Inexpensive High-Throughput Multiplexed Biomarker Detection Using Enzymatic Metallization with Cellphone-Based Computer Vision

[Image: see text] Multiplexed biomarker detection can play a critical role in reliable and comprehensive disease diagnosis and prediction of outcome. Enzyme-linked immunosorbent assay (ELISA) is the gold standard method for immunobinding-based biomarker detection. However, this is currently expensive, limited to centralized laboratories, and usually limited to the detection of a single biomarker at a time. We present a low-cost, smartphone-based portable biosensing platform for high-throughput, multiplexed, sensitive, and quantitative detection of biomarkers from single, low-volume drops (<1 μL) of clinical samples. Biomarker binding to spotted capture antigens is converted, via enzymatic metallization, to the localized surface deposition of amplified, dry-stable, silver metal spots whose darkness is proportional to biomarker concentration. A custom smartphone application is developed, which uses real-time computer vision to enable easy optical detection of the deposited metal spots and sensitive and reproducible quantification of the biomarkers. We demonstrate the use of this platform for high-throughput, multiplexed detection of multiple viral antigen-specific antibodies from convalescent COVID-19 patient serum as well as vaccine-elicited antibody responses from uninfected vaccine-recipient serum and show that distinct multiplexed antibody fingerprints are observed among them.