A Versatile DNAzyme‐Amplified Protease‐Sensing Platform for Accurate Diagnosis of SARS‐CoV‐2 and Reliable Classification of Colorectal Cancer

Peptide-based biosensors are widely used for in vitro detection of protease activity but often suffer from the limited sensitivity, poor accuracy, and incompatibility with point-of-care testing (POCT) devices. Herein, we developed a versatile deoxyribozyme (DNAzyme)-amplified protease-sensing (DP) platform that integrates the positively charged oligopeptides with a negatively charged DNAzyme biocatalyst for highly-sensitive protease detection. The system leverages the electrostatic peptide-DNAzyme interactions to inhibit DNAzyme catalytic activity, which is reactivated upon the protease-triggered peptide hydrolysis, thus enabling an efficient signal amplification via the successive cleavage of DNAzyme substrate. Compared to conventional peptide-based sensing platform, our DP system offers an enhanced sensitivity and signal-to-noise ratio and is highly modular for detecting various clinically relevant proteases through a simple replacement of the peptide blocker. By introducing a dual-enzyme recognition mechanism, we developed a dual-protease-triggered DP platform for enabling the accurate detection of SARS-CoV-2 proteases in saliva. We also applied the DP platform to differentiate between normal and cancerous colon cells and tissues by detecting colorectal cancer (CRC)-associated proteases. Overall, this work introduces a universal and scalable biosensing strategy for activity-based protease detection with potential applications in both infectious disease diagnostics and cancer classification, advancing the field of DNAzyme-based POCT technologies.