Magnetofluidic immuno-PCR for point-of-care COVID-19 serological testing

Abstract

Serological tests play an important role in the fight against Coronavirus Disease 2019 (COVID-19), including monitoring the dynamic immune response after vaccination, identifying past infection and determining community infection rate. Conventional methods for serological testing, such as enzyme-linked immunosorbent assays and chemiluminescence immunoassays, provide reliable and sensitive antibody detection but require sophisticated laboratory infrastructure and/or lengthy assay time. Conversely, lateral flow immunoassays are suitable for rapid point-of-care tests but have limited sensitivity. Here, we describe the development of a rapid and sensitive magnetofluidic immuno-PCR platform that can address the current gap in point-of-care serological testing for COVID-19. Our magnetofluidic immuno-PCR platform automates a magnetic bead-based, single-binding, and one-wash immuno-PCR assay in a palm-sized magnetofluidic device and delivers results in ∼30 min. In the device, a programmable magnetic arm attracts and transports magnetically-captured antibodies through assay reagents pre-loaded in a companion plastic cartridge, and a miniaturized thermocycler and a fluorescence detector perform immuno-PCR to detect the antibodies. We evaluated our magnetofluidic immuno-PCR with 108 clinical serum/plasma samples and achieved 93.8% (45/48) sensitivity and 98.3% (59/60) specificity, demonstrating its potential as a rapid and sensitive point-of-care serological test for COVID-19.

Keywords: COVID-19; Immuno-PCR; Magnetofluidic device; Point-of-care; Serological testing.

Fig. 1

Overview of the magnetofluidic immuno-PCR platform. (A) The magnetofluidic immuno-PCR features a streamlined immuno-PCR assay performed in a disposable 3-well cartridge using a portable magnetofluidic device, which automates magnetic bead transfer and PCR thermal cycling and communicates results via Bluetooth to a smartphone app. (B) Receptor-binding domain (RBD)-coated magnetic beads capture anti-SARS-CoV-2 IgG together with oligo-goat anti-human IgG antibody (oligo-anti-IgG) conjugates before being loaded into the sample well of the cartridge. The IgG-captured magnetic beads are then transferred to the washing well to remove nonspecifically-adsorbed oligo-anti-IgG conjugates followed by another bead transfer to the PCR well to initiate PCR. The entire process is completed in ∼0.5 h, including 15 min immunobinding, 2 min washing, and 15 min for rapid PCR.

Fig. 2

Development of a streamlined immuno-PCR assay. (A) Five different recombinant SARS-CoV-2 antigens were tested using ELISA for assessing anti-SARS-CoV-2 IgG capture efficiency. (B) A dilution series of anti-SARS-CoV-2 IgG spike-in in diluted human serum were tested using benchtop immuno-PCR to evaluate the assay sensitivity. (C) The 1-step, one-pot immunobinding showed differentiable C_t values between background and 10 ng/mL spike-in sample even though the ΔC_t decreased compared with the 2-step workflow. (D) The immunobinding time was reduced from 60 min to 15 min to shorten the assay time. (E) The immuno-PCR was further simplified by reducing the number of washing steps from 5 times to 1 time. The error bars are standard deviations from duplicated experiments.

Fig. 3

Implementation of SARS-CoV-2 immuno-PCR assay in the magnetofluidic device. (A) The real-time amplification curves of the magnetofluidic immuno-PCR tests showed earlier amplification with higher spike-in anti-S1 IgG concentrations. (B)As a result, the ΔC_t values of IgG spike-in series in diluted human serum increased linearly with the IgG concentrations across three orders of magnitude with an estimated LOD of 2 ng/mL. (C) Our magnetofluidic immuno-PCR was also compatible with whole blood sample input though the LOD (∼22 ng/mL) was inferior to that of serum.

Fig. 4

Clinical evaluation of the magnetofluidic immuno-PCR using convalescent serum/plasma samples. We tested two independent cohorts, 48 in total, of convalescent serum/plasma samples from SARS-CoV-2 patients and 60 pre-pandemic plasma/serum samples to characterize the sensitivity and specificity of our magnetofluidic immuno-PCR. (A) ΔC_t values from magnetofluidic immuno-PCR analysis of clinical specimens. Negative specimens (black) are from archived samples collected prior to the pandemic. Red and blue dots indicate test-positive samples from ECLIA and custom Luminex analyses, respectively. (B) Receiver operating curve (ROC) analysis was performed on the ΔC_t values of 60 pre-pandemic samples and 48 convalescent samples, resulting in an area under the curve (AUC) of 0.968 (95% CI: [0.933, 1.000]). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)