key: cord-0996912-fag49da4
authors: Nakano, Ryuichi; Yamaguchi, Akira; Sunada, Kayano; Nagai, Takeshi; Nakano, Akiyo; Suzuki, Yuki; Yano, Hisakazu; Ishiguro, Hitoshi; Miyauchi, Masahiro
title: Inactivation of various variant types of SARS-CoV-2 by indoor-light-sensitive TiO(2)-based photocatalyst
date: 2022-04-14
journal: Sci Rep
DOI: 10.1038/s41598-022-09402-7
sha: af4f7e8939f77f0e2a9f1f0756c1e1fdde60d28b
doc_id: 996912
cord_uid: fag49da4

Photocatalysts are promising materials for solid-state antiviral coatings to protect against the spread of pandemic coronavirus disease (COVID-19). This paper reports that copper oxide nanoclusters grafted with titanium dioxide (Cu(x)O/TiO(2)) inactivated the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus, including its Delta variant, even under dark condition, and further inactivated it under illumination with a white fluorescent bulb. To investigate its inactivation mechanism, the denaturation of spike proteins of SARS-CoV-2 was examined by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) and enzyme-linked immunosorbent assay (ELISA). In addition to spike proteins, fragmentation of ribonucleic acids in SARS-CoV-2 was investigated by real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR). As a result, both spike proteins and RNAs in the SARS-CoV-2 virus were damaged by the Cu(x)O/TiO(2) photocatalyst even under dark condition and were further damaged under white fluorescent bulb illumination. Based on the present antiviral mechanism, the Cu(x)O/TiO(2) photocatalyst will be effective in inactivating other potential mutant strains of SARS-CoV-2. The Cu(x)O/TiO(2) photocatalyst can thus be used to reduce the infectious risk of COVID-19 in an indoor environment, where light illumination is turned on during the day and off during the night.

Characterisation and photocatalytic activity of Cu x O/TiO 2 . The Cu x O nanoclusters were facilely grafted onto TiO 2 powder by a simple impregnation method based on a previous report 25 . Briefly, rutile TiO 2 powder was dispersed in an aqueous solution of copper chloride (CuCl 2 ) under stirring at 90 °C, and then sodium hydroxide and glucose were added to reduce Cu(II) species to Cu(I) species. After washing, filtration, and drying, Cu x O/TiO 2 powder was obtained. For antiviral evaluation, Cu x O/TiO 2 powder was coated onto a glass substrate. Figure 1a shows a transmission electron microscopy (TEM) image of Cu x O/TiO 2 . The size of the TiO 2 powder ranged from approximately 100 to 200 nm, on which small Cu x O nanoclusters of a few nanometres in size were grafted. Our energy dispersive X-ray spectrometer (EDS) equipped in a TEM apparatus, scanning electron www.nature.com/scientificreports/ microscope (SEM) image with its EDS mapping, and X-ray diffraction (XRD) pattern indicated that the grafted small nanoclusters were composed of amorphous copper oxide (see Supplementary Fig. 1-3) . Figure 1b shows the ultraviolet-visible (UV-Vis) absorption spectra of Cu x O/TiO 2 and bare TiO 2 . Bare TiO 2 absorbs UV light below 400 nm, which is attributed to the band-to-band transition in TiO 2 . On the other hand, Cu x O/TiO 2 exhibits a broad visible light band. The absorption band in the range of 400-500 nm is assigned to interfacial charge transfer (IFCT) from the valence band of TiO 2 to the unoccupied orbital of Cu(II) species 22, 23 , which was confirmed by our in situ electron spin resonance (ESR) analysis under light irradiation (see Supplementary Fig. 4 ). In addition, the absorption in the range of 500-600 nm originates from the band-to-band transition in Cu x O, while the absorption over 650 nm is assigned to the d-d transition of the Cu(II) species 34 . According to a previous report, IFCT transition induced by blue light (around 400-500 nm) irradiation only contributed to the photocatalytic oxidation activity among the observed visible-light absorption bands 25 . Indeed, the present Cu x O/TiO 2 exhibited photocatalytic activity under blue light, while the pristine TiO 2 did not show any activity under the same blue light (see Supplementary Fig. 5a ). The Cu x O/TiO 2 photocatalyst could completely oxidise gaseous 2-propanol molecules into carbon dioxide under visible-light irradiation ( Supplementary  Fig. 5b ). On the basis of its strong oxidising power, the Cu x O/TiO 2 photocatalyst is expected to have high antiviral function. Furthermore, its antiviral activity under dark condition was examined and discussed below.

Inactivation of SARS-CoV-2. The photocatalytic antiviral activity of Cu x O/TiO 2 against SARS-CoV-2 virus was evaluated by the method reported in our previous study 15 . Figure 2a shows the inactivation properties of the SARS-CoV-2 virus of wild-type strain under dark and visible light irradiation conditions. Visible light irradiation was conducted using a commercial white fluorescence bulb (see its spectrum in Supplementary Fig. 6 ), which is usually used as an indoor lighting apparatus. For light irradiation, the UV region was cut off by inserting an optical filter, and the light intensity was set at 1000 lx. We investigated the antiviral properties of the glass substrate without photocatalyst as a control group and found that the virus titer on the glass did not decrease under dark and visible light irradiation conditions. In contrast, the virus titer on the Cu x O/TiO 2 photocatalyst drastically decreased even under dark condition. After 3 h of exposure to Cu x O/TiO 2 in the dark, the virus titer decreased to the detection limit. Furthermore, the antiviral property of the photocatalyst was improved under visible light irradiation, and the virus titer decreased to the detection limit after only 2 h. In other words, four orders of magnitude of the virus were inactivated by the Cu x O/TiO 2 photocatalyst even after 2 h under regular indoor lighting. Figure 2b ,c show photographs of SARS-CoV-2 plaques for the glass substrate and Cu x O/TiO 2 photocatalyst under visible light irradiation, where white spots indicate active SARS-CoV-2 viruses. These photos also demonstrate the efficient photocatalytic antiviral properties of our Cu x O/TiO 2 photocatalyst.

In addition to the wild-type strain of SARS-CoV-2, we investigated the inactivation properties of Cu x O/TiO 2 versus Alfa (α), Beta (β), Gamma (γ), and Delta (δ) variants. Figure 3a shows the virus titer of Alfa, Beta, and Gamma variants on bare glass and those on the Cu x O/TiO 2 coated sample. The virus titer was examined before light irradiation (dark) and after visible light irradiation for 2 h. Similar to the wild-type strain of SARS-CoV-2 (b) (c) 1 35 , but it did not work well under dark conditions. In this report, the virus titer was reduced by only two orders of magnitude after 3 h under 3000 lx white light irradiation, which was much stronger than the present illumination conditions. TiO 2 based photocatalyst used in this study has an advantage over these previous studies because of its more efficient anti-SARS-CoV-2 property under visible light irradiation. In addition, our TiO 2 based material is more chemically stable under alkaline and acidic conditions and is more economical than WO 3 based photocatalysts. Furthermore, the present Cu x O/TiO 2 exhibits an efficient antiviral activity even in the dark condition, while most of the reported photocatalysts were not functioned under the dark. We previously investigated the antiviral mechanism of Cu x O/TiO 2 using bacteriophage Qβ without an envelope as a virus. It is well understood that the organic substances in bacteriophage Qβ are oxidised to carbon dioxide, causing inactivation of viruses under visible light irradiation. In addition, Cu(I) species in Cu x O nanoclusters cause denaturation of protein even in dark condition by the strong adsorption between protein and Cu x O 27 . Sunada et al. 20, 24 investigated the antiviral mechanism of copper oxides and found that the inactivation property of Cu(I) species was due to its solid-state property toward strong protein adsorption, rather than by the generation of reactive oxygen species or leached copper ions. These previous studies used bacteriophage Qβ without envelope for their mechanism studies; thus, we carefully investigated the denaturation of both spike proteins and ribonucleic acids (RNAs) in SARS-CoV-2 by using SDS-PAGE, ELISA, and RT-qPCR analyses, as presented in the next section.

coronavirus-spike (S) glycoproteins promote entry into cells and are the main target of antibodies 31 . Figure 4a ,b show the results of SDS-PAGE analysis for Cu x O/TiO 2 and bare glass under dark condition (a) and under visible light irradiation condition (b). It is noteworthy that the band signal of S1 proteins and receptor binding domain (RBD) at S1 proteins were obviously decreased after exposure to the Cu x O/TiO 2 catalyst under dark conditions www.nature.com/scientificreports/ as well as under visible light irradiation. As RBD recognizes ACE2, a receptor on the surface of host cells 36, 37 , the signal disappearance of RBD suggests a decrease in virus infectivity. We also quantitatively evaluated the denaturation of S1 spike proteins by ELISA, and the results are shown in Fig. 4c . The spike proteins were not denatured on a control glass substrate under dark and light illumination conditions. On the other hand, the spike proteins were denatured on the Cu x O/TiO 2 photocatalyst even in the dark, and its denaturation property was further enhanced by visible light irradiation. These results indicate that the Cu x O/TiO 2 photocatalyst denaturalises S1 spike proteins, which play an essential role in entry into lung cells, even under dark conditions as well as under visible light irradiation. Previously, similar denaturation properties to albumin, haemagglutinin, and neuraminidase were observed by Cu(I) species in copper oxides 20, 24 . According to these previous studies, the strong antiviral ability of Cu(I) species is owing to its strong adsorption ability towards proteins, rather than the effects of reactive oxygen species or leached copper ions. Therefore, the efficient antiviral activity of Cu x O/TiO 2 photocatalyst even under the dark condition is attributed to its strong adsorption property yielding denaturation of proteins. Protein molecules were further oxidised by the Cu x O/TiO 2 photocatalyst under visible light illumination, because the photogenerated holes in the valence band of TiO 2 generated through the interfacial charge transfer have strong oxidative power for complete decomposition into carbon dioxide molecules, as shown in the Supplementary Information (Fig. 5 ).

In addition to spike protein denaturation, we also investigated the fragmentation of RNA in the SARS-CoV-2 virus under exposure to our catalyst. Figure 5 shows the changes in the RNA copies of SARS-CoV-2. Similar to the trends of virus titer and spike proteins shown above, RNA copies were also decreased by exposure to the Cu x O/TiO 2 catalyst, even under dark condition. Furthermore, visible light irradiation of the Cu x O/TiO 2 photocatalyst enhanced the fragmentation of RNAs in SARS-CoV-2. Based on these results, the fragmentation of RNA by the present photocatalyst also contributes to its efficient antiviral activity. 25 . Therefore, we speculate that the strong adsorption ability of Cu(I) species also causes fragmentation of RNAs even under the dark condition. On the other hand, the second step reduction (4-8 h) would be further driven by the photocatalytic oxidation process, which is able to oxidise organic molecules into water and carbon dioxide molecules. However, we would like to emphasise that complete oxidation is not necessary to inactivate the SARS-CoV-2 virus to the detection limit. As shown in Figs. 2 and 3 , an exposure time of 3 h was sufficient for Cu x O/TiO 2 to inactivate SARS-CoV-2 below the detection limit, and even under dark condition. These results indicate that the 3 h exposure to Cu x O/TiO 2 denaturalises spike proteins and also causes fragmentation of RNA, as proven by our SDS-PAGE, ELISA, and RT-qPCR analyses. Figure 6 shows the antiviral mechanism of the Cu x O/TiO 2 photocatalyst. The Cu(I) species in Cu x O denaturalises spike proteins and also causes RNA fragmentation of SARS-CoV-2, even under dark condition, yielding inactivation under the detection limit after only 3 h. Furthermore, light irradiation causes the photocatalytic oxidation of the organic molecules of SARS-CoV-2. Based on this antiviral mechanism, involving denaturation of proteins, fragmentation of RNAs, and oxidation of organic substances by photocatalysis, the Cu x O/TiO 2 photocatalyst is not limited to a specific variant of the virus. It will be effective to inactivate other types of a mutant strain of SARS-CoV-2, such as Omicron strain 29 . It is noted that in the case of vaccines and/or oral drugs, there is a possibility that resistant mutants will emerge in the future. In contrast to vaccines or drugs, the Cu x O/TiO 2 photocatalyst is very useful because it has potential effectiveness against various mutants broadly.

The present study mainly focused on the inactivation of SARS-CoV-2 which consists of an envelope membrane. We also investigated the antiviral properties of feline calicivirus (FCV), which does not have an outer www.nature.com/scientificreports/ envelope, in contrast to SARS-CoV-2 (see Supplementary Fig. 7) . Notably, the present Cu x O/TiO 2 catalyst also inactivated FCVs even under the dark condition, and its performance was further improved under visible-light irradiation. Our previous studies also revealed that the Cu x O/TiO 2 inactivated bacteriophage Qβ, which does not have an envelope membrane 25, 27 . These results strongly indicate that the Cu x O/TiO 2 photocatalyst is effective for inactivation of various kinds of viruses by its denaturation and/or strong oxidation ability. Furthermore, Cu x O/TiO 2 exhibited a significant antibacterial effect on Escherichia coli and Staphylococcus aureus, as well as viruses 25 . Thus, the Cu x O/TiO 2 will be one of the valuable anti-microorganism materials with wide spectrum. It is important to discuss the toxicity of the present Cu x O/TiO 2 for its practical use. It has been reported the cytotoxic risk of TiO 2 and CuO nanoparticles towards Zebrafish or colon cells [38] [39] [40] . On the other hand, the previous studies directly examined the influence of TiO 2 and CuO particles on human skin 41, 42 . These papers concluded that the TiO 2 and CuO have extremely low risk to human skin. In the present study, we have conducted the Salmonella reverse mutation assay test (Ames test) 43 on Cu x O/TiO 2 to discuss its genotoxic risk (Supplementary Material, Table 1 and 2). The test results indicate its extremely low risk. We suppose that the present antiviral particles will be mainly applied as a solid-state coating material on a substrate. Under such applications, concentrated particles are not exposed to human for long term. Therefore, we expect that our antiviral material can be safely used for various coating applications.

The Cu x O/TiO 2 photocatalyst inactivated SARS-CoV-2 even under dark condition, and its antiviral performance was improved by white light illumination, which is usually used as an indoor light apparatus. Thus, the antiviral function of the Cu x O/TiO 2 photocatalyst can be maintained in an indoor atmosphere, where light illumination is turned on during the day and off during the night without any maintenance, such as spraying of antiviral liquid or wipe-off procedures. The present photocatalyst denaturalises spike proteins and also causes fragmentation of RNAs in the SARS-CoV-2 virus, as proven by SDS-PAGES, ELISA, and RT-qPCR analyses. The Cu x O/TiO 2 photocatalyst has already been commercialised (NAKA CORPORATION, Tokyo Japan) and is expected to be applied to various antiviral industrial items in indoor environments, such as hospitals, airports, metro stations, and schools, as coating materials for air filters, respiratory face masks, and antifungal fabrics to prevent the COVID-19 spread.

The Cu x O nanoclusters were grafted onto rutile TiO 2 powder using an impregnation technique. In a typical preparation, one gram of TiO 2 was dispersed in 10 mL of aqueous CuCl 2 solution in a vial reactor. The weight fraction of Cu relative to TiO 2 was 0.25%. During stirring, the temperature of the aqueous solution was maintained at 90 °C for 1 h. Then, sodium hydroxide (NaOH) and glucose were added to the solution (molar ratio of NaOH/glucose/CuCl 2 = 8/4/1) at the same temperature to graft Cu x O nanoclusters onto TiO 2 particles 25 .

The Cu x O/TiO 2 powder was suspended in 99% ethanol at a concentration of 1 mg/ml. Then, 0.6 ml of the suspension was uniformly loaded onto a soda-lime glass substrate (50 mm × 50 mm) and dried at 100 °C for 15 min. By repeating this operation twice, the glass substrate was thus coated with Cu x O/TiO 2 powder (1.2 mg).

Transmission electron microscopy (TEM) images were obtained using a JEM-2100F TEM/STEM (JEOL, Japan) operated at an acceleration electron beam voltage of 200 kV. Field-emission scanning electron microscopy (FE-SEM) images and energy-dispersive X-ray (EDS) analysis were performed using an S4700 (Hitachi High-Tech, Japan). X-ray diffraction (XRD) patterns were recorded using a SmartLab diffractometer (Rigaku, Japan) with Cu Kα radiation (λ = 1.5418 Å). A Si non-reflective plate was used as the substrate. UV-visible (UV-Vis) diffuse reflectance spectra were recorded using a spectrophotometer (V-670, JASCO, Japan) equipped with an integration sphere unit. Optical absorption spectra were obtained using the Kubelka-Munk function 44 calculated from raw reflection data, where a white BaSO 4 plate was used as the reflectance standard. Electron spin resonance (ESR) spectra were recorded using an in situ ESR system under light irradiation (EMX Nano, Bruker). For ESR measurements, the photocatalyst powder was placed into a quartz tube filled with nitrogen gas at 90 K with a microwave frequency (X-band) of 9.629 GHz to 9.633 GHz.

Photocatalytic oxidation activity test. The photocatalytic oxidation activity was evaluated by monitoring the oxidation of gaseous 2-propanol into acetone and carbon dioxide under visible-light irradiation, as the oxidation pathway was well studied in a previous paper 45 . A 100 mg Cu x O/TiO 2 sample was uniformly spread over a glass dish (5.5 cm 2 ). Before each photocatalysis test, pre-irradiation was performed overnight in a 500 mL reaction vessel filled with fresh synthetic air to eliminate organic contaminants on the sample surface. Next, the gas inside the reactor was replaced with fresh synthetic air, and 4.1 μmol gaseous 2-propanol was injected into the reactor. Before light irradiation, the system was kept in the dark for 1 h to allow 2-propanol gas to reach absorption equilibrium. Visible light irradiation was performed using a blue light emission diode (LED) with an intensity of 20 mW/cm 2 measured using a spectro-radiometer (USR-40D, Ushio, Japan), which could drive the interfacial charge transfer (IFCT) in Cu x O/TiO 2 . Furthermore, a relatively higher intensity of visible light source (85 mW/cm 2 by a 150 W Xe lamp with a UV cutoff filter below 420 nm) was also used to investigate whether our photocatalyst could completely oxidise 2-propanol molecules to carbon dioxide. Concentrations of 2-propanol and produced gases of acetone and carbon dioxide were measured using a photoacoustic gas monitor (1412i, INNOVA). www.nature.com/scientificreports/ Virus strain. The SARS-CoV-2 virus reference strains used in this study were the wild-type strain 2019-nCoV JPN/TY/WK-521 and variants of concern (VOCs), including the Alpha variant (B. 

After being illuminated with Cu x O/TiO 2 -coated glass or control glass for a certain period (0-8 h), all samples were collected and quantitative alterations of the proteins were analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and enzyme-linked immunosorbent assay (ELISA). For the SDS-PAGE analysis, collected samples were extracted by EzApply (ATTO, Japan) and the protein of the SARS-CoV-2 virus was separated by 10% SDS-polyacrylamide gels as previously described 48 . Protein bands were stained with SYPRO Ruby (Thermo Fisher Scientific, USA) and visualised using a Chemidoc imaging system (BioRad, France). The quantification of SARS-CoV-2 spike S1 protein of the collected sample was determined using a SARS-CoV-2 Spike S1 Protein ELISA Kit (RK04154; ABclonal, USA), according to the manufacturer's instructions. The calibration standards were assayed at the same time as the samples and allowed the operator to produce a standard curve of optical density versus SARS-CoV-2 spike S1 protein concentration. The concentration of the samples was then determined by comparing the O.D. of the samples to the standard curve. Absorbance was measured at 450 nm using a spectrophotometer. The samples were tested in duplicates.

Detection of SARS-CoV-2 N gene by RT-qPCR. The quantity of viral RNA was analysed using RT-qPCR. Briefly, RNA was extracted from the samples using the QIAamp Viral RNA Mini Kit (QIAGEN, Hilden, Germany), according to the manufacturer's instructions. RT-qPCR was performed using the QuantiTect Probe RT-PCR Kit (QIAGEN) on the QuantStudio 5 Real-Time PCR System (ThermoFisher, USA) and the following set of primers/probes specific for the viral N gene: The forward primer, 5'-AAA TTT TGG GGA CCA GGA AC-3'; the reverse primer, 5'-TGG CAG CTG TGT AGG TCA AC-3'; and the probe, 5'-(FAM) ATG TCG CGC ATT GGC ATG GA (BHQ)-3′ 49 . The cycle threshold (Ct) values of RT-qPCR were converted into viral RNA copy numbers based on a standard curve prepared from tenfold serial dilutions of known copy numbers of SARS-CoV-2 RNA.

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. www.nature.com/scientificreports/

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This work was funded by the New Energy and Industrial Technology Development Organization (NEDO) in Japan (project name: Project to Create Photocatalyst Industry for Recycling-Oriented Society). This 

The authors declare no competing interests.

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