Rapid and Sensitive Detection of miRNA by Single‐Molecule Fluorescence Dequenching Assay with Target Recycled CRISPR/Cas12a Amplification System

Abstract Dysregulated miRNAs play a critical role in the development of cancers. A rapid and sensitive single‐molecule fluorescence dequenching assay combined with a CRISPR/Cas12a‐based target recycling amplification system for miRNA detection is developed. This single‐molecule assay detects miRNAs down to ≈10 fM within 10 min. An automated single‐molecule fluorescent puncta analysis procedure is also created, improving the signal‐to‐noise ratio by 3.76‐fold compared to traditional hidden Markov model (HMM)based methods. The clinical applicability of this technique is demonstrated. Two key miRNA targets associated with non‐small cell lung cancer (NSCLC) and ovarian cancer (OC) from 2867 datasets of the TCGA database are screened. Validation is initially conducted at the cell line level, followed by testing with tissue and blood samples from 10 patients with NSCLC and OC. The assay demonstrated high diagnostic accuracy, with receiver operating characteristic curves (area under the curve (AUC) > 0.93) and significant statistical differentiation ( p < 0.001) between cancer and healthy samples. This method's exceptional sensitivity and speed highlight its potential for early cancer diagnostics and personalized medicine.