ACS Sensors, 26 February, 2026, DOI：https://doi.org/10.1021/acssensors.5c03979
Amplification-Free Ribonucleic Acid Detection via Cas13-Csm6-Horseradish Peroxidase Cascade with Single-Molecule Sensitivity
Huimin Zhong; Xin Zhang; Jiayan Zhou; Rui Gong; Minghai Chen; Dong Men; Xiaojun Feng; Xian-En Zhang*
Abstract
Achieving a single-molecule level for RNA detection remains a significant challenge in point-of-care settings. We present CCHP, a Cas13-Csm6-horseradish peroxidase three-step enzymatic cascade assay that enables amplification-free RNA detection with a simple colorimetric readout at 450 nm within 1 h under standard conditions. Across diverse viral and non-coding targets, CCHP spans a dynamic range of 10?107 copies per reaction, showing strong agreement with RT-qPCR on serial dilutions (|r| ≈ 0.95). In a small blinded feasibility comparison using extracted RNA from infected versus uninfected cells, CCHP showed concordance with the reference method (κ = 1.0). In representative matrices spiked with synthetic RNA and without nucleic acid extraction or purification, positive events were observed near the single-molecule input level for H1N1-NP in oropharyngeal swabs and for SARS-CoV-2-N in oropharyngeal swabs and plasma, consistent with stochastic initiation of the cascade by single-molecule inputs under Poisson-limited conditions. Under these conditions, the assay maintains stable low-copy detection performance. A physics-guided minimal model integrating deterministic kinetics and stochastic partition statistics informed assay design and sensitivity projection, which were subsequently validated experimentally. By coupling single-molecule?level initiation with ultralow-copy detection and operational simplicity, CCHP advances point-of-care RNA diagnostics and surveillance.
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