The Study of Enzyme Mechanisms by Single Molecule Optoplasmonic Whispering Gallery Mode Biosensing

Abstract

Whispering Gallery Mode sensors are emerging with unprecedented sensing power and ever-expanding applications such as the detection and analysis of biomolecules and of biological processes such as DNA interactions, neurotransmitter-membrane interactions and biocatalysis. The capacity of these sensors for real-time detection of native biological processes, without the need for addition of tags that may affect mechanisms, has been demonstrated in several publications. These optoplasmonic sensors rely on signal enhancement using plasmonic nanostructures to detect molecule-induced changes in polarisability occurring in the near-field enhancement region; this allows detection of enzyme immobilisation and conformational changes of a single enzyme molecule. This thesis describes the development of an optoplasmonic sensor for the detection of enzyme immobilisation and conformational change, exploring the capabilities of these sensors for analysing protein phenomena. It first outlines the procedures for enzyme production and bulk immobilisation, ensuring activity is maintained once bound to the surface of plasmonic gold nanorods, before exploring the observation of thermo-optoplasmonic effects from single-molecule immobilisation and how these can be used to calculate absorption cross-sections. Detection of turnover events is then described for several enzymes, before discussing the generation of optical forces using this optoplasmonic system and how further developments of this sensor could allow control of single enzyme molecules. The optoplasmonic sensor is applied to a temperature-dependent regime, providing additional evidence for the macromolecular rate theory of enzyme catalysis to enable further understanding of biocatalytic mechanisms. Several areas of optoplasmonic sensing are thereby explored, demonstrating numerous applications and indicating an exciting future for the technique. This thesis contains published works from the Vollmer and Bagby Laboratories, including Houghton and Vartabi Kashanian et al. (2024),1 Toropov and Houghton et al. (2024),2 and Houghton and Toropov et al. (2024).3 All reproductions of materials are with the permissions of the authors.