Sensitive analysis of viral proteases is crucial for early infection diagnosis and antiviral drug development; however, developing activity-based assays with high sensitivity and broad adaptability remains a significant challenge. To address this, we developed a cascade signal amplification strategy that enhances protease detection sensitivity through rational coupling of two modular, protease-responsive enzyme switches. Upon recognition of the target protease, this upstream switch triggers the activation of a downstream, protease-responsive CRISPR/Cas12a effector, which converts the specific proteolytic event into an amplified fluorescence signal. This cascaded enzymatic amplification generates a robust signal output, achieving an order-of-magnitude improvement in detection sensitivity compared to single-stage CRISPR/Cas12a assays. The modular nature of the enzyme switches renders the system highly expandable, allowing for the specific detection of diverse viral proteases. We demonstrated the platform's utility in complex biological samples by sensitively monitoring 3C protease activity within enterovirus 71-infected cells. Furthermore, dose-dependent inhibition analysis using the HRV 3C protease inhibitor rupintrivir validated the system's potential for evaluating antiviral drug efficacy. Collectively, this work establishes a versatile and modular analytical platform for the sensitive detection and functional study of viral proteases.
