BACKGROUND: The precise detection of tumor markers is crucial for early cancer diagnosis and monitoring. Existing unimodal detection methods are susceptible to interference in complex biological samples, making it difficult to simultaneously achieve high sensitivity and reliability. MicroRNAs (miRNAs), as a key class of cancer-related biomarkers, necessitate novel detection methods capable of multi-layered verification. This study aims to develop an biosensing platform with multi-signal outputs to address the critical challenge of balancing sensitivity, specificity, and result credibility in current tumor marker detection.
RESULTS: We successfully constructed a tri-modal biosensor based on a dual CRISPR/Cas12a system for the highly sensitive and specific detection of miRNA let-7a. The sensor generates a trigger strand via an exponential amplification reaction (EXPAR), which concurrently regulates three independent signaling pathways: ① It initiates the first CRISPR/Cas12a to suppress G-quadruplex/hemin DNAzyme (G4/hemin DNAzyme) formation, turning off the ABTS colorimetric signal. ② It also activates duplex-specific nuclease (DSN) to inhibit the hybridization chain reaction (HCR), thereby blocking FAM fluorescence coupling to streptavidin-coated magnetic nanoparticles (SMBs) and turning off the fluorescence signal. ③ The absence of HCR products on the SMBs inhibits the second CRISPR/Cas12a system, thereby preserving the electrode's P1 probe for binding with P2-3D-CdCo-ONSs@AuNPs and maintaining a high "turn-on" electrochemical signal from the nanocomposite. Thus, the concentration of miRNA let-7a, ranging from 50 fM to 1 pM, can be precisely quantified and validated through colorimetric, fluorescent, and electrochemical signals.
SIGNIFICANCE: This study integrates a dual CRISPR/Cas12a system with a tri-modal output strategy encompassing colorimetric, fluorescent, and electrochemical detection, thereby constructing a detection platform featuring a cross-verification mechanism. This design not only significantly enhances detection accuracy and anti-interference capability but also lays a solid foundation for developing next-generation, highly reliable molecular diagnostic tools. It holds considerable application potential in the fields of early cancer screening and precision medicine.
