With our drop-cast phage templating system, we are able to create precisely defined biomimetic nanomaterials which can be developed into novel materials with improved strength, toughness and functionality. Along with the Lee Lab, (https://leelab.berkeley.edu/ ) we use the bacterial virus M13 phage as a building block for the templating process. We use this viral macromolecule because of its helical, nanofibrous shape, for its monodispersity and for its ability to display multiple functional motifs (Fig. 1a). We then assemble phage into large-area films by creating an apparatus to pull substrates vertically from phage suspensions at precisely controlled speeds. As the substrates are pulled, evaporation proceeded fastest near the air–liquid–solid contact line resulting in the local accumulation and deposition of phage particles on the substrate at the meniscus (Fig. 1b). By controlling the phage concentration, the pulling speed, the ionic concentration and the functional motif on the virus itself, we are able to create an array of chiral liquid-crystal phase transitions at the meniscus. These include nematic orthogonal twist, cholesteric helical ribbon, and smectic helicoidal Nanofilament (Fig. 2).
Figure 1 | Schematic diagram of the phage-based self-templating process. A) Schematic illustration of the phage structure covered by about 2,700 a-helical major coat (pVIII) protein subunits having five-fold helical symmetry and a two-fold screw rotation axis (red and green arrows). The phage has a helical angle (w) of 41u with periodic subunit spacing of 3.3 nm. B) Schematic illustration of the helical self-templating assembly of phage particles controlled by competing interfacial forces at the meniscus where liquid crystal phase transitions occur. The polarized optical microscopy image shows iridescent colours originating from liquid crystal phase formation at the air–liquid–solid interface.
Figure 2 | Self-templated helical supramolecular structures. A) Diagram of the nematic orthogonal twist structure. B) Scanning electron microscopy (SEM) image of a periodic, striped pattern. C), Atomic force microscopy (AFM) image depicting left-handed rotation of phage fibre bundles when progressing from a groove to ridge area. D) Diagram of alternating nematic grooves (1, black) and cholesteric ridges (2, red). E) Diagram of the cholesteric helical ribbon (CHR) F) Photograph of CHR structures showing the curved meniscus (dashed red line), which induces the rolling of phage supramolecular structures (blue arrows). G), SEM images of the CHR structure on the left and right sides, which show right and left handedness, respectively. H) AFMimage showing that theCHR is composed of twisted cholesteric phase (red dashed lines indicate the cholesteric arch morphology). I) Diagram of the smectic helicoidal nanofilament (SHN) structure. J,K) AFM images of the SHN structure (j), which is composed of smectic C bundles (k). L) Proposed model of the SHN structure composed of left-handed and right-handed helicoidal nanofilaments. M) Grazing-incidence small-angle X-ray scattering measurement perpendicular to the pulling direction, showing pseudo-hexagonally packed phage structures within the SHN.
Biologically inspired hierarchically self-assembled structures: Bone, cornea, skin tissues and collagen are organic polymers with stiffness related to its helical structure. We can recreate this structure using genetically engineered M13 system. 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 (Fig. 3A) 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. 3B). We also found that the stiffness (Young’s modulus) of the phage film significantly increased ~20x after mineralization (Fig. 3C). 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 building a diverse set of functional materials with increased force protection properties.
Figure 3| Fabrication of hierarchical tissue matrices. A) SEM image of the dental enamel-like helicoidal nanofilament phase phage film (1:1). B) SEM image of the phage based hard tissue composite materials mineralized using Ca2+ and PO43- 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.
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 (Figure 4). 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 4| 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)
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. 5). 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 5| Schematic diagram of phage display against a TNT target
