# An Optimized Graphene-Based Surface Plasmon Resonance Biosensor for Detecting SARS-CoV-2
## 3 Results and Discussions
### Application of the Optimized SPR Biosensor for SARS-CoV-2 Sensing  
Table S8 presents the optimized parameters for \(\text{Sys}_{\text{8}}\). The structure comprises a BK7 prism with a refractive index of 1.5151, forming the base substrate for efficient SPR excitation [29]. Over this, a 50 nm silver layer (refractive index 0.056253 + 4.2760i) is used to generate the SPR effect, achieving a balance between signal strength and resonance sharpness. A 15 nm layer of silicon nitride, with a refractive index of 2.0394, serves as a dielectric spacer, enhancing the sensor's sensitivity by optimizing the evanescent field interaction. Single-layer graphene (0.34 nm, refractive index 2.7611 + 1.6987i) is then added to amplify the SPR signal without significant attenuation, while a 5 nm thiol-tethered ssDNA layer (refractive index 1.462) provides selectivity for SARS-CoV-2 detection through specific binding interactions. We point out that the sensor is calibrated with PBS (refractive index 1.334) as the sensing medium, which closely mimics physiological conditions. To detect SARS-CoV-2, the refractive index of the virus in PBS is presented for varying concentrations, ranging from 1.340 at 150 mM to 1.355 at 525 mM. These values, based on a study by [PERSON] et al. [19], demonstrate a linear relationship between refractive index and viral concentration, enabling quantitative analysis of SARS-CoV-2 levels. Figure 7a shows the SPR reflectance curves for various SARS-CoV-2 concentrations (150 to 525 mM) in PBS, with the black curve representing the baseline optimized system in PBS without virus adsorption (refractive index of 1.334). As the virus concentration increases, corresponding to refractive indices of 1.340, 1.345, 1.350, and 1.355, the SPR dip shifts toward higher angles, demonstrating the sensor's response to changes in the refractive index due to virus presence [30]. This shift indicates successful detection, as increased virus concentration enhances the binding events on the sensor surface. Figure 6: (**a**) SPR curves as a function of the angle of incidence ranging from 70\({}^{\circ}\) to 80\({}^{\circ}\), increasing the thickness of ssDNA layers. ssDNA\({}_{3\,2\,\text{base}}\) denotes \(\text{Sys}_{\text{8}}\) in water with the optimized silver thickness of 50 nm, the optimized silicon nitride thickness of 15 nm, the optimized single-layer graphene, and the initial ssDNA thickness of 3.2 nm. (**b**) Attenuation percentage, (**c**) FWHM, and (**d**) sensitivity enhancement percentage for all ssDNA thickness tested. Figure 7b and Table S9 demonstrate the increasing trend in attenuation as the virus concentration rises. Starting from 18.11% at 150 mM, attenuation escalates to 78.47% at 525 mM. This high attenuation suggests that higher concentrations of SARS-CoV-2 significantly dampen the reflected light, indicating a strong interaction between the evanescent field and the viral particles. However, this also highlights a drawback: excessive attenuation at higher concentrations can lead to signal saturation, reducing the sensor's efficiency in detecting further increases in virus concentration. This saturation effect is visible as a plateau in sensitivity at higher concentrations. The FWHM values in Figure 7c and Table S9 indicate broadening of the SPR dip as virus concentration increases. Starting from 5.64\({}^{\circ}\) at 150 mM, the FWHM rises to 8.62\({}^{\circ}\) at 525 mM. This broadening suggests a decrease in dip sharpness, which may affect detection precision. While some increase in FWHM is expected with higher analyte concentrations due to enhanced binding, excessive broadening could reduce the sensor's ability to resolve small changes in virus concentration, as the signal becomes less distinct. Figure 7d and Table S9 show sensitivity enhancement percentages for each concentration. Sensitivity initially rises from 2.25% at 150 mM to 5.11% at 400 mM, following a linear trend up to this concentration, as observed in Figure S5. Beyond 400 mM, sensitivity enhancement plateaus, reaching 4.74% at 525 mM.