Mechanisms of Plasma Ozone and UV-C Sterilization of SARS-CoV-2 Explored through Atomic Force Microscopy

Ultraviolet-C (UV-C) radiation and ozone gas are potential mechanisms employed to inactivate the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), each exhibiting distinct molecular-level modalities of action. To elucidate these disparities and deepen our understanding, we delve into the intricacies of SARS-CoV-2 inactivation via UV-C and ozone gas treatments, exploring their distinct molecular-level impacts utilizing a suite of advanced techniques, including biological atomic force microscopy (Bio-AFM) and single virus force spectroscopy (SVFS). Whereas UV-C exhibited no perceivable alterations in virus size or surface topography, ozone gas treatment elucidated pronounced changes in both parameters, intensifying with prolonged exposure. Furthermore, a nuanced difference was observed in virus–host cell binding post-treatment: ozone gas distinctly reduced SARS-CoV-2 binding to host cells, while UV-C maintained the status quo. The results derived from these methodical explorations underscore the pivotal role of advanced Bio-AFM techniques and SVFS in enhancing our understanding of virus inactivation mechanisms, offering invaluable insights for future research and applications in viral contamination mitigation.

(C) Within this chamber, a specialized electrode for ozone gas (O 3 ) production is installed.deposited onto mica slides (1 cm × 1 cm) placed either in 48-well plates or on slide glass, and then immediately exposed to ozone gas for 10 or 30 minutes, respectively.The ozone gas concentrations measured on the slide glass were 0.28 mg/L for 10 minutes and 1 mg/L for 30 minutes, while those in the 48-well plate were 0.18 mg/L for 10 minutes and 0.55 mg/L for 30 minutes.Following the ozone treatment, 300 µL of PBS was added to the wells to collect the bacteria for colony counting.

Figure S3
. Bio-AFM images of SARS-CoV-2 particles on a mica surface following UV-C and ozone gas (O 3 ) treatments.Panel (A) displays images of three different virus particles that have not been treated with either UV-C or ozone gas.Panels (B) through (E) display the effects of UV-C treatment at 1, 3, 5, and 7 minutes, respectively, while panels (G) through (J) show the effects of ozone gas treatment for the same intervals.In each treatment group, four distinct virus particles were analyzed.This figure illustrates the topographic images of three different particles per condition, while images of the fourth particle are presented in Fig. 3D and 3E.Height profiles and line analyses were conducted on each particle to quantify morphological changes and maintain consistent measurement standards across all samples.These analyses are crucial for supporting the height variation data shown in Fig. 3F and 3G.
UV absorption spectroscopy.The ozone concentration was measured using ultraviolet absorption spectroscopy.Light from a mercury lamp (BHK 90-0005-01, spectral line: 253.65 nm) was directed through a lens and optical fiber, then passed through a cuvette containing a buffer in which the ozone was dissolved.The intensity of the transmitted light was measured using a spectrometer (AvaSpec-2048L, Avantes, Apeldoorn, The Netherlands).Given that the absorption cross section of ozone is broad near 253.65 nm and known to be 1.137 × 10⁻¹⁷ cm ² /molecule, the ozone concentration was calculated using the Beer-Lambert law, as described in Equation ( 1) below: Absorbance is defined as -log(I/I₀), where I and I₀ represent the light intensities of the ozonetreated sample and the non-treated sample, respectively.In this context, ε is the ozone molar absorption coefficient, which is 2900 M⁻¹ cm⁻¹, and l is the optical path length, set at 1 cm in the region of interest. 1The measurements were repeated three time.
HS-AFM setup.HS-AFM (RIBM, Ibaraki, Japan) was employed to obtain the topography of SARS-CoV-2 after UV-C and ozone treatments.We used an ultra-short cantilever USC-F1.2-k0.15(NanoWorld, Neuchâtel, Switzerland) with a nominal spring constant of 0.15 N/m, a resonance frequency approximating 500 kHz, and a quality factor close to 2 in a liquid environment.

Figure S1 .
Figure S1.Optical images showcase the UV-C device (A) and the ozone gas (O 3 ) generator components (B, C). (A) The UV-C device contains four LED modules, each with a 275 nm

Figure S2 .
Figure S2.Infectivity test results for E. coli O157:H7 on slide glass and 48-well plates: (A) optical

Figure S4 .
Figure S4.Bio-AFM images of SARS-CoV-2 particles on silicon surface utilizing the same

Figure S5 .
Figure S5.Example of force-distance curves.(A) Tip with PEG linker only but without inactivated