Hybridization Chain Reaction Lateral Flow Assays for Amplified Instrument-Free At-Home SARS-CoV-2 Testing

Abstract

The lateral flow assay format enables rapid, instrumentfree, at-home testing for SARS-CoV-2. Due to the absence of signal amplification, this simplicity comes at a cost in sensitivity. Here, we enhance sensitivity by developing an amplified lateral flow assay that incorporates isothermal, enzyme-free signal amplification based on the mechanism of hybridization chain reaction (HCR). The simplicity of the user experience is maintained using a disposable 3-channel lateral flow device to automatically deliver reagents to the test region in three successive stages without user interaction. To perform a test, the user loads the sample, closes the device, and reads the result by eye after 60 min. Detecting gamma-irradiated SARS-CoV-2 virions in a mixture of saliva and extraction buffer, the current amplified HCR lateral flow assay achieves a limit of detection of 200 copies/μL using available antibodies to target the SARS-CoV-2 nucleocapsid protein. By comparison, five commercial unamplified lateral flow assays that use proprietary antibodies exhibit limits of detection of 500 copies/μL, 1000 copies/μL, 2000 copies/μL, 2000 copies/μL, and 20,000 copies/μL. By swapping out antibody probes to target different pathogens, amplified HCR lateral flow assays offer a platform for simple, rapid, and sensitive at-home testing for infectious diseases. As an alternative to viral protein detection, we further introduce an HCR lateral flow assay for viral RNA detection.

Bottom‐up DNA nanostructure‐based paper as point‐of‐care diagnostic: From method to device

“…Reproduced with permission. 65 Copyright 2023, American Chemical Society. (F) Detection of known mutations in SARS-CoV-2 variants using a specially designed TEprobe on a 3D origami device.…”

“…The antibody currently in use, which targets the nuclear capsid protein of SARS-CoV-2, achieved an LOD of 2 copies/μL (Figure 3E). 65 An enzymatic colorimetric assay called MARVE (for multiplexed, nucleic acid-amplification-free, singlenucleotide-resolved viral evolution) has been developed by Zhang et al based on a toehold exchange DNA probe (TEprobe). Its main advantage is its ability to detect SARS-CoV-2 while distinguishing specific mutations.…”

“…(E) HCR‐based 3D folding card device to detect SARS‐CoV‐2. Reproduced with permission 65 . Copyright 2023, American Chemical Society.…”

“…The antibody currently in use, which targets the nuclear capsid protein of SARS‐CoV‐2, achieved an LOD of 2 copies/μL (Figure 3E). 65 …”

Signal Amplification by Spatial Concentration for Immunoassay on Cellulose Media

“…[4] It can help to obtain more accurate diagnostic results by correcting false-negative results [5] and/or facilitate biological studies in research laboratories. [6] Various strategies to amplify signals from bioassays have been investigated, such as hybridization chain reaction for lateral flow DOI: 10.1002/smll.202307556 assay, [7] Tyramide-mediated [8] and CRISPR/Cas13a signal amplification for enzyme-linked immunosorbent assay (ELISA), [9] and rolling circle amplification (RCA) for immunosensors. [10] These strategies can be sorted into two, pre-assay and post-assay approach, to name.…”