Entropy-driven catalysis cycle assisted CRISPR/Cas12a amplification photoelectrochemical biosensor for miRNA-21 detection

Herein, a CRISPR/Cas12a-mediated photoelectrochemical (PEC) biosensor based on entropy-driven catalysis (EDC) cycle amplification technology was presented for identification of miRNA-21. On the one hand, sensitizer methylene blue (MB) was introduced by double-strand DNA (H1-H2) on the electrode modified by ZnIn2S4 nanoflowers and CdS quantum dots (QDs), which further optimized initial photocurrent. On the other hand, compared with traditional work, the two single strands generated by EDC cycle triggered by miRNA-21 were fully utilized, the problem of low utilization of EDC cycle products was cleverly solved and the multiple signal amplification with simpler operation was realized by combining with CRISPR/Cas12a. A signal strand S1 generated by EDC cycle made the MB-labeled single strand (H2-MB) bend into a hairpin structure to approach electrode, maximizing the PEC signal. Another signal strand S2 formed a ternary complex with CRISPR/Cas12a to activate the trans-cleavage characteristics, the MB-labeled hairpin structure was cut randomly, which made MB left electrode to significantly reduce the signal, realizing sensitive identification of miRNA-21. The designed PEC biosensor presented a satisfactory linear relationship within the scope of 10 fM-100 nM, and detection line as low as 1.5 fM, supplying a new idea in biomarkers assay and disease diagnosis.