| In the chapter one of this thesis, the studies on the detection of single molecules were reviewed detailedly. Then we introduced several methods and techniques of the detection of single biomolecules. Emphases of this chapter were total internal reflection fluorescence microscopy (TIRFM) and its application in detection of single biomolecules. Finally, the fluorescent probe and the prospects of single molecule detection were summarized. In this chapter, 79 references were cited.In the chapter two, a highly rapid new cathodal adsorptive voltammetric method was developed for the determination of trace amount of Rhdamine 6G at ITO electrode. Suitable conditions were optimized for the detection of R6G R6G was adsorbed on the surface of ITO electrode in 0.20 mol/L KCl buffer solution(pH 6.5) yielding one reductive peak at -0.725 V (vs. Ag/AgCl) when scanning from -0.4 V to -0.95 V. The linear relationship between the peak current and R6G concentration is in the range of 2.0×10-7-8.0×10-10 mol/L. The correlation coefficient is 0.998. Its detection limit is 4.0×10-10 mol/L.In the chapter three, a novel method for determination of R6G single molecules was developed by a combination of cathodal adsorptive voltammetry and TIRFM. Some electrochemistry suitable conditions were followed in the chapter two. Several parameters including laser power, exposure time were optimized for imaging of single R6G molecules. The following conditions were suitable for the imaging of single molecules: laser power, 9 mW; exposure time, 100 ms. To reduce background fluorescence, the filtrated KCl solution and the ITO electrode were pre-photobleached for 24 h by ultraviolet radiation. Then the images of single R6G molecules were acquired by EMCCD with high detection sensitivity. The molecular number was liner with R6G concentration in the range of 5.0×10-12-5.0×10-13 mol/L and 2.0×10-13-5.0×10-15 mol/L. Its detection limit was 5.0×10-15 mol/L.In the chapter four, we described a procedure, which was the design and the fabrication of nanochannels on ITO glass substrates with PDMS in a conventional chemical laboratory. PDMS and ITO were used as the up layer and the down layer of the channels respectively. In order to reduce resistance of solution in the channels, the channels were designed with the deep head and tail as well as the narrow middle. The width of the head and tail was about 150 μm and the middle was about 150 nm. To carry out our idea, First of all, The channels were designed with a CAD program (Adobe Illustrator 8.0). This design was transferred onto a sheet of Konica film by a high resolution laser setter. The Konica film was used as a mask for ultraviolet exposure in the photolithographic procedure. Cr photoresist layer on the commercially available glass substrate was used as a sacrificial mask , effective for etching with 1.0 mol/L HF +1.0 mol/L NaF . After rigorously cleaning the etched and blank substrate, the channels were casted with PDMS. Then ITO was etched a line of 150μm in length. Finally the ITO etched and the PDMS were brought into closly contact with each other. We can detect single molecules in nanometer segment when solution flowed in the channels.
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