Ultrafast and specific miRNA quantification via single-molecule fluorescence quenching kinetics

Rapid and precise detection of multiple nucleic acid biomarkers is crucial for the development of clinically practical molecular diagnostics. The analytic specificity of biomarker detection can be greatly enhanced by utilizing the information of single-molecule probe binding kinetics. However, existing single-molecule fluorescence methods require up to 10 minutes per biomarker due to the slow binding rates of detection probes to their targets. To enable high-throughput profiling of multiple biomarkers within a short timeframe, faster detection techniques are essential. Here, we introduce Q-FISH (Quenching-based Fluorescence In-Situ Hybridization), a technology capable of detecting nucleic acid biomarkers in sub-second timeframes-achieving speeds over 600 times faster than the previously reported methods. Using Q-FISH, we demonstrated the rapid discrimination of highly homologous miRNAs and the precise quantification of endogenous miRNAs.