CRISPR CLAMP: Attomolar level of multiple miRNAs

Simultaneous detection of multiple microRNAs (miRNAs) is crucial for diagnosing major diseases. Conventional miRNA detection methods face limitations in multiplexing, detection cycle, sensitivity and specificity. Herein, we present a novel CRISPR-linked amplification-free miRNA photoelectrochemical (CLAMP) biosensor for the direct and simultaneous analysis of multiple miRNAs with high sensitivity and specificity. The biosensor employs multi-channel screen-printed electrodes with vertical reduced graphene oxide as the substrate for the working electrode. In particular, based on the surface plasmon resonance effect of Au nanoparticles, ZnO/Au heterojunctions with an excellent photoelectric response are constructed and modified on the surface of vertical graphene to generate photocurrent signals. The probes containing protospacer adjacent motif sites, immobilised on the Au nanoparticle surface, enable the dCas9/sgRNA complex to act as a clamp to specifically recognise and bind target sequences. This biosensor translates miRNA recognition into the effect of the dCas9/sgRNA complex on the ultrahigh photoelectric response of ZnO/Au heterojunction to “CLAMP” the target miRNAs, with no need for reverse transcription or amplification. The biosensor achieves multi-channel miRNA detection and an amol-level detection limit while demonstrating single-base recognition specificity as well as excellent stability and reproducibility during the direct detection of miRNA-10b, miRNA-155 and miRNA-21. Clinical tests revealed higher miRNA concentrations in malignant pleurisy than in non-malignant pleurisy. The CLAMP biosensor presents a substantial advancement in miRNA detection and is a promising tool for clinical diagnostics and the comprehensive analysis of multiple miRNA targets.