Quantitative detection of the diabetic retinopathy-associated microRNA-1281 via a CRISPR/Cas12a-RCA ratiometric...

Diabetic retinopathy (DR) is a major microvascular complication of diabetes, yet accessible molecular tools for early-stage screening remain limited. Circulating microRNAs are promising non-invasive biomarkers for DR, but their reliable quantification in serum is hindered by low abundance, matrix interference, and limited robustness of single-signal assays. Here, we report a Cas12a-RCA ratiometric sensing platform (CRRSP) for the quantitative detection of the DR-associated biomarker miR-1281. The assay is constructed using a target-triggered cascaded strand-displacement circuit that enforces sequential molecular recognition prior to amplification, thereby improving specificity. Upon target recognition, rolling circle amplification (RCA) generates DNA products that simultaneously produce a thioflavin T (ThT) fluorescence signal via G-quadruplex (G4) formation and sequester the Cas12a/gRNA Activator strand to suppress ROX fluorescence. This dual-channel ThT/ROX readout enables intrinsic ratiometric normalization, effectively compensating for matrix effects and experimental variability. The workflow operates under sequential constant-temperature steps without PCR-type thermal cycling. The assay achieves a detection limit of 0.83 pM with high selectivity and shows excellent agreement with RT-qPCR in clinical serum samples from healthy controls, glaucoma patients, and patients with non-proliferative diabetic retinopathy (NPDR) or proliferative diabetic retinopathy (PDR).