## 3 The Results and Discussion
### Synthesis and Characterization of AS-MNPs  
In our diagnostic assay, the immuno-capture of viral particles, during the enrichment step, was performed using superparamagnetic MNPs coated with rabbit polyclonal anti-spike antibody. Such superparamagnetic NPs respond rapidly to an applied magnetic field but exhibit negligible residual magnetism away from the magnetic field, making them particularly attractive for biodetection applications [32]. To assess their quality, AS-MNPs were characterized using SEM, TEM, and flow cytometry. SEM and TEM imaging clearly showed that the majority of AS-MNPs had a regular spherical shape with an average diameter of 500 nm (Figures 3A and S2). The efficiency of AS-MNPs in the immuno-capturing of the virus-mimicking particles is tightly dependent on both the quantity of antibodies on their surface and the quantity of spike proteins on the surface of VM-SPs. To ensure that they were efficiently coated, AS-MNPs were stained with an APC-labeled secondary anti-rabbit antibody and analyzed using flow cytometry. Non-stained MNPs were used as a control for gate setting (Figure 3B). Flow cytometry analysis showed that 100% of the stained AS-MNPs were coated with anti-spike antibodies (all events were APC-positive) (Figure 3C). Since different antibodies were used in MICaFVi (FITC-labeled monoclonal anti-spike antibody, APC-labeled secondary anti-rabbit antibody, and anti-spike rabbit polyclonal antibody) (Figure 1B), we performed different tests to ensure that no non-specific interactions existed between these elements, as such interactions would have increased false positive events during diagnosis and subsequently affected the assay specificity and accuracy. To exclude any possible non-specific interaction between AS-MNPs and the FITC-labeled monoclonal anti-spike antibody, used for the detection of immuno-captured viral particles, these two components were incubated together and AS-MNPs were analyzed using flow cytometry. The results showed that almost all AS-MNPs were FITC-negative, which excludes any non-specific interaction between these  
Figure 2: Characterization of virus-mimicking silica particles (VM-SPs). **(A)** SEM and TEM images show uniform average sizes of \(\sim\)0.5–1 μm. **(B)** EDX spectroscopy showing the chemical composition of naked and spike-protein-coated SPs. SPs showed an extra peak corresponding to the nitrogen element which indicates the successful attachment of the spike protein on their surface. **(C)** Western blotting analysis was used to quantify the spike protein in the reaction supernatant before and after conjugation. After conjugation, the spike protein was not detected in the supernatant, indicating complete protein conjugation. **(D)** Flow cytometry analysis of VM-SPs using an FITC-labeled anti-spike protein clearly showed that almost all SPs were FITC-positive (coated with spike protein). A non-specific APC-labeled antibody was also used as a negative control. **(E)** To eliminate any possible non-specific binding of the FITC-anti-spike antibody to SPs directly, we used this antibody to stain the naked SPs. The results showed that all events were negative.  
two components (Figure 3D). To exclude the hypothesis that FITC-labeled monoclonal anti-spike antibody could bind to AS-MNPs through the APC-labeled secondary anti-rabbit antibody, these three elements were incubated together and then AS-MNPs were analyzed using flow cytometry. The results showed again the absence of FITC-positive events, which indicates that the FITC-labeled monoclonal anti-spike antibody interacted neither with AS-MNPs nor with the APC-labeled secondary anti-rabbit antibody (Figure 3E).