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
出版年:2011
期:3
页码:201-206
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.
资源类型:期刊论文
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