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.

Fig 1. Schematic of optical system used for measurements.

Fluorescence intensity is detected as a photon pulse series when the fluorescent dye molecules traverse the confocal volume scanned in the sample solution.

Fig 2. Raw signals of photon time series data.

A typical photon pulse sequence for an aqueous solution of ATTO 647N scanned at a linear scanning speed of 77 mm/s (top). A photon pulse series showing the passage of a single fluorescent dye molecule (bottom).

Fig 3. Evaluation of single molecule detection by scanning of confocal region.

(A) Typical photon pulse series obtained by scanning a 1 pM ATTO 647N solution. From top to bottom, the speeds are 51 mm/s, 77 mm/s, 102 mm/s, and 128 mm/s, respectively. (B) Relationship between scanning speed and FWHM of signal pulse. Measuring 1 pM ATTO 647N solution at scanning speeds (V_s) of 45 to 110 mm/s. The average FWHM of signals fitting the Gaussian function was plotted. Error bars are the SD calculated from around 100,000 peaks of three measurements.

Fig 4. Comparison of LOD between conventional methods and SSMC analysis.

(A) Log-log plot of ATTO 647N measurement by SSMC method. Raw traces of photon count for each concentration are shown as S3 Fig. (B) Plot for 0 to 1000 aM ATTO 647N. The number of peaks detected was converted for a measurement time of 600 s. Error bars are the SD of three measurements. The detection limit was 62 aM based on 3SD of the background. (Linear fit y = 0.24x + 28, R² = 0.996 by SSMC analysis) (C) Log-log plot of the dye concentration in the range of ATTO 647N 10⁻¹⁷ to 10⁻¹² and the countrate. (D) Plots in the range of ATTO 647N 0 to 1 pM (y = 16.6x + 7.55, R2 = 0.995) and the detection limit was 12 fM.

Fig 5. Comparison of signal-to-noise ratio for oligonucleotide detection using Eprobe.

The signal values of each method were detected in the absence/presence of target oligonucleotide in 100 pM Eprobe solution. (A) Oligonucleotide detection by conventional method. Count rates in the absence and presence of target oligonucleotide are 1.2 kHz and 9.1 kHz, respectively. (B) Oligonucleotide detection by SSMC. Numbers of peaks in the absence and presence of target oligonucleotide is 2 and 4652, respectively.