Abstract
Circulating tumor DNA (ctDNA) serves as a pivotal biomarker for non-invasive cancer diagnostics, yet its low physiological concentration in blood serum poses severe analytical challenges. Here, we report a highly sensitive and selective CRISPR-Cas12a-powered electrochemical biosensor designed for the rapid quantification of the EGFR L858R point mutation in human serum without requiring target pre-amplification. The biosensor utilizes a screen-printed gold electrode functionalized with electrochemically deposited gold nanoparticles (AuNPs) and methylene blue (MB)-labeled single-stranded DNA (ssDNA) reporter probes. Upon target ctDNA recognition, the activated Cas12a–crRNA ribonucleoprotein complex exhibits robust trans-cleavage activity, rapidly degrading the surface-immobilized MB-ssDNA reporters. This cleavage induces a sharp reduction in the peak current signal measured via square wave voltammetry (SWV). Under optimized reaction conditions, the assay demonstrated a broad dynamic linear range from 10 aM to 10 pM and an ultra-low limit of detection of 8.5 aM. Furthermore, the platform displayed exceptional single-nucleotide specificity, effectively discriminating target mutant ctDNA from abundant wild-type genomic backgrounds at ratios as low as 0.01%. Validation in spiked human serum specimens yielded recoveries between 95.4% and 104.2% within 35 minutes. Overall, this biosensing strategy successfully bridges the specificity of CRISPR-Cas platforms with the portability of electrochemical detection, offering strong promise for point-of-care liquid biopsy applications.
