| Description |
This dissertation focuses on the study of surface biomolecular interactions using second harmonic generation (SHG) spectroscopy, surface SHG imaging (SSHGI), and SH correlation spectroscopy (SHCS). The binding kinetics and energetics of four biotinbound proteins, avidin, streptavidin, neutrAvidin, and anti-biotin antibody were compared and data revealed significant differences in their apparent binding affinities and nonspecific binding. Specifically, protein-protein interactions were found to play an important role in the apparent binding affinity, making the streptavidin-biotin interaction the most energetically favorable. The details of the binding properties of these frequently employed tether/linker protein-biotin complexes provide valuable information for biosensors, immunoassays, and medical diagnostics. As most biosensor platforms are designed for high throughput detection, the resolution and planar wave-front of the SSHGI system was thoroughly analyzed. It was demonstrated that the coherent plane wave generated by SHG followed Gaussian beam propagation, enabling SSHGI to image without a lens system at rather long distances. Lens-less imaging simplifies the detection method, increases photon collection efficiency, and increases the detection area. These advantages could potentially make SSHGI a simple, label-free high throughput detection method for surface biomolecular interactions. The versatility and sensitivity of SHG were further probed by coupling SHG with correlation spectroscopy, a statistical fluctuation time-dependent method. SHCS was established as a viable and valuable option for the detection of surface binding kinetics for small molecule and protein-ligand interactions at the surface of lipid bilayers. First, the simple binding kinetics of a small molecule, (s)-(+)-1,1'-bi-2-napthol (SBN), incorporating into a lipid bilayer was determined using SHCS and results were statistically similar to those obtained from a traditional binding isotherm. Next, SHCS was used to examine the binding kinetics of a more complex interaction between the multivalent proteins, cholera toxin subunit b (CTb) and peanut agglutinin (PnA), and a GM1 doped lipid bilayer. SHCS was able to obtain the binding kinetics for these surface biomolecular interactions with more efficiency, less analyte, and less sensitivity to mass transport effects. Cumulatively, the studies of this dissertation showcase SHG, SSHGI, and SHCS as valuable label-free detection methods with incredible sensitivity for investigation of surface biomolecular interactions. |
