Marrying Protein Synthetic Biology and Bioelectronic Transistors for the Rapid Detection of Single Protein Molecules (SARS-CoV-2 or otherwise)

The COVID-19 pandemic highlighted the need for rapid protein detection and quantification at the single-molecule level in a format that is simple and robust enough for widespread point-of-care applications. We here introduce a modular nanobody-organic electrochemical transistor (OECT) architecture that enables the fast and specific detection and quantification of single-molecule to nanomolar protein antigen concentrations in complex bodily fluids. The sensor combines advances in OECT design with a novel organic semiconductor material, a miniaturized antigen receptor and a modular biological immobilization strategy. Protein antigens are recognized by nanobody-spyCatcher fusion proteins that autocatalytically self-assemble onto a disposable gate electrode with perfectly controlled orientation and at their maximal physically possible density. The resulting sensor provides results after a 10 minutes exposure to 5 µL of unprocessed samples, maintains high specificity and single-molecule sensitivity in human saliva or serum, is mostly re-usable and can be rapidly reprogrammed towards any protein target for which nanobodies exist. We demonstrate the use of this highly modular platform for the detection of green fluorescent protein, SARS-CoV-1/2, and MERS-CoV spike proteins and validate the sensor for COVID-19 screening in unprocessed clinical nasopharyngeal swab and saliva samples.