The Research On Fluorescence Propreties Of Quantum Dots And Microarray Chip At Single Molecules Level

Single molecules detection (SMD) is one of the highest sensitive techniques. It can be used to study on the activity of single molecules and it is the last milestone in the filed of detecting minute sample. Compared with traditional fluorescence dyes, quantum dots (QDs), as fluorescence labels, have many excellent optical properties. They have been playing important roles in the fields of molecular biology, cell biology, medicine, etc. Study on the properties of single quantum dots can provide a mentality to improve the properties of quantum dots, and contribute to a better application of QDs in various fields, such as, the application of microarray at single molecules level. Toward minute sample and low abundant gene expression, traditional microarray requires sample amplification. However, microarray at single molecules level, which doesn't need sample amplification, can futher improve the sensitivity and quantitative accuracy of gene chip. This thesis studied the properties difference of QDs in water/air and quantitative method on microarray chip at single molecules level. The main work were as follows:1. The properties difference of single quantum dots at liquid-solid interface and gas-solid interface.For the QD at liquid-solid interface (in water, for short: wet sample) and gas-solid interface (in air, for short: dry sample), we studied on five phenomenons in the bleaching, brightening, blinking, bluing and fluorescence lifetime.Compared with the wet sample in properties, we found the dry sample showed higher resistance bleaching; higher fluorescene intensity; shorter fluorescene lifetime and lower frequency of blinking, that is to say, the power-law distribution exponential (αoff/αon) in dry sample is smaller than that in wet sample. The blue-shift wavelength and rate of dry sample is smaller than wet sample's. Compared with QD585, the rate of blue-shift in QD525/QD655 is more. With the increase of the QD size, the absolute value of blue-shift wavelength is on the rise.2. Quantitative method on microarray chip at the single molecules levelA single molecules counting method was developed to quantitatively analyze miRNA expression on a microarray chip, in which a general fluorescence microscope excited with a mercury lamp and an electron multiplied CCD were used. After considering molecule numbers in each hybridize spot by tracking the photobleaching steps, the molecule density was calculated. The compared results of molecule density and fluorescence signal of commercial platform indicated our single molecules quantitative method was feasible for microarray chip and a better limit of detection can be obtained.