Scanning single-molecule counting system for Eprobe with highly simple and effective approach.

Here, we report a rapid and ultra-sensitive detection technique for fluorescent molecules called scanning single molecular counting (SSMC). The method uses a fluorescence-based digital measurement system to count single molecules in a solution. In this technique, noise is reduced by conforming the signal shape to the intensity distribution of the excitation light via a circular scan of the confocal region. This simple technique allows the fluorescent molecules to freely diffuse into the solution through the confocal region and be counted one by one and does not require statistical analysis. Using this technique, 28 to 62 aM fluorescent dye was detected through measurement for 600 s. Furthermore, we achieved a good signal-to-noise ratio (S/N = 2326) under the condition of 100 pM target nucleic acid by only mixing a hybridization-sensitive fluorescent probe, called Eprobe, into the target oligonucleotide solution. Combination of SSMC and Eprobe provides a simple, rapid, amplification-free, and high-sensitive target nucleic acid detection system. This method is promising for future applications to detect particularly difficult to design primers for amplification as miRNAs and other short oligo nucleotide biomarkers by only hybridization with high sensitivity.

Déclaration de conflit d'intérêts

The authors have read the journal's policy and the authors of this manuscript have the following competing interests: Authors TT, T. Hanashi, MY, HN, and SK are paid employees of Olympus Corporation. Authors YM and YK are paid employees of K.K. DNAFORM, a company holding commercial rights to Eprobe technology. T. Hanami and TS are inventors of Japanese patent applications [JP2012-091400] and [JP2012-158229]. YK is an inventor of Japanese patent application [JP2012-158229]. YH is an inventor of patents [JP2009-171935], [US8067162], [JP2012-091400], [JP2012-158229]. TT, MY and SK are patent inventors for SSMC device [US8710413/JP 5250152]. TT, T. Hanashi, HN are patent inventors for SSMC assay [US9395357 /JP5877155]. The other authors declare no further patents, products in development, or marketed products. This does not alter our adherence to PLOS ONE policies on sharing data and materials.