| In chapter one, we mainly make a brief overview of the single molecule detection, single molecule detection techniques can be used for a number areas of bioanalytical detection, also there are many technologies used in the detection of single molecules. In the filed of analytical chemistry, the single molecule is the limits of analytical detection, until now, it mainly includes two major categories of chemical and optical method. Next, we mainly introduce principle and the application of confocal fluorescence microscopy, total internal reflection fluorescence microscopy and epi-fluorescence microscopy.In chapter two, we mainly talk about the principle and the methord of the ultra-sensitive assay for quantification of DNA based on single-molecule detection coupled with hybridization accumulation. and the DNA assay was applied to measure the beta-2-microglobulin messenger RNA level in single human breast cancer cells without a need for PCR amplification. When the accumulation time was9h, the same linear dynamic range (8.0×10-18to1.0×10-15mol/L) was obtained. A distinct advantage of the single-cell mRNA analysis based on the DNA assay is no cDNA or mRNA amplification steps, which improves the reliability of the results.In chapter three, An ultra-sensitive DNA microspot assay was developed that required1.8nL samples and was based on single-molecule detection. The solution of the target DNA (tDNA) was spotted onto the coverslip modified with capture DNA (DNA1) and blocked with ethanolamine and bovine serum albumin using a pintool type microspoting robot. The microspot had a diameter of～300μm. The tDNA was captured by the DNA1, and the tDNA was then labeled with a detection DNA that previously was labeled with a quantum dot. Next, a fluorescence microscopic image of the microspot was acquired using a single-molecule microspot reader during total internal reflection fluorescence excitation. As little as4×10-22mole (240molecules) of tDNA can be detected by this method. The response is linear in the range from6.0×10-22to1.2×10-19mole of tDNA. All operations (including the acquisition of microspot images and single-molecule counting) were performed using the MetaMorph software. The assay was applied to the determination of osteopontin messenger RNA in single decidual stromal cells without the need for PCR amplification.In chapter four, We modify the AMCA, the FAM and CY5dye-labled DNA to the streptavidin-advin-modified magnetic beads with the particle size of300nm, according to the primary colors of light principle, using the epifluorescence microscopy to get the fluorescence images blue, green and red of the magnetic beads, and then obtain the actual fluorescence images by overlaying the primary color images. We have got21kinds of coded microspheres through controlling the ratio of the dye, and the coded microspheres can be used in the simultaneous determination of biological molecules.In chapter five, Anultrasensitive solid-phase fluorescence resonance energy quenching (FREQ)method for determination of1,4-dihydroxybenzene (DHB) using mercuptosuccinic acid (MSA)-capped CdTe quantum dots (QDs) immobilized on silica nanoparticles (NPs) as donors was developed. In the method, silica NPs were first modified with3-aminopropyltriethoxysilane (APTS). Then, MSA-capped CdTe QDs were immobilized on the surface of the APTS-modified silica NPs. Finally, DHB in the solution was attached to the empty sites on the surface of silica NPs with QDs through electrostatic interaction. The fluorescence emission of the QDs was quenched by the proximal DHB molecules on the silica NPs. The quenching efficiency of the solid-phase FREQ method was200-times higher than that of the solution-phase FREQ method. Using the ultrasensitive solid-phase FREQ method, DHB as low as2.4×10-12mol/L could be detected. The method was applied to quantify trace DHB in water samples. |
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