Surface Plasmon Resonance Biosensors: Novel N-Heterocylic Carbene Based Surfaces
Surface plasmon resonance (SPR) biosensing is a technique that is increasingly being used to investigate interactions between biomolecules due to its ability to be monitored in real-time and the lack of labelling needed. Traditionally, SPR biosensor chips utilized thiol-based self-assembled monolayers (SAMs) as linking layers on a gold surface, however, the use of thiols has been limited due to their tendency to oxidize in ambient conditions. Thus, a new and emerging class of surface modification using N-heterocyclic carbenes (NHCs) as surface ligands is being explored as an alternative to thiol-based SAMs for biosensing surfaces. In this thesis, chemical modification of SPR biosensing surfaces using NHCs as a SAM on gold is examined. The first part of this thesis focuses on developing an NHC SPR biosensing chip comparable to the most commonly used biosensor, the Biacore general-purpose carboxymethylated (CM) 5 chip. Using an interaction involving bovine serum albumin (BSA) and anti-BSA antibody, the capabilities of the homemade NHC-based sensor chip were examined using a 50 cycle analysis of antibody binding to the BSA surface. A response was observed for all binding interactions; however, the reproducibility of the interactions was inadequate due to non-specific binding. This limited the use of the NHC sensor chip in applications involving kinetic studies. The second part of my thesis describes the creation of a more stable NHC sensing surface using a high-affinity capture approach, circumventing non-specific binding. Protein A was immobilized as the surface ligand and immunoglobin G (IgG) was used as the analyte. Highly reproducible interactions over 80 cycles involving little to no deterioration of the response were observed. This facilitated the study of kinetic analysis of the protein A – IgG system; however, due to mass transfer limitations (MTL), the rate constants derived were inaccurate. Finally, NHC-based sensors were used in the creation of a lipid bilayer surface. This bilayer surface demonstrated instability as observed from its low binding ability and ease of being removed from the sensor surface.