A Photoelectrochemical Biosensor Mediated by CRISPR/Cas13a for Direct and Specific Detection of MiRNA-21

Direct detection of miRNA is currently limited by the complex amplification and reverse transcription processes of existing methods, leading to low sensitivity and high operational demands. Herein, we developed a CRISPR/Cas13a-mediated photoelectrochemical (PEC) biosensing platform for direct and sensitive detection of miRNA-21. The direct and specific recognition of target miRNA-21 by crRNA-21 eliminates the need for pre-amplification and reverse transcription of miRNA-21, thereby preventing signal distortion and enhancing the sensitivity and precision of target detection. When crRNA-21 binds to miRNA-21, it activates the trans-cleavage activity of CRISPR/Cas13a, leading to the non-specific cleavage of biotin-modified DNA with uracil bases (biotin-rU-DNA). This cleavage prevents the biotin-rU-DNA from being immobilized on the electrode surface. As a result, streptavidin cannot attach to the electrode via specific biotin binding, reducing spatial resistance and causing a positively correlated increase in the photocurrent response. This Cas-PEC biosensor has good analytical capabilities, linear responses between 10 fM and 10 nM, a minimum detection limit of 9 fM, and an excellent recovery rate in the analysis of real human serum samples. This work presented an innovative solution for detecting other biomarkers in bioanalysis and clinical diagnostics.