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.
