Aptamer Sandwich Lateral Flow Assay (AptaFlow) for Antibody-Free SARS-CoV-2 Detection

Abstract

The COVID-19 pandemic is among the greatest health and socioeconomic crises in recent history. Although COVID-19 vaccines are being distributed, there remains a need for rapid testing to limit viral spread from infected individuals. We previously identified the SARS-CoV-2 spike protein N-terminal domain (NTD) binding DNA aptamer 1 (SNAP1) for detection of SARS-CoV-2 virus by aptamer-antibody sandwich enzyme-linked immunoassay (ELISA) and lateral flow assay (LFA). In this work, we identify a new aptamer that also binds at the NTD, named SARS-CoV-2 spike protein NTD-binding DNA aptamer 4 (SNAP4). SNAP4 binds with high affinity (<30 nM) for the SARS-CoV-2 spike protein, a 2-fold improvement over SNAP1. Furthermore, we utilized both SNAP1 and SNAP4 in an aptamer sandwich LFA (AptaFlow), which detected SARS-CoV-2 UV-inactivated virus at concentrations as low as 10⁶ copies/mL. AptaFlow costs <$1 per test to produce, provides results in <1 h, and detects SARS-CoV-2 at concentrations that indicate higher viral loads and a high probability of contagious transmission. AptaFlow is a potential approach for a low-cost, convenient antigen test to aid the control of the COVID-19 pandemic.

Figure 1.

A naïve aptamer library is enriched for S-2P binders by cell SELEX then protein SELEX. In cell SELEX, HEK293_S-2P cells are used for positive selection and HEK293 cells are used for negative selection. In protein SELEX, S-2P recombinant protein is used for positive selection and bovine serum albumin (BSA) is used for negative selection. Aptamer pools are evaluated for binding to targets and sequenced.

Figure 2.

A) Cy5-labeled SNAP4 at various concentrations was incubated with HEK293_S-2P cells to generate a binding curve. The graph shows the total (blue) and non-specific (gray) binding of aptamer based on a one-site binding model (dashed lines). K_D was determined by non-linear regression (1 biological replicate.) B-C) Biotinylated aptamer was loaded on SA biosensors and associated with protein. The gray lines indicate the switch from analyte association to dissociation. K_on values (mean±s.d., n= 4–6) were determined from a global fit (dark gray line) of the kinetic data at various concentrations of proteins for the indicated binding model. B) SNAP4 aptamer binding to S-2P protein fitted to a 1:1 model for association only. C) SNAP4 aptamer (left) or SNAP4.74 aptamer (right) binding to S1 protein with 1:2 model fit (top) or NTD protein with 1:1 model fit (bottom).

Figure 3.

A) Step-by-step process for AptaFlow. (i) incubating reagents with SARS-CoV-2 sample. (ii) dipping of the test strip into the sample. (iii) washing to reduce non-specific signals. (iv) enhancing signal to increase sensitivity. B) Limit of detection of LFA system with UV-inactivated virus (UV) at various concentrations (copies/mL) and heat-inactivated (HI) virus (negative control). Bars indicate mean. Brackets indicate 95% CI. Statistical significance was determined by one-way ANOVA with Dunnett correction. * indicates p < 0.05 and **** indicates p < 0.0001 when compared to HI 1 × 10⁷ copies/mL; n = 3–4 per experimental group. A representative image is shown.