One-Step, Wash-free, Nanoparticle Clustering-Based Magnetic Particle Spectroscopy Bioassay Method for Detection of SARS-CoV-2 Spike and Nucleocapsid Proteins in the Liquid Phase

Abstract

With the ongoing global pandemic of coronavirus disease 2019 (COVID-19), there is an increasing quest for more accessible, easy-to-use, rapid, inexpensive, and high-accuracy diagnostic tools. Traditional disease diagnostic methods such as qRT-PCR (quantitative reverse transcription-PCR) and ELISA (enzyme-linked immunosorbent assay) require multiple steps, trained technicians, and long turnaround time that may worsen the disease surveillance and pandemic control. In sight of this situation, a rapid, one-step, easy-to-use, and high-accuracy diagnostic platform will be valuable for future epidemic control, especially for regions with scarce medical resources. Herein, we report a magnetic particle spectroscopy (MPS) platform for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) biomarkers: spike and nucleocapsid proteins. This technique monitors the dynamic magnetic responses of magnetic nanoparticles (MNPs) and uses their higher harmonics as a measure of the nanoparticles’ binding states. By anchoring polyclonal antibodies (pAbs) onto MNP surfaces, these nanoparticles function as nanoprobes to specifically bind to target analytes (SARS-CoV-2 spike and nucleocapsid proteins in this work) and form nanoparticle clusters. This binding event causes detectable changes in higher harmonics and allows for quantitative and qualitative detection of target analytes in the liquid phase. We have achieved detection limits of 1.56 nM (equivalent to 125 fmole) and 12.5 nM (equivalent to 1 pmole) for detecting SARS-CoV-2 spike and nucleocapsid proteins, respectively. This MPS platform combined with the one-step, wash-free, nanoparticle clustering-based assay method is intrinsically versatile and allows for the detection of a variety of other disease biomarkers by simply changing the surface functional groups on MNPs.

A comparison of conventional and advanced electroanalytical methods to detect SARS-CoV-2 virus: A concise review

“…This strategy could have a big impact in regions where early identification is needed to stop SARS-CoV-2 outbreaks from spreading. Recently, “Dual-mode lateral flow immunoassay” (LFIA) biosensor based on magnetic quantum dots ( Wang et al, 2021a ), and “magnetic particle spectroscopy” (MPS) bioassay method ( Wu et al, 2021a , Wu et al, 2021b ), for spike and nucleocapsid detections were reported.…”

“…A reaction mixture incorporating a cresol red (pH-dependent indicator) dye was tested for rapid RT-LAMP based real time COVID-19 detection ( Lu et al, 2020a , 2020b ). The cresol red changes color from red to orange-yellow as an outcome of a +ve reaction ( Tanner et al, 2015 ; Wu et al, 2021a ). The pH of the LAMP reaction mix generally decreases in positive samples due to increased DNA polymerase activity ( Augustine et al, 2020 ).…”

Highly Stable Buffer-Based Zinc Oxide/Reduced Graphene Oxide Nanosurface Chemistry for Rapid Immunosensing of SARS-CoV-2 Antigens

“…With the outbreak of the COVID-19 pandemic globally, the urgent need for rapid and accurate diagnostic tests has been underlined to prevent further disease spread. − While detecting viral RNAs through real-time reverse-transcriptase-polymerase chain reaction (rRT-PCR) is the gold standard diagnostic technique, , growing demands for rapid diagnosis, through the integration of detection techniques and fluid handing chips, are highlighted for controlling the spread, which can be enabled through the advancement of immunoassays − and nanodiagnostics . In this regard, methods working based on the distinct antibody (Ab)/antigen viral biomarkers have become prevalently adopted, especially when rapid and early detection is of interest. ,, Taking advantage of the structural proteins of the SARS-CoV-2 virus, highly specific monoclonal antibodies , are becoming commonly employed for the development of immunoassays. , Among different structural proteins, Nucleocapsid proteins (N-protein) exist in the blood, saliva, serum, and nasopharyngeal (NP) swab samples of infected patients and proved to be the most predictive with less susceptibility to mutation for COVID-19 detection in the early phase of infection . It has been used as a reliable disease indicator when used for Rapid Antigen Testing along with the incorporation of Artificial Intelligence and the Internet of Things can provide big data and predictive models for pandemic management. − …”

“…12 In this regard, methods working based on the distinct antibody (Ab)/antigen viral biomarkers have become prevalently adopted, especially when rapid and early detection is of interest. 5,13,14 Taking advantage of the structural proteins of the SARS-CoV-2 virus, highly specific monoclonal antibodies 15,16 are becoming commonly employed for the development of immunoassays. 17,18 Among different structural proteins, Nucleocapsid proteins (Nprotein) exist in the blood, 19 saliva, 20 serum, 21 and nasopharyngeal (NP) swab samples of infected patients 22 and proved to be the most predictive with less susceptibility to mutation for COVID-19 detection in the early phase of infection.…”

Recent Advances in CRISPR/Cas-Based Biosensors for Protein Detection

“…In particular, the immunoassays based on enzyme-linked immunosorbent assay (ELISA) [ 2 , 7 ] and chemiluminescence immunoassay (CLIA) [ 8 ] are currently the most commonly used protein assays, in which ELISA serves as the gold standard for protein detection in the fields of clinical diagnosis and biosafety [ 9 ]. However, the levels of protein biomarkers in clinical samples are generally very low, while a large amount of matrix interference exists in samples [ 10 ], so ELISA assays in clinical use are generally limited by their sensitivity, reliability, and specificity [ 11 ]. Therefore, it is urgent to develop new methods for the rapid, sensitive, portable, and highly specific detection of protein biomarkers.…”