Electrochemical-Fluorescent\nBimodal Biosensor Based\non Dual CRISPR-Cas12a Multiple Cascade Amplification for ctDNA Detection

Circulating tumor DNA (ctDNA) is a critical biomarker\nfor early\ntumor detection. However, accurately quantifying low-abundance ctDNA\nin human serum remains a significant challenge. To address this challenge,\nwe introduce a bimodal biosensor tailored for detecting the epidermal\ngrowth factor receptor (EGFR) mutation L858R in specific nonsmall\ncell lung cancer (NSCLC) patients. This biosensor utilizes dual CRISPR-Cas12a\nsystems to quantify the target via fluorescence and electrochemical\nsignals. In our system, the EGFR L858R exhibits resistance to digestion\nby the restriction enzyme MscI, which activates the first CRISPR-Cas12a\nprotein and inhibits the binding of magnetic beads with fluorescein\n(FAM)-labeled hybridization chain reaction (HCR) products, thereby\nreducing the fluorescence signal. This activation also inhibits the\ncleavage activity of the second CRISPR-Cas12a protein, allowing the\nelectrode to sustain a higher electrochemical signal from nanomaterials.\nThe wild-type EGFR (wt EGFR) produces the opposite effect. Consequently,\nthe concentration of EGFR L858R can be accurately quantified and verified\nusing both fluorescence and electrochemical signals. The biosensor\noffers a dynamic detection ranging from 10 fM to 1 μM, with\na detection limit of 372 aM. It demonstrates excellent specificity,\nreproducibility, stability, and recovery rates. Moreover, the sensor’s\nenhanced analytical sensitivity highlights its critical role in biosensing\napplications and early disease diagnosis.