Nanozyme chemiluminescence paper test for rapid and sensitive detection of SARS-CoV-2 antigen

Abstract

COVID-19 has evolved into a global pandemic. Early and rapid detection is crucial to control of the SARS-CoV-2 transmission. While representing the gold standard for early diagnosis, nucleic acid tests for SARS-CoV-2 are often complicated and time-consuming. Serological rapid antibody tests are characterized by high rates of false-negative diagnoses, especially during early infection. Here, we developed a novel nanozyme-based chemiluminescence paper assay for rapid and sensitive detection of SARS-CoV-2 spike antigen, which integrates nanozyme and enzymatic chemiluminescence immunoassay with the lateral flow strip. The core of our paper test is a robust Co-Fe@hemin-peroxidase nanozyme that catalyzes chemiluminescence comparable with natural peroxidase HRP and thus amplifies immune reaction signal. The detection limit for recombinant spike antigen of SARS-CoV-2 was 0.1 ng/mL, with a linear range of 0.2-100 ng/mL. Moreover, the sensitivity of test for pseudovirus could reach 360 TCID₅₀/mL, which was comparable with ELISA method. The strip recognized SARS-CoV-2 antigen specifically, and there was no cross reaction with other coronaviruses or influenza A subtypes. This testing can be completed within 16 min, much shorter compared to the usual 1-2 h required for currently used nucleic acid tests. Furthermore, signal detection is feasible using the camera of a standard smartphone. Ingredients for nanozyme synthesis are simple and readily available, considerably lowering the overall cost. In conclusion, our paper test provides a high-sensitive point-of-care testing (POCT) approach for SARS-CoV-2 antigen detection, which should greatly facilitate early screening of SARS-CoV-2 infections, and considerably lower the financial burden on national healthcare resources.

Keywords: Antigen detection; Chemiluminescence; Nanozyme; Paper test; SARS-CoV-2.

Fig. 1

Characterization of Co–Fe@hemin-peroxidase nanozyme. (A) TEM image of Co–Fe@hemin. (B) SEM image of Co–Fe@hemin. (C) Dynamic light scattering (DLS) analysis. (D) The UV–Vis absorbance spectrum of Co–Fe@hemin, Co–Fe NPs and hemin.

Fig. 2

Catalytic activity of Co–Fe@hemin-peroxidase nanozyme. (A) Chemiluminescence curve for luminol-H₂O₂ system. (B) Effects of pH on chemiluminescence catalytic activity of Co–Fe@hemin and HRP. (C) Effects of temperature on chemiluminescence catalytic activity of Co–Fe@hemin and HRP. (D) Effects of H₂O₂ concentration on Co–Fe@hemin-luminol system. Data were collected in triplicates.

Fig. 3

Design of the nanozyme chemiluminescent paper test. (A) Schematic illustration of the nanozyme chemiluminescence paper test for SARS-CoV-2 S-RBD antigen. Recognition, separation and catalytic amplification by nanozyme probes. (B) ELISA analysis of antibodies binding activity for S-RBD protein. (C) Screening of paired antibodies using nanozyme colorimetric strip. Positive signal was evaluated by 100 ng/mL of S-RBD protein. Sample dilution buffer was used as the negative control. Data were collected in triplicates.

Fig. 4

Rapid testing of recombinant SARS-CoV-2 spike antigen. (A) Gradient paper testing of SARS-CoV-2 S-RBD protein, before and after adding luminol substrate. A total of 100 μl sample solution was loaded for each test. (B) The calibration curve of SARS-CoV-2 S-RBD detection by nanozyme chemiluminescence test strip. The Y value was defined as the chemiluminescent intensity ratio of T-line to C-line. Concentrations of S-RBD protein ranged from 0~6400 ng/mL. Samples were measured in triplicates. (C) Calibration curve of ELISA detection for S-RBD protein. Samples were measured in triplicates. (D) Nanozyme chemiluminescence test paper specifically recognized the spike antigen of SARS-CoV-2, remaining negative for other human coronaviruses (SARS, MERS, HCoV-HKU1 and HCoV-OC43).

Fig. 5

Rapid testing of pseudo-SARS-CoV-2. (A) Calibration curve of pseudovirus test. (B) Gradient detection of pseudo-SARS-CoV-2 samples using nanozyme chemiluminescence test paper. (C) ELISA detection of the pseudo-SARS-CoV-2 samples. Samples were measured in triplicates. (D) Nanozyme chemiluminescence strip specifically recognized the pseudo-SARS-CoV-2. The inactivated samples of H1N1, H3N2, H5N1 and H7N9 with the same titer were tested positively using the commercial Influenza A antigen testing kits.