## 4 Results and Discussion
### Detection of SARS-CoV-2 in Clinical Samples  
The clinical sensitivity of the assay for detecting SARS-CoV-2 in nasal samples was evaluated. The assay was able to detect SARS-CoV-2-positive samples with cycle thresholds (Ct) of up to 14.3 with statistical significance (\(p\)-value \(<\) 0.0001 vs. negative sample; one-way ANOVA) (Figure 5). However, the signals for Ct = 23.4 and Ct = 28.5 were not statistically significant (\(p\)-values = 0.9980 and 0.9997, respectively, vs. negative sample; one-way ANOVA). We hypothesized that by utilizing a larger-volume aliquot of the nasal sample, we could utilize the MB-based preconcentration to increase the clinical sensitivity of our assay. Hence, we repeated the assay on a 250 \(\mu\)L aliquot (out of the 1 mL nasal samples) and performed EC (DPV) detection. One of the key advantages of immunomagnetic separation over the BinaxNow and similar lateral flow detection mechanisms is the volumetric range that the assay can intake. The BinaxNow detection kit is limited to a sample volume equal to that captured by the nasal swab, which is approximately 50 \(\mu\)L. Using immunomagnetic separation, our assay can reasonably intake up to 500 \(\mu\)L of the sample input. Increased sample intake increases the total amount of analyte and consequently improves the LOD.  
Figure 4: (**a**) EC DPV peak current corresponding to varying concentrations (0–10 ng/mL) of N protein in 500 \(\mu\)L of PBS buffer (linear regression curve). (**b**) Subset of the EC DPV peak current corresponding to N protein concentration (in 500 \(\mu\)L of PBS buffer) in a 0–0.5 ng/mL concentration range. Error bars represent standard deviation (n = 3).  
Figure 5: EC (DPV) peak currents corresponding to human nasal samples obtained from SARS-CoV-2-infected patients (three different cycle thresholds representing varying viral load; analyzed volume = 50 \(\mu\)L). The negative samples (nasal samples from healthy patients) represent the control. Error bars represent standard deviation (n = 3). The mean values of peak current are labeled above the columns. The asterisk (****) denotes different probability values as per the GraphPad style (see Supporting Information, Table S1).  
Figure 6 shows that the peak currents associated with all the positive samples tested were above those of the negative samples. The assay detected SARS-CoV-2-positive samples with a viral load down to Ct = 25 with statistical significance (_p_-value \(<\) 0.0001 vs. negative sample 1). The peak current associated with Ct = 31 was 21% higher than that for negative samples. However, this difference was not statistically significant (_p_-value = 0.0976 vs. negative sample 1). It is important to note that we achieved an improved LOD by increasing the sample aliquot from 50 to 250 \(\mu\)L. For nasal samples, the swabs were placed in 1 mL of viral transport media for SARS-CoV-2 commercial PCR assays [43]. Hence, by utilizing a larger sample volume (volumes higher than the 250 \(\mu\)L used in our study) for the MB preconcentration, the assay can, in principle, achieve increased sensitivity. Alternatively, the modification of the collection protocol by reducing the initial sample dilution volume from 1 mL to 250 \(\mu\)L could further improve the clinical sensitivity. The future integration and automation of various steps described in this work utilizing microfluidic devices can lead to the assay being run at the point-of-care [44; 45].