Cell-free immuno-profiling on a genetically programmed biochip

Cell-free synthetic biology approaches offer biosafe, low-cost and versatile genetic tools to advance therapeutic research and development. Measuring the antibody response to a range of target and off-target proteins is essential for deep immuno-profiling of therapeutic antibodies and individual patient immune responses. Here we extend a previously developed microfluidic-free biochip platform to quantitatively reconstitute interactions of cell-free synthesized antigens with antibodies in miniaturized, photolithographically patterned compartments from localized gene brushes. This creates a continuous density gradient of antigens displayed on the surface, generating multiple antibody binding curves, one in each single nanolitre-volume compartment for affinity determination. We used SARS-CoV-2 antigens to profile the specificity and affinity of monoclonal antibodies to more than 30 viral epitopes, which were synthesized simultaneously on a single chip. We also profiled polyclonal antibodies in a total of 1 μl of human serum, revealing patient-specific epitope profiles that are difficult to detect by conventional approaches. By spatially separating gene brushes in the compartment, we extended the gradient approach to reconstitute the interaction of on-chip cell-free expressed human ACE2 receptor with the viral receptor-binding domain in a specific manner. This on-chip genetically programmed approach enables rapid and quantitative interrogation of complex protein-protein interactions, without protein purification steps, for human immuno-profiling and preparedness for emerging pathogens.