What to Look for When Comparing UV Sterilization Devices

In this study, a cost-effective sterilization box is developed and the synergistic effect of UV and temperature over sanitization was examined. For this purpose, the effect of UV and heat sanitization was performed, and its effect on glycoprotein viz., IgG and bacterial cells were observed. The detailed results are described in the sequel.

3.1. Effect of heat and UV treatment over IgG

The effects of temperature alone and UV incubation along with temperature were analyzed on IgG model protein. The hydrodynamic size of the native IgG was found to be ~5 nm, in agreement with the literature (Hawe et al., 2011). The absorbance of native IgG was 0.172, which corresponded to 0.1 mg/mL of IgG using Beer Lambert's law (Reader et al., 2019). The effects of varying temperature and combined UV and temperature with time were evaluated at the conditions suggested by Design-Expert software (). For this purpose, the absorbance values at 280 nm and hydrodynamic sizes were measured after treating the protein at different temperatures with and without UV as discussed in Section 2.3. The responses (hydrodynamic size and absorbance) at these conditions are shown in . In the figure, unfilled symbols pertain to heat only and filled symbol correspond to heat with UV-C.

The absorbance values at 280 nm were found to increase with the increase in temperature above 70 °C. Protein solution incubated at 76.21 °C exhibited the maximum absorbance value of 0.94 within 10 min followed by absorbance at 70 °C. The increase in absorbance is related to the exposure of aromatic amino acids present in IgG because of the conformational changes (Sharma and Pandey, 2021). The denaturation temperatures of IgG is reported as 61 and 71 °C (Vermeer and Norde, 2000). Moreover, the melting point, Tm of the IgG was found to be 69 °C (Martin et al., 2014); hence, the observed increase in the absorbance values indicated the deformation in the IgG's native conformation. However, the effect of time was found not to be significant below the temperature of 70 °C.

Similar to the absorbance values at a higher temperature, the hydrodynamic sizes of the IgG protein was found to increase with the increase in temperature and time of exposure. The increase in the hydrodynamic size at a higher temperature indicated the unfolding (increase in the size) of IgG at elevated temperatures (Martin et al., 2014; Zhang and Topp, 2012). IgG incubated at 76.21 °C under UV incubation resulted in the maximum hydrodynamic size of 309.02 nm (); at the same temperature without UV the hydrodynamic size was found to be lower i.e., 293.93 nm (). It indicates the significant role of UV incubation on the unfolding/denaturation of the IgG at elevated temperatures (Zhang and Topp, 2012). It has been observed that a 4 W UV-C lamp under 0.930–0.932 mW/cm2 and 65 °C dry heat for 20 min was sufficient to inactivate the entire swine coronavirus from N95 mask surfaces (Chotiprasitsakul et al., 2020). In the present study, two 11 W UV-C lamps resulted in the unfolding of IgG protein.

The effect of temperature below a critical time was found not to be significant at the conditions selected in this study. Similarly, the effect of time up to 15 min was also ineffective below a critical temperature. Hence, model equations relating input variables with the responses as predicted by Design-Expert software were not significant. However, the experimental data were fitted to a sigmoidal expression as shown in A (dotted line). The data fitted very well with R2 value of 0.99. The critical point (lag time) was estimated from the sigmoidal expression and was found to be 70 ± 1 °C. This temperature also agreed to the denaturation temperatures of IgG (Vermeer et al., 1998). Therefore, a temperature of 70 °C appears to be optimal for the unfolding of this model glycoprotein. It has been reported that unfolding/conformation changes of the surface glycoprotein of a virus leads to its disintegration and finally its inactivation (Hsu et al., 2011). The loss of viral surface protein due to external factors like a higher temperature and the strong surface-protein interactions disintegrates the virus assembly (Liu et al., 2015; Pandey, 2020b). It is also observed that UV-C light enhances the hydrodynamic size, but not drastically. Heat alone can perform well in the denaturation of IgG. ( B Absorbance280 of IgG at various conditions.)

To examine the combined effect of heat and UV treatments on the conformational changes of IgG, the intrinsic fluorescence values were recorded by exciting the protein at 290 nm and the emission values were scanned from 300 nm to 450 nm. The intrinsic fluorescence is produced due to the presence of aromatic amino acids (monomers) of the protein. The native IgG exhibited the maxima at 327 nm, whereas a peak-shift was observed at 332 nm and 331 nm at 76.21 °C for 10 min and 70 °C for 15 min, respectively (A). This indicates the unfolding and deformation in the native structure of the IgG at temperatures above a critical temperature of 70 °C (Arfat et al., 2014; Pandey, 2020b). Further, in the case of combined UV and heat exposure, the maximum fluorescence intensity was observed at 70 °C for 15 min (B). This indicates the synergistic effective role of UV in the unfolding and conformational changes in the IgG structure (Arfat et al., 2014). All these results suggested that the incubation of heat and UV together at 70 °C for 15 min unfolds the glycoprotein to a required extent and can be useful for the inactivation of viruses for sanitization purposes.

Further, the conformation changes of the combined heat and UV treated IgG at the above optimized condition (70 °C for 15 min) were analyzed as compared to the native IgG using Fourier Transform Infrared Spectroscopy (FTIR) analysis. The FTIR spectra of native and heat treated IgG are shown in , which were de-convoluted and the area of de-convoluted peaks have been used to deduce the contents of the secondary structure (Sharma et al., 2020). The contents of α-helix, β-sheet and β-turn in native IgG were found to be 32, 55 and 13%, respectively, which agreed with the reported data (Hasan et al., 2018). In the case of heat treated IgG, the contents of β-sheet increased to 70% and α-helix decreased to 22%. This complemented the DLS, absorbance and fluorescence data.

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