Precision Medicine and Diagnostics
Introduction: Highly sensitive, specific, and point-of-care (POC) serological assays are an essential tool to manage COVID-19. We report on a passive microfluidic POC test that profiles the antibody response against multiple SARS-CoV-2 antigens—spike S1 (S1), nucleocapsid (N), and the receptor binding domain (RBD)—simultaneously from 60 μL of undiluted blood, plasma, or serum. This platform builds upon our group’s previously developed D4 assay—an “all-in-one” immunoassay built upon a non-fouling POEGMA polymer brush which removes background noise and stabilizes assay reagents without refrigeration. While the D4 offers advantages over standard ELISA, the user must still manually time the incubation, and wash/ dry the test by hand prior to analysis, increasing the potential for human error, exposure to biohazardous materials, and hands-on time. Here, we introduce a capillary and semi-gravity driven microfluidic cassette to automate the D4 assay, which eliminates all user intervention after sample introduction, and does not require actuators or pumps. We paired the microfluidic cassette with our lab’s D4Scope—a handheld fluorescent scanner that instantly quantifies and reports the results from the microfluidic cassette with similar performance metric compared to bulkier, and more expensive laboratory devices.
Methods: We used a double antigen assay to detect anti-SARS-CoV-2 antibodies on the microfluidic D4 platform. Physically immobilized capture antigens (S1, RBD, and N) and soluble detection antigens (S1 and N-terminus of N) were inkjet-printed onto the POEGMA substrate. Once printed, the printed assay was interfaced with a microfluidic flow cell constructed from complementary layers of laser ablated adhesives and acrylics to create a series of inlets, outlets, reaction chambers, and timing channels that automate the steps of D4. The D4Scope consists of a laser, band-pass filter, lens, camera, and Raspberry Pi 4 housed in a 3D-printed chassis making a low cost ( < $1k) device in a small form factor ( < 5 lbs., < 0.2 ft3)
Results: We tested plasma from 31 individuals with severe COVID-19, 41 healthy individuals, and 18 individuals with seasonal coronavirus infections on the microfluidic D4. The assay achieved 100% sensitivity and specificity 15+ days post-symptom onset. Further, we quantitatively tracked seroconversion in longitudinal sample sets from 6 patients and found good concordance (Pearson’s r > 0.70 (P < 0.0001)) with a live virus microneutralization assay. The assay also functioned from whole blood samples, where we observed 100% sensitivity and 100% specificity (5 positive / 4 negative) in a proof-of-concept study.
Broader Impact: Our platform is a promising approach to democratize access to healthcare by enabling rapid, sensitive, quantitative, and decentralized protein/antibody detection with minimal user intervention at the point-of-care.