 0.82 nm, which corresponds to the interlayer distance between (100) surfaces (d(100)) (Fig. 2D and E).
  5. HAP crystal surfaces are terminated by phosphate groups, and their surface charges switch from negative to positive through protonation and deprotonation of these groups. We monitored pH dependent (100) step height profiles between pH 5-11 and found that step heights shorter than d(100) were not observed during dissolution. This observation excludes the coexistence of calcium and phosphate terminated layers at any given pH within this range (Fig. 2E).

Figure 2. Structure of bone model surfaces and their microstructure characteristics. (A, B) SEM images of (100) and (001) hydroxyapatite surfaces synthesized by molten salt and hydrothermal synthesis methods, respectively. Schematic insets represent the (100) and (001) surfaces (Scale bars: 2 um). (C) AFM image (1 um x 1um) of hexagonal etch pits formed in pH 6 buffer with 1 M NaCl. (D) The vertical cross-sectional trace along the yellow line in (C) displays the quantized nature of the step heights (E) Atomic models viewed along the [001] direction. The conventional unit cell is represented by the blue diamond (a = b = 0.9410 nm, c = 0.6883 nm, and d(100) 0.82 nm) (Ca: green; O: red; P: purple; H: white). Red dotted line represents the possible phosphate termination surface of the (100) HAP

A.2. High ionic concentrations of NaCl suppress bone demineralization: Na+ and Cl- ions are the major contributors to the ionic concentration in our bodies. Our recent publication (Kwon et al, JPC 2009) showed that high ionic concentrations of NaCl play a critical role in stabilizing bone crystals. Our findings suggest that typical Na+ and Cl- ion concentrations (140 mM) in extracellular body fluids may play a significant role in bone mineral stability.

A.3. Bone defects accelerate demineralization of bone crystal: Defects in bone minerals play a critical role in the bone remodeling processes. Our recent paper (Kwon et al, Langmuir, 2008) demonstrated that local structural defects on the model bone surfaces significantly accelerated demineralization (Fig. 3A). We characterized demineralization both in the presence and absence of defects. Our characterization showed that defects in bone can be easily removed by exposure to acidic buffer secreted by osteoclasts. Crystal surfaces with defects are dissolved ~100 times faster than non-defected surfaces.

A.4. Anti-cariogenic property of fluoride originates from the specific binding of fluoride to specific HAP steps: Fluoride ions play a critical role in preventing tooth decay. The work reported in our recent manuscript (Kwon et al, Langmuir 2010 in revision) verified the molecular mechanism behind the anti-cariogenic properties of fluoride. Through in situ dynamic characterization of steps on the (100) HAP surfaces under medically relevant fluoride concentrations (ranging from those found in tap water and mouth rinses), we measured the dynamic evolution of the crystal morphology. Changes to individual steps across a wide range of fluoride concentrations suggested that the anti-cariogenic property of fluoride ions originates from their strong interactions with the molecular steps in a structure dependent manner (Fig. 3B).

Figure 3. Structure dependent demineralization morphology of the (100) HAP bone model surfaces. (A) Defect-induced hexagonal shaped etch pit. (B) Fluoride-induced triangle morphology. Schematic inset shows that fluoride ions can bind to steps in a structure dependent manner. The triangle exhibited mirror symmetry through the horizontal plane.

B. Molecular Level Interactions at Bone Biointerfaces

Jump to section:

A. Atomic Level Investigation of Biomolecular Interfaces on Bone
C. Microscopic Level Interactions at Bone Biointerfaces
D. Macroscopic Level Interactions with Proteins and Cells
E. Virus-based Piezoelectric Energy Generation
F. Molecular recognition based biomimetic biosensor development
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We investigated the molecular level interactions between well-defined model bone surfaces (HAP (100) surfaces) and HAP-binding peptides discovered through phage display. The formation of natural bone is thought to occur by the templated mineralization of HAP by surrounding proteins, which include collagen and highly acidic phosphoproteins associated with the collagen scaffold. It has been proposed that the acidic groups serve as binding sites for calcium ions and align them in an orientation that matches the HAP crystal lattice. However, the biological mineralization process is not understood at the molecular level. An appealing strategy for biomimetic bone synthesis would be to employ HAP-binding peptides to facilitate HAP crystal growth. Unfortunately, the rational design of such peptides is deterred by a lack of knowledge regarding the sequences that guide HAP mineralization in vivo. Using phage display, we first discovered (100) HAP specific peptides and investigated their role in HAP growth at the molecular level (Langmuir 2011). We also measured the surface specific binding interactions between (100) HAP and the identified HAP peptides. Phage display is a combinatorial process used to identify specific binding peptides through a rapid, directed evolution process involving phage and bacterial biology. To identify the best peptide binding sequence for a given bone model surface, a library of engineered phage goes through several rounds of a selection process commonly called biopanning. Initially, the phage library is allowed to bind to the (100) HAP surfaces (90% purity). Non-bound phage are then washed away, and the bound ones are eluted and amplified through E. coli bacterial host infection. This approach of selecting the best binding peptide sequences is repeated several times to enrich the eluted phage with those that have the best affinities for the target. Finally, the DNA of the dominant binding phage are sequenced to identify the peptides that have the greatest affinities for target surfaces.

We discovered a collagen-like peptide that nucleated HAP crystals: In our recent work (Chung et al, Langmuir 2011), we presented a HAP-binding peptide that was discovered through phage display and the effects of this peptide on bone crystal mineralization. Using phage display, we identified a 12-residue peptide that bound to single crystal (100) HAP surfaces under physiological pH conditions (pH 7.5) (Fig. 4). This peptide was able to template the nucleation and growth of crystalline HAP mineral in a sequence- and composition-dependent manner (Fig. 5). The sequence responsible for the mineralizing activity resembled the tripeptide repeat (Gly-Pro-Hyp; Hyp: Hydroxyproline) of type I collagen, a major component of bone extracellular matrix. Using a panel of synthetic peptides, we defined the structural features required for mineralizing activity. The results suggest a model for the cooperative non-covalent interaction of the peptide with HAP and support the theory that native collagen has a mineral-templating function in vivo.

Figure 4. The identified HAP-binding peptide sequences from phage display and corresponding bone- and tooth-associated protein sequences from protein databases. The best binding peptide (CLP12) for the (100) HAP surface at pH 7.5 possessed repeating proline residues at i and i+3n positions (positions 2, 5 and 8) and periodic hydroxylated residues (positions 3, 6 and 10) resembling the major repeat sequence of type I collagen and amelogenin.

Figure 5. Transmission electron micrograph of mineralized HAP crystal templated by CLP12 peptide.

C. Microscopic Level Interactions at Bone Biointerfaces

Jump to section:

A. Atomic Level Investigation of Biomolecular Interfaces on Bone
B. Molecular Level Interactions at Bone Biointerfaces
D. Macroscopic Level Interactions with Proteins and Cells
E. Virus-based Piezoelectric Energy Generation
F. Molecular recognition based biomimetic biosensor development
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We investigated microscopic bone protein-crystal interactions to develop biomimetic, organic-inorganic nanocomposite materials.The organic component of bone materials is comprised of collagenous and non-collagenous protein matrices. Hierarchical collagenous protein matrices are known to provide a structural framework for templating mineralized bone crystals, whereas non-collagenous proteins in bone bind the mineral phase, transfer strain, and dissipate energy through the breaking of "sacrificial bonds". These proteins form various bonds through non-covalent intra-chain, inter-chain, and interfacial interactions. Because of their importance in nature, it would be extremely beneficial to further explore the rational design of synthetic organic phase analogues. Because conventional approaches of utilizing off-the-shelf polymers and naturally derived biological materials are very limited and fail to emulate the highly tuned sequences of natural proteins, we designed sequence specific biomacromolecules that closely emulated the function of bone protein matrices using biological approaches. Specifically, we developed sequence specific elastin-based protein matrices (Wang et al Biomacromolecules 2011) and phage based collagen-mimetic nanofiber matrices.

We developed elastin-based sequence-specific bone protein nanocomposite materials: Non-collagenous protein matrices play multiple critical roles in bone mechanical properties. Our recent paper (Wang et al, Biomacromolecules 2011) presented our approach of emulating the mechanical enhancements observed in nature by designing sequence specific protein-based polymers and incorporating them into calcium phosphate bone cement materials. We developed novel elastin-like polymers (ELP) to construct mechanically enhanced calcium phosphate cement (CPC)-protein nanocomposites. By controlling ELP primary structures at the molecular level through genetic engineering, we could synthesize protein matrices to mimic the adhesive proteins of bone. We incorporated octaglutamic acid, a previously characterized HAP binding motif, at the protein termini and positively, negatively, or neutrally charged amino acids along the polymer backbone to control intramolecular, intermolecular, and interfacial interactions with HAP. By imparting ELPs with varying modes of inter/intra molecular bonding and interfacial bonding, we were able to demonstrate sequence specific property changes in ELP-HAP mixtures (Fig. 6A and B). Our results showed that interfacial binding to HAP through octaglutamic acid motifs was critical for imparting functionalities such as nanoparticle dispersion and mechanical property improvements in ELP-CPC composites (Fig. 6C). In the absence of binding, the physical properties of the protein had no effect. We also demonstrated the injectability of the ELP-CPC composites, which transformed into nanocomposites at body temperature in vitro. We believe that our results are not limited to applications involving HAP but can be expanded to explore composites containing other metals and minerals. Our design and synthesis approaches may yield strong and tough materials for future load-bearing applications such as hard-tissue replacements.

Figure 6. Sequence specific elastin-based bionanocomposite (A) A: Backbone-backbone Van der Waals interactions or hydrogen bonding. B: ELP-HAP binding C: Backbone-backbone ionic interactions. D: Ion-mediated crosslinks. (B) MALDI mass spec-results show the the presence of monodisperse ELP protein matrices (C) Mechanical characterization of ELP-CPC composites. Results of compressive strength tests show enhanced strength for V125-E8 and V125-E8E8 containing cements. (D) ELP (V125E8) stabilizes calcium phosphate composites in 37C HEPES buffer after mixing. The ELP-free mixtures were unstable and disintegrated, whereas the ELP containing mixtures remained cohesive.

We developed collagen-mimetic hierarchically self-assembled structures: The helical structure of collagen results in the creation of diverse hierarchical structures that are made in a self-assembled manner (e.g., bone, cornea, skin tissues). However, the molecular mechanism by which these structures are formed is not understood at the molecular level. In our recent report (Nature 2011), we used phage-based nanofibers to determine the mechanism by which the hierarchical organization of self-assembled structures occurs. We used M13 phage as a model system because it possesses collagen-like structural features with genetic flexibility (helical nanofiber-like shape, monodispersity, and the ability to display functional peptides on its surfaces). To mimic natural hard tissue molecular structures, we genetically engineered M13 to display RGD (integrin binding peptide)- and EEEE(tetra glutamic acid; negatively charged protein domains in osteogenic proteins)-peptides on its major coat proteins. Using these engineered phages, we fabricated biomimetic dental enamel-like helicoidal structures through phage self-assembly processes. When we treated dental enamel-like hierarchical zig-zag structure films composed of both RGD-phage and EEEE-phage (1:1) (Fig. 7A) with a precursor solution containing Ca2+ and PO43-, the phage films templated mineralization of calcium phosphate that exhibited hierarchically organized tooth-like organic-inorganic composite structures (Fig. 7B). We also found that the stiffness (Young's modulus) of the phage film significantly increased ~20x after mineralization (Fig. 7C). Thus, we induced desired tissue functions by constructing self-assembled micropatterns through the display of biochemical functional motifs using genetically engineered phages. Our approach may provide a means for understanding the helical self-assembly of the hierarchical structures of bones and give insight into hierarchical structure-function relationships in nature. Currently, we are applying our self-assembly techniques to other biomacromolecules (collagen and cellulose) and investigating their molecular assembly processes for the development of biomimetic hierarchical structures for tissue regeneration.

Figure 7. Fabrication of hierarchical tissue matrices. (A) SEM image of the dental enamel-like helicoidal nanofilament phase RGD-/EEEE-phage film (1:1). (B) SEM image of the RGD-/EEEE-phage (1:1) based hard tissue composite materials mineralized using Ca2+ and PO43- solutions. Blue arrows indicate zig-zag structures of the composite materials. (C) Young's modulus (stiffness) of the phage film significantly increased (~18 times) after mineralization using Ca2+ and PO43- solutions.

D. Macroscopic Level Interactions with Proteins and Cells

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A. Atomic Level Investigation of Biomolecular Interfaces on Bone
B. Molecular Level Interactions at Bone Biointerfaces
C. Microscopic Level Interactions at Bone Biointerfaces
E. Virus-based Piezoelectric Energy Generation
F. Molecular recognition based biomimetic biosensor development
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We have developed novel phage-based materials that can regulate cell behavior to study macroscopic events at the interfaces of biomacromolecules and cells. Phage possess many desirable features that make them useful as biomedical materials for various therapeutic purposes: 1) Phage are safe and have little harmful effects. Phage are easily removed from the body through lysosomal degradation and the liver, causing few known side-effects (Wadih Arap et al Cell 2006). Recently, the Food and Drug Administration approved the use of phage for the disinfection of frozen foods to prevent food poisoning (FDA Report 2006). 2) Phage can be readily modified to display functional peptide motifs on their minor (pIII, pIX) and major (pVIII) coat proteins. Functional peptide sequences can be identified through directed evolutionary screening processes (phage display). 3) Large quantities of identical phage building blocks can be easily prepared through amplification (non-lytic phage reproduction) using bacterial host cells (E. coli). 4) Due to their monodispersity and high aspect ratio, phage can be self-assembled by controlling their concentration and external force fields. 5) Additional gene engineering can be performed to further modify the biological properties of the phage. By exploiting the unique material features of phage, we have developed various novel bionanomaterials including phage-based tissue matrix materials and topical therapeutics.

Phage-based nanofiber matrices for regulating cell behavior: Designing biomimetic materials with precisely controlled structural organization that closely mimics the natural tissue environment is critical for the development of regenerative medicines. Many attempts have been made to mimic natural tissue microenvironments in vivo by using extracted extracellular matrix (ECM) or synthetic ECM-mimetic materials to manipulate the ECM's biological, chemical and mechanical properties. Although the resulting structures provide great promise for improving cell behavior, emulating complex tissue microenvironments and precisely controlling biochemical and physical cues using conventional approaches is still challenging. Any modification of chemical structures requires labor-intensive chemical synthesis, and the resulting chemical structures affect other important physical and mechanical parameters. A material that mimics the nanofibrous structure of the ECM and whose biochemical structure can be conveniently modified with little change in physical structure and assembly processes would be very desirable for investigating various biochemical cues and their effects on cellular growth processes. Recently, we developed novel phage-based biomimetic nanofiber matrices that could easily be engineered to display various biochemical cues and form self-assembled nanostructures for regulating cellular behaviors (Fig. 7). Using this phage-based tissue matrix system, we investigated various protein and cellular interfaces and discovered new findings on the interactions between protein and cells. In our recent work (Yoo et al, Biomacromolecules 2011 in Press), we examined the effects of a collagen-derived biochemical cue (DGEA) on bone cells. We engineered M13 phage to display the DGEA-peptide in high densities on their major coat proteins and studied their effects on mouse derived bone stem cells (preosteoblasts, MC3T3-E1). Through our study, we verified that the DGEA-peptides could stimulate bone stem cells to outgrow, an event that is linked to osteogenic differentiation. We expanded our protein-cell interface investigation by using other cell types and biochemical cues. Our recent papers (Merzlyak et al, Nano Letters 2009 and Chung et al, Langmuir 2010) reported that phage engineered to express RGD (integrin binding peptide) and IKVAV (neural cell stimulating peptide) peptides formed two- and three-dimensional matrices that regulated the directional growth of neural cells in a chemical and physical cue specific manner. In addition, our recent paper (Yoo et al, Soft Matter 2010) presented a facile strategy for immobilizing growth factors on genetically engineered phage matrices for tissue regeneration. We modified M13 phages to express streptavidin-binding peptides (HPQ) and/or integrin binding peptides (RGD) on their major and minor coat proteins. The resulting phages formed nanofibrous matrices that could easily immobilize the streptavidin-conjugated growth factors FGF-b and NGF for neural cell proliferation and differentiation. We verified that the immobilized growth factors possessed a prolonged ability to stimulate the target cells. We demonstrated the synergistic roles of the growth factors and integrin binding peptides in controlling cell morphologies and the growth of neural cells. Our phage matrices, which could be easily functionalized with various ligands and growth factors, can be used as convenient test beds for investigating the functions of various biochemical stimulants on numerous cell types.

Figure 7. Schematic diagram of the development of novel biomedical engineering materials using genetically engineered phage. After creating the functional motif-displaying phage, we can construct nanofibrous matrices that can control and guide cell behavior. These matrix materials can be used for the development of topical therapeutics.

E. Virus-based Piezoelectric Energy Generation

Jump to section:

A. Atomic Level Investigation of Biomolecular Interfaces on Bone
B. Molecular Level Interactions at Bone Biointerfaces
C. Microscopic Level Interactions at Bone Biointerfaces
D. Macroscopic Level Interactions with Proteins and Cells
F. Molecular recognition based biomimetic biosensor development

Piezoelectric materials can convert mechanical energy into electrical energy, and piezoelectric devices made of various inorganic materials and organic polymers have been demonstrated. However, synthesizing such materials often requires toxic materials, harsh conditions and/or complex procedures. Alternatively, it was shown that hierarchically organized natural materials, such as bones, collagen fibrils and peptide nanotubes, can display piezoelectric properties. Therefore, we are exploring the piezoelectric properties of M13 bacteriophage (phage). Using piezoresponse force microscopy, we characterized the structure-dependent piezoelectric properties of phage at the molecular level. We then showed that self-assembled thin films of phage can exhibit piezoelectric strengths of up to 7.8 pm/V. We also demonstrate that it is possible to modulate the dipole strength of phage, and hence tune their piezoelectric response by genetically engineering the phage's major coat proteins. Finally, we developed a phage-based piezoelectric generator that produced up to 6 nA of current and 400 mV of potential, and used it to operate a liquid crystal display. Because biotechnology techniques enable large-scale production of genetically modified phages, phage-based piezoelectric materials potentially offer a simple and environment-friendly approach to piezoelectricity generation.

Figure 8. The M13 bacteriophage has a rod-like shape and is coated with approximately 2700 charged proteins (right) that enable it to act as a piezoelectric nanofiber.

Figure 9. Out of plane piezoelectric properties of the phage. (A) AFM topography image of a region (3um x 3um) of highly ordered phages. (B) The effective piezoelectric coefficient deff image of the same region as (A). The deff shows relatively lower values on the grooves. The piezoresponse of phage film in comparison to periodically poled lithium niobate (PPLN) and collagen film. We obtained the effective piezoelectric coefficient deff ~ 3.86 pm/V for the phage film. The PPLN and collagen showed deff ~ 6.61 pm/V and ~0.57 pm/V, respectively. (D) Trends of the piezoelectric response dependent on additional negative surface charge incorporation from one glutamate (1E) up to four glutamates (4E)

F. Molecular recognition based biomimetic biosensor development:

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A. Atomic Level Investigation of Biomolecular Interfaces on Bone
B. Molecular Level Interactions at Bone Biointerfaces
C. Microscopic Level Interactions at Bone Biointerfaces
D. Macroscopic Level Interactions with Proteins and Cells
E. Virus-based Piezoelectric Energy Generation
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Miniaturized smart sensors that can perform sensitive, selective, and real-time monitoring of explosives and biological toxins are tremendously valuable to our nation's ability to deploy effective homeland security measures and to protect civilians and our military forces throughout the world. Current sensing devices are still far from being able to offer selective, sensitive, and real-time point-detection. They are also lacking in multi-analyte assessment, ease-of-use, and low manufacturing costs. To address these critical issues, we have developed a new approach, whereby the principles of molecular recognition in biology are mimicked to achieve highly selective binding to small molecular targets such as explosives and biotoxins. We discovered molecular recognition elements (MREs) against explosives (TNT and DNT) and biological and environmental toxins (Cholera toxin, PBDEs, and pesticides) by using directed evolution of phage peptide libraries (Fig. 8). Using these MREs, we developed multiple nanocoatings for cantilever and quartz crystal microbalance sensing platforms which showed highly selective and sensitive multimodal detection (Jaworski et al Langmuir 2008 and Anal Chem 2009) through the collaboration with Professors Arun Majumdar and Roya Maboudian (UC Berkeley). In addition, we have recently developed a novel selective and sensitive biomimetic nanocoating by combining TNT receptors bound to conjugated polydiacetylene (PDA) polymers. PDA is a lipid-like polymer comprised of a conjugated polymer backbone with carboxylic acid and alkyl side-chains. The amphiphilic nature of PDA monomers facilitates its formation into supramolecular assemblies such as vesicles and membranes. PDA's conjugated polymer backbone can serve as a stable and sensitive colorimetric sensor due to changes in its conjugated electronic band structure resulting from interactions between target analytes and specific functional motifs on PDA's head-groups. In our recent work (Jaworski Langmuir 2011 In Press), we described our colorimetric PDA-based TNT sensor development. Furthermore, we applied these PDA-TNT receptor coupled nanocoating materials into CNT-FET devices through collaboration with Professor Seunghun Hong (Physics, Seoul National University). Our recent paper (Kim et al ACS Nano 2010 submitted) reported that selective binding events between the TNT molecules and phage display-derived TNT receptors were effectively transduced to sensitive SWNT-FET conductance sensors through the PDA coating layers. The resulting sensors exhibited unprecedented 1 fM sensitivity toward TNT in real time, with excellent selectivity over various similar aromatic compounds. Our biomimetic receptor coating approach may be useful for the development of sensitive and selective micro- and nanoelectronic sensor devices for various other target analytes.

Figure 10. Schematic diagram of phage display against a TNT target