Biosensors based on potent miniprotein binder for sensitive testing of SARS-CoV‑2 variants of concern.

The miniprotein binder TRI2-2 was employed as an antibody alternative to build a single antibody-coupled TRI2-2 based gold nanoparticle-based lateral flow immunoassay (AT-GLFIA) biosensor. The biosensor provides high specificity and affinity binding between TRI2-2 and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) spike antigen receptor binding domain (S-RBD). It also enables rapid testing of wild-type (WT), B.1.1.7 (Alpha), B.1.351 (Beta), B.1.617.2 (Delta), P.1 (Gamma), and B.1.1.529 (Omicron) SARS-CoV-2 S-RBD and is at least ~ 16-fold more sensitive than conventional antibody pair-based GLFIA (AP-GLFIA). Besides, we developed a wireless micro-electrochemical assay (WMECA) biosensor based on the TRI2-2, which demonstrates an excellent VOCs testing capability at the pg mL -1 level. Overall, our results demonstrate that integrating miniprotein binders into conventional immunoassay systems is a promising design for improving the testing capabilities of such systems without hard-to-obtain antibody pair, complex reporter design, laborious signal amplification strategies, or specific instrumentation.