ABSTRACT The objective of this proposal is to develop and validate a new set of low-cost, easy-to-use, point-of-care tests (POCTs) that can simultaneously detect multiple biomarkers within 30 minutes with high sensitivity and specificity to enable early diagnosis, prognosis, and serology of COVID19 patients. The multiplexed D4-POCT employs antibodies for antigen detection in the diagnosis and prognosis chips, and antigens for antibody detection in the serology chip. Capture and detection antibodies will be used to generate assays for S1 and N antigens from nasal/throat swab collection media for diagnosis, D-dimer, IL-2R, IL-6, IL-10 and TNF-α in human blood or serum for prognosis, and S1, N, and RBD viral antigens will be used as capture and detection reagents in a double- antigen serological assay to detect anti-SARS-CoV-2 antibodies in human blood and serum. Optimal reagents for assay development will be identified by leveraging the multiplexing capabilities of the D4-POCT to perform high-throughput screening and pairing of patient derived antibodies and viral antigens. The D4-POCT has three components. The first component—the D4 chip—is fabricated by inkjet printing microspots of capture and fluorescently labeled detection reagents (antibodies for antigen detection or antigens for antibody detection) directly on a protein- and cell-resistant poly(oligoethylene glycol methacrylate) brush that is grafted from the surface of glass, which reduces background noise, yielding pg/mL limit-of-detection and over 4 logs of dynamic range. The second component is a gravity-driven microfluidics cassette that encases the D4 chip and is fabricated by low-cost and high-throughput injection molding. The microfluidics cassette reduces user exposure to potentially infectious material and minimizes user interaction, as it only requires the addition of a drop of sample into the sample inlet and two drops of wash buffer into the wash buffer inlet —thereby mimicking the procedure used to carry out lateral flow assays, the gold standard of immunoassay for field use. The chip automatically performs a timed incubation and rinse step that removes the sample and any unbound detection reagent from the surface of the D4 chip, leaving the central area of the D4 chip ready for imaging by the third component—the D4Scope—a customized, low-cost, hand-held fluorescence detector that we have developed. The fluorescent spot intensities on the D4 chip are converted to analyte concentration by a built-in “App” on the D4Scope, and analyte concentrations and the raw data —the images— are both automatically uploaded to a secure cloud server. All components of the D4-POCT are easy to fabricate at high volumes as the chips are rapidly manufactured by inkjet printing picograms of reagents per test, the microfluidics cassette is fabricated by injection molding, and the D4Scope is assembled from off-the shelf parts. The impact of this proposal will be the development and clinical validation ...