A semi-quantitative upconversion nanoparticle-based immunochromatographic assay for SARS-CoV-2 antigen detection

Abstract

The unprecedented public health and economic impact of the coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has been met with an equally unprecedented scientific response. Sensitive point-of-care methods to detect SARS-CoV-2 antigens in clinical specimens are urgently required for the rapid screening of individuals with viral infection. Here, we developed an upconversion nanoparticle-based lateral flow immunochromatographic assay (UCNP-LFIA) for the high-sensitivity detection of SARS-CoV-2 nucleocapsid (N) protein. A pair of rabbit SARS-CoV-2 N-specific monoclonal antibodies was conjugated to UCNPs, and the prepared UCNPs were then deposited into the LFIA test strips for detecting and capturing the N protein. Under the test conditions, the limit of detection (LOD) of UCNP-LFIA for the N protein was 3.59 pg/mL, with a linear range of 0.01-100 ng/mL. Compared with that of the current colloidal gold-based LFIA strips, the LOD of the UCNP-LFIA-based method was increased by 100-fold. The antigen recovery rate of the developed method in the simulated pharyngeal swab samples ranged from 91.1 to 117.3%. Furthermore, compared with the reverse transcription-polymerase chain reaction, the developed UCNP-LFIA method showed a sensitivity of 94.73% for 19 patients with COVID-19. Thus, the newly established platform could serve as a promising and convenient fluorescent immunological sensing approach for the efficient screening and diagnosis of COVID-19.

Keywords: SARS-CoV-2; antigen detection; fluorescence immunochromatography assay; semi-quantitative; upconversion nanoparticles.

Figure 1

Schematic illustration of SARS-CoV-2 detection using UCNP-LFIA. (A) Preparation process for fabricating the mAb-conjugated UCNP. (B) Procedure for the detection of SARS-CoV-2 using UCNP-LFIA.

Figure 2

(A) TEM images of unconjugated UCNPs. (B) The fluorescence spectra of UCNPs. (C) DLS distributions of unconjugated UCNPs. (D) Optimization of the concentration of mAb-conjugated UCNPs. (E) Optimization of the drying time for prepared UCNP-LFIA.

Figure 3

Binding kinetics of capture mAb (RM3166) (A) and detection mAb (RM3165) (B) with SARS-CoV-2 N protein as measured using Biacore 3,000. Colored lines were original curves, while the black lines were the fitted curves. K_D apparent values are shown for the capture or detection of monoclonal mAb binding to the N protein using a 1:1 global fit model.

Figure 4

The fluorescence signal (A) and corresponding test line intensities (B) of UCNP-LFIA between the T/C values and the SARS-CoV-2 N protein antigen detection. (C) Corresponding calibration curve for detecting SARS-CoV-2 N protein antigen with the UCNP-LFIA. (D) The representative results of colloidal gold-based LFIA detection of SARS-CoV-2 N protein at different concentrations ranging from 0 to 100 ng/mL with 10-fold serial dilution.

Figure 5

(A) Specificity of UCNP-LFIA. Reproducibility of UCNP-LFIA at 500 pg/mL (B) and 100 ng/mL (C) for 15 random assays.

Figure 6

(A) Application of UCNP-LFIA to detect the T/C value of SARS-CoV-2 N protein in simulated pharyngeal swab samples. (B) Comparison of the T/C values measured by UCNP-LFIA and Ct values measured by RT-PCR in patients with different times of infection.