School of Chemistry and Chemical Engineering, Shandong University, Jinan, 250100, China.
Analyst. 2011 Oct 7;136(19):3950-5. doi: 10.1039/c1an15405b. Epub 2011 Aug 5.
An ultra-sensitive assay for quantification of DNA based on single-molecule detection coupled with hybridization accumulation was developed. In this assay, target DNA (tDNA) in solution was accumulated on a silanized substrate blocked with ethanolamine and bovine serum albumin (BSA) through a hybridization reaction between tDNA and capture DNA immobilized on the substrate. The tDNA on the substrate was labeled with quantum dots which had been modified with detection DNA and blocked with BSA. The fluorescence image of single QD-labeled tDNA molecules on the substrate was acquired using total internal reflection fluorescence microscopy. The tDNA was quantified by counting the bright dots on the image from the QDs. The limit of detection of the DNA assay was as low as 6.4 × 10(-18) mol L(-1). Due to the ultra-high sensitivity, 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.
基于单分子检测与杂交积累相结合的超高灵敏 DNA 定量分析方法。在该方法中，溶液中的靶 DNA（tDNA）通过 tDNA 与固定在基底上的捕获 DNA 之间的杂交反应在经乙醇胺和牛血清白蛋白（BSA）封闭的硅烷化基底上累积。tDNA 用经检测 DNA 修饰并用 BSA 封闭的量子点进行标记。通过全内反射荧光显微镜获取基底上单量子点标记的 tDNA 分子的荧光图像。通过对 QD 上的亮点进行计数，定量 tDNA。DNA 分析的检测限低至 6.4×10(-18)mol/L(-1)。由于超高灵敏度，该 DNA 分析方法无需 PCR 扩增即可应用于测量单个人类乳腺癌细胞中的β-2-微球蛋白信使 RNA 水平。
