Capillary flow-driven immunoassay platform for COVID-19 antigen diagnostics

Abstract

Over the last few years, the SARS-CoV-2 pandemic has made the need for rapid, affordable diagnostics more compelling than ever. While traditional laboratory diagnostics like PCR and well-plate ELISA are sensitive and specific, they can be costly and take hours to complete. Diagnostic tests that can be used at the point-of-care or at home, like lateral flow assays (LFAs) are a simple, rapid alternative, but many commercially available LFAs have been criticized for their lack of sensitivity compared to laboratory methods like well-plate ELISAs. The Capillary-Driven Immunoassay (CaDI) device described in this work uses microfluidic channels and capillary action to passively automate the steps of a traditional well-plate ELISA for visual read out. This work builds on prior capillary-flow devices by further simplifying operation and use of colorimetric detection. Upon adding sample, an enzyme-conjugated secondary antibody, wash steps, and substrate are sequentially delivered to test and control lines on a nitrocellulose strip generating a colorimetric response. The end user can visually detect SARS-CoV-2 antigen in 15-20 min by naked eye, or results can be quantified using a smartphone and software such as ImageJ. An analytical detection limit of 83 PFU/mL for SARS-CoV-2 was determined for virus in buffer, and 222 PFU/mL for virus spiked into nasal swabs using image analysis, similar to the LODs determined by traditional well-plate ELISA. Additionally, a visual detection limit of 100 PFU/mL was determined in contrived nasal swab samples by polling 20 untrained end-users. While the CaDI device was used for detecting clinically relevant levels of SARS-CoV-2 in this study, the CaDI device can be easily adapted to other immunoassay applications by changing the reagents and antibodies.

Keywords: COVID-19; Capillary-driven microfluidics; Diagnostic testing; Immunoassays; Point-of-care; SARS-CoV-2.

Figure 1.

The workflow for nasal swab samples is shown above. After a swab is collected, it is submerged in extraction buffer to lyse any virus present. The buffer/sample is then pushed through a syringe filter before adding to the device.

Figure 2.

(A) Molecular level representation of the immunoassay steps at the test line of the nitrocellulose membrane using an HRP-labeled antibody. The steps shown here occur at the test line on the nitrocellulose and correspond to the steps simulated with dye in B (B) Simulation of reagent addition and washing steps using yellow food dye to represent the HRP-antibody and blue food dye to represent the TMB substrate. (C) 3D rendering of the CaDI device channels that show how each plug of buffer in the channels is used in the assay. To see the assay and sequential delivery, refer to video S1 and video S2.

Figure 3.

(A) Buffer conditions and 2°Ab-HRP concentrations were held constant (20 μg/mL) while the volume of TMB dried on the conjugate release pad was varied. (B) Buffer conditions and TMB volume were held constant (22.5 μL) while the concentration of 2°Ab-HRP was varied. Each condition was run with 0 and 550 PFU/mL of inactivated virus to determine the condition that would give the greatest signal without increasing the signal of the blank.

Figure 4.

Inactivated virus diluted in extraction buffer comparing the CaDI devices (blue triangles and dashed trace) to a well-plate ELISA (red circles and solid trace). The limits of detection were 86 PFU/mL and 8 PFU/mL for the CaDI and ELISA respectively. Each point for the well-plate ELISA and CaDI devices were run as n=3 for standard deviation, except for the blank in the CaDI devices which was run as n=5.

Figure 5.

50 μL of varying virus concentrations was added to nasal swabs after collected (n=3 for each concentration). The swabs were submerged in extraction buffer, filtered, and added to the device. The LOD from this study was 222 PFU/mL.