标题：DNA microspot assay using single-molecule detection and requiring 1.8 nL samples only
作者：Benhui Sui;Lu Li;Xincang Li
作者机构：School of Chemistry and Chemical Engineering, Shandong University, 27 Shanda Road, 250100, Jinan, China;College of Chemistry, Ch
来源：Mikrochimica Acta: An International Journal for Physical and Chemical Methods of Analysis
关键词：Single-molecule detection;Deoxyribonucleic acid assay- Messenger ribonucleic acid determination;Quantum dot-labeling;Single-cell analysis
摘要：An ultra-sensitive DNA microspot assay was developed that required 1.8 nL 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 ～300 μm. 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 as 4 x10~(-22) mole (240 molecules) of tDNA can be detected by this method. The response is linear in the range from 6.0 x 10~(-22) to 1.2 x 10~(-19) mole of tDNA. All operations (including the acquisition ofmicrospot 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.