Decoding Antibody Repertoires—Large-Scale Profiling Enabled by Highly Multiplexed Metasurfaces and Digitized Acoustic Bioprinting

The human adaptive immune system can generate over a quintillion unique immunoglobulin antibodies, which are crucial for combating infections, regulating immunity, and serving as the foundation for various immunotherapies. Although the recent breakthrough of AlphaFold has significantly accelerated the in silico discovery and design of antibody therapeutics, empirical assays that provide experimental insights into antibody-antigen interactions have not kept pace. Conventional bioanalytical techniques for probing antibody binding to target molecules (antigens or peptides) typically screen only a few hundred samples simultaneously due to the constraints imposed by the multiplexing capacities of surface-plasmon-resonance spectroscopy and microfluidics. These methods may also introduce potential bias in the selection process, failing to recover all target-specific binders present in phage display libraries.<br/><br/>Here, we present a scalable multiplexed assay utilizing ultra-densely pixelated metasurfaces and digitized acoustic bioprinting enabling large-scale antibody profiling. Our high-throughput metasurface antibody screening (HT-MAbS), incorporating over 10 million sensors per cm², capitalize on the ultrasharp resonance modes driven by the physics of the quasi-bound state in the continuum (qBIC). With quality factors exceeding 2000 in physiological buffers and subwavelength mode volume, this array of nanoresonators provides a sensing figure of merit of approximately 400. Our nozzle-free acoustic bioprinting system deposits picoliter droplets of antigens at rates up to 25,000 droplets per second with micrometer precision, allowing highly customizable surface functionalization. Our hyperspectral imaging technique captures two-dimensional spatially resolved images of thousands of nanosensors across contiguous spectral bands, revealing subtle differentiation of spectral signatures associated with a few tens of linked biomolecules.<br/><br/>We characterize the binding properties and epitope landscapes of antibodies targeting two potential "Disease X" antigens: the SARS-CoV-2 receptor binding domain and avian Influenza hemagglutinin. This characterization includes quantitative analyses of specificity, kinetic binding rates, and equilibrium affinity constants. The antibody panels tested include neutralizing antibodies against SARS-CoV-2 and Influenza A&B subtypes, alongside therapeutic antibodies such as Herceptin and Cetuximab, serving as specificity controls. By visualizing the epitope binning results using dendrograms and heatmaps, we assess the diversity and redundancy of a panel of monoclonal antibodies targeting various functional epitopes on the H5N1 strain of avian influenza A. Our HT-MAbS, with a femtomolar detection limit, demonstrates a linear dynamic range in the concentration range from picomolar to micromolar, and unlocks binding kinetics within a 30-minute timeframe. This high-throughput, sample-efficient, and spectrometer-free assay platform can significantly accelerate biotherapeutic discovery by enabling efficient screening of millions of protein-protein interactions on a single miniaturized chip.