Rolling circle amplification and CRISPR/Cas14a with nanozyme for electrochemical detecting miRNA-205 in NPC-derived exosomes

Nasopharyngeal carcinoma (NPC) is a highly malignant tumor, and early detection of biomarkers like miRNA-205 (miR-205) is critical for improving prognosis. However, accurate detection of miR-205 remains challenging due to its low abundance and matrix interference. Herein, an ultrasensitive electrochemical biosensor integrating Pt nanowires/MXene (PtNWs/MXene), rolling circle amplification (RCA), and CRISPR/Cas14a was developed for detecting exosomal miR-205. The dual characteristics of PtNWs/MXene (differential adsorption capacity for intact and cleaved DNA; HRP-like nanozyme activity) enable the conversion of the miR-205-triggered RCA-Cas14a cascade reaction into significant electrochemical signal changes. This biosensor eliminates the requirement for signal probe labeling of the electrode-modified DNA. Moreover, the enzyme-mimicking catalytic activity of PtNWs/MXene enables the catalysis of numerous 3,3′,5,5′-tetramethylbenzidine (TMB) molecules, realizing a “one-to-many” signal amplification effect that significantly improves detection sensitivity. The biosensor achieves a detection limit of 4.6 aM (50 aM-10 pM linear range) and distinguishes single-base mismatches. Clinical validation confirmed its ability to differentiate NPC patients from healthy individuals, aligning with qRT-PCR results. By adjusting the RCA template, this strategy can be adapted for diverse RNA/DNA targets, offering a versatile platform for early disease diagnosis.