Development of a Novel Nitric Oxide Sensor Using Nitrophorin 4 on an Attenuated Total Reflectance Platform

摘要

Nitric oxide plays a major role in physiology and disease pathology. There are many available methods for the detection of NO; however, these techniques typically detect products of nitric oxide decomposition. Herein, I present a novel method of direct nitric oxide detection using a nitrophorin mutant to capture NO on an optical waveguide platform. Nitrophorins are unique among ferric proteins for their ability to bind NO strongly. The spectral shift of the Soret band of the Nitrophorin was used to monitor NO concentration. The limit of detection was found to be 18 nM, and a linear response to 225 nM. The sensor is highly specific, non-destructive and reusable. Detection of NO was demonstrated in a solution of BSA at serum level concentration, and cell culture solution containing 10% FBS. This method allows for direct NO with high specificity, low detection limit and good temporal resolution. Also described herein are the investigations of the structure of the NO receptor, soluble guanylate cyclase (sGC). sGC is a 150 kDa heterodimeric protein that catalyzes the production of cyclic guanosine monophosphate from guanosine triphosphate, which leads to many downstream affects such as vasodilation. The structural analysis was performed with transmission electron microscopy (TEM). Data presented indicate that the protein is too heterogeneous to be reconstructed with TEM. This is either the result of the sample preparations examined, the purity of the sample, the inherent flexibility or conformational heterogeneity of the protein after applying the sample to the TEM grid.The final project presented describes the use of silica colloidal crystals for the enhancement of the sensitivity of protein microarrays as a function of silica particle size. Protein microarrays are a tool used to discover biomarkers of diseases, and increasing the sensitivity lowers the limit of detection of the method. This is accomplished by an increase of the surface area available for proteins to bind, and by using silica so that the surface chemistry of the microarray is maintained. It was concluded that 510 nm colloids provide the greatest enhancement of the fluorescence of a BSA ant-BSA-FITC system, providing a 17-fold enhancement over the control.