Inactivation of SARS-CoV-2 and photocatalytic degradation by TiO2 photocatalyst coatings

The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causative agent of the COVID-19, which is a global pandemic, has infected more than 552 million people, and killed more than 6.3 million people. SARS-CoV-2 can be transmitted through airborne route in addition to direct contact and droplet modes, the development of disinfectants that can be applied in working spaces without evacuating people is urgently needed. TiO2 is well known with some features of the purification, antibacterial/sterilization, making it could be developed disinfectants that can be applied in working spaces without evacuating people. Facing the severe epidemic, we expect to fully expand the application of our proposed effective approach of mechanical coating technique (MCT), which can be prepared on a large-scale fabrication of an easy-to-use TiO2/Ti photocatalyst coating, with hope to curb the epidemic. The photocatalytic inactivation of SARS-CoV-2 and influenza virus, and the photocatalytic degradation of acetaldehyde (C2H4O) and formaldehyde (CH2O) has been investigated. XRD and SEM results show that anatase TiO2 successfully coats on the surface of Ti coatings, while the crystal structure of anatase TiO2 can be increased during the following oxidation in air. The catalytic activity towards methylene blue of TiO2/Ti coating balls has been significantly enhanced by the followed oxidation in air, showing a very satisfying photocatalytic degradation of C2H4O and CH2O. Notably, the TiO2/Ti photocatalyst coating balls demonstrate a significant antiviral activity, with a decrease rate of virus reached 99.96% for influenza virus and 99.99% for SARS-CoV-2.

www.nature.com/scientificreports/ to application technology development, focusing, and antibacterial/sterilization, self-cleaning, energy-saving air conditioning, and so on [14][15][16] . Various volatile organic compounds (VOCs) such as acetaldehyde, benzene and formaldehyde are considered as air toxins and known for their adverse effects on health, and the VOCs could be subject to disruption by an oxidation process, caused by TiO 2 photocatalyst [17][18][19][20] . In 2021, Xie et al. demonstrated that intermediates accumulation was primarily responsible for the deactivation of the TiO 2 photocatalyst, which expected to overcome the fundamental issues to be addressed for photodegrading VOCs in practical applications caused by poor efficiency and stability of photocatalysts 18 . Herein, this study demonstrates the inactivation of SARS-CoV-2 and photocatalytic degradation of C 2 H 4 O and CH 2 O, with the presented TiO 2 /Ti photocatalyst coatings, formed on 2 mm diameter Al 2 O 3 balls. Notably, the TiO 2 /Ti photocatalyst coatings balls show a very satisfying photocatalytic degradation of C 2 H 4 O and CH 2 O, and a significant inactivation towards influenza virus and SARS-CoV-2.

Experimental method
Fabrication of TiO 2 /Ti photocatalyst coatings on Al 2 O 3 balls. TiO 2 /Ti photocatalyst coatings were formed on Al 2 O 3 balls using the previously established MCT [21][22][23] . First, Al 2 O 3 balls (approximately 2 mm diameter) and Ti powder (particle size less than 45 µm, purity 99.4%) were filled in sequence into an alumina pot, with a covered alumina lid. The Ti coatings were formed on Al 2 O 3 balls by MCT, with a planetary ball mill (P-6; FRITSCH) at a rotational speed of 480 rpm for 3 h, named as "Ti". Then, TiO 2 photocatalyst coatings were formed on the Ti coatings by MCT, with filling the Ti sample and TiO 2 powder (ST-01, particle size of 7 nm) in an alumina pot at a MCT rotational speed of 300 rpm for 3 h, named as "TiO 2 /Ti". To enhance the photocatalytic activity of the coatings, the TiO 2 /Ti sample were subjected to heat treatment at 500 °C for 5 h in air using an electric furnace. After the oxidation, the sample is marked as "TiO 2 /Ti-O".
Characterization of TiO 2 /Ti photocatalyst coatings on Al 2 O 3 balls. The crystal structure of the fabricated TiO 2 /Ti photocatalyst coatings were analyzed by an X-ray diffractometer (XRD, Rigaku Ultima IV) with Cu-Kα radiation, the surface and cross-section were observed by a scanning electron microscopy (SEM, Hatachi-8030). X-ray photoelectron spectroscopy (XPS, PHI Quantes) measurements was used to observe the change in the chemical composition on the surface. According to ISO 10678-2010, a wet decomposition performance test under UV irradiation towards methylene blue (MB) was used to evaluate the photocatalytic function of the TiO 2 /Ti photocatalyst coatings on Al 2 O 3 balls. The test cells (inner diameter Φ 40 mm × 30 mm, cylindrically shaped with a bottom) were spread over one-layer sample and filled with MB solution (20 μmol/L, 35 mL), then eliminated any possible absorption by keep the cells in dark for 18 h for adsorption. Followed by the adsorption, the cells were refreshed with a test MB solution (10 μmol/L, 35 mL) and irradiated with UV light of 1.0 mW/cm 2 intensity for 3 h. The absorbance of the MB solution at 640 nm was measured within every 20 min using a digital colorimeter (mini photo 10; Sanshin Kogyo).
Evaluation tests of the environmental purification. The environmental function evaluation test was conducted by the Kanagawa Institute of Industrial Science and Technology, a public research institute. Acetaldehyde (C 2 H 4 O), which causes sick house syndrome, etc. due to its odor and irritation, and formaldehyde (CH 2 O), a toxic substance that causes inflammation of the human respiratory system, eyes, and throat, contained in adhesives used in building materials such as furniture and wallpaper, were used as the targets. The decomposition and removal performance tests of C 2 H 4 O and CH 2 O were conducted at approximately 25 °C, as per JIS R 1701-2:2016 (Testing methods for air purification performance of fine ceramics-Photocatalytic materials-Part 2: Removal performance of acetaldehyde) and JIS R 1701-4:2016 (Fine ceramics-Air purification performance test method for photocatalytic materials-Part 4: Formaldehyde removal performance), with spreading the TiO 2 / Ti-O sample over a 100 × 50 mm cell to be a single layer. For the decomposition and removal performance test of C 2 H 4 O, the concentration of the target gas was 5 ppm at a flowing rate of 1.0 L/min, and the UV irradiation was 10 W/m 2 , then the C 2 H 4 O concentration and CO 2 concentration were measured. While the CH 2 O decomposition removal performance test, the concentration of test gas was set to 1.02 ppm at a flowing rate of 3.0 L/min, and the UV irradiation to 1.0 mW/cm 2 .

Inactivation performance tests. In compassion of the currently inactivation of new coronaviruses by
sheet and plate photocatalysts, we tested the inactivation performance of influenza virus and SARS-CoV-2 on TiO 2 /Ti photocatalytic coatings on balls. The inactivation test of influenza virus was conducted by requesting a test from the Kanagawa Institute of Industrial Science and Technology. The tests were conducted at approximately 25 °C, according to JIS R 1706:2020 (UV-responsive photocatalyst, antiviral, film adhesion method). Influenza A virus (H3N2) was used as the viral strain, ATCC CCL-34 as the host cell, and the irradiation conditions were UV irradiation of 0.25 mW/cm 2 with a black light fluorescent lamp, or 0 mW/cm 2 (in dark). The samples were sterilized and pre-irradiated with UV rays at 1.0 mW/cm 2 for 24 h, then aseptic treated at 80 °C for 15 min. The samples were spread in a sterile petri dish with a diameter of 60 mm to form a single layer. Then, 2.4 ml of sterile water and 0.1 ml of the virus solution were added and covered with a glass plate for moisture retention. After 8 h of UV irradiation and storage in dark, the infectivity titer of the virus was determined by the plaque method.
Furthermore, the inactivation test of SARS-CoV-2 was conducted according to JIS R 1706 (Test method for antiviral activity of fine ceramic photocatalytic materials), at Nara Medical University. Infected Vero E6 cells with SARS-CoV-2 were used as the target, stored in a − 80 °C freezer before the test. The UV irradiation conditions were 0.25 mW/cm 2 with a black light fluorescent lamp, or 0 mW/cm 2 (in dark). After the operation time,  (Fig. S2). Then, the TiO 2 coatings formed on the surface of the Ti coatings show grainy textured surface structure ( Fig. 1b-2). Interesting, the uneven part of the Ti coatings has been filled with TiO 2 coatings ( Fig. 1b-4), which make the surface to be smooth ( Fig. 1b-1). In addition, the thicknesses of the Ti and TiO 2 coatings are approximately 97 μm and 3 μm, respectively, according to the abbreviated calculations from SEM photographs. However, with comparison of the samples of TiO 2 /Ti and TiO 2 / Ti-O, the influence of followed oxidation in air at 500 °C for 5 h on the surface structure and cross sections is insignificant. Figure 2a shows the XRD patterns of the samples of Ti, TiO 2 /Ti, and TiO 2 /Ti-O. In general, the Ti peaks and TiO 2 peaks mean that Ti coatings and TiO 2 coatings successfully form on Al 2 O 3 balls. After oxidation in air, the Al 2 O 3 peaks disappear and the Ti and anatase TiO 2 peaks significantly increase, which indicates that the crystallinity of anatase TiO 2 has been greatly enhanced. XPS spectra has been used to investigate the change of chemical bonding on the surface of the samples, as shown in Fig. 2b-d. For comparison, Fig. 2b shows the O 1 s peak at around 529.4 eV of the samples, which could be corresponded to the Ti-O bonding from the anatase TiO 2 24,25 . Although the O 1 s shift hardly could be found from the samples of TiO 2 /Ti and TiO 2 /Ti-O, but the peak at around 530.8 eV from the TiO 2 /Ti-O sample decrease, compared with that of the TiO 2 /Ti-O sample, which hints that the crystallinity of anatase TiO 2 has been greatly enhanced, matching with the XRD results. Figure 2e reveals that the samples of TiO 2 /Ti and TiO 2 /Ti-O exhibit excellent photocatalytic activity, compared with that of Ti coatings. In general, the TiO 2 coatings clearly shows the photocatalytic activity, and the photocatalytic activity could be further enhanced with an increased crystallinity of anatase TiO 2 .    Furthermore, Fig. 3c shows the decomposition and removal performance of the TiO 2 /Ti-O sample for CH 2 O. When UV light turns on, the concentration of CH 2 O rapidly decreases from 1 ppm, then keeps approximately 0.43 ppm. It has believed that the formed hydroxyl radicals transfer on the surface of TiO 2 can not only directly react with CH 2 O molecules, but also can suppress the recombination of electron-hole during the transfer process to further enhance the photocatalytic activity [29][30][31] . When the UV light stops, the concentration of CH 2 O quickly returns to 1 ppm of the supplied concentration. These results also reveal the high decomposition ability of the TiO 2 /Ti-O sample for CH 2 O. In the case of the degradation process of CH 2 O, the generated OH ⋅ and O 2 −⋅ will firstly attack the C-H bonds in CH 2 O, then react with the liberated hydrogen atoms to form new free radicals 29,30 . In general, the initial stage of the degradation process will produce formic acid, then ultimately decompose CH 2 O molecules into H 2 O and CO 2 . Figure 4 shows the setup of inactivation test for influenza virus of H3N2, according to JIS R 1706:2020. Table 1 shows the infectious value and antiviral activity value of the samples under UV irradiation and in dark. The antiviral activity values are calculated by the following equations of (1) and (2).    Figure 5a clearly shows the setup of inactivation test. The infection titer of the control under UV light irradiation tends to decrease, whereas the infection titer of the TiO 2 /Ti-O sample significantly decreases, with an infectious value below than the detection limit after 6 h, as shown in Fig. 5b. In addition, the decrease rate of virus has been calculated and shown in Fig. 5c. The inactivation function of the TiO 2 /Ti-O sample is satisfactory in UV irradiation, and the decrease virus rate rapidly increases to 96% in short time, with reaching 99.99% within 6 h. These results mean that the TiO 2 /Ti-O sample are with a high inactivation function against the SARS-CoV-2.   www.nature.com/scientificreports/ It is well known that TiO 2 is a semiconductor metal oxide photocatalyst with a wide band gap of 3.2 eV (anatase type) 32 . TiO 2 when exposed to UV light of energy equal to or greater than its band gap, there is an excitation of electrons from valance band (VB) to conduction band (CB) of TiO 2 . These charge carriers move onto the surface of TiO 2 , then interact with the ambient oxygen (O 2 ) and water (H 2 O) molecules. Holes oxidizes H 2 O molecules into highly reactive hydroxyl radicals (superoxide radical anion (O 2 −⋅ ), which is further reduced to OH ⋅ . Since these radicals are highly reactive, thus known as reactive oxygen species (ROSs). These formed ROSs on the surface of TiO 2 react with the viruses and result into its degradation to CO 2 and H 2 O 33 , as shown in the proposed schematic diagram of Fig. 6. Photocatalysis is a surface phenomenon, which oxidizes/reduces or degrades the organic pollutants. Therefore, the TiO 2 /Ti photocatalyst coating balls with a large specific surface area are easy to use, showing high environmental purification and virus inactivation functions.

Conclusion
In present work, the TiO 2 /Ti photocatalyst coatings has been formed on Al 2 O 3 balls using a simple and effective approach of mechanical coating method and followed oxidation in air. After oxidation in air, the larger amount of anatase TiO 2 forms on the surface of Ti coatings, confirmed with XRD, SEM and XPS results. TiO 2 /Ti photocatalyst coatings on Al 2 O 3 balls are effective for environmental purification, owing to their high decomposition function for C 2 H 4 O and CH 2 O. Notably, TiO 2 /Ti photocatalyst coatings also show significant viral inactivation capability, reaching 99.96% inactivation rate for influenza virus and 99.99% inactivation rate for new coronavirus.

Data availability
The data that support the findings of this study are available from the article and Supplementary Information files, or from the corresponding authors upon reasonable request.   Figure 6. Proposed mechanisms of viral inactivation induced by TiO 2 /Ti photocatalyst coatings.