Single Molecule Detection Based On ITO And The Preliminary Study Of Electric Transfer Using ITO

The thesis includes four parts: The introduction; Detection of single antibody molecules in solution by total internal reflection fluorescence microscopy; Detection of single molecules fixed on the silanized ITO by total internal reflection fluorescence microscopy; The preliminary study of electric transfer using ITO.In the introduction, we intrduced western blot, single molecule detection, and the usage of ITO. First, the development and apply of western blot. Second we introduce the application of single molecule detection. The third part is about ITO used in optical detection and biological detection.In the chapter one of this dissertation, the method of detecting single goat anti-rat IgG (H+L) molecules in solution by total internal reflection fluorescence microscopy were investigated. Several parameters including laser power, exposure time were optimized for imaging of single goat anti-rat IgG (H+L) molecules. The following conditions were suitable for the imaging of single molecules: laser power is 10 mW; exposure time is 100 ms. Reducing background fluorescence by pre-photoblanching buffer after filtration, pre-photoblanching ITO, reducing excited volume, imaging single goat anti-rat IgG (H+L) molecules by EMCCD. And we proved what we detected are single molecules. The molecular number is linear with goat anti-rat IgG (H+L) concentration in the range of 5 ×10^-12 mol/L～1 ×10^-10 mol/L.In the chapter two, we detected single molecules fixed on the silanized ITO by total internal reflection fluorescence microscopy. ITO was dealed with glycidoxypropyltrimethoxysilane and epoxy group was created on the surface of ITO to combine with the antigen. We discuss the silanization time infaction on the adsorption of the antigen, and the best time for the antigen absorbed onto the ITO, blocking out the unspecific active position with BSA, washing time to remove the unwanted reagent, the incubate time and concentration of the antigen and antibody. From these experiments, we got the goal of detecting the antigen at single molecular level. The molecular number we detected is linear with the antigen concentration in the range of 5×10^-14 mol/L～5×10^-13 mol/L. And this result show the feasibility to reduce the detection limit in other biochemical experiments.In the chapter three, we improved the western blot using the silanized ITO. We use the silanized ITO as the film to absorb the antigen, and as the anode in the electrical transfer at the same time. We discussed the appropriate transfer voltage and transfer time to transfer the antigen molecular from the PAGE to ITO anode. Then we used the method studied in the chapter two to detect the antigen transferred to the ITO. The result explain that the novel method we presented is feasible. And compared with the traditional western blot, the new method using the silanized ITO may decrease the detection limit.