2.5. NanoLuc Luciferase (NanoBiT) Assay

For the analysis of biosensor activity in living cells in vivo, 2 × 10⁴ HEK293T cells were seeded in triplicate in a 96-well plate 24 h before transfection. We transfected 100 ng of Lg-RBD, RBD-Lg, Sm-ACE2, or ACE2-Sm plasmids alone or together into HEK293T cells using PolyJet transfection reagent (SignaGen, Frederick, MD, USA). At 24 h after transfection, the medium was transferred to new wells. The attached cells were lysed in 20 µL of 1 × passive lysis buffer (1 × PLB, Promega) at room temperature (RT) for 15 min. We subjected 20 µL of cell lysate or medium to a NanoLuc luciferase assay using Nano-Glo Live Cell Reagent containing 1/50 diluted furimazine substrate (Promega, Madison, WI, USA). Relative Luminescence Unit (RLU) was measured using GloMax Navigator Microplate Luminometer (Promega).

For analysis of biosensor activity in vitro, 500 ng/well of wild-type (WT) or mutant (deletions or point mutations) Sm-ACE2 or RBD-Lg were transfected into 12-well plates using PolyJet transfection reagent (SignaGen). Two days after transfection, the cells were lysed in 1 × PLB at RT for 15 min. Protein concentrations were quantified using a RC DC Protein Assay kit (Bio-Rad, Mississauga, Canada).

Protein lysates were diluted into 1 µg/µL and kept at −80 °C. For in vitro biosensor analysis, equal amounts of WT or mutant RBD-Lg and Sm-ACE2 were mixed together in 10 µL and incubated at RT for 30 min. We added 10 µL of 1/50 diluted furimazine substrate, followed by measurement of the RLUs using GloMax Microplate luminometer. All experiments were repeated at least two to three times. The means and standard deviations (S.D.) of the RLUs of triplicate samples are shown.