Multiplexed proteomic biosensor platform for label-free real-time simultaneous kinetic screening of thousands of protein interactions.

Studying protein interactions in high throughput (HTP) is critical for advancing many aspects of drug discovery, biomarker identification, and diagnostic development. However, existing methods for producing functional protein libraries are costly, time-consuming, and lack real-time kinetic screening capabilities. To address these limitations, we developed an automated platform for HTP production and screening of a library of proteins on biosensor surfaces, facilitating large-scale measurement of interaction kinetics. This technology, Sensor-Integrated Proteome On Chip (SPOC®), involves cell-free protein expression in nanowells directly from customizable libraries of DNA, facilitating simultaneous capture-purification of up to 2400 unique full-length folded proteins or proteoforms onto a single gold biosensor chip. SPOC protein biosensors can then be screened by real-time label-free analysis, including surface plasmon resonance (SPR) to generate kinetic data. Fluorescent and SPR assays were used to demonstrate zero crosstalk between protein spots. The functionality of SPOC proteins was validated using a variety of assays. Monoclonal antibodies were found to selectively bind their SPOC protein targets and anti-RBD antibody was used to demonstrate discriminated binding kinetics to numerous SARS-CoV-2 RBD variants on-chip. With advantages of HTP, flexibility, low-cost, and real-time kinetic profiling, the SPOC platform addresses challenges of interrogating protein interactions at scale for research and clinical applications.