In each row of Determine ?Figure66a, by adding the same amount of spike protein molecules, the 3rd harmonic signal drop decreases from I to IV, which agrees with the results shown in Physique ?Physique44eCi. binding says. By anchoring polyclonal antibodies (pAbs) onto MNP surfaces, these nanoparticles function as nanoprobes to specifically bind to target analytes Rotundine (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 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. 0.001; ** 0.01; * 0.05. For the scenario of coating four pAbs onto each MNP (Group I, MNP/pAb = 1:4, Physique ?Physique44a), adding 400 nM (equivalent to 32 pmole) of spike protein to vial #I-1 causes the 3rd harmonic to be significantly lower ( 0.001) than the 3rd harmonics from other vials in Group I. This weaker harmonic signal is due to the abundancy of target spike protein molecules from the sample that causes the nanoparticle clustering, as explained in Section 2.4 and Determine ?Figure22c,d. However, vials #I-2 to #I-8 show no significant differences. In Group II (MNP/pAb = 1:3, Physique ?Figure44b), vials #II-1 to #II-4 show comparable harmonic amplitudes and are significantly lower than other vials from the same group, which indicates that in this scenario (MNP/pAb = 1:3), adding 32 pmole (400 nM), 8 pmole (100 nM), 2 pmole (25 nM), and 1 pmole (12.5 nM) SARS-CoV-2 spike protein can completely block the Brownian relaxation of MNPs and cause the lowest achievable MPS spectra (or weakest dynamic magnetic responses). By reducing the number of spike protein in vials #II-5 to #II-8, the 3rd harmonic amplitudes gradually increase due to the less amount of the target spike protein molecule present in the sample that can cause the nanoparticle clustering. From Group II, a clear linear concentration-response curve is usually observed from vials #II-4 to #II-8. Indicating that this bioassay design could be used for quantitative assays of spike protein samples within a concentration range of 0C12.5 nM (corresponding to the spike protein amount of 0C1 pmole) and qualitative assays of spike protein samples with concentrations above 12.5 nM. On the other hand, by functionalizing two pAbs (Group III, MNP/pAb = 1:2, Physique ?Physique44c) and one pAb (Group IV, MNP/pAb = 1:1, Physique ?Determine44d) per MNP, no significant response curves are observed. There is a clear trend showing that this harmonic signal amplitude decreases, as we add a IL4 higher number of spike protein molecules. Figure ?Physique44e compares the 3rd harmonic amplitudes from vials #X-1 (X = Rotundine I, II, III, and IV), bare MNP (vial #9), and blank (no IPG30 MNP, i.e., noise floor). All the vials added with IPG30 MNPs show significantly higher harmonic signal intensities than the blank sample, of which the bare MNP (vial #9) sample shows the highest harmonic signal because no pAbs are functionalized and the Brownian relaxation of MNPs is not hindered even by adding 400 nM (32 pmole) spike protein to it, which confirms that MNPs without pAb functionalization will not form clusters in the presence of target analytes. In addition, the 3rd harmonics from vials #X-3, #X-5, #X-7, and #X-8 (X = I, II, III, and IV) are compared in Figure ?Physique44fCi, respectively. A clear Rotundine trend is observed that by adding the same amount of spike protein molecules, MNPs.