Consequently, the flow could be maintained without air bubble problems. detect SARS-CoV-2 antibodies in clinical samples. This work describes a microfluidic sequential flow device that is as simple to use as Allopurinol a lateral flow assay, but as sensitive as a well-plate ELISA through sequential delivery of reagents to the detection area using only capillary flow. The device utilizes a network of microfluidic channels made of transparency film and double-sided adhesive combined with paper pumps to drive flow. The geometry of the channels and storage pads enables automated sequential washing and reagent addition actions with two simple end-user actions. An enzyme label and Lepr colorimetric substrate produce an amplified, visible signal for increased sensitivity, while the integrated washing actions decrease false positives and increase reproducibility. Naked-eye detection can be used for qualitative results or a smartphone camera for quantitative analysis. The device detected antibodies at 2.8 ng mL?1 from whole blood, while a well-plate ELISA using the same capture and detection antibodies could detect 1.2 ng mL?1. The performance of the capillary-driven immunoassay (CaDI) system developed here was confirmed by demonstrating SARS-CoV-2 antibody detection, and we believe that the device represents a fundamental step forward in equipment-free point-of-care technology. A capillary-driven microfluidic sequential flow device, designed for eventual at-home or doctor’s office use, was developed to perform an enzyme-linked immunosorbent assay (ELISA) for serology assays. 1.?Introduction Serology assays are traditionally performed in laboratory settings using enzyme-linked immunosorbent assays (ELISA) or at the point of care using lateral flow assays (LFAs).1,2 While traditional ELISAs have superior analytical performance LFAs, they require expensive gear and samples must be shipped to a centralized laboratory for testing. The volume of serology assessments needed in the US alone for a robust serosurvey of different populations can overwhelm clinical laboratory resources and samples may take days or weeks to process for COVID-19 and other diseases. Additionally, at >$100 per sample the cost of a test represents a significant financial burden to patients.3 Furthermore, when access to clinical laboratories is limited, and purified by nickel affinity and size exclusion chromatography in 50 mM HEPES buffer (pH 7.4) and 500 mM NaCl throughout purification to reduce aggregation. Protein purity and quality was verified by SDS-PAGE gel electrophoresis. N protein was striped onto the nitrocellulose strip with a reagent dispenser (Claremont Bio). The striping solution contained 45 mM trehalose, 4.5% glycerol, and 0.5 mg mL?1 N protein. The trehalose and glycerol were used to improve storage stability. Roughly Allopurinol 120 ng of N protein was added to each 3 mm nitrocellulose strip. The detection antibody was an anti-mouse-IgG conjugated to horse radish peroxidase (HRP) (Abcam ab97040, lot no. 3327554). The antibody was diluted to 5.0 g mL?1 in a solution of 0.01 M FeSO4CEDTA, 4% trehalose, and 0.1% BSA to improve long term storage.18 Two 5.00 L aliquots of the detection antibody solution (50 ng) were sequentially dried onto a 3 5 glass fiber pad. The colorimetric substrate used was 3,3-diaminobenzidine (DAB). Pierce? DAB Substrate kit from ThermoFisher, which included a 10 solution of DAB and a peroxide buffer, was used for the substrate and washing buffer. 15.00 L of the 10 DAB solution was added to a 3 5 glass fiber pad in three 5.00 L aliquots and air dried at 37 C for 20 min. The peroxide buffer was adjusted to a pH of 6.5 with sodium hydroxide and Tween-80 (Fisher Scientific) was added to a concentration of 0.1%. To start the assay, 10.0 L of sample Allopurinol is added to the.