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

Abstract

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.

Development of a Highly Sensitive β-Glucan Detection System Using Scanning Single-Molecule Counting Method

“…The SSMC method is an ultra-sensitive analytical method with high sensitivity and a good signal-to-noise ratio, which can detect fluorescent molecules even at concentrations of several tens of aM [ 30 ]. To improve the measurement efficiency, we labeled the binding protein with the fluorescent dye Alexa Fluor 647, which is resistant to photobleaching [ 31 ], and adopted a method to rapidly count fluorescent molecules in a limited volume of the confocal site.…”

“…To improve the measurement efficiency, we labeled the binding protein with the fluorescent dye Alexa Fluor 647, which is resistant to photobleaching [ 31 ], and adopted a method to rapidly count fluorescent molecules in a limited volume of the confocal site. The SSMC method has also been applied to the sensitive detection of oligonucleotide interactions hybridized to specific sequences [ 30 ]. The SSMC method has the potential to become an analytical method with excellent specificity and sensitivity for the detection of interactions between macromolecules and small molecules.…”

“…Optical scanning was performed at a scanning speed of 69 mm sec -1 and with an excitation light of 1 mW. Fluorescence time series data were acquired for 600 s and analyzed as previously described [ 30 ].…”

A handheld luminometer with sub-attomole limit of detection for distributed applications in global health

“…Nonetheless, there are unique engineering challenges related to the low radiant power density resulting from kinetically-limited enzymatic light generation. While a single fluorescent dye molecule is capable of emitting tens of thousands of photons per second from a nanoscopic volume [ 10 , 11 ], luminescence is generated gradually by enzymatic turnover on the order of a few photons per second per enzyme, even in optimized flash-mode assay conditions [ 12 ]. Consequently, a much larger number of emitting molecules is required to generate comparable photon rates by catalytic turnover as compared with fluorescence, placing a much greater demand on the design and sensitivity of luminometers relative to fluorescence readers.…”