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

Area of Science:

• Analytical Chemistry
• Biotechnology
• Molecular Diagnostics

Background:

• Accurate detection of low-concentration molecules is crucial for diagnostics.
• Existing methods often require amplification or complex statistical analysis.
• Developing sensitive and rapid detection techniques remains a challenge.

Purpose of the Study:

• To introduce a novel, rapid, and ultra-sensitive detection technique for fluorescent molecules.
• To demonstrate the capability of counting single molecules in solution without amplification.
• To establish a highly sensitive nucleic acid detection system.

Main Methods:

• Scanning Single Molecular Counting (SSMC) utilizes a fluorescence-based digital system.
• Noise reduction is achieved by conforming signal shape to excitation light intensity via circular scanning.
• Single fluorescent molecules diffuse freely through the confocal region and are counted individually.

Main Results:

• Detection of 28 to 62 aM fluorescent dye within 600 s.
• Achieved a high signal-to-noise ratio (S/N = 2326) for 100 pM target nucleic acid.
• Demonstrated amplification-free detection using hybridization-sensitive fluorescent probes (Eprobe).

Conclusions:

• SSMC combined with Eprobe offers a simple, rapid, and highly sensitive nucleic acid detection system.
• The method is promising for detecting difficult-to-amplify biomarkers such as miRNAs and short oligonucleotides.
• This approach eliminates the need for amplification and complex statistical analysis.