MicroRNAs (miRNAs) are promising biomarkers for clinical diagnosis and disease monitoring. However, current CRISPR/Cas-based miRNA sensors generally require reverse transcription or nucleic acid amplification to improve sensitivity, which complicates the workflow and increases the risk of contamination, nonspecific amplification, and false-positive results. Herein, we developed a one pot amplification-free Cas13a/Cas12a cascade platform based on a designed dual-functional molecular bridge probe, Conv HP-3, which served as both a substrate for Cas13a-mediated trans-cleavage and an activator for Cas12a-mediated trans-cleavage, thereby linking target recognition to cascade signal amplification for miRNA-21 detection. Following the introduction of miRNA-21, Cas13a was specifically activated through target-crRNA recognition and cleaved the Conv HP-3 probe to release a Cas12a-activating DNA fragment. This fragment subsequently triggered Cas12a-mediated cleavage of the ssDNA reporter, generating a markedly enhanced fluorescence signal. This one-pot Cas13a/Cas12a cascade fluorescence biosensor enabled quantitative detection of miRNA-21 over a concentration range of 1-1000 pmol/L within 60 min, with a low detection limit of 0.66 pM. Notably, the assay achieved average recoveries ranging from 95.97% to 108.59%, with a variation between 0.6% and 1.78%, demonstrating its good accuracy and precision. This biosensing platform shows great promise for the rapid and sensitive detection of miRNAs in clinical applications.
Jing Yuan, Meng Shen, Lihua Ding et al.· Talanta: The International J...· 0 citations
DNA methylation is an important epigenetic biomarker for early disease screening and prognosis evaluation, but its reliable detection remains challenging because methylated DNA is often present at low abundance in complex biological backgrounds. Here, we report a methylation-sensitive bioelectronic sensing platform that integrates AciI-assisted target discrimination, CRISPR/Cas12a-mediated trans-cleavage, and vertical organic electrochemical transistors (vOECTs) amplification for highly sensitive methylated DNA detection. In this strategy, unmethylated DNA is selectively digested by AciI, while intact methylated DNA activates the crRNA-guided Cas12a system, triggering collateral cleavage of ssDNA reporters immobilized on the Au gate electrode. The resulting interfacial changes are efficiently amplified by the vOECTs through coupled electric-double-layer gating. The platform achieved quantitative methylated DNA detection from 100 fM to 100 pM with a sensitivity of 267.6 μA/dec and a detection limit of 100 fM. The sensor also exhibited good operational stability, reproducibility, and reliable recovery performance in artificial serum samples. This work demonstrates the potential of CRISPR/vOECTs bioelectronics for sensitive epigenetic analysis and presents a promising proof-of-concept for future non-invasive screening strategies.
Kun Xu, Sibo Wang, Kejie Zhang et al.· Talanta: The International J...· 0 citations
A controllable CRISPR/Cas12a-based signal logic gate that enables simultaneous detection of KRAS G12C mutation and Septin9 promoter methylation in a single reaction and offers a simple and reliable platform for simultaneous genetic and epigenetic analysis in point-of-care cancer screening.
