Nanoyeast-based impedimetric biosensor with mutated single chain antigen-binding fragment anchoring for SARS-CoV-2 detection.

Impedimetric biosensors are useful for pathogen detection as they combine electrical impedance spectroscopy with the specificity of immunological reactions. These devices can be engineered to detect minute changes in electrical impedance caused by interactions between immobilized recognition elements and target antigens in a sample. They are advantageous in allowing for label-free and real-time detection, with the ability to operate without electroactive materials. Herein, we report an impedimetric biosensor containing nanoyeast expressing SARS-CoV-2 antibody fragments as the active layer. Using nanoyeast offers key advantages such as biocompatibility and stability. The single-chain antigen-binding fragment (scFab) against receptor binding domain of SARS-CoV-2 was mutated according to in silico predictions. It was expressed in Saccharomyces cerevisiae fused to the agglutinin 2 (Aga2), where the binding to Aga1 on the yeast cell wall displays the scFab on the surface of nanofragmented yeast (NY). Electrical impedance monitoring confirmed the successful immobilization of NY onto an adsorbed chitosan layer. This biosensor architecture detected SARS-CoV-2 spike protein with a limit of detection (LoD) of 5 × 10⁻¹⁸ g/mL. It distinguished viral concentrations ranging from 0.3 to 80 plaque-forming units per milliliter (PFU/mL) and demonstrated selectivity for SARS-CoV-2 over H1N1 influenza and Dengue virus. These findings suggest that this biosensing technology could be further adapted for other biomedical and clinical analyses, being promising to improve current pathogen detection methods.