CRISPR/Cas12a-based tag-free fluorescent biosensor using G-quadruplex specific thioflavin T for detection of oncogenic microRNAs.

MicroRNAs (miRs) are central regulators of tumor initiation and progression, and their aberrant expression patterns have been identified as clinically valuable biomarkers for the early diagnosis of malignancies and prognostic evaluation. Here, we report tag-free fluorescence biosensing platform for the detection of circulating miRs in serum, targeting microRNA-21 (miR-21) and microRNA-10b (miR-10b) as clinically relevant oncogenic markers. The assay integrates CRISPR/Cas12a-mediated signal regulation with complementary strand (CS)-mediated target recognition. In this strategy, target miRs hybridize with the CS, thereby preventing CS-mediated activation of the Cas12a-crRNA complex. As a result, Cas12a collateral cleavage is suppressed, the G-quadruplex reporter remains intact, and Thioflavin T fluorescence is enhanced. The platform demonstrated excellent sequence discrimination capability, effectively distinguishing closely related and mismatched targets. Sensitive quantification was achieved with limits of detection of 1.4 nM for miR-21 and 852 pM for miR-10b. Importantly, robust analytical performance was maintained in complex biological matrices, confirming its applicability to serum samples. Collectively, this CRISPR/Cas12a-enabled fluorescent biosensor provides a simple approach for circulating miR detection.