Keywords: Lateral flow assay, Hybridization Chain Reaction (HCR), SARS-CoV-2

Background: Currently available lateral flow assays enable rapid, instrument-free, at-home testing for SARS-CoV-2. The simplicity of this test format comes at a cost in sensitivity, generating only one signal for each detected protein.

Objective: We hypothesized that signal amplification with hybridization chain reaction (HCR) would be well-suited for the lateral flow assay format, enabling improved sensitivity while maintaining simplicity. We set firm design criteria for an amplified HCR lateral flow assay: the test must be simple (remaining as easy to use as an unamplified lateral flow test), inexpensive (not requiring at-home instrumentation), robust (avoiding reagents that require cold storage, such as enzymes), rapid (returning results within 60 minutes), and sensitive (exhibiting a limit of detection of less than 1000 virions/μL).

Methods: A disposable folding card device was created with a 3D printer. The device was functionalized with a sample pad; three conjugate pads loaded with anti-nucleocapsid initiator-labeled signal and biotinylated capture antibodies (Channel 1), digoxin-labeled HCR hairpins (Channel 2), or a carbon black-labeled anti-digoxin reporter antibody (Channel 3); a three-channel nitrocellulose membrane; and a wicking pad. The nitrocellulose membrane test line was spotted with polystreptavidin R. To validate the assay, irradiated SARS-CoV-2 was added to saliva and extraction buffer to create a 0.3 mL sample, and the sample was deposited on the sample pad before closing the folding card device. After 60 minutes, the test region was photographed.

Results: The amplified HCR lateral flow assay for SARS-CoV-2 nucleocapsid protein demonstrates a limit of detection of 200 copies/μL. By comparison, five unamplified commercial lateral flow assays exhibit limits of detection that are 2.5, 5, 10, 10, and 100 times higher.

Conclusions: Amplified HCR lateral flow assays for viral protein detection are simple to use (requiring only one step), inexpensive (disposable and not requiring any instrumentation), robust (enzyme-free and using reagents that may be stored at room temperature), rapid (delivering a result in 60 minutes), and sensitive (with a limit of detection between 2.5 and 100 times lower than that of currently available lateral flow assays).