Woo-Jae Chung

Biomimetic virus-based colorimetric sensors

Many materials in nature change colours in response to stimuli, making them attractive for use as sensor platform. However, both natural materials and their synthetic analogues lack selectivity towards specific chemicals, and introducing such selectivity remains a challenge. Here we report the self-assembly of genetically engineered viruses (M13 phage) into target-specific, colourimetric biosensors. The sensors are composed of phage-bundle nanostructures and exhibit viewing-angle independent colour, similar to collagen structures in turkey skin. On exposure to various volatile organic chemicals, the structures rapidly swell and undergo distinct colour changes. Furthermore, sensors composed of phage displaying trinitrotoluene (TNT)-binding peptide motifs identified from a phage display selectively distinguish TNT down to 300 p.p.b. over similarly structured chemicals. Our tunable, colourimetric sensors can be useful for the detection of a variety of harmful toxicants and pathogens to protect human health and national security.

Polydiacetylene incorporated with peptide receptors for the detection of trinitrotoluene explosives.

Because of their unique optical and stimuli-response properties, polydiacetylene-based platforms have been explored as an alternative to complex mechanical and electrical sensing systems. We linked chromic responsive polydiacetylene (PDA) onto a peptide-based molecular recognition element for trinitrotoluene (TNT) molecules in order to provide a system capable of responding to the presence of a TNT target. We first identified the trimer peptide receptor that could induce chromic changes on a PDA backbone. We then investigated the multivalent interactions between TNT and our peptide-based receptor by nuclear magnetic resonance (NMR) spectroscopy. We further characterized various parameters that affected the conjugated PDA system and hence the chromic response, including the size of end-group motifs, the surface density of receptors, and the length of alkane side chains. Taking these necessary design parameters into account, we demonstrated a modular system capable of transducing small-molecule TNT binding into a detectable signal. Our conjugated PDA-based sensor coupled with molecular recognition elements has already proven useful recently in the development of another sensitive and selective electronic sensor, though we expect that our results will also be valuable in the design of colorimetric sensors for small-molecule detection.

Assembly of Bacteriophage into Functional Materials

For the last decade, the fabrication of ordered structures of phage has been of great interest as a means of utilizing the outstanding biochemical properties of phage in developing useful materials. Combined with other organic/inorganic substances, it has been demonstrated that phage is a superior building block for fabricating various functional devices, such as the electrode in lithium-ion batteries, photovoltaic cells, sensors, and cell-culture supports. Although previous research has expanded the utility of phage when combined with genetic engineering, most improvements in device functionality have relied upon increases in efficiency owing to the compact, more densely packable unit size of phage rather than on the unique properties of the ordered nanostructures themselves. Recently, self-templating methods, which control both thermodynamic and kinetic factors during the deposition process, have opened up new routes to exploiting the ordered structural properties of hierarchically organized phage architectures. In addition, ordered phage films have exhibited unexpected functional properties, such as structural color and optical filtering. Structural colors or optical filtering from phage films can be used for optical phage-based sensors, which combine the structural properties of phage with target-specific binding motifs on the phage-coat proteins. This self-templating method may contribute not only to practical applications, but also provide insight into the fundamental study of biomacromolecule assembly in in vivo systems under complicated and dynamic conditions.

Evolutionary screening of collagen-like peptides that nucleate hydroxyapatite crystals.

The biogenesis of inorganic/organic composite materials such as bone typically involves the process of templated mineralization. Biomimetic synthesis of bone-like materials therefore requires the development of organic scaffolds that mediate mineralization of hydroxyapatite (HAP), the major inorganic component of bone. Using phage display, we identified a 12-residue peptide that bound to single-crystal HAP and templated the nucleation and growth of crystalline HAP mineral in a sequence- and composition-dependent manner. The sequence responsible for the mineralizing activity resembled the tripeptide repeat (Gly-Pro-Hyp) 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 support a model for the cooperative noncovalent interaction of the peptide with HAP and suggest that native collagen may have a mineral-templating function in vivo. We expect this short HAP-binding peptide to be useful in the synthesis of three-dimensional bone-like materials.

Genetically Engineered Liquid-Crystalline Viral Films for Directing Neural Cell Growth

Designing biomimetic matrices with precisely controlled structural organization that provides biochemical and physical cues to regulate cell behavior is critical for the development of tissue-regenerating materials. We have developed novel liquid-crystalline film matrices made from genetically engineered M13 bacteriophages (viruses) that exhibit the ability to control and guide cell behavior for tissue-regenerating applications. To facilitate adhesion between the viruses and cells, 2700 copies of the M13 major coat protein were genetically engineered to display integrin-binding peptides (RGD). The resulting nanofiber-like viruses displaying RGD motifs were biocompatible with neuronal cells and could be self-assembled to form long-range-ordered liquid-crystalline matrices by a simple shearing method. The resulting aligned structures were able to dictate the direction of cell growth. Future use of these virus-based materials for regenerating target tissues in vivo would provide great opportunities for various tissue therapies.

Facile patterning of genetically engineered M13 bacteriophage for directional growth of human fibroblast cells

We report a facile strategy for the patterning of cells that utilizes nanofibrous RGD-engineered phages in conjunction with microcontact printing methods to provide human fibroblast cells with specific biochemical and physical cues. This approach can be used for high-throughput screening assays as well as for energy and biosensor development.

Fabrication of engineered M13 bacteriophages into liquid crystalline films and fibers for directional growth and encapsulation of fibroblasts

We report on a novel method to utilize genetically engineered M13 phages as functional nano building blocks that can form structurally aligned film and fiber matrices for tissue engineering scaffolds. Two- and three-dimensional directionally aligned long range ordered structures were constructed using shearing and polyionic complexation with cationic polymers. Further we have demonstrated that aligned phage-based tissue engineering materials can guide and stimulate the growth of the target fibroblasts. Our phage-based tissue engineering scaffolds can be used for providing micro and macroscopic control of cell behaviors.

Biomimetic Self-Templated Hierarchical Structures of Collagen-Like Peptide Amphiphiles

Developing hierarchically structured biomaterials with tunable chemical and physical properties like those found in nature is critically important to regenerative medicine and studies on tissue morphogenesis. Despite advances in materials synthesis and assembly processes, our ability to control hierarchical assembly using fibrillar biomolecules remains limited. Here, we developed a bioinspired approach to create collagen-like materials through directed evolutionary screening and directed self-assembly. We first synthesized peptide amphiphiles by coupling phage display-identified collagen-like peptides to long-chain fatty acids. We then assembled the amphiphiles into diverse, hierarchically organized, nanofibrous structures using directed self-assembly based on liquid crystal flow and its controlled deposition. The resulting structures sustained and directed the growth of bone cells and hydroxyapatite biominerals. We believe these self-assembling collagen-like amphiphiles could prove useful in the structural design of tissue regenerating materials.

2.206 – Phages as Tools for Functional Nanomaterials Development

Protein-based ‘bottom-up’ synthesis of nanoscale functional materials and devices is one of the most promising areas in the newly emerging field of nanotechnology. However, identifying active basic building blocks from biological examples is still a challenge because of their complex and encrypted sequence structure. Genetic engineering of phage viruses provides opportunities for building novel bio-nanomaterials by integrating biology, chemistry, physics, materials science, and electric engineering. By mimicking the evolutionary process in nature, phages can be used as an information-mining tool for identifying functional peptide (or protein) sequences that can specifically recognize desired materials at the molecular level. These recognition elements can be used to guide the design of unprecedented materials such as semiconductor and metallic materials. Moreover, phages are unique in their intrinsic ability to self-replicate within a cellular host and self-assemble into highly ordered two- and three-dimensional nanostructures. By combining these self-replicating and self-assembling functions, virus-based materials can be used to construct nanomaterials and devices with novel structure and function that could be useful in applications including energy, biosensors, electronics, and tissue-regenerating materials. In this chapter, we introduce the unique features of phages and recent accomplishments in the development of virus-based materials for use as tools to fabricate functional nanomaterials, and review the potential future applications of this emerging technology.

Phage display for the discovery of hydroxyapatite-associated peptides.

In nature, proteins play a critical role in the biomineralization process. Understanding how different peptide or protein sequences selectively interact with the target crystal is of great importance. Identifying such protein structures is one of the critical steps in verifying the molecular mechanisms of biomineralization. One of the promising ways to obtain such information for a particular crystal surface is to screen combinatorial peptide libraries in a high-throughput manner. Among the many combinatorial library screening procedures, phage display is a powerful method to isolate such proteins and peptides. In this chapter, we will describe our established methods to perform phage display with inorganic crystal surfaces. Specifically, we will use hydroxyapatite as a model system for discovery of apatite-associated proteins in bone or tooth biomineralization studies. This model approach can be generalized to other desired crystal surfaces using the same experimental design principles with a little modification of the procedures.