Catalytic BiFeO3/g-C3N4 Heterojunction Nanosheets Coupled with DNAzyme/Low-Leakage Sequential DNA Circuit for Ultrasensitive Electrochemical Aptamer-Based Detection of Colorectal Cancer Exosomes

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Tumor-derived exosomes are promising circulating biomarkers for early colorectal cancer (CRC) diagnosis, yet their low abundance and the complexity of biological fluids demand sensing strategies with both stringent specificity and amplified signal output. Here, we report an ultrasensitive electrochemical aptasensor for SW620 cell-derived exosomes (SW620-Exo) that integrates aptamer proximity ligation, a three-dimensional DNAzyme walker, an integrated sequential activation DNA (ISD) molecular circuit, and highly catalytic BiFeO3/g-C3N4 heterojunction nanosheets. Dual aptamer recognition of distinct epitopes on SW620-Exo brings split DNAzyme segments into proximity, displacing an inhibitory blocker and assembling an active Mg2+-dependent DNAzyme walker. The walker cyclically cleaves hairpin substrates immobilized on magnetic nanobeads to release abundant single-stranded primers to initiate the ISD circuit, in which the second hybridization chain reaction (HCR) module is preconfined within the first to prevent premature activation and reduce background leakage. The resulting methylene blue (MB)-tagged DNA assemblies are captured on the electrode through sequence hybridization, where BiFeO3/g-C3N4 nanosheets efficiently catalyze MB oxidation to generate amplified electrochemical currents. This multilevel design combines cooperative exosome recognition, low-leakage DNA circuitry, and catalytic signal enhancement, affording a linear response for SW620-Exo from 1 × 102 to 1 × 108 particles/mL and a detection limit of 119 particles/mL. The sensor also enables reliable SW620-Exo detection in diluted human sera, indicating its potential as a programmable bioanalytical platform for trace exosome analysis and proof-of-concept for liquid biopsy applications.

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