Multiplex quantitative detection of SARS-CoV-2 specific IgG and IgM antibodies based on DNA-assisted nanopore sensing

Abstract

The coronavirus disease (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread into a global pandemic. Early and accurate diagnosis and quarantine remain the most effective mitigation strategy. Although reverse transcriptase polymerase chain reaction (RT-qPCR) is the gold standard for COVID-19 diagnosis, recent studies suggest that nucleic acids were undetectable in a significant number of cases with clinical features of COVID-19. Serologic assays that detect human antibodies to SARS-CoV-2 serve as a complementary method to diagnose these cases, as well as to identify asymptomatic cases and qualified convalescent serum donors. However, commercially available enzyme-linked immunosorbent assays (ELISA) are laborious and non-quantitative, while point-of-care assays suffer from low detection accuracy. To provide a serologic assay with high performance and portability for potential point-of-care applications, we developed DNA-assisted nanopore sensing for quantification of SARS-CoV-2 related antibodies in human serum. Different DNA structures were used as detection reporters for multiplex quantification of immunoglobulin M (IgM) and immunoglobulin G (IgG) antibodies against the nucleocapsid protein of SARS-CoV-2 in serum specimens from patients with conformed or suspected infection. Comparing to a clinically used point-of-care assay and an ELISA assay, our technology can reliably quantify SARS-CoV-2 antibodies with higher accuracy, large dynamic range, and potential for assay automation.

Keywords: Antibody; COVID-19; In vitro diagnostics; Nanopore; SARS-CoV-2.

Fig. 1

Schematic representation of the DNA-assisted Nanopore Assay for multiplex quantification of SARS-CoV-2 antibodies. Step 1: IgG and IgM captured by the N-protein modified MBs. Step 2: Formation of the sandwich structure between MBs, IgG or IgM antibody, and probe DNA modified AuNPs. Step 3: Dehybridization of the probe DNAs from the AuNPs. Step 4: Magnetic separation of probe DNAs from the remaining sandwich complex. Step 5: Quantification of probe DNAs to derive the concentration of IgG and IgM, respectively.

Fig. 2

Statistical characterization of signals by two different probe DNAs. a: Signal A is generated by DNA-A. b: Histograms of current blockades of level 1 and level 2 in Signal A. The solid lines are Gaussian fit to the histograms. c: Histograms of dwell times of level 1 and level 2 in Signal A. The solid lines are single exponential fit to the histograms. d: Signal B is generated by DNA-B. e: Histograms of current blockades of level 1 and level 2 in Signal B. The solid lines are Gaussian fit to the histograms. f: Histograms of dwell times of level 1 and level 2 in Signal B. The solid lines are single exponential fit to the histograms. All electrical resistive pulse nanopore sensing data was acquired in 3 M KCl, 10 mM tris buffer, pH 8.0, n = 3.

Fig. 3

Quantification of SARS-CoV-2 IgG and IgM in human serum samples. a: Standard curve of correlation between signal frequencies and concentrations of humanized IgG spiked in blank human serum (0.01–100 μg/mL). Inset shows the curve at 0–1000 μg/mL. b: Standard curve of correlation between signal frequencies and concentrations of humanized IgM spiked in blank human serum (0.05–10 μg/mL). Inset shows the curve at lower concentration ranges. c: IgG and IgM concentrations measured by the DNA-assisted Nanopore Assay for 1 negative sample, 3 positive samples, and 18 possible samples. If the calculated concentration was below the LOD, the concentration is marked as 0 μg/mL. Data recording conditions are the same as used in Fig. 2d: Qualitative IgG and IgM results for matching samples measured by the ELISA kit and the LFA kit.