## 3 The Results and Discussion
### MICaFVi Detection of MERS-CoV Pseudoviral Particles (MERSpp)  
We showed that MICaFVi detects coronavirus spike proteins on the surface of virus-mimicking particles efficiently and specifically. To demonstrate that our assay is also applicable to the detection of viral particles, we tested its ability in detecting a pseudovirus expressing the MERS-CoV spike protein on its surface (i.e., MERSpp) (Figure 6). The results of the flow cytometry analysis of AS-MNPs incubated with a solution of MERSpp showed that 19% of events were APC/FITC double-positive, confirming that our method successfully detected MERSpp present in the used sample. Virus-free cell culture medium was used as a negative control (Figure 6A). To further confirm the immuno-capture of the pseudoviral particles, we performed a luciferase assay using an MERSpp-carrying luciferase reporter gene. Huh7 cells were infected with pseudovirus solution before or after depletion with AS-MNPs. The results of luminescence quantification, after viral infection,  
Figure 5: MICaFVi specificity. (**A**) Legend. (**B**) MERS-CoV AS-MNPs failed in capturing VSV-Gpp. (**C**) VSV-Gpp viral titer (fluorescence unit) remained unchanged before and after incubation with MERS-CoV AS-MNPs. showed that incubation with AS-MNPs reduced the luminescence to 11.5% compared to the control (Figure 6B). This indicates that the majority (88.5%) of MERSpp in the depleted solution was immuno-captured. The turnaround time of diagnostic tests is extremely important for patient management, particularly during outbreaks and pandemics. In such a context, high-throughput systems along with rapid diagnostic testing are essential. Thus, to define the minimum incubation time needed for optimal sensitivity, we incubated AS-MNPs with a solution of MERSpp, and MICaFVi was then performed at different time points of incubation. The results showed that MICaFVi was able to detect viral particles (positive signal) with an incubation period as short as half an hour (% double-positive events ~14%). The highest double-positive events (50% MICaFVi) were reached after 6-8 h of incubation (Figure 6C). However, even with a reduced incubation time, MICaFVi can still detect viral particles, albeit with lower sensitivity. This means that for samples with a high viral titer, half an hour will be sufficient to detect the virus in the sample; however, samples with a lower viral titer will need an extended incubation time to reach the optimal sensitivity. The sensitivity of MICaFVi was experimentally determined using the free recombinant spike protein. MICaFVi was carried out to capture the free spike protein from different known dilutions. The results showed that the minimum concentration detected was 3.9 \(\mathrm{\SIUnitSymbolMicro g}\)/mL of spike protein (MW ~200 kDa), resulting in a limit of detection (LOD) equal to 20 pmol/mL (Figure 6D). It has to be noted that several factors can contribute to the specificity and sensitivity of MICaFVi detection. These factors encompass the quality and specificity of the capture antibodies utilized, the quality and stability of the sample or pseudoviral particles being analyzed, the efficiency of the immuno-capture process, and, most importantly, the physiochemical features of the functional MNPs that capture the virus (i.e., size, stability, appropriate functionality, and magnetic responsiveness). The optimization of  
Figure 6: MICaFVi detection of pseudovirus. (**A**) Detection of MERS-CoV pseudoviral particles using MICaFVi. Complete cell culture medium was used as a negative control. (**B**) Evaluation of AS-MNPs’ immuno-capture potential via their viral depleting effect. (**C**) Time-dependent sensitivity of MICaFVi. (**D**) MICaFVi limit of detection (LoD) was determined using different concentrations of MERS-CoV-free spike protein. (**E**) Legend. all of these factors is necessary to attain the precise, accurate, and dependable detection of the target virus.