Bio-Inorganic Interface Engineering via Solid-Binding Peptides toward Nano-sensing Applications
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Bridging the worlds of molecular biology and inorganic materials is a crucial step in many of today's medical, nano-technology, energy, catalysis and renewable technology fields. By its very nature, this task is highly multidisciplinary, incorporating aspects from surface physics, organic and inorganic chemistry, the tools of nano-technology, and of course, biology from genetic to molecular to structural to organismal levels. Solid-binding peptides are a unique class of biomolecules perfectly suited to build this bridge at the scale consistent with both organic and inorganic systems. These 7-12 amino acid long peptides are biological in origin (consisting of 20 natural amino acids), bind strongly through a number of non-covalent interactions, self-assemble into dense ordered structures, are selected to be highly specific to their target solid materials, and can be fused with other biomolecules through many chemical and biological means to create entities with multiple functionalities. These properties make solid-binding ideal for functionalizing simple and complex inorganic surfaces to impart broad chemical or functional properties to the system in an environmentally friendly and biologically viable fashion, with minimal effect on the properties of the underlying substrate. This thesis demonstrates the design of surface biofunctionality in proof-of-principle biotechnological and nanotechnological implementations in a variety of applications. As an example of the utility of engineered peptides with state-of-the art nanomaterials systems, the thesis addresses the design and manufacture of a graphene based, peptide enabled nanobio-sensor for molecular detection in a biological medium. The present report, therefore, focuses on a design and application of solid-binding peptides to control the bio-inorganic interface, and simultaneously to impart bio-functionality to an inorganic system. More specifically, in this research group, one of our goals has been is to develop a graphene biosensor for specific, ultra-sensitive detection of cancer markers against a background of serum proteins, through a combination of newly developed molecular biomimetic functionalization and nano-fabrication approaches. In order to build such a sensor three broad steps must be taken: (1) The surface of the sensor must be passivated to prevent non-specific adsorption of undesired proteins. (2) Target-specific bio-functionality must be imparted to the sensor to enable it to capture the targets in the sample. (3) A modular system, the bio-functionality of which can be easily modified, must be developed to simplify detection of a variety of targets. These steps must be taken in such a way as to not negate the sensitivity of the sensor, a graphene field effect transistor (gFET). The work covers experiments aimed at achieving bio-inert and bio-active interfaces (i.e. interfaces which do not interact with cells/proteins, and ones that interact with them in a predictable manner, respectively) via peptide functionalization, as well as sensor construction, peptide sequence optimization for assembly and property display, and selective detection of proteins from a complex mixture. As an example, using an optimized gFET sensor system, the detection of streptavidin binding against a background of serum albumin at less than 50ng/ml is demonstrated. Additionally, using a bi-functional graphene binding peptide - antibody binging peptide construct, we demonstrate a modular detection system, which can be modified to detect a given biomolecule, for which an anti-body has been selected, in a single step, by replacing the probing module (the anti-body). Cancer markers are large biomolecules present in the blood or other tissues, which can be used for early diagnosis. Their detection is challenging due to the low concentrations at which they are present, and the difficulty of selecting them against the complex background of other proteins present in the serum. Using our system we demonstrated selective cancer marker detection, specifically immobilizing antibodies as the probe on the sensor surface, using engineered peptide chimeras to detect cancer markers in a serum environment. The system we demonstrate is the first significant step toward creating a fast, sensitive methodology for profiling the low levels of multiple markers simultaneously, a must for accurate, reliable, and rapid diagnosis. Our modular approach, which allows for easy switching of targets, can also be used for other clinical and research applications in general, such as, ligand-receptor interaction studies, screening for drug candidates, and developing vaccines against small viruses.