Capillary driven microfluidic sequential flow device for point-of-need ELISA: COVID-19 serology testing

Abstract

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. Serology assays that detect SARS-CoV-2 antibodies can be used to determine prior infection, immunity status, and/or individual vaccination status and are typically run using well-plate ELISAs in centralized laboratories, but in this format SARs-CoV-2 serology tests are too expensive and/or slow for most situations. Instead, a point-of-need device that can be used at home or in doctor's offices for COVID-19 serology testing would provide critical information for managing infections and determining immune status. Lateral flow assays are common and easy to use, but lack the sensitivity needed to reliably detect SARS-CoV-2 antibodies in clinical samples. This work describes a microfluidic sequential flow device that is as simple to use as 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 steps with two simple end-user steps. An enzyme label and colorimetric substrate produce an amplified, visible signal for increased sensitivity, while the integrated washing steps 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.

Fig. 1. (A) Designs for each layer of the device. Blue layers are transparency sheets and black layers are double-sided adhesive. (B) Three-dimensional representation of the channels and reagent pads within the device. (C) Top view device schematic. (D) Cross-sectional view of device cut along the dashed line in C. (E) Top view of a real device.

Fig. 2. (A) Molecular level representation of detection zone during different stages of the assay. (B) Simulation of assay steps in the device using blue dye to mimic the sample and substrate, and yellow to mimic the detection antibody. (C) Three-dimensional representation of the channels in the device and how the reagents and buffer in the channels are used in different stages of the assay.

Fig. 3. Optimization results for (A) Commercial DAB solution concentration dried on pad 2; (B) mass of detection antibody dried on pad 1; (C) concentration of N-protein striped onto the nitrocellulose; (D) pH of washing buffer. (n = 3) Star represents the condition used for final assay.

Fig. 4. (A) Dose-response curve of anti-N protein spiked into whole human blood using the dELISA. (n = 3). Images are of the test line and show robust visual detection below 10 ng mL⁻¹. (B) Dose-response curve of anti-N protein obtained with a well-plate ELISA. (n = 3) The data for both curves was fit to a 4-parameter logistic curve.