despite the importance of rapid and accurate detection of sars-cov-2 in controlling the covid-19 pandemic, current diagnostic methods are static, unable to distinguish between viable/non-viable virus, or directly reflect viral replication activity. real-time imaging of protease activity specific to sars-cov-2 can overcome these issues but remains lacking. herein, we report a near-infrared fluorescence (nirf) activatable molecular probe (sars-cycd) for detection of sars-cov-2 protease in living mice. the probe comprises a hemicy-anine fluorophore caged with a protease peptide substrate and a cyclodextrin unit, which functions as a nirf signaling moiety and renal-clearable enabler, respectively. the peptide substrate of sars-cycd can be specifically cleaved by the main protease (mpro) of sars-cov-2, resulting in nirf signal activation and liberation of the renal-clearable fluorescent frag-ment (cycd). such a design allows not only sensitive detection of mpro in the lungs of living mice after intratracheal administration, but also permits optical urinalysis of sars-cov-2 infection. thus, this study presents an in vivo sensor that holds potential in preclinical high throughput drug screening and clinical diagnostics for respiratory viral infections.
