School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100, China.
Biosens Bioelectron. 2011 Apr 15;26(8):3688-91. doi: 10.1016/j.bios.2011.01.040. Epub 2011 Mar 2.
A novel ultra-sensitive single-molecule-counting microarray assay (SMCMA) with a 1.8-nL sample volume for quantification of proteins was provided using total internal reflection fluorescence microscopy coupled with quantum dot (QD)-labeling. In the SMCMA, the microarray consisting of ∼ 300 μm diameter microspots with the spot-to-spot pitch distance of 500 μm was fabricated by spotting 1.8 nL of solutions containing the target protein onto the substrate which was modified with primary antibody of the protein and blocked with ethanolamine and BSA using a pin-tool type microarraying robot. Then, biotinylated secondary antibody of the protein was bound to the protein to form sandwich immunocomplexes. After labeling with streptavidin-coated QDs, the whole image of the microarray was acquired using a homemade single-molecule microarray reader. The target protein was quantified based on the number of bright dots from the QDs corresponding to single target protein molecules on the microarray. Using the SMCMA, an amount as low as 1.5 × 10(-21) mole (904 molecules) for proteins could be detected. The SMCMA was applied to measure dynamic expression of osteopontin in living cells.
提供了一种新型的超灵敏单分子计数微阵列分析（SMCMA），其样品体积为 1.8nL，用于使用全内反射荧光显微镜和量子点（QD）标记定量蛋白质。在 SMCMA 中，通过使用针式微阵列机器人将含有目标蛋白质的溶液点样到经蛋白质的一抗修饰并用乙醇胺和 BSA 封闭的基底上，制造出由约 300μm 直径的微点组成的微阵列，点与点之间的间距为 500μm。然后，将生物素化的蛋白质的二级抗体结合到蛋白质上，形成三明治免疫复合物。用链霉亲和素包被的 QD 标记后，使用自制的单分子微阵列读取器获取整个微阵列的图像。基于对应于微阵列上单个目标蛋白质分子的 QD 的亮点数，定量目标蛋白质。使用 SMCMA，可以检测到低至 1.5×10(-21)摩尔（904 个分子）的蛋白质。SMCMA 被应用于测量活细胞中骨桥蛋白的动态表达。
