Multiple DNA cycle amplification-assisted one-pot isothermal Cas12a for ultrasensitive nucleic acid detection

Rapid and ultrasensitive nucleic acid detection is essential for environmental monitoring and biomedical diagnostics. Here, we report a modular one-pot isothermal platform that integrates catalytic hairpin assembly (CHA), rolling circle amplification (RCA), and CRISPR-Cas12a to construct a self-reinforcing multilayer DNA circuit (CRC). In this system, Cas12a cis-cleavage generates short DNA fragments that recursively activate CHA and RCA, forming a self-sustained cascade amplification loop, while trans-cleavage enables real-time fluorescence signal readout. Using this one-pot platform, ultralow detection limits of 62 aM and 58 aM were achieved for the SARS-CoV-2 S and N genes, respectively, with a total assay time ranging from 20 to 120 min depending on the required sensitivity. Furthermore, functionalizing single-stranded DNA probes on gold nanoparticles (AuNPs) allowed the cleaved DNA to restore fluorescence of fluorophore-quencher reporters, and freeze-thaw-induced AuNP aggregation produced visible colorimetric changes and measurable photothermal signals, enabling trimodal readout without sophisticated instruments. The system demonstrated effective discrimination in controlled experiments, indicating its potential suitability for point-of-care applications. This integrated, one-pot, and scalable platform provides a versatile strategy for fast, sensitive, and multimodal nucleic acid detection applicable to diverse targets.