## 3 The Results and Discussion
### Synthesis and Characterization of VM-SPs  
MERS-CoV and SARS-CoV-2 are highly pathogenic viruses that require specific and strict handling precautions (CDC guidelines for MERS-CoV handling). Using these viruses for the design and optimization of a diagnostic assay will be very challenging in terms of safety. Since our detection assay only targets the surface glycoprotein \"spike\", and as proof-of-principle, we prepared silica particles coated with this protein (denoted as virus-mimicking silica particles (VM-SPs)) to be used for the optimization of the different steps of our diagnostic technology.  
To synthesize VM-SPs, acid-functionalized mesoporous silica particles (APMS-COOH) were first prepared followed by amide coupling with the free amine groups of the MERS-CoV spike protein, leading to spike-protein-conjugated SPs. The as-synthesized viral particles were characterized using electron microscopy (SEM and TEM) to examine their morphology and size. As expected, VM-SPs showed \(\sim\)0.5-1 \(\upmu\)m uniform sizes (Figures 2A and S2). We confirmed the conjugation of the spike protein to the silica particles via several methods. The results of the comparative analysis of VM-SPs before and after coating via energy-dispersive X-ray (EDX) spectroscopy showed the presence of an extra element in the composition of the coated particles corresponding to nitrogen. The presence of this element is indirect proof of the presence of the spike protein on SPs (Figure 2B). Moreover, the detection of the spike protein via Western blotting analysis showed that the spike disappeared completely from the supernatant after the coating reaction, indicating quantitative protein conjugation to acid-functionalized silica microparticles (Figure 2C). To provide direct evidence of spike protein attachment, SPs were stained with an FITC-labeled anti-spike monoclonal antibody and then analyzed using flow cytometry. The results showed that all of the SPs were coated with the spike protein (FITC-positive) (Figure 2D). To exclude the hypothesis that the FITC-antibody stains the core of SPs via non-specific binding, we used naked non-functionalized SPs that were stained similarly, and no FITC-positive events were detected. This confirms that the FITC-labeled anti-spike antibody was attaching to SPs via the spike protein (Figure 2E). It is noteworthy that the anti-rabbit APC-labeled secondary antibody that was used in the subsequent steps of MICaFVi was added during these tests to exclude any non-specific interaction between either the FITC-labeled antibody or the silica particles (Figure 2D,E).  
Figure 1: Schematic representation of MICaFVi method. (**A**) In the first step, magnetic-capturing antibody-conjugated MNPs (AS-MNPs) are added to virus-mimicking silica particles (VM-SPs) or spike-protein-expressing pseudoviral particles, incubated, and magnetically captured (enrichment). In the second step, the complex AS-MNP/viral particles are stabilized and neutralized using PFA (neutralization). In the last step, after staining with highly specific fluorescent antibodies, the enumeration of positive AS-MNPs (NPs harboring captured viral particles) is performed via flow cytometry (detection). (**B**) Schematic representation of a positive AS-MNP complexed with a captured virus and stained with two fluorescent antibodies: an APC-labeled antibody used for the staining of AS-MNP and an FITC-labeled anti-spike antibody specific to the staining of the captured virus.