Abstract
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 × 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.
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.
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Acknowledgments
This project was supported by the National Natural Science Foundation of China (Grant Nos. 90713016 and 20975061). We are grateful to Prof. Xun Qu (Institute of Basic Medical Sciences of Qilu Hospital and Key Laboratory of Cardiovascular Proteomics of Shandong Province) for providing DSCs.
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Sui, B., Li, L., Li, X. et al. DNA microspot assay using single-molecule detection and requiring 1.8 nL samples only. Microchim Acta 174, 201–206 (2011). https://doi.org/10.1007/s00604-011-0640-1
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DOI: https://doi.org/10.1007/s00604-011-0640-1