Clustering based magnetic assays for SARS-CoV-2 detection with scFv-functionalized magnetic nanoparticles

Abstract

Magnetic nanoparticles (MNP) can be used in magnetic immunoassays (MIA) by functionalizing them with antibodies. In homogeneous assays that follow a "mix-and-measure" approach, it is then sufficient to add the sample in question and evaluate the response of the MNPs using a magnetic measurement method. Magnetic Particle Spectroscopy (MPS) is a fast and sensitive method for this purpose, capable of determining the binding state by measuring changes in Brownian relaxation. For further analysis, alternating current susceptometry (ACS) is considered, although it is significantly more time-consuming and therefore no option for applications interested in point of care detection. In this study, we aim to further improve the approach of MIA evaluated by MPS. To this end, we use self-synthesized scFv fragments instead of whole IgG antibodies during functionalization in order to keep the size of the BNF-Dextran MNP with 80 nm nominal diameter as small as possible, which allows for greater relative size changes upon binding to an analyte. Virus-like particles (VLP) and the N protein of SARS-CoV-2, which were also produced in-house, are used as analytes. Due to multiple binding sites, these form cluster structures, which, in the case of VLP, were investigated in greater depth ACS to assess their field dependence. In addition, the sensitivity of the assays was analyzed as a function of MNP concentration, and the detection limit for both analytes was estimated. We found that, using scFv-functionalized MNPs, we were able to detect low concentrations of just 490 fM of SARS-CoV-2 VLP and 1.7 nM of the N protein. However, the slightly above-proportional increase in sensitivity as the MNP concentration decreases does not automatically imply a better detection limit.