Antiviral E ﬀ ect of Visible Light-Sensitive Cu x O / TiO 2 Photocatalyst

: Photocatalysis is an e ﬀ ective technology for preventing the spread of pandemic-scale viruses. This review paper presents an overview of the recent progress in the development of an e ﬃ cient visible light-sensitive photocatalyst, i.e., a copper oxide nanoclusters grafted titanium dioxide (Cu x O / TiO 2 ). The antiviral Cu x O / TiO 2 photocatalyst is functionalised by a di ﬀ erent mechanism in addition to the photocatalytic oxidation process. The Cu x O nanocluster consists of the valence states of Cu(I) and Cu(II); herein, the Cu(I) species denaturalizes the protein of the virus, thereby resulting in signiﬁcant antiviral properties even under dark conditions. Moreover, the Cu(II) species in the Cu x O nanocluster serves as an electron acceptor through photo-induced interfacial charge transfer, which leads to the formation of an anti-virus Cu(I) species and holes with strong oxidation power in the valence band of TiO 2 under visible-light irradiation. The antiviral function of the Cu x O / TiO 2 photocatalyst is maintained under indoor conditions, where light illumination is enabled during the day but not during the night; this is because the remaining active Cu(I) species works under dark conditions. The Cu x O / TiO 2 photocatalyst can thus be used to reduce the risk of virus infection by acting as an antiviral coating material. of antiviral activity of textile products, and ISO 18071:2016 Fine ceramics—Determination of antiviral activity of semiconducting photocatalytic materials under indoor lighting environment—Test method using bacteriophage Q-beta). cleave the plasmid DNA; however, conversion of the plasmid DNA from the supercoiled to the open circular form was clearly observed in the systems of the hybrid Cu x O / TiO 2 nanocomposites. Notably, the degradation activity was enhanced as the ratio of Cu(I) / Cu(II) in the hybrid Cu x O / TiO 2 nanocomposites increased. The complete conversion of supercoiled DNA was achieved using a Cu x O / TiO 2 [Cu(I) / Cu(II) = 1.3] sample. These results suggest that the hybrid Cu x O / TiO 2 nanocomposites can destroy the critical biomolecules of viruses, leading to their death and inactivation, even under dark conditions.


Introduction
Human beings have suffered from numerous kinds of pandemic viruses, such as SARS [1], Ebola virus [2], H1N2/2009 influenza [3], and COVID-19 (SARS-CoV-2) [4]. These viruses spread through direct person-to-person contact and/or indirect contact via virus-containing airborne droplets or contaminated surfaces of objects such as floors, handrails, touch panel/buttons, or furniture [5]. Therefore, antiviral chemicals and/or materials are useful for protecting against the spread of pandemic-scale viruses. For example, alcohol [6], hydrogen peroxide [7], and hypochlorous acid [8] have been widely used to disinfect various objects against bacteria or viruses. These chemicals deactivate viruses by denaturising their proteins [9]. However, the antiviral effect of these chemicals is not sustainable over the long term because of their evaporation and/or dissipation. Conversely, solid-state antiviral metal compounds could be useful because of their robustness and feasibility for use as coating materials. Although the biocidal properties of copper and silver have been reported previously [10], their antiviral effects are insufficient and do not last over the long term. Once their We anticipated three plausible reasons for the efficient antiviral properties of Cu2O, as shown in Figure 2: (a) reactive oxygen species (ROS) [41], (b) leached copper ions [10], and (c) the solid-state compound itself [34,35]. Based on our careful investigation, we excluded ROS by evaluating the antiviral properties under nitrogen atmosphere. The antiviral activity of Cu2O under nitrogen was consistent with that under oxygen atmosphere, indicating that ROS did not contribute to the antiviral activity of Cu2O. It was also found that leached copper ions did not influence the antiviral activity of Cu2O according to a control experiment using a copper ion solution [34]. Therefore, the most plausible reason for the efficient antiviral properties of Cu2O is the solid-state Cu2O compound itself involving Cu(I) species. There are several experimental results that support the importance of direct physical contact between Cu2O and viruses [34]. For example, we inserted a 105 µm thickness of filter paper (pore size = 30 nm) between the Cu2O-coated glass substrate and the viral suspension, which inhibited the antiviral properties of the Cu2O [34]. Furthermore, we chemically modified the Cu2O surface with 1H-benzotriazole (BTA), which strongly coordinates with surface copper atoms via the nitrogen atoms of its triazole ring [42], and the results showed that the antiviral properties of Cu2O treated with BTA were significantly worse than those of untreated Cu2O [34]. These results strongly imply that the surface of Cu2O causes the denaturation or degradation of biomolecules in viruses, which results in their inactivation.  We anticipated three plausible reasons for the efficient antiviral properties of Cu 2 O, as shown in Figure 2: (a) reactive oxygen species (ROS) [41], (b) leached copper ions [10], and (c) the solid-state compound itself [34,35]. Based on our careful investigation, we excluded ROS by evaluating the antiviral properties under nitrogen atmosphere. The antiviral activity of Cu 2 O under nitrogen was consistent with that under oxygen atmosphere, indicating that ROS did not contribute to the antiviral activity of Cu 2 O. It was also found that leached copper ions did not influence the antiviral activity of Cu 2 O according to a control experiment using a copper ion solution [34]. Therefore, the most plausible reason for the efficient antiviral properties of Cu 2 O is the solid-state Cu 2 O compound itself involving Cu(I) species. There are several experimental results that support the importance of direct physical contact between Cu 2 O and viruses [34]. For example, we inserted a 105 µm thickness of filter paper (pore size = 30 nm) between the Cu 2 O-coated glass substrate and the viral suspension, which inhibited the antiviral properties of the Cu 2 O [34]. Furthermore, we chemically modified the Cu 2 O surface with 1H-benzotriazole (BTA), which strongly coordinates with surface copper atoms via the nitrogen atoms of its triazole ring [42], and the results showed that the antiviral properties of Cu 2 O treated with BTA were significantly worse than those of untreated Cu 2 O [34]. These results strongly imply that the surface of Cu 2 O causes the denaturation or degradation of biomolecules in viruses, which results in their inactivation. We anticipated three plausible reasons for the efficient antiviral properties of Cu2O, as shown in Figure 2: (a) reactive oxygen species (ROS) [41], (b) leached copper ions [10], and (c) the solid-state compound itself [34,35]. Based on our careful investigation, we excluded ROS by evaluating the antiviral properties under nitrogen atmosphere. The antiviral activity of Cu2O under nitrogen was consistent with that under oxygen atmosphere, indicating that ROS did not contribute to the antiviral activity of Cu2O. It was also found that leached copper ions did not influence the antiviral activity of Cu2O according to a control experiment using a copper ion solution [34]. Therefore, the most plausible reason for the efficient antiviral properties of Cu2O is the solid-state Cu2O compound itself involving Cu(I) species. There are several experimental results that support the importance of direct physical contact between Cu2O and viruses [34]. For example, we inserted a 105 µm thickness of filter paper (pore size = 30 nm) between the Cu2O-coated glass substrate and the viral suspension, which inhibited the antiviral properties of the Cu2O [34]. Furthermore, we chemically modified the Cu2O surface with 1H-benzotriazole (BTA), which strongly coordinates with surface copper atoms via the nitrogen atoms of its triazole ring [42], and the results showed that the antiviral properties of Cu2O treated with BTA were significantly worse than those of untreated Cu2O [34]. These results strongly imply that the surface of Cu2O causes the denaturation or degradation of biomolecules in viruses, which results in their inactivation.   To verify the distinctive antiviral mechanism of Cu 2 O, we investigated the adsorption properties of model protein molecules [bovine serum albumin (BSA)] on the surface of Cu 2 O, because the outer capsids of bacteriophage Qβ are composed of protein molecules. Figure 3a shows the adsorption properties of Cu 2 O in comparison with those of CuO and silver (Ag) as control groups. We used Ag for comparison because metallic Ag compounds have also been reported as effective anti-bacterial materials [43][44][45][46]. As shown in Figure 3a, the incubation of a 130 ng/mL solution of BSA with Cu 2 O for 8 h resulted in a 30% decrease in the supernatant concentration, revealing strong protein adsorption onto the solid-state Cu 2 O. Conversely, BSA adsorption onto CuO and Ag was limited. Furthermore, we investigated the protein denaturation by measuring the enzyme activity of alkaline phosphatase as a model enzyme, and the results are shown in Figure 3b. After exposure of the enzyme to Cu 2 O for 1 h, the enzyme activity decreased to 30% and 50% of the original activity at enzyme concentrations of 148 and 240 ng/mL, respectively. However, after exposure to CuO or Ag, the active enzyme concentration did not decrease from that of its original state. These results strongly imply that the protein adsorption and denaturation abilities of solid-state Cu 2 O are significantly higher than those of CuO and Ag, resulting in strong deactivation of bacteriophage Qβ. To verify the distinctive antiviral mechanism of Cu2O, we investigated the adsorption properties of model protein molecules [bovine serum albumin (BSA)] on the surface of Cu2O, because the outer capsids of bacteriophage Qβ are composed of protein molecules. Figure 3a shows the adsorption properties of Cu2O in comparison with those of CuO and silver (Ag) as control groups. We used Ag for comparison because metallic Ag compounds have also been reported as effective anti-bacterial materials [43][44][45][46]. As shown in Figure 3a, the incubation of a 130 ng/mL solution of BSA with Cu2O for 8 h resulted in a 30% decrease in the supernatant concentration, revealing strong protein adsorption onto the solid-state Cu2O. Conversely, BSA adsorption onto CuO and Ag was limited. Furthermore, we investigated the protein denaturation by measuring the enzyme activity of alkaline phosphatase as a model enzyme, and the results are shown in Figure 3b. After exposure of the enzyme to Cu2O for 1 h, the enzyme activity decreased to 30% and 50% of the original activity at enzyme concentrations of 148 and 240 ng/mL, respectively. However, after exposure to CuO or Ag, the active enzyme concentration did not decrease from that of its original state. These results strongly imply that the protein adsorption and denaturation abilities of solid-state Cu2O are significantly higher than those of CuO and Ag, resulting in strong deactivation of bacteriophage Qβ.  [34]. These data are based on average of triplicate measurements.
To further verify the disinfection of influenza viruses by Cu2O, we focused on the viral surface proteins that are highly involved in the infection process. Influenza viruses consist of hundreds of haemagglutinin (HA) and neuraminidase (NA) protein groups on the envelope surface. HA is a glycosylated lectin protein that recognizes sialic acid residues on the receptor proteins of the host cells [47]. Once influenza viruses bind through the HA-sialic acid interaction, they can enter the host cells through endocytosis. NA is an endoglycosidase that is necessary for the release of viruses from the surfaces of host cells; it is also involved in the initiation of influenza infection [48]. Both proteins play important roles in the spread of influenza infection. To determine HA activity after exposure to copper oxides, the HA protein was incubated and mixed with chicken red blood cells [49]. To determine NA activity, the 1,2-dioxetane derivative of sialic acid (NA-STAR) was used as a chemiluminescence substrate for highly sensitive detection [50]. Figure 4a,b show the changes in HA and NA activity. After exposure to Cu2O, the HA titer drastically decreased and fell below the detection limit within 30 min. Conversely, the HA titer after exposure to CuO did not change over 30 min. Similarly, NA activity decreased after exposure to Cu2O after 10 min, whereas NA activity was not influenced by exposure to CuO. These results reveal that both the haemagglutination ability of HA and the enzymatic activity of NA are disrupted by exposure to Cu2O. Based on these results, we can conclude that the protein denaturation property of Cu2O yields efficient antiviral function, even under dark conditions. shows enzyme activities of these materials after 1 h exposure [34]. These data are based on average of triplicate measurements.
To further verify the disinfection of influenza viruses by Cu 2 O, we focused on the viral surface proteins that are highly involved in the infection process. Influenza viruses consist of hundreds of haemagglutinin (HA) and neuraminidase (NA) protein groups on the envelope surface. HA is a glycosylated lectin protein that recognizes sialic acid residues on the receptor proteins of the host cells [47]. Once influenza viruses bind through the HA-sialic acid interaction, they can enter the host cells through endocytosis. NA is an endoglycosidase that is necessary for the release of viruses from the surfaces of host cells; it is also involved in the initiation of influenza infection [48]. Both proteins play important roles in the spread of influenza infection. To determine HA activity after exposure to copper oxides, the HA protein was incubated and mixed with chicken red blood cells [49]. To determine NA activity, the 1,2-dioxetane derivative of sialic acid (NA-STAR) was used as a chemiluminescence substrate for highly sensitive detection [50]. Figure 4a,b show the changes in HA and NA activity. After exposure to Cu 2 O, the HA titer drastically decreased and fell below the detection limit within 30 min. Conversely, the HA titer after exposure to CuO did not change over 30 min. Similarly, NA activity decreased after exposure to Cu 2 O after 10 min, whereas NA activity was not influenced by exposure to CuO. These results reveal that both the haemagglutination ability of HA and the enzymatic activity of NA are disrupted by exposure to Cu 2 O. Based on these results, we can conclude that the protein denaturation property of Cu 2 O yields efficient antiviral function, even under dark conditions. . Hemagglutinin (HA) titer and neuraminidase (NA) activity exposed to Cu2O and CuO suspensions. Effect on (a) HA titer and (b) NA activity of Cu2O (red squares) and CuO (blue circles) as determined by a hemagglutination test and chemiluminescence using the NA-Star method, respectively. N0 in panel (b) is the initial NA amount [35]. These data are based on an average of triplicate measurements.
Although Cu2O exhibits strong antiviral properties, Cu(I) is easily oxidized to Cu(II) states under ambient humid atmosphere. In fact, the antiviral properties of Cu2O exposed to humid air (relative humidity 90% at 25 °C) for one week or two weeks significantly worsened compared to those of fresh Cu2O ( Figure 5). These results indicate that the antiviral activity of Cu2O is decreased by its selfoxidation [51]. Platzman et al. reported that the Cu2O surface transformed to a copper hydroxide [Cu(OH)2] metastable state with several nanometres in thickness, due to the interactions of Cu ions with hydroxyl groups present at the surface [52]. Further, the metastable Cu(OH)2 phase transformed into a stable CuO layer [51,52]. Therefore, keeping Cu(I) species on the surface of Cu2O under ambient conditions is important for achieving the sustained antiviral activity of Cu2O. Figure 5. Antiviral properties of Cu2O after a week storage in 90% humid air atmosphere (green circles), those after two weeks storage in 90% humid air (blue triangles), and those of as-prepared sample using fresh Cu2O powder (FUJIFILM Wako Pure Chemical Corporation) taken from a commercial bottle (red squares). The data were based on averages of triplicate measurements for asprepared sample, while duplicate measurements for 1 and 2 weeks after samples.  is the initial NA amount [35]. These data are based on an average of triplicate measurements.

Visible Light-Sensitive Cu(II)/TiO2 Photocatalyst
Although Cu 2 O exhibits strong antiviral properties, Cu(I) is easily oxidized to Cu(II) states under ambient humid atmosphere. In fact, the antiviral properties of Cu 2 O exposed to humid air (relative humidity 90% at 25 • C) for one week or two weeks significantly worsened compared to those of fresh Cu 2 O ( Figure 5). These results indicate that the antiviral activity of Cu 2 O is decreased by its self-oxidation [51]. Platzman et al. reported that the Cu 2 O surface transformed to a copper hydroxide [Cu(OH) 2 ] metastable state with several nanometres in thickness, due to the interactions of Cu ions with hydroxyl groups present at the surface [52]. Further, the metastable Cu(OH) 2 phase transformed into a stable CuO layer [51,52]. Therefore, keeping Cu(I) species on the surface of Cu 2 O under ambient conditions is important for achieving the sustained antiviral activity of Cu 2 O. . Hemagglutinin (HA) titer and neuraminidase (NA) activity exposed to Cu2O and CuO suspensions. Effect on (a) HA titer and (b) NA activity of Cu2O (red squares) and CuO (blue circles) as determined by a hemagglutination test and chemiluminescence using the NA-Star method, respectively. N0 in panel (b) is the initial NA amount [35]. These data are based on an average of triplicate measurements.
Although Cu2O exhibits strong antiviral properties, Cu(I) is easily oxidized to Cu(II) states under ambient humid atmosphere. In fact, the antiviral properties of Cu2O exposed to humid air (relative humidity 90% at 25 °C) for one week or two weeks significantly worsened compared to those of fresh Cu2O ( Figure 5). These results indicate that the antiviral activity of Cu2O is decreased by its selfoxidation [51]. Platzman et al. reported that the Cu2O surface transformed to a copper hydroxide [Cu(OH)2] metastable state with several nanometres in thickness, due to the interactions of Cu ions with hydroxyl groups present at the surface [52]. Further, the metastable Cu(OH)2 phase transformed into a stable CuO layer [51,52]. Therefore, keeping Cu(I) species on the surface of Cu2O under ambient conditions is important for achieving the sustained antiviral activity of Cu2O. Figure 5. Antiviral properties of Cu2O after a week storage in 90% humid air atmosphere (green circles), those after two weeks storage in 90% humid air (blue triangles), and those of as-prepared sample using fresh Cu2O powder (FUJIFILM Wako Pure Chemical Corporation) taken from a commercial bottle (red squares). The data were based on averages of triplicate measurements for asprepared sample, while duplicate measurements for 1 and 2 weeks after samples.

Visible Light-Sensitive Cu(II)/TiO 2 Photocatalyst
The previous section suggests that maintaining the Cu(I) species is critical for sustaining antiviral properties over the long term. The main goal of this paper is to introduce the combination of a TiO 2 photocatalyst with Cu x O nanoclusters containing Cu(I) and Cu(II) species to achieve sustained antiviral properties. Before providing a detailed explanation of the Cu x O/TiO 2 system, we describe the role of the Cu(II) species attached to the TiO 2 photocatalyst.
We previously reported Cu(II) nanoclusters grafted onto TiO 2 [Cu(II)/TiO 2 ] as an efficient visible light-sensitive photocatalyst for the oxidation of organic molecules [26,27]. Cu(II) nanoclusters could be grafted onto TiO 2 (rutile, MT-150A, TAYCA Corporation) by wet chemical impregnation method using copper chloride dissolved aqueous media (0.1 wt % versus TiO 2 ) as reported in our previous studies [26,27]. Figure 6a shows a transmission electron microscope (TEM) image of Cu(II)/TiO 2 , where Cu(II) clusters a few nanometres in size were grafted onto the TiO 2 surface. Although the size of the Cu(II) nanocluster was too small to detect its X-ray diffraction, a previous study determined the local chemical structure of the Cu(II) nanoclusters by X-ray absorption near-edge structure (XANES) and extended X-ray absorption fine structure (EXAFS) [27]. Figure 6b shows the XANES spectra of Cu(II)/TiO 2 and commercial reference powders. The spectrum of Cu(II)/TiO 2 resembles that of Cu(OH) 2 , indicating that the valence number of the nanoclusters is in the 2+ state and that the Cu(II) species are likely to be in the five-coordinate square pyramidal form [53][54][55]. Figure 6c shows the EXAFS results of Cu(II)/TiO 2 and commercial powder references of Cu(OH) 2 and CuO. In contrast to the XANES results, the local chemical environment of the Cu(II) nanoclusters resembles that of CuO. The EXAFS data were carefully analysed using the REX2000 (Rigaku Corporation) and the FEFF program [56], and a one-coordinate Cu-O bond length (2.1-2.2 Å) was observed in Cu(OH) 2 and Cu(II)/TiO 2 . Thus, the grafted Cu(II) nanoclusters are in the five-coordinate environment, which is consistent with the XANES results. In addition, one four-coordinate Cu-Cu and three types of two-coordinate Cu-Cu were observed, and the Cu-Cu bond lengths were similar to those in CuO, and so it can be considered that the grafted Cu(II) nanoclusters resemble the chemical environment of Cu(II) in CuO. That is, the local structure of the Cu(II) nanoclusters is distorted CuO, wherein the apical oxygen approaches Cu(II), forming a five-coordinate square pyramid attached to the TiO 2 surface [27].
Catalysts 2020, 10, x FOR PEER REVIEW 6 of 19 The previous section suggests that maintaining the Cu(I) species is critical for sustaining antiviral properties over the long term. The main goal of this paper is to introduce the combination of a TiO2 photocatalyst with CuxO nanoclusters containing Cu(I) and Cu(II) species to achieve sustained antiviral properties. Before providing a detailed explanation of the CuxO/TiO2 system, we describe the role of the Cu(II) species attached to the TiO2 photocatalyst.
We previously reported Cu(II) nanoclusters grafted onto TiO2 [Cu(II)/TiO2] as an efficient visible light-sensitive photocatalyst for the oxidation of organic molecules [26,27]. Cu(II) nanoclusters could be grafted onto TiO2 (rutile, MT-150A, TAYCA Corporation) by wet chemical impregnation method using copper chloride dissolved aqueous media (0.1 wt % versus TiO2) as reported in our previous studies [26,27]. Figure 6a shows a transmission electron microscope (TEM) image of Cu(II)/TiO2, where Cu(II) clusters a few nanometres in size were grafted onto the TiO2 surface. Although the size of the Cu(II) nanocluster was too small to detect its X-ray diffraction, a previous study determined the local chemical structure of the Cu(II) nanoclusters by X-ray absorption near-edge structure (XANES) and extended X-ray absorption fine structure (EXAFS) [27]. Figure 6b shows the XANES spectra of Cu(II)/TiO2 and commercial reference powders. The spectrum of Cu(II)/TiO2 resembles that of Cu(OH)2, indicating that the valence number of the nanoclusters is in the 2+ state and that the Cu(II) species are likely to be in the five-coordinate square pyramidal form [53][54][55]. Figure 6c shows the EXAFS results of Cu(II)/TiO2 and commercial powder references of Cu(OH)2 and CuO. In contrast to the XANES results, the local chemical environment of the Cu(II) nanoclusters resembles that of CuO. The EXAFS data were carefully analysed using the REX2000 (Rigaku Corporation) and the FEFF program [56], and a one-coordinate Cu-O bond length (2.1-2.2 Å) was observed in Cu(OH)2 and Cu(II)/TiO2. Thus, the grafted Cu(II) nanoclusters are in the five-coordinate environment, which is consistent with the XANES results. In addition, one four-coordinate Cu-Cu and three types of twocoordinate Cu-Cu were observed, and the Cu-Cu bond lengths were similar to those in CuO, and so it can be considered that the grafted Cu(II) nanoclusters resemble the chemical environment of Cu(II) in CuO. That is, the local structure of the Cu(II) nanoclusters is distorted CuO, wherein the apical oxygen approaches Cu(II), forming a five-coordinate square pyramid attached to the TiO2 surface [27].   Figure 7a shows the UV-vis absorption spectra of pristine TiO 2 and Cu(II)/TiO 2 . The pristine TiO 2 exhibited strong UV light absorption shorter than 400 nm owing to its bandgap excitation. Meanwhile, Cu(II)/TiO 2 exhibited additional visible-light absorption around 400-480 nm and over 650 nm. The former absorption is owing to the inter facial charge transfer (IFCT) excitation from the valence band of TiO 2 to the Cu(II) nanocluster [26,27], whereas the latter originates in the d-d transition in the Cu(II) species [57]. The IFCT process is theoretically feasible between a semiconductor and ligand under photon irradiation [58], and visible-light absorption through IFCT was experimentally observed in previous studies [59][60][61]. The IFCT transition was also observed in the iron oxide-based Fe(III) nanocluster-grafted TiO 2 [31,62].
Catalysts 2020, 10, x FOR PEER REVIEW 7 of 19 Figure 7a shows the UV-vis absorption spectra of pristine TiO2 and Cu(II)/TiO2. The pristine TiO2 exhibited strong UV light absorption shorter than 400 nm owing to its bandgap excitation. Meanwhile, Cu(II)/TiO2 exhibited additional visible-light absorption around 400-480 nm and over 650 nm. The former absorption is owing to the inter facial charge transfer (IFCT) excitation from the valence band of TiO2 to the Cu(II) nanocluster [26,27], whereas the latter originates in the d-d transition in the Cu(II) species [57]. The IFCT process is theoretically feasible between a semiconductor and ligand under photon irradiation [58], and visible-light absorption through IFCT was experimentally observed in previous studies [59][60][61]. The IFCT transition was also observed in the iron oxide-based Fe(III) nanocluster-grafted TiO2 [31,62].  Figure 7b shows the photocatalytic oxidation activities of gaseous 2-propanol to carbon dioxide (CO2) under visible-light irradiation. As control groups, we also evaluated the photocatalytic activities of bare TiO2 and nitrogen-doped TiO2 (TiO2-xNx). The TiO2-xNx photocatalyst, which is recognized as an efficient visible-light photocatalyst [63], was prepared by a wet chemical method using titanium tetrachloride and ammonia, similar to a previous report [64]. The activity of pristine TiO2 was limited because of the lack of its visible-light absorption. In the case of TiO2-xNx, CO2 molecules were generated by the oxidation of 2-propanol; however, its activity was worse than that of Cu(II)/TiO2 because of the lower oxidation power of the holes excited in the nitrogen orbital [65][66][67]. It is noted that the Cu(II)/TiO2 photocatalyst decomposed 2-propanol with an initial amount of 5 μmol, producing approximately 15 μmol of CO2, showing that complete decomposition was achieved under visible-light irradiation. The quantum efficiency of the Cu(II)/TiO2 system reached over 80% by the optimization of the fabrication process [29], and thus it was significantly superior to that of TiO2-xNx [65,66].
The mechanism of the photocatalytic reaction by Cu(II)/TiO2 was previously investigated by various spectroscopic analyses. For example, Nosaka et al. examined the in situ electron spin resonance (ESR) of Cu(II)/TiO2 under visible-light irradiation [68]. Cu(II) species involve unpaired electrons, thus exhibiting an ESR signal, whereas Cu(I) is ESR-inactive. Furthermore, the photogenerated electrons and holes in TiO2 can be detected by ESR. When the Cu(II)/TiO2 sample was irradiated by visible light under vacuum conditions, the ESR signal of the Cu(II) species decreased and that of photogenerated holes in the valence band of TiO2 appeared. These results strongly suggest that the electron transition occurs from the valence band of TiO2 to the Cu(II) species through their interface under visible-light irradiation to generate Cu(I) species and holes in TiO2. The signal of the photogenerated holes decreased by the introduction of gaseous 2-propanol into the ESR chamber, whereas that of Cu(II) recovered by exposure to oxygen [68]. These results also indicate that the photogenerated holes oxidize 2-propanol, whereas excited electrons in the copper ion species  Figure 7b shows the photocatalytic oxidation activities of gaseous 2-propanol to carbon dioxide (CO 2 ) under visible-light irradiation. As control groups, we also evaluated the photocatalytic activities of bare TiO 2 and nitrogen-doped TiO 2 (TiO 2-x N x ). The TiO 2-x N x photocatalyst, which is recognized as an efficient visible-light photocatalyst [63], was prepared by a wet chemical method using titanium tetrachloride and ammonia, similar to a previous report [64]. The activity of pristine TiO 2 was limited because of the lack of its visible-light absorption. In the case of TiO 2-x N x , CO 2 molecules were generated by the oxidation of 2-propanol; however, its activity was worse than that of Cu(II)/TiO 2 because of the lower oxidation power of the holes excited in the nitrogen orbital [65][66][67]. It is noted that the Cu(II)/TiO 2 photocatalyst decomposed 2-propanol with an initial amount of 5 µmol, producing approximately 15 µmol of CO 2 , showing that complete decomposition was achieved under visible-light irradiation. The quantum efficiency of the Cu(II)/TiO 2 system reached over 80% by the optimization of the fabrication process [29], and thus it was significantly superior to that of TiO 2-x N x [65,66].
The mechanism of the photocatalytic reaction by Cu(II)/TiO 2 was previously investigated by various spectroscopic analyses. For example, Nosaka et al. examined the in situ electron spin resonance (ESR) of Cu(II)/TiO 2 under visible-light irradiation [68]. Cu(II) species involve unpaired electrons, thus exhibiting an ESR signal, whereas Cu(I) is ESR-inactive. Furthermore, the photogenerated electrons and holes in TiO 2 can be detected by ESR. When the Cu(II)/TiO 2 sample was irradiated by visible light under vacuum conditions, the ESR signal of the Cu(II) species decreased and that of photogenerated holes in the valence band of TiO 2 appeared. These results strongly suggest that the electron transition occurs from the valence band of TiO 2 to the Cu(II) species through their interface under visible-light irradiation to generate Cu(I) species and holes in TiO 2 . The signal of the photogenerated holes decreased by the introduction of gaseous 2-propanol into the ESR chamber, whereas that of Cu(II) recovered by exposure to oxygen [68]. These results also indicate that the photogenerated holes oxidize 2-propanol, whereas excited electrons in the copper ion species react with oxygen molecules. Formation of Cu(I) species on TiO 2 under light irradiation was also reported in the other previous literature [69]. The redox potential of Cu(II)/Cu(I) is approximately 0.16 V [versus a normal hydrogen electrode (NHE)] [26,27], which is more negative than that of the multi-electron reduction reaction of oxygen molecules to hydrogen peroxide (0.68 V vs. NHE) [70][71][72]. Therefore, excited electrons in the Cu(I) species react with oxygen molecules through a multi-electron reduction process under an oxygen-abundant atmosphere. A similar electron transition trend was seen in the XANES results [27]. Furthermore, Osako et al. visualized the reduction and oxidation sites in a Cu(II)/TiO 2 system by using an ultrathin CuO film with a well-defined pattern coated onto a TiO 2 single crystal prepared by pulsed laser deposition and photolithography [73]. Using an atomic force microscope (AFM), the authors observed the formation of metal Ag particles on the film resulting from the photoreduction of Ag + ions, and Ag particles were selectively deposited on the edge of a CuO film under visible-light irradiation [74]. These results also suggest that the IFCT transition occurs by visible light and that the Cu(II) species acts as reduction sites. The concept of an IFCT transition for the development of visible light-sensitive photocatalysts has been extended to semiconductor systems other than TiO 2 , such as ZnO [75,76], SrTiO 3 [77,78] and Ag-based compounds [84]. The concept of an IFCT transition was also adopted for impurity-doped TiO 2 , such as Ti(III) self-doped TiO 2 [28], Nb(IV)-doped TiO 2 [85], and W(IV) and Ga(III)-codoped TiO 2 [86]. Figure 8 shows the antiviral bacteriophage Qβ activity of TiO 2-x N x and Cu(II)/TiO 2 under white-light irradiation and dark conditions. Among these samples, the antiviral activity of Cu(II)/TiO 2 under white-light irradiation was the most significant. Even though TiO 2-x N x exhibited photocatalytic oxidation activity for 2-propanol [ Figure 7b], its antiviral activity was negligible, attributed to its limited oxidation power [65][66][67]. In contrast, the number of bacteriophage Qβ on contact with Cu(II)/TiO 2 under white-light irradiation decreased more than two orders of magnitude after 60 min of exposure. The antiviral properties of Cu(II)/TiO 2 under dark conditions, however, were limited because the Cu(II) species was not as effective for the disinfection of viruses, as described in the previous section.  [26,27], which is more negative than that of the multi-electron reduction reaction of oxygen molecules to hydrogen peroxide (0.68 V vs. NHE) [70][71][72]. Therefore, excited electrons in the Cu(I) species react with oxygen molecules through a multielectron reduction process under an oxygen-abundant atmosphere. A similar electron transition trend was seen in the XANES results [27]. Furthermore, Osako et al. visualized the reduction and oxidation sites in a Cu(II)/TiO2 system by using an ultrathin CuO film with a well-defined pattern coated onto a TiO2 single crystal prepared by pulsed laser deposition and photolithography [73].
Using an atomic force microscope (AFM), the authors observed the formation of metal Ag particles on the film resulting from the photoreduction of Ag + ions, and Ag particles were selectively deposited on the edge of a CuO film under visible-light irradiation [74]. These results also suggest that the IFCT transition occurs by visible light and that the Cu(II) species acts as reduction sites. The concept of an IFCT transition for the development of visible light-sensitive photocatalysts has been extended to semiconductor systems other than TiO2, such as ZnO [75,76], SrTiO3 [77,78], SnO2 [79], Nb3O8 - [80], Ag3PO4, Bi2O3 [81], BiOCl [82], BiVO4 [83], and Ag-based compounds [84]. The concept of an IFCT transition was also adopted for impurity-doped TiO2, such as Ti(III) self-doped TiO2 [28], Nb(IV)doped TiO2 [85], and W(IV) and Ga(III)-codoped TiO2 [86]. Figure 8 shows the antiviral bacteriophage Qβ activity of TiO2-xNx and Cu(II)/TiO2 under whitelight irradiation and dark conditions. Among these samples, the antiviral activity of Cu(II)/TiO2 under white-light irradiation was the most significant. Even though TiO2-xNx exhibited photocatalytic oxidation activity for 2-propanol [ Figure 7b], its antiviral activity was negligible, attributed to its limited oxidation power [65][66][67]. In contrast, the number of bacteriophage Qβ on contact with Cu(II)/TiO2 under white-light irradiation decreased more than two orders of magnitude after 60 min of exposure. The antiviral properties of Cu(II)/TiO2 under dark conditions, however, were limited because the Cu(II) species was not as effective for the disinfection of viruses, as described in the previous section. Through the IFCT transition in Cu(II)/TiO2, the Cu(I) species are created, in addition to the generation of holes in the valence band of TiO2. The produced Cu(I) species are effective for protein denaturation, and the holes, which have strong oxidation power, causing protein decomposition, and leading to virus disinfection. The contribution of the Cu(I) species generated by an IFCT transition to the antiviral properties was suggested by a previously reported "pre-irradiation" experiment [32].  Through the IFCT transition in Cu(II)/TiO 2 , the Cu(I) species are created, in addition to the generation of holes in the valence band of TiO 2 . The produced Cu(I) species are effective for protein denaturation, and the holes, which have strong oxidation power, causing protein decomposition, and leading to virus disinfection. The contribution of the Cu(I) species generated by an IFCT transition to the antiviral properties was suggested by a previously reported "pre-irradiation" experiment [32]. Figure 9 shows the antiviral activities of Cu(II)/TiO 2 under dark conditions without/with pre-irradiation. As a pre-irradiation treatment, the Cu(II)/TiO 2 sample was placed under a white fluorescence lightbulb passed through a UV cut-off film below 400 nm before the evaluation of the antiviral effect. After the pre-irradiation treatment, the Cu(II)/TiO 2 film was subjected to antiviral activity testing using bacteriophage Qβ under dark conditions. As shown in Figure 9, the pre-irradiation treatment improved the antiviral activity of Cu(II)/TiO 2 . This result suggests that pre-irradiation produced the Cu(I) species through the IFCT process, and some of them reacted with oxygen molecules in air, but the others remained even in the dark for a while, causing an antiviral effect. The previous study also showed that pre-irradiation with UV light improved the antiviral activity of Cu(II)/TiO 2 [32], indicating that the excited electrons in the conduction band of TiO 2 would also be injected into Cu(II) nanoclusters to form Cu(I) species.
Catalysts 2020, 10, x FOR PEER REVIEW 9 of 19 Figure 9 shows the antiviral activities of Cu(II)/TiO2 under dark conditions without/with preirradiation. As a pre-irradiation treatment, the Cu(II)/TiO2 sample was placed under a white fluorescence lightbulb passed through a UV cut-off film below 400 nm before the evaluation of the antiviral effect. After the pre-irradiation treatment, the Cu(II)/TiO2 film was subjected to antiviral activity testing using bacteriophage Qβ under dark conditions. As shown in Figure 9, the preirradiation treatment improved the antiviral activity of Cu(II)/TiO2. This result suggests that preirradiation produced the Cu(I) species through the IFCT process, and some of them reacted with oxygen molecules in air, but the others remained even in the dark for a while, causing an antiviral effect. The previous study also showed that pre-irradiation with UV light improved the antiviral activity of Cu(II)/TiO2 [32], indicating that the excited electrons in the conduction band of TiO2 would also be injected into Cu(II) nanoclusters to form Cu(I) species. Figure 9. Inactivation of bacteriophage Qβ by Cu(II)/TiO2 under dark conditions without preirradiation (black) and after pre-irradiation treatment (red) [32]. The pre-irradiation treatment was conducted using a white fluorescence lightbulb passed through a UV cut-off film below 400 nm.

Antiviral CuxO/TiO2 Photocatalyst
Although Cu(II)/TiO2 exhibited efficient antiviral properties under visible-light irradiation, its antiviral function under dark conditions was limited as shown in Figure 8. Here, we introduce the CuxO (1 < x < 2) nanoclusters grafted TiO2 for efficient antiviral properties even under dark conditions. CuxO nanoclusters were facilely grafted onto TiO2 powder by a method similar to that used for the fabrication of Cu(II)/TiO2. Different from the case of Cu(II)/TiO2 synthesis, we added sodium hydroxide and glucose to the aqueous solution of copper chloride for the grafting process [33]. Glucose dissolved in an alkaline solution acts as a reducing agent of Cu(II) into Cu(I) species; thus, we could control the ratio of Cu(II)/Cu(I) in the CuxO nanoclusters by the concentration of glucose and sodium hydroxide in the aqueous solution [33]. Figure 10a shows the TEM image of CuxO/TiO2. Nanoclusters of CuxO were well dispersed on the surfaces of TiO2. In the X-ray diffraction (XRD) pattern of CuxO/TiO2 [33], no additional peaks other than those of TiO2 were observed, indicating the amorphous nature of the CuxO nanoclusters. Figure 10b shows the XANES spectra of CuxO/TiO2 with the reference data of commercial Cu2O and Cu(OH)2 powders. Peaks I and II are assigned to Cu(I) and Cu(II) species, respectively. The CuxO nanoclusters contained both Cu(I) and Cu(II) species. The ratio of Cu(I)/Cu(II) was estimated by their peak intensities in XANES, and the Cu(I)/Cu(II) ratio of the sample was 1.3, which is the optimum ratio to maintain efficient photocatalytic visible-light activity and sustain antiviral properties, which will be discussed later.   [32]. The pre-irradiation treatment was conducted using a white fluorescence lightbulb passed through a UV cut-off film below 400 nm.

Antiviral Cu x O/TiO 2 Photocatalyst
Although Cu(II)/TiO 2 exhibited efficient antiviral properties under visible-light irradiation, its antiviral function under dark conditions was limited as shown in Figure 8. Here, we introduce the Cu x O (1 < x < 2) nanoclusters grafted TiO 2 for efficient antiviral properties even under dark conditions. Cu x O nanoclusters were facilely grafted onto TiO 2 powder by a method similar to that used for the fabrication of Cu(II)/TiO 2 . Different from the case of Cu(II)/TiO 2 synthesis, we added sodium hydroxide and glucose to the aqueous solution of copper chloride for the grafting process [33]. Glucose dissolved in an alkaline solution acts as a reducing agent of Cu(II) into Cu(I) species; thus, we could control the ratio of Cu(II)/Cu(I) in the Cu x O nanoclusters by the concentration of glucose and sodium hydroxide in the aqueous solution [33]. Figure 10a shows the TEM image of Cu x O/TiO 2 . Nanoclusters of Cu x O were well dispersed on the surfaces of TiO 2 . In the X-ray diffraction (XRD) pattern of Cu x O/TiO 2 [33], no additional peaks other than those of TiO 2 were observed, indicating the amorphous nature of the Cu x O nanoclusters. Figure 10b shows the XANES spectra of Cu x O/TiO 2 with the reference data of commercial Cu 2 O and Cu(OH) 2 powders. Peaks I and II are assigned to Cu(I) and Cu(II) species, respectively. The Cu x O nanoclusters contained both Cu(I) and Cu(II) species. The ratio of Cu(I)/Cu(II) was estimated by their peak intensities in XANES, and the Cu(I)/Cu(II) ratio of the sample was 1.3, which is the optimum ratio to maintain efficient photocatalytic visible-light activity and sustain antiviral properties, which will be discussed later.  Figure 11a shows the optical absorption spectrum of CuxO/TiO2. In addition to the intrinsic interband absorption below 400 nm of TiO2, the absorption band assigned to the IFCT in the range of 400-500 nm [26,27], and the absorption over 650 nm attributable to the d-d transition of the Cu(II) species [57], all of which were observed with Cu(II)/TiO2, as described in the previous section. The CuxO/TiO2 nanocomposites showed an additional absorption band in the range of 500-600 nm, owing to the inter-band transition of Cu2O [87].  [33]. Visible-light irradiation was conducted using a xenon lamp passed through optical filters to set the wavelength at 400-530 nm with an illuminance of 1 mW/cm 2 . The ratio of Cu(I)/Cu(II) in CuxO was 1.3. Figure 11b shows the photocatalytic oxidation activities of gaseous 2-propanol to carbon dioxide (CO2) under visible-light irradiation. In addition to the CuxO/TiO2 composite, we evaluated the photocatalytic activities of TiO2 and nitrogen-doped TiO2 (TiO2-xNx) as control groups. The photocatalytic oxidation activity of CuxO/TiO2 was superior to those of TiO2 and TiO2-xNx and comparable to the Cu(II)/TiO2 result [ Figure 7b]. It is noted that the photocatalytic oxidation activity depends on the ratio of Cu(I)/Cu(II) [33]. A higher content of Cu(II) is better for photocatalytic oxidation activity. The ratio of Cu(I)/Cu(II) in the study sample was 1.3 [33], which optimized to exhibit high photocatalytic activity as well as antiviral activity under dark conditions, which is discussed below.    Figure 11a shows the optical absorption spectrum of CuxO/TiO2. In addition to the intrinsic interband absorption below 400 nm of TiO2, the absorption band assigned to the IFCT in the range of 400-500 nm [26,27], and the absorption over 650 nm attributable to the d-d transition of the Cu(II) species [57], all of which were observed with Cu(II)/TiO2, as described in the previous section. The CuxO/TiO2 nanocomposites showed an additional absorption band in the range of 500-600 nm, owing to the inter-band transition of Cu2O [87].  [33]. Visible-light irradiation was conducted using a xenon lamp passed through optical filters to set the wavelength at 400-530 nm with an illuminance of 1 mW/cm 2 . The ratio of Cu(I)/Cu(II) in CuxO was 1.3. Figure 11b shows the photocatalytic oxidation activities of gaseous 2-propanol to carbon dioxide (CO2) under visible-light irradiation. In addition to the CuxO/TiO2 composite, we evaluated the photocatalytic activities of TiO2 and nitrogen-doped TiO2 (TiO2-xNx) as control groups. The photocatalytic oxidation activity of CuxO/TiO2 was superior to those of TiO2 and TiO2-xNx and comparable to the Cu(II)/TiO2 result [ Figure 7b]. It is noted that the photocatalytic oxidation activity depends on the ratio of Cu(I)/Cu(II) [33]. A higher content of Cu(II) is better for photocatalytic oxidation activity. The ratio of Cu(I)/Cu(II) in the study sample was 1.3 [33], which optimized to exhibit high photocatalytic activity as well as antiviral activity under dark conditions, which is discussed below.  Figure 11b shows the photocatalytic oxidation activities of gaseous 2-propanol to carbon dioxide (CO 2 ) under visible-light irradiation. In addition to the Cu x O/TiO 2 composite, we evaluated the photocatalytic activities of TiO 2 and nitrogen-doped TiO 2 (TiO 2-x N x ) as control groups. The photocatalytic oxidation activity of Cu x O/TiO 2 was superior to those of TiO 2 and TiO 2-x N x and comparable to the Cu(II)/TiO 2 result [ Figure 7b]. It is noted that the photocatalytic oxidation activity depends on the ratio of Cu(I)/Cu(II) [33]. A higher content of Cu(II) is better for photocatalytic oxidation activity. The ratio of Cu(I)/Cu(II) in the study sample was 1.3 [33], which optimized to exhibit high photocatalytic activity as well as antiviral activity under dark conditions, which is discussed below. Figure 12 shows the antiviral properties of Cu x O/TiO 2 under white-light irradiation and dark conditions in comparison with TiO 2-x N x . The Cu x O/TiO 2 displayed a 4-log reduction (i.e., a 99.9 9% reduction of bacteriophage Qβ) after 1 h of contact time under dark conditions, which was significantly superior to the antiviral activity of Cu(II)/TiO 2 under dark conditions (Figure 8, black circles). The antiviral activity of Cu x O/TiO 2 was further improved under visible-light irradiation as a 7.5-log reduction of bacteriophage was achieved after 40 min. The Cu(I) species in Cu x O nanoclusters can denature proteins and lose virus activity under dark conditions. Also, the Cu(II) species in the Cu x O nanocluster accepts electrons from the valence band of TiO 2 to form a Cu(I) species through photo-induced IFCT transition. Therefore, both antiviral active species, i.e., the Cu(I) species and holes in the valence band of TiO 2 , are simultaneously created in the Cu x O/TiO 2 system under visible-light irradiation, exhibiting efficient antiviral function under both visible-light irradiation and dark conditions. Figure 12 shows the antiviral properties of CuxO/TiO2 under white-light irradiation and dark conditions in comparison with TiO2-xNx. The CuxO/TiO2 displayed a 4-log reduction (i.e., a 99.9 9% reduction of bacteriophage Qβ) after 1 h of contact time under dark conditions, which was significantly superior to the antiviral activity of Cu(II)/TiO2 under dark conditions (Figure 8, black circles). The antiviral activity of CuxO/TiO2 was further improved under visible-light irradiation as a 7.5-log reduction of bacteriophage was achieved after 40 min. The Cu(I) species in CuxO nanoclusters can denature proteins and lose virus activity under dark conditions. Also, the Cu(II) species in the CuxO nanocluster accepts electrons from the valence band of TiO2 to form a Cu(I) species through photo-induced IFCT transition. Therefore, both antiviral active species, i.e., the Cu(I) species and holes in the valence band of TiO2, are simultaneously created in the CuxO/TiO2 system under visiblelight irradiation, exhibiting efficient antiviral function under both visible-light irradiation and dark conditions.  [33]. Light irradiation was conducted using a commercial 10 W cylindrical white fluorescent lightbulb with a UV cut-off film at an illuminance of 800 lux.
Here, we discuss the optimum ratio of Cu(I)/Cu(II) for both photocatalytic visible light-activity and antiviral properties under dark conditions. We previously evaluated the visible-light activities of CuxO/TiO2 samples with Cu(I)/Cu(II) ratios of 0.13, 0.2, and 1.3 [33], and those activities were comparable to that of Cu(II)/TiO2. We also investigated the degradation activity of DNA, which is an essential component of viruses, for the various CuxO/TiO2 samples with different Cu(I)/Cu(II) ratios and pristine TiO2 as a control group [33]. Figure 13 shows the resulting agarose gel electrophoresis patterns after the exposure of supercoiled plasmid pBR322 DNA to various samples for 2 h under dark conditions. Among the examined samples, bare TiO2 did not cleave the plasmid DNA; however, conversion of the plasmid DNA from the supercoiled to the open circular form was clearly observed in the systems of the hybrid CuxO/TiO2 nanocomposites. Notably, the degradation activity was enhanced as the ratio of Cu(I)/Cu(II) in the hybrid CuxO/TiO2 nanocomposites increased. The complete conversion of supercoiled DNA was achieved using a CuxO/TiO2 [Cu(I)/Cu(II) = 1.3] sample. These results suggest that the hybrid CuxO/TiO2 nanocomposites can destroy the critical biomolecules of viruses, leading to their death and inactivation, even under dark conditions.  [33]. Light irradiation was conducted using a commercial 10 W cylindrical white fluorescent lightbulb with a UV cut-off film at an illuminance of 800 lux.
Here, we discuss the optimum ratio of Cu(I)/Cu(II) for both photocatalytic visible light-activity and antiviral properties under dark conditions. We previously evaluated the visible-light activities of Cu x O/TiO 2 samples with Cu(I)/Cu(II) ratios of 0.13, 0.2, and 1.3 [33], and those activities were comparable to that of Cu(II)/TiO 2 . We also investigated the degradation activity of DNA, which is an essential component of viruses, for the various Cu x O/TiO 2 samples with different Cu(I)/Cu(II) ratios and pristine TiO 2 as a control group [33]. Figure  Next, we investigated the long-term antiviral properties of CuxO/TiO2 [Cu(I)/Cu(II) = 1.3] according to the following procedure using bacteriophage Qβ. First, the as-prepared CuxO/TiO2 sample was initially examined under dark conditions [label (i) in Figure 14]. Second, the sample stored under ambient air conditions for more than 6 years was examined [label (ii) in Figure 14]. Third, the stored sample was irradiated with white light for 4 days, and its antiviral properties were evaluated under dark conditions [label (iii) in Figure 14]. The initial activity of CuxO/TiO2 decreased under ambient air exposure by self-oxidation [(i)→(ii)], similar to the results for bare Cu2O shown in Figure 5. However, the deteriorated activity after air exposure was significantly recovered by light irradiation for 4 days. These results imply that the oxidized Cu(II) species in CuxO can be recovered to Cu(I) species by light irradiation. Such a recovery function has never been observed in a pristine Cu2O sample or other solid-state antiviral materials. In contrast to conventional antiviral solid materials, our CuxO/TiO2 maintains its efficient antiviral function, even when light illumination is turned on during the day and off during the night.   Figure 14]. Second, the sample stored under ambient air conditions for more than 6 years was examined [label (ii) in Figure 14]. Third, the stored sample was irradiated with white light for 4 days, and its antiviral properties were evaluated under dark conditions [label (iii) in Figure 14]. The initial activity of Cu x O/TiO 2 decreased under ambient air exposure by self-oxidation [(i)→(ii)], similar to the results for bare Cu 2 O shown in Figure 5. However, the deteriorated activity after air exposure was significantly recovered by light irradiation for 4 days. These results imply that the oxidized Cu(II) species in Cu x O can be recovered to Cu(I) species by light irradiation. Such a recovery function has never been observed in a pristine Cu 2 O sample or other solid-state antiviral materials. In contrast to conventional antiviral solid materials, our Cu x O/TiO 2 maintains its efficient antiviral function, even when light illumination is turned on during the day and off during the night. Next, we investigated the long-term antiviral properties of CuxO/TiO2 [Cu(I)/Cu(II) = 1.3] according to the following procedure using bacteriophage Qβ. First, the as-prepared CuxO/TiO2 sample was initially examined under dark conditions [label (i) in Figure 14]. Second, the sample stored under ambient air conditions for more than 6 years was examined [label (ii) in Figure 14]. Third, the stored sample was irradiated with white light for 4 days, and its antiviral properties were evaluated under dark conditions [label (iii) in Figure 14]. The initial activity of CuxO/TiO2 decreased under ambient air exposure by self-oxidation [(i)→(ii)], similar to the results for bare Cu2O shown in Figure 5. However, the deteriorated activity after air exposure was significantly recovered by light irradiation for 4 days. These results imply that the oxidized Cu(II) species in CuxO can be recovered to Cu(I) species by light irradiation. Such a recovery function has never been observed in a pristine Cu2O sample or other solid-state antiviral materials. In contrast to conventional antiviral solid materials, our CuxO/TiO2 maintains its efficient antiviral function, even when light illumination is turned on during the day and off during the night.    Figure 15 shows a schematic illustration of the working principle of the present antiviral CuxO/TiO2 photocatalyst. Cu(I) species disinfect viruses by denaturalizing their protein under dark conditions. Under light irradiation, photogenerated holes oxidize the organic components of the viruses. Further, light irradiation continuously produces Cu(I) species to suppress the self-oxidation of CuxO, resulting in sustained antiviral properties.  Table 1 summarizes the comparison of the antiviral properties of various copper-based compounds. The antiviral activity of pristine CuO is negligible. Conversely, pristine Cu2O exhibits efficient antiviral properties at its initial use; however, its initial red colour turns black by selfoxidation to change into Cu(II) inactive species [51,52]. Further, Cu(II)/TiO2 shows photocatalytic oxidation activity under visible light because of the IFCT transition, but its antiviral activity is limited because of the lack of Cu(I) species. Among these samples, the CuxO/TiO2 composite exhibited good antiviral activity under both light irradiation and dark conditions.

Viruses Droplet Splash Test of CuxO/TiO2 Photocatalyst
Considering the practical application of the CuxO/TiO2 photocatalyst, we conducted antiviral tests on the CuxO/TiO2-coated sheet fabrics using the pseudo splash-containing bacteriophage Qβ. Figure 16 shows a photograph of the experimental setup for the antiviral splash test. An atomizer generated an aerosol that contained 6 × 10 7 pfu/h of bacteriophage Qβ, and the particle size of the aerosol was approximately 0.3 μm. The virus aerosol from the atomizer attached to the photocatalyst sheets on a desk of 1 m high from the floor under white fluorescence light at an illuminance of 1000 lux. After 4 h, the number of bacteriophages was counted using the same procedure with the previous studies [33][34][35]. Bacteriophages on a control sheet without CuxO/TiO2 coating were also sampled at 1 h and 2 h.  Table 1 summarizes the comparison of the antiviral properties of various copper-based compounds. The antiviral activity of pristine CuO is negligible. Conversely, pristine Cu 2 O exhibits efficient antiviral properties at its initial use; however, its initial red colour turns black by self-oxidation to change into Cu(II) inactive species [51,52]. Further, Cu(II)/TiO 2 shows photocatalytic oxidation activity under visible light because of the IFCT transition, but its antiviral activity is limited because of the lack of Cu(I) species. Among these samples, the Cu x O/TiO 2 composite exhibited good antiviral activity under both light irradiation and dark conditions.

Viruses Droplet Splash Test of Cu x O/TiO 2 Photocatalyst
Considering the practical application of the Cu x O/TiO 2 photocatalyst, we conducted antiviral tests on the Cu x O/TiO 2 -coated sheet fabrics using the pseudo splash-containing bacteriophage Qβ. Figure 16 shows a photograph of the experimental setup for the antiviral splash test. An atomizer generated an aerosol that contained 6 × 10 7 pfu/h of bacteriophage Qβ, and the particle size of the aerosol was approximately 0.3 µm. The virus aerosol from the atomizer attached to the photocatalyst sheets on a desk of 1 m high from the floor under white fluorescence light at an illuminance of 1000 lux. After 4 h, the number of bacteriophages was counted using the same procedure with the previous studies [33][34][35]. Bacteriophages on a control sheet without Cu x O/TiO 2 coating were also sampled at 1 h and 2 h.  Figure 17 shows the changes in the number of bacteriophages on the photocatalyst sheet and control sheet. It is noteworthy that the number of bacteriophages on the CuxO/TiO2 sheet was negligible, indicating its strong antiviral function against the virus attached to the surface. A CuxO/TiO2-coated material can thus potentially disinfect viruses on any surface derived from droplets and aerosol to protect against viral disease spread by contact infection. Figure 17. Antiviral properties of the CuxO/TiO2-coated sheet and the control sheet without the photocatalyst using splash-containing bacteriophage Qβ. In the case of CuxO/TiO2 to avoid the overestimation of its antiviral property, the number of experiments was set to 1 time (after 4h) in order to exclude the influence of air flow due to human's entering into the room for measurement.

Conclusions
This review paper introduces the recent progress in the development of CuxO/TiO2 as an efficient visible light-sensitive photocatalyst for antiviral applications. The CuxO nanocluster consists of the valence states of Cu(I) and Cu(II). Cu(I) species in CuxO nanoclusters can denature viral proteins, resulting in significant antiviral properties even under dark conditions. Unfortunately, the Cu(I)    Figure 17 shows the changes in the number of bacteriophages on the photocatalyst sheet and control sheet. It is noteworthy that the number of bacteriophages on the CuxO/TiO2 sheet was negligible, indicating its strong antiviral function against the virus attached to the surface. A CuxO/TiO2-coated material can thus potentially disinfect viruses on any surface derived from droplets and aerosol to protect against viral disease spread by contact infection. Figure 17. Antiviral properties of the CuxO/TiO2-coated sheet and the control sheet without the photocatalyst using splash-containing bacteriophage Qβ. In the case of CuxO/TiO2 to avoid the overestimation of its antiviral property, the number of experiments was set to 1 time (after 4h) in order to exclude the influence of air flow due to human's entering into the room for measurement.

Conclusions
This review paper introduces the recent progress in the development of CuxO/TiO2 as an efficient visible light-sensitive photocatalyst for antiviral applications. The CuxO nanocluster consists of the valence states of Cu(I) and Cu(II). Cu(I) species in CuxO nanoclusters can denature viral proteins, resulting in significant antiviral properties even under dark conditions. Unfortunately, the Cu(I)

Control sheet
Time (h) Virus titer (pfu/125cm 2 ) Cu x O/TiO 2 sheet Figure 17. Antiviral properties of the Cu x O/TiO 2 -coated sheet and the control sheet without the photocatalyst using splash-containing bacteriophage Qβ. In the case of Cu x O/TiO 2 to avoid the overestimation of its antiviral property, the number of experiments was set to 1 time (after 4h) in order to exclude the influence of air flow due to human's entering into the room for measurement.

Conclusions
This review paper introduces the recent progress in the development of Cu x O/TiO 2 as an efficient visible light-sensitive photocatalyst for antiviral applications. The Cu x O nanocluster consists of the valence states of Cu(I) and Cu(II). Cu(I) species in Cu x O nanoclusters can denature viral proteins, resulting in significant antiviral properties even under dark conditions. Unfortunately, the Cu(I) species in Cu x O are easily oxidized to inactive Cu(II) in ambient air. However, the combination of Cu x O with the TiO 2 photocatalyst maintained its antiviral function by visible-light irradiation. In the Cu x O/TiO 2 photocatalyst, electron transition occurs by visible-light irradiation through the IFCT process; this results in the generation of antiviral Cu(I) species and holes in the valence band of TiO 2 , which are effective in disinfecting viruses. Once the Cu(I) species in Cu x O turn into Cu(II) by self-oxidation, antiviral active Cu(I) species can be regenerated by visible light like a white fluorescence bulb. Therefore, the antiviral function of Cu x O/TiO 2 can be maintained, even under indoor conditions, where light illumination is turned on during the day and off during the night. It is also noted that the Cu x O/TiO 2 composite samples have been commercialized (NAKA CORPORATION, Tokyo Japan). We expect the Cu x O/TiO 2 material to be applied to various antiviral industrial items in indoor circumstances, 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. Furthermore, the present concept contributes to the design of various antiviral materials, such as bimetallic catalysts [88][89][90].