| Description |
Many biological processes occur at a surface, such as the plasma membrane of a cell. Detection of adsorbed proteins or small-molecules is relevant for clinical and forensic purposes. Therefore, it is of utmost importance to examine these biomolecular interactions using biologically relevant models. Second harmonic generation (SHG) is a highly sensitive label-free spectroscopic technique that is well-suited for investigating interfacial biomolecular interactions. The utility of SHG for quantitative detection of biomolecular interactions is demonstrated throughout this dissertation. First, SHG was used to quantitatively determine the components necessary for the initial association of HIV-1 Gag protein with model lipid bilayers. It was found that interaction of Gag with the lipid PI(4,5)P2 prolonged its residence at the membrane. The N-terminal myristoyl group on Gag also anchored the protein to the membrane. Mutations in the matrix domain of Gag prevented these specific interactions, thereby limiting the membrane association of Gag. The ability of SHG for quantitative protein detection was applied to investigate the G-quadruplex structural dependency of apurinic/apyrimidinic endonuclease 1 (APE1) binding to DNA. Specifically, disruption or loss of this hydrogen-bonded G-quadruplex reduced APE1 capture efficiency, even when an abasic site was present. Cleavage activity was restored upon addition of Mg2+, and the rate of protein release was measured directly by SHG. iv Finally, a label-free direct detection SHG immunoassay was developed for the detection of small-molecule drugs, which was shown to have higher sensitivity and selectivity than commercial competitive enzyme-linked immunosorbent assays (ELISAs). The selectivity of competitive immunoassays was shown to be driven by the kinetics of association and dissociation, limited by a conformational isomerization. The direct detection of these drugs using a direct SHG primary immunoassay was demonstrated here for the first time. Combined, the investigations of these protein-membrane, protein-DNA, and drug-antibody interactions with SHG detailed here have revealed quantitative information about each of these processes. They have also shown that the label-free and surface specific nature of SHG is extremely advantageous for biosensing. |
