Active three-dimensional protein microstructures

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Hill, Ryan Toler

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Direct-write multiphoton-excited photocrosslinking of chemically active threedimensional (3D) protein microstructures could potentially extend in situ cellular analysis to include more interactive studies of neural networks, signal transduction, and neural response to local chemical gradients. This dissertation presents a strategy that has been developed for highly specific functionalization of 3D protein microstructures with protein-coated nanoparticles, an approach that is demonstrated to be appropriate for imparting these materials with desired electronic and chemical/biochemical properties. Once targeted with protein-gold conjugates, these protein scaffolds can serve as conductive bio-wires after electroless deposition is used to fuse the nanoparticles together. Unlike earlier approaches for templating metals with biomolecules, the current strategy can be used to construct scaffolds with precise spatial control in three dimensions, offering new opportunities to construct advanced bioelectronic architectures. This protein-gold conjugate functionalization technique has also proven to be an excellent way to localize high concentrations of active molecules with minimal nonspecific adsorption. Sequential functionalization steps can be used in conjunction vii with direct crosslinking of active enzymes, such as cytochrome c (cyt c), to produce enzyme suites capable of quantifying substrates with low micromolar detection limits. Also presented here are detailed electrochemical and spectroscopic studies that are aimed at quantitatively characterizing the native heme integrity and enzyme activity of directly photocrosslinked cyt c. Finally, this work presents initial studies focused on adapting gold conjugate functionalization of protein microstructures for compatibility with cultures of neurons and other cell types, a goal that would substantially expand capabilities for constructing in situ bioelectronics, localized dosing sources, and biochemical sensors for monitoring and stimulating biological processes. Use of this electrostatically driven approach towards functionalization combined with direct crosslinking of active molecules offers flexibility in the creation of highly definable, 3D reactive regions that are capable of sensing chemical gradients and/or modifying electric fields in sensitive aqueous environments.