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
Early detection of both genetic mutations and epigenetic modifications is critical for cancer diagnosis, but current methods often require separate assays or suffer from bisulfite-induced DNA damage. Here, we present a controllable CRISPR/Cas12a-based signal logic gate that enables simultaneous detection of KRAS G12C mutation and Septin9 promoter methylation in a single reaction. The strategy converts target information into two distinct ssDNA activators (T1 for methylation and T2 for mutation) via orthogonal enzymatic cascades (GlaI- and FEN1-mediated cleavage followed by strand displacement amplification). By limiting crRNA concentrations, the CRISPR/Cas12a trans-cleavage activity produces three well-resolved fluorescence kinetic states: low (mutation only), medium (methylation only), and high (both targets), respectively, which could be distinguished using three predefined threshold values. Validation with clinical samples from colorectal cancer patients and healthy controls showed complete concordance with Sanger sequencing and qPCR. And the proposed method successfully detected Septin9 methylation in peripheral blood. This isothermal, single-tube, bisulfite-free strategy offers a simple and reliable platform for simultaneous genetic and epigenetic analysis in point-of-care cancer screening.