| The single-molecule behavior and the interaction between molecules can be real-time monitored by single-molecule detection (SMD) to unveil molecular properties of the static inhomogeneities and dynamic fluctuations, and the molecular properties are hidden in averaged ensemble. Single-molecule detection has been widely used in the molecular conformational change, biomolecular interactions and ultrasensitive detection and so on. In this paper, based on total internal reflection fluorescence microscopy (TIRFM), the aptamers conformation change and quantum dots imaging were studied, include:1. The configuration change of aptamers fixed on slide was studied at the single- molecule level. The adenosine triphosphate (ATP) aptamers were labled with fluorophore tetramethylrhodamine (TMR) and biotin at 5' end, quencher Dabcyl at 3' end. The aptamers were fixed on the slide with the interaction of biotin and avidin. Based on TIRFM, the aptamers were studied in the presence of ATP or cDNA. The results showed that without ATP and cDNA, fluorescence intensity of the aptamers was weak, and the majority of the aptamers were closed-loop, and a few of aptamers were random coil. In the presence of ATP, aptamers changed into closed loop from random coil, and the fluorescence intensity declined. In the presence of cDNA, the aptamers formed double helix, and fluorescence intensity increased. In the presence of cDNA and ATP, with the concentrations of ATP increased, the aptamers changed into closed loop from double helix, and the fluorescence intensity declined. The method provides a reference to develop the biosensors based on aptamer.2. The method for the ultrasensitive detection of protein was developed by quantum dots labeled aptamers with single-molecule imaging. Thrombin as target protein has two aptamers, and the binding sites are different. In this paper, an aptamer was fixed on slide with the interaction of biotin and avidin, and another aptamer was labeled with quantum dots or TMR. Sandwich assay was formed by thrombin and the aptamer fixed on the slide and the fluorescent-labeled aptamer, and then detected the fluorescence signal by TIRFM. The results showed that using the quantum dots as fluorescent labeling, the linear relationship for thrombin detection was obtained in the range of 0.1 to 10 pM, and the detection limit reached 60 fM. Using TMR as fluorescent labeling, the linear relationship for thrombin detection was obtained in the range of 5 to 98 pM, and the detection limit was 2.8 pM. So, compared to the TMR, quantum dots can decrease the detection limit to 47 times. It is attributed the quantum-dots superior optical properties of the large molar absorptivity and strong photostability.3. The single-particle fluorescence properties of water-soluble amino-CdTe quantum dots treated with 1-butyl-3-ethylimidazolium tetrafluoroborate ([BEIm]?BF4) ionic liquids were studied with single-molecule imaging. Quantum dots were extracted into the ionic liquids, the fluorescence intensity of quantum dots and anti-photobleaching were enhanced in the first hour, because of the protection of hydrophobic ionic liquids. Then, the blinking became apparent and anti-photobleaching capacity decreased with the time. Infrared spectroscopy, fluorescence spectroscopy and the results of quantum dots treated with NaBF4 solution showed that the changes of blinking and anti-photobleaching may be due to the BF4- binding to the surface of quantum dots and the BF4- etches quantum dots, which lead to the increase of surface defects of quantum dots. This work is contributed to investigate the interaction mechanism of quantum dots and ionic liquids.
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