caa a22 4500 445881178 CHVBK 20180317145543.0 cr unu---uuuuu 170323e20110901xx s 000 0 eng 10.1007/s00604-011-0640-1 doi (NATIONALLICENCE)springer-10.1007/s00604-011-0640-1 DNA microspot assay using single-molecule detection and requiring 1.8 nL samples only [Elektronische Daten] [Benhui Sui, Lu Li, Xincang Li, Jinxing Wang, Xiaoli Zhang, Wenrui Jin] An ultra-sensitive DNA microspot assay was developed that required 1.8nL 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 ~300μm. 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 × 10−22 mole (240 molecules) of tDNA can be detected by this method. The response is linear in the range from 6.0 × 10−22 to 1.2 × 10−19 mole of tDNA. All operations (including the acquisition of microspot 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. Figure A DNA microspot assay with a limit of detection of 240 molecules was developed that requires 1.8 nL samples and is based on single-molecule detection. The assay was applied to the determination of osteopontin messenger RNA in single decidual stromal cells without the need for PCR amplification. Springer-Verlag, 2011 Single-molecule detection nationallicence Deoxyribonucleic acid assay nationallicence Messenger ribonucleic acid determination nationallicence Quantum dot-labeling nationallicence Single-cell analysis nationallicence Sui Benhui School of Chemistry and Chemical Engineering, Shandong University, 27 Shanda Road, 250100, Jinan, China aut Li Lu College of Chemistry, Chemical Engineering and Materials Science, Key Laboratory of Molecular and Nano Probes, Shandong Normal University, 88 Wenhua East Road, 250014, Jinan, China aut Li Xincang School of Life Sciences, Shandong University, 27 Shanda Road, 250100, Jinan, China aut Wang Jinxing School of Life Sciences, Shandong University, 27 Shanda Road, 250100, Jinan, China aut Zhang Xiaoli School of Chemistry and Chemical Engineering, Shandong University, 27 Shanda Road, 250100, Jinan, China aut Jin Wenrui School of Chemistry and Chemical Engineering, Shandong University, 27 Shanda Road, 250100, Jinan, China aut Microchimica Acta Springer Vienna 174/3-4(2011-09-01), 201-206 0026-3672 174:3-4<201 2011 174 604 https://doi.org/10.1007/s00604-011-0640-1 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1007/s00604-011-0640-1 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Sui Benhui School of Chemistry and Chemical Engineering, Shandong University, 27 Shanda Road, 250100, Jinan, China aut NATIONALLICENCE 700 1- Li Lu College of Chemistry, Chemical Engineering and Materials Science, Key Laboratory of Molecular and Nano Probes, Shandong Normal University, 88 Wenhua East Road, 250014, Jinan, China aut NATIONALLICENCE 700 1- Li Xincang School of Life Sciences, Shandong University, 27 Shanda Road, 250100, Jinan, China aut NATIONALLICENCE 700 1- Wang Jinxing School of Life Sciences, Shandong University, 27 Shanda Road, 250100, Jinan, China aut NATIONALLICENCE 700 1- Zhang Xiaoli School of Chemistry and Chemical Engineering, Shandong University, 27 Shanda Road, 250100, Jinan, China aut NATIONALLICENCE 700 1- Jin Wenrui School of Chemistry and Chemical Engineering, Shandong University, 27 Shanda Road, 250100, Jinan, China aut NATIONALLICENCE 773 0- Microchimica Acta Springer Vienna 174/3-4(2011-09-01), 201-206 0026-3672 174:3-4<201 2011 174 604 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer