| dc.description.abstract | Controlling protein adsorption at solid surfaces is critical for a large variety of applications such as biocatalysis, biomedicine, food safety, and environmental monitoring. The importance of controlling the orientation and conformation of the protein at the surface has been recognized as key to the successful implementation of such applications. Metal nanostructure platforms have potential applications not only in protein-based biosensing but also in electronics and energy harvesting applications. This works investigates the specificity and selectivity of proteins binding to metal surfaces for incorporation into metal nanostructure arrays for such applications. The specificity and selectivity of the coupling in this case is achieved through a genetically engineered peptide tag with a high affinity for a gold surface. Controlling the protein orientation on selected areas of surfaces is challenging due to the inability to control the selectivity and specificity of the desired molecules. This work mainly focuses on the utilization of affinity peptides to control the protein orientation on selected areas of metal-organic hybrid films. Chapters 1 and 2 provide an introduction to the project and the methods employed. Investigations into the control of binding and orientations of a model protein (PutOX) at surfaces are described in Chapters 3 and 4. Chapter 3 describes the utilization of an affinity peptide towards the selective immobilization of proteins on surfaces. Affinity peptides are specific sequences of amino acids that have a high affinity for a material, and in this study, we investigate gold affinity peptides. The specific attachment of functionally active PutOx via gold affinity peptide sequence (AuBP) to gold surfaces has been demonstrated using QCM, activity assays, temperature treatment, and AFM investigations of coverage and shape of individual molecules. The temperature treatment studies show that the peptide tagged protein shows higher stability on the surface than in the solution. Chapter 4 reports the behavior of both wild type and PutOX-AuBP enzymes on a variety of surfaces including TSG (template stripped gold), mica, Si(111), OTS-Si-SAM, Graphite, COOH-TSG-SAM, and OH-TSG-SAM. This part of the study addresses the effect of properties of the surface in the attachment of protein in the presence of affinity peptide tags to understand the binding specificity and selectivity when using the AuBP. Once the selectivity and specificity of the gold binding peptide sequence towards the gold surface were demonstrated, methods to create Au nanostructures to bind the protein selectively on the metal nanostructures were required to create protein nanoarrays. Therefore, Chapters 5-7 investigate the fabrication of gold nanostructures. The development of gold plating solutions for electroless deposition is described in Chapter 5. The approach we used is electroless deposition (ELD), which is a well-established process on silicon and other semiconductor surfaces to deposit metal films. This chapter further discusses the effects of plating components and how self-assembled monolayers are used to selectively deposit metal on Si surfaces. Here, the effect of plating solution components, pH, and deposition time was studied to develop mild plating solutions for fabrication of gold nanostructures in Chapters 6 and 7. Two different methods for gold nanostructure fabrication are explored in these Chapters. Chapter 6 describes an AFM-based method for making gold nanowires using the combined techniques including self-assembled monolayer (SAM) resist formation, AFM nanoshaving, and electroless gold plating (whose deposition conditions were optimized in Chapter 5). Here, an OTS SAM on a silicon surface was utilized as a compelling molecular resist film for gold nanowire formation. The AFM nanoshaving was used to remove part of a molecular resist to expose the underlying silicon to facilitate gold nanostructure fabrication during ELD. In Chapter 7, a combination of particle lithography (PL), self-assembly, and electroless deposition was used to develop large, periodic arrays of gold nanodots on the silicon surface. The PL-based method addresses the limitations associated with the throughput of the AFM-based nanoshaving strategy discussed in Chapter 6. Here, the nanohole arrays are produced using a nanosphere mask and the formation of a self-assembled monolayer (SAM) film, and the nanohole array is then filled with metal via the ELD process. The metallic nanostructures developed using the PL provides the advantages of controllability of size and interparticle distance by changing the plating time and nanosphere diameter. The combination of developed nanostructures and the affinity peptide-tagged proteins can be used to develop materials for the fabrication of nanoscale bionanodevices, which display a range of surface chemistries in the device. The developed nanoarrays could be useful towards plasmonic biosensing applications, with localized plasmonic resonance wavelength tunability, as well as platforms capable of sensitive electrochemical detection. Finally, this dissertation addresses several critical concepts for biomaterials research including orientation control of proteins, precise placement of nanostructures and nanoarrays, modification of the surface, ease of fabrication, and cost. In the future, this study will be extended to develop multiple metal nanoarrays of silver and gold on the same surface to study cell signaling pathways or coupled enzyme reactions using multiple affinity peptides, which specifically bind to different metals (e.g., Au and Ag). | |
