Single Molecule Counting Quantitative Methods

This thesis includes three parts: Detection of single biomolecules. Detection of single antibody molecules in solution by total internal reflection fluorescence microscopy. Determination of goat anti-rat IgG (H+L) by counting single molecules.In the chapter one of this dissertation, the detail review of detection of single biomolecules was delivered. Significance, principle and basic content of single molecules detection and structure, principle of microscope in single molecules detection were introduced. Actuality, method of single molecules detection were introduced in aspect of tracing single molecules, single molecules enzymology, dynamics of molecular conformation, motion of motor protein, ion channels, cell signal conduction, detection of specific DNA sequences at the single-molecule level, counting of single molecules. In the chapter, 81 references have been referred.In the chapter two, the method of detection single goat anti-rat IgG (H+L) molecules by total internal reflection fluorescence microscopy was investigated. Several parameters including laser power, exposure time was 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, 2.4 kW; exposure time, 100 ms. Reduce background fluorescence by pre-photoblanching buffer after filtration, pre-photoblanching coverslip, reducing excited volume, making use of fitting cubes, increase detection sensitivity, then imaging single goat anti-rat IgG (H+L) molecules by ICCD. There are two evidences for proving single molecules. The molecular number is liner with goat anti-rat IgG (H+L) concentration in the range of 1.0×10^-10 mol/L-4.0×10^-9mol/L, This is not only a potent evidence for single molecules, but also a method for determination of protein.In the chapter three, A novel method for determination of protein was presented, coverlips were dealed with glycidoxypropyltrimethoxysilane and epoxy group was created on the surface of coverlips, Alexa488 goat anti-rat IgG (H+L) in phosphate-buffered saline (PBS) buffer were added on a coverslip, 30 minute later, protein molecules were deposited to the surface of coverslips by evaporation solvent,distilled water ^ as added to the surface of coverslips to dissolve the salt deposited to the surface of covesrlips, reduce background fluorescence efficiently, then imaging and counting single goat anti-rat IgG(H+L) molecules absorbed to the surface of a coverslip by total internal reflection fluorescence microscope and ICCD, calculate concentration directly. Determination range is 5.0X 10'15 mol/L-5.0X 10"l4mol/L.