Bioluminescent label-free molecular switch for SARS-CoV-2 detection

Simple, single-step platforms for virus detection are still important for improving diagnostic testing beyond laboratory settings. Molecular switch platforms, which generate measurable signals upon specific target binding, represent a promising strategy for virus detection due to their modularity, high sensitivity, and potential adaptability to emerging pathogens.

Here, we present CovSwitch, a novel one-step bioluminescent molecular switch platform engineered for rapid, sensitive, and potentially point of care detection-compatible detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). CovSwitch is a chimeric construct combining aequorin and a peptide sequence derived from human angiotensin-converting enzyme 2 (ACE2), which interacts with the SARS-CoV-2 spike receptor binding domain (RBD). It enables specific viral target recognition through enhanced bioluminescence intensity that increases significantly with viral load in a dose-dependent manner. The molecular switch demonstrated high sensitivity within the linear range of cycle threshold (Ct) values from 19 to 37, corresponding to 2.7 × 10⁷ down to 10 copies/μl, with a detection limit of Ct 40.54, corresponding to fewer than 10 copies/μL. In addition, CovSwitch showed high selectivity and was successfully used to detect SARS-CoV-2 in clinical nasal fluid samples. Site-directed mutagenesis and immunoassays revealed that the enhanced bioluminescence signal results from the specific interaction between the switch and SARS-CoV-2 RBD. Furthermore, in silico analyses showed that the interaction enhances switch flexibility, leading to an optimized conformation for binding coelenterazine and Ca²⁺ ions.

These results highlight the potential of photoprotein-based switch systems as highly sensitive, selective, and label-free platforms for SARS-CoV-2 detection. This single-step platform offers the potential for adaptation to other viral targets.