Fabricate Enzymatic Micropatterns By SECM And Detect Rat IgG Molecules By Total Internal Reflection Fluorescence Microscopy

This thesis includes three parts. The introduction; the fabrication of micropatterns of HRP by the means of SECM "Dip-Pen" technique; the detection of rat IgG molecules by total internal reflection fluorescence microscopy.In the introduction,we introduced the evelepment of the fabrication of micropatters by SECM and single molecule detection.In chapter one, micrometer dots of HRP were fabricated by the means of SECM "Dip-Pen" technique. We utilized the elements of "Dip-Pen nanolithography" (DPN) of AFM in SECM in this work. First, the adsorption and desorption of Bio-HRP on platinum electrode were investigated by electrochemistry method. when the platinum electrode was dipped into the HRP solution for five times, and each time for 4 min, the Bio-HRP can be best absorbed; when the electrode which absorbed Bio-HRP was dipped into the PBS buffer for 60 min, the Bio-HRP can be best desorbed. Second, Bio-HRP was adsorbed on self-made platinum microelectrode in the best condition which was selected before. Then the electrode was approached to the streptomycin avidin modified substrate with -0.3 V (vs. Ag/AgCl) which was selected previously. Finally, Bio-HRP was desorbed from platinum microelectrode and reacted with streptomycin avidin, therefore the micropatterns of HRP were fabricated. The activity of the pattern was characterised with SECM. In addition, a thin film made between the microelectrode and the substrate was used to minimize the diffusion of the HRP in solution.In chapter two, the rat IgG molecules were detected by total internal reflection fluorescence microscopy. First, coverlips were dealed with glycidoxypropyltrimeth-oxysilane and epoxy group was created on the surface of coverlips; second, the rat IgG solution was added on a coverlip hatching by which the rat IgG molecules were fixed onto the coverlip; third, the coverlip was dipped into BSA solution hathing by which the remanent where having no rat IgG molecules was obturated; fourth, Alexa 488 goat anti-rat IgG(H+L) solution was added on the coverlip where the rat IgG solution was added hatching; fifth, the excrescent Alexa 488 goat anti-rat IgG(H+L) molecules was washed. After these approaches, where there is a rat IgG molecule there is a goat anti-rat IgG(H+L) one. At last, the Alexa 488 goat anti-rat IgG(H+L) molecules which were fixed onto the coverlip were detected by total internal reflection fluorescence microscopy. We can educe the amount of rat IgG molecules via the amount of goat anti-rat IgG(H+L) molecules. In this chapter we reduced all the material's background fluorescence which was used in the experiment. using total internal reflection fluorescence microscopy, reducing excited volume, making use of fitting cubes, using ICCD to image molecules which was very sensitive, increasing detection sensitivity. The molecule number is line with rat IgG concentration in the range of 2.0×10^-14 mol/L-30×10^-14mol/L.