标题：Single-molecule-counting protein microarray assay with nanoliter samples and its application in the dynamic protein expression of living cells
作者：Li, Lu; Qu, Xun; Sun, Jintang; Yang, Meixiang; Song, Bingfeng; Shao, Qianqian; Zhang, Xiaoli; Jin, Wenrui
作者机构：[Zhang, Xiaoli; Jin, Wenrui] Shandong Univ, Sch Chem & Chem Engn, Jinan 250100, Peoples R China.; [Qu, Xun; Sun, Jintang; Yang, Meixiang; Song, Bing 更多
通讯作者地址：[Jin, WR]Shandong Univ, Sch Chem & Chem Engn, Jinan 250100, Peoples R China.
来源：BIOSENSORS & BIOELECTRONICS
关键词：Single-molecule detection of proteins; Protein microarray assay with; quantum dot-labeling; Measurement of dynamic protein expression in; living cells
摘要：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 similar to 300 mu m diameter microspots with the spot-to-spot pitch distance of 500 mu m 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 home-made 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 x 10(-21) mole (904 molecules) for proteins could be detected. The SMCMA was applied to measure dynamic expression of osteopontin in living cells. (C) 2011 Elsevier B.V. All rights reserved.