Nanoporous gold electrode-assisted CRISPR/Cas12a electrochemical detection of synthetic methylated DNA models for breast cancer liquid-biopsy development

Abstract

Circulating tumor DNA (ctDNA) methylation is a promising liquid-biopsy signal for early breast cancer, but low target abundance, fragmented cell-free DNA (cfDNA), and matrix fouling remain major barriers to electrochemical implementation. Here, a nanoporous gold (NPG) electrode-assisted CRISPR/Cas12a electrochemical assay was developed for synthetic methylated DNA models that mimic breast-cancer-associated cfDNA promoter fragments. The assay integrates HpaII methylation-sensitive restriction-enzyme (MSRE) digestion, recombinase polymerase amplification (RPA), Cas12a trans-cleavage, and methylene-blue (MB)-labeled single-stranded DNA (ssDNA) reporters immobilized on NPG. A three-marker panel targeting RASSF1A, APC, and FOXA1 was selected from breast cancer cfDNA methylation literature. The nanoporous electrode exhibited a 9.4-fold electrochemically active surface-area (ECSA) enhancement, and the assay achieved linear responses from 10 fM to 10 nM with limits of detection (LODs) of 3.4, 5.2, and 4.6 fM for RASSF1A, APC, and FOXA1, respectively. The platform distinguished 1% methylated DNA in a high wild-type background, produced 92.4–106.8% recovery in an artificial plasma matrix, and retained 91.8% of response after 14 days of storage. Blinded validation in 60 methylation-positive and 45 methylation-negative contrived specimens yielded an area under the curve (AUC) of 0.966, positive-call sensitivity of 86.7%, specificity of 88.9%, and accuracy of 87.6% at a fixed panel-score threshold. The results support NPG-CRISPR electrochemistry as a compact strategy for nonclinical methylated-DNA model analysis and define experimental benchmarks for subsequent ethically approved translational studies.