Ultrasensitive Chemiluminescence Probes Designed from Covalent Inhibitors for SRAS-CoV-2 M^pro Detection

In the postpandemic era, the emergence of "long COVID" from SARS-CoV-2 has brought ongoing negative impacts on individual health and society. The development of more efficient methods for drug screening and monitoring viral activity remains a critical need. The main protease (M^pro), due to its important role in the virus lifecycle, high conservation, and specificity, is considered an ideal biomarker for SARS-CoV-2. Herein, we have developed several chemiluminescence probes based on different substrates modified from covalent inhibitors targeted at M^pro. Among these, the best probe, MPCL-2, exhibits a rapid response (<20 min), an extremely low limit of detection (LoD; 0.11 nM), great selectivity, and chemical stability. After validating the probe's mechanism of action, MPCL-2 can also be used for real-time, in-situ imaging of enzymes in cells infected with the authentic virus and has the potential for real-time, in-situ M^pro imaging <i>in vivo</i>. Compared to other methods reported to date, the probe demonstrates superior performance and broader applicability, such as in drug screening or virus activity monitoring. Further, the unique design strategy for the substrate can be adopted to develop sensitive probes for other pathogens.