Ultra-sensitive and specificity quantitative detection of miRNA using a combined CRISPR/Cas13a and DNA-PAINT

Abstract

MicroRNAs (miRNAs) are considered to be important biomarkers for the diagnosis of numerous diseases and basic biomedical research. Therefore, a novel rapid diagnostic technique with high sensitivity and specificity is essential for miRNAs detection in clinical trials. Herein, we proposed a sensing platform based on CRISPR/Cas13a and DNA point accumulation in nanoscale topology (DNA-PAINT) to enable an ultra-sensitive analysis of miRNA at the single-molecule level. In this platform, mismatch bases are artificially introduced into the crRNA spacer region, which reduces the mismatch tolerance of the Cas13a/crRNA complex, thus providing high specificity for miRNA detection. Furthermore, signal amplification can be achieved by the dynamic repetitive process of binding and unbinding between fluorescent probes and capture probes in DNA-PAINT imaging, with a detection limit (LOD) as low as 1.12 fM. Convincingly, the practical application capability of the platform for miRNA quantification in complex biological samples is validated. More importantly, given the flexible design of the crRNA spacer region sequence, the strategy could be easily applied to the rapid detection of other miRNAs with excellent reliability, sensitivity and specificity, which has great potential for early diagnosis of miRNA-related diseases.