Protein cage-based assemblies and characterization of their electrical properties

Abstract

Proteins naturally self-assemble to function. Protein cages result from the self-assembly of multiple protein subunits that interact to form hollow symmetrical structures with functions that range from cargo storage to catalysis. In recent years, proteins, as building blocks or as components, have been assembled into higher-order structures via various methods. However, as a part of the protein family, architectures based on protein cages yet to be fully explored Furthermore, while the functional aspects of these structures have been part of the designs, the experimental proofs are still lacking. Therefore, my projects are dedicated to enhancing structural regularity by leveraging metal surface interactions, incorporation within carbohydrate hydrogel matrix, and integration into conductive polymer. By improving the organization of superstructures, functional properties, especially electrical properties, are expected to be enhanced. Therefore, potential applications can be explored based on the potential functional performances. The model protein cage in this thesis is ferritin (AfFtnAA). The first work leveraged surface interactions for assembling higher-order protein structures on copper substrate using ferritin as building blocks. This was achieved by introducing cysteines on ferritin surface with thiol groups as the active moiety. Using Raman spectroscopy, the mechanism of the assembly was determined to be copper-induced. Functional characterization showed that HER performance of the copper-assembled AfFtnAA/E94C is 3.8 times higher compared to the amorphous samples of AfFtnAA group at -1.6 V, suggesting that the higher-order structures composed of AfFtnAA/E94C contribute to the improvement of HER behavior. The second project, ferritin was incorporated within carbohydrate hydrogel matrix which was achieved by incorporating ferritin in chitosan network via freeze-melting-neutralization method. The morphology of the hydrogel composed of chitosan and ferritin was found to have near-uniform particle sizes and pores under SEM. The variation of cyclic voltammetry (CV) performances of samples using different approaches to add AfFtnAA was demonstrated. The peak current (Ip) of samples indicates that AfFtnAA and the porous structure synergistically contributed to the enhanced electrochemical behaviors. Further CV tests for S1 (i.e., sample prepared by adding AfFtnAA prior to chitosan dissolution) of different concentration confirms the influence of AfFtnAA to electron transfer kinetics and shows S1 with 0.1 mg/ml AfFtnAA has the best electrochemical behaviors among all the S1 samples. This material was planned to explore further applications related to bioelectronics. In the third part of this dissertation, hydrogel of polypyrrole-ferritin complex was prepared by mixing ferritins during in situ polymerization stage of pyrrole. The morphology of the hydrogel of different content of components, including different ratio of FeCl3 and pyrrole, different content of pyrrole, and different content of protein, was explored under SEM. The material was shown to have the lead ion sensing ability, therefore, suggesting potential application related to biosensor.

The incorporation of ferritin into various conductive surfaces and matrices demonstrated in this thesis provides insights into higher-order structures based on protein cages for future bioelectrochemical applications.