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

Abstract

AbstractPeptide‐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.