## 4 Results and Discussion
### Analytical Sensitivity of the Assay for the Detection of SARS-CoV-2 Nucleocapsid Protein in PBS  
Analytical sensitivity refers to the ability of an assay to detect the smallest amount of a target analyte, in this case, the SARS-CoV-2 nucleocapsid protein [21]. The assay's performance in detecting different concentrations of SARS-CoV-2 N protein in PBS was evaluated. As described in Section 3.3, AuNP conjugation and immunomagnetic capture were performed on different concentrations of SARS-CoV-2 N protein suspended in 500 \(\mu\)L of PBS (ranging from 1 to 10 ng/mL). Figure 4 shows that a linear calibration plot was observed in the 1-10 ng/mL range with a regression equation: \(y~{}=~{}0.1~{}x+0.1388\) (R\({}^{2}~{}=~{}0.98\)). The calculated LOD was 2.64 ng/mL (calculated as LOD = 3.3 (\(\sigma\)/S), where \(\sigma\) is the standard deviation of the calibration curve, and S is the slope of the calibration curve). Figure 3: Absolute EC peak current (DPV) was obtained for 20 nm AuNPs in serial 10x dilutions (D0 = 1.4 \(\times\) 10\({}^{8}\), D1 = 1.4 \(\times\) 10\({}^{7}\), D2 = 1.4 \(\times\) 10\({}^{6}\), and D3 = 1.4 \(\times\) 10\({}^{5}\) AuNPs in 2 \(\mu\)L of DI water). Error bars represent standard deviation (n = 3). The blank (2 \(\mu\)L of DI water, no AuNPs) represents the control. The mean values of peak current are labeled above the columns. The inset shows the calibration curve generated for AuNP dilutions in a range of D0–D3. In total, 50 \(\mu\)L of 0.1 M HCl was used as the supporting electrolyte (deposition time = 120 s). The asterisk (**) denotes different probability values as per the GraphPad style (see Supporting Information, Table S1). The 0 ng/mL blank control provided a peak current of 0.065 uA. This signal corresponds to AuNP conjugation and immunomagnetic capture performed on 500 uL of PBS (no analyte present). While we observed large standard deviations in our current results (as seen in Figure 4), we recognize the importance of assay precision. To address this and further validate our findings, we plan to conduct a spike-recovery test in subsequent studies. Our current assay comprises a 120 min incubation period, followed by a cumulative washing duration of 19 min and a 2 min electrochemical signal acquisition. The overall duration of the entire procedure is approximately 141 min. Table 1 lists the performance of EC immunoassay that utilize separation with immunomagnetic particles (either antibody- or peptide-conjugated) and an EC tag for the detection of SARS-CoV-2. Our LOD is comparable [41] or better [13] than the results described in the literature. [PERSON] et al. utilized peroxidase-loaded magnetic beads to capture and detect the spike protein of COVID-19 in serum, urine, and saliva. The study utilized human samples spiked with different concentrations (3.12-200 ng/mL) of SARS-CoV-2 spike protein and did not utilize the study on actual clinical samples [41]. [PERSON] et al. developed an EC immunoassay for N protein using primary-antibody-conjugated MBs and a secondary phosphatase EC tag. The device provided a LOD of 10 ng/mL and was utilized to detect N protein in SARS-CoV-2-positive human saliva samples [13]. An EC immunoassay utilizing peptide-conjugated MBs and a secondary AuNP EC tag was utilized by [PERSON] et al. for the detection of the spike protein of SARS-CoV-2 in clinical saliva samples. The assay reported a LOD of 0.35 ag/mL spike protein in saliva. This is about ten orders of magnitude better than what is currently reported in the literature (Table 1). A LOD of 0.35 ag/mL for the spike protein, using a 50 uL aliquot (used in the study) and a 3 mm working electrode dia., translates into the detection of less than one AuNP (20 nm) on a given SPCE (see Supporting Information). We observed a LOD of \(2.8\times 10^{6}\) (20 nm) for bare, immobilized AuNPs on the working electrode of the SPCE. A recent study reported a LOD of \(6.1\times 10^{3}\) (60 nm) for AuNPs on an SPCE (2.64 mm\({}^{2}\) geometric working electrode area) [42].