Enhanced Photocatalytic Antibacterial Properties of TiO2 Nanospheres with Rutile/Anatase Heterophase Junctions and the Archival Paper Protection Application

TiO2 has been generally studied for photocatalytic sterilization, but its antibacterial activities are limited. Herein, TiO2 nanospheres with rutile/anatase heterophase junctions are prepared by a wet chemical/annealing method. The large BET surface area and pore size are beneficial for the absorption of bacteria. The rutile/anatase heterojunctions narrow the bandgap, which enhances light absorption. The rutile/anatase heterojunctions also efficiently promote the photogenerated carriers’ separation, finally producing a high yield of radical oxygen species, such as •O2− and •OH, to sterilize bacteria. As a consequence, the obtained TiO2 nanospheres with rutile/anatase heterojunctions present an improved antibacterial performance against E. coli (98%) within 3 h of simulated solar light irradiation, exceeding that of TiO2 nanospheres without annealing (amorphous) and TiO2 nanospheres annealing at 350 and 550 °C (pure anatase). Furthermore, we design a photocatalytic antibacterial spray to protect the file paper. Our study reveals that the TiO2 nanospheres with rutile/anatase heterojunctions are a potential candidate for maintaining the durability of paper in the process of archival protection.


Introduction
Archives are a non-renewable important source. Archival entities, like other materials, will be inevitably degraded and aged with the prolongation of saving time. Moreover, microorganisms and bacteria in the air of the archive store can fall into the archival entity, thereby affecting the durability of the archival entity. Paper is one of the most important carrier materials in existing archival entities. In the process of archival protection, maximizing the maintenance of paper's durability is vital.
Traditional disinfection methods, such as UV disinfection, ozonation, and chlorination, are widely used [1]. However, they have the disadvantage of being operationally intensive, having an energy cost, and forming hazardous disinfection byproducts [2]. However, photocatalytic antibacterial technology using sustainable solar energy with no disinfection byproducts is an attractive technology for oxidative disinfection to various bacteria [3]. TiO 2 as a photocatalyst is widely studied owing to being chemically stable and inexpensive [4]. After light irradiation, the nearby O 2 molecules can react with the electrons to form •O 2 − . Meanwhile, the H 2 O molecules can capture holes to form •OH. •O 2 − and •OH are highly reactive oxygen species (ROS); these ROS have strong oxidation potentials and can degrade bacteria [5]. Yet, the photocatalytic antibacterial performance of TiO 2 is limited due to the fast carrier recombination [6]. To improve TiO 2 's photocatalytic performance, reducing the carrier recombination is needed.
Semiconductor heterojunctions, such as band-structure matching heterojunction, p-n junction, and Schottky junction, are designed to enhance the carrier separation [7]. TiO 2 possesses rutile, brookite, TiO 2 (B), and anatase polymorphs [8]. TiO 2 (B) and anatase in TiO 2 nanobelts present different band structures and form a heterophase junction, which can promote the separation of photoinduced carriers [9]. Among the four polymorphs, rutile and anatase are the most studied in photocatalysis [10], which can also construct the heterophase junction and promote carrier separation [11]. The preparation of TiO 2 nanomaterials can generally be divided into four strategies, including the hydrothermal strategy, sol-gel strategy, template strategy, and template-free (wet chemical) strategy [12]. The hydrothermal strategy needs high production costs and strict equipment requirements for high pressure and high temperature [13]. The sol-gel strategy easily causes the agglomeration of TiO 2 [14]. However, the template-free (wet chemical) strategy only requires one step of reaction, such as annealing, which avoids the impurities and the affecting of the inherent properties and structural stability of the material caused by template methods [15]. Thus, the template-free (wet chemical) strategy is more effective and flexible for large-scale manufacturing than other methods [13].
In this paper, we use a wet chemical/annealing method to prepare TiO 2 nanospheres with rutile/anatase heterophase junctions. The construction of rutile/anatase heterophase junctions in TiO 2 nanospheres and large BET surface areas/pore sizes can efficiently improve the light absorption, enhance the absorption of bacteria, and promote carrier separation. Hence, compared with amorphous TiO 2 (without annealing) and pure anatase TiO 2 (annealing at 350 and 550 • C), the obtained TiO 2 nanospheres with rutile/anatase heterojunctions (annealing at 750 • C) present the enhanced photocatalytic antibacterial performance against Escherichia coli (E. coli). Inspired by this, we design a photocatalytic antibacterial spray with TiO 2 nanospheres with rutile/anatase heterojunctions as the photocatalysts. The TiO 2 nanospheres with rutile/anatase heterojunctions spray can maintain the durability of paper in the process of archival protection.

Synthesis of TiO 2 Nanospheres
Amorphous TiO 2 nanospheres were firstly synthesized by a wet chemical procedure. Twenty milliliters of Ti(OC 4 H 9 ) 4 (TBOT) as the Ti source was quickly injected into 200 mL deionized water with 6 g NH 3 ·H 2 O. After reacting for 30 s, the white precipitate was washed thoroughly and dried at 60 • C for 10 h. The amorphous TiO 2 (TiO 2 -A) was obtained. Thermal annealing of the amorphous TiO 2 at 250, 350, 550, and 750 • C for 4 h led to the production of TiO 2 nanospheres, labeled as TiO 2 -250, TiO 2 -350, TiO 2 -550, and TiO 2 -750, respectively.

Characterization
XRD patterns were carried out using a D8 Advance (Bruker, Berlin, Germany) powder X-ray diffractometer. TEM and SEM with an EDS were performed using a JEM 2100F (JOEL, Tokyo, Japan) and NanoSEM 450 (FEI, Portland, OR, USA) microscope, respectively. XPS was performed with the ESCALAB 250 instrument. The UV-Vis DRS spectra were examined on the UV-3101 UV-Vis spectrophotometer (SHIMADZU, Tokyo, Japan). The BET specific surface area was measured with the ASAP2020 (Micromeritics, Norcross, GA, USA) instrument. The electric paper board breaking resistance tester (Yq-zb-1) was manufactured by Hangzhou Light Industry Testing Instrument Co., LTD, Hangzhou, China.

Photocatalytic Activity Tests
Escherichia coli (E. coli, ATCC 25922) was selected for photocatalytic sterilization, in which the Xe arc lamp (300 W) with a simulated solar light filter was the simulated solar light source. The bacterial suspension was diluted to~5 × 10 4 colony forming units (CFU)/mL, and 100 µL of which was mixed with TiO 2 nanospheres (each at 30 mg) in a test tube containing 10 mL of distilled water. Then, a 0.1 mL aliquot of the bacterial suspension mixtures was collected at given time intervals after the light turned on, diluted with the PBS buffer solution, and then spread on nutrient agar at 37 • C for 24 h. Error bars represent the standard deviation of three independent experiments.

Paper Breakage Resistance Tests
Potassium permanganate and formaldehyde fumigation: We adopted a 40% formaldehyde solution of 3 mL/m 3 and potassium permanganate of 1.5 g/m 3 to calculate the amount of disinfection in the archives, and the fumigation time was 15 h. The doors and windows were kept closed during disinfection. The doors and windows were opened for 48 h after the fumigation.
Photocatalytic bactericidal spray: The photocatalytic bactericidal spray was composed of photocatalysts (TiO 2 -750) and epichlorohydrin, in which water:TiO 2 -750:epichlorohydrin = 1000:1:5. The photocatalytic bactericidal spray solution was also 3 mL/m 3 to calculate the amount of disinfection in the archives, and disinfection time was 15 h. The doors and windows were kept closed during disinfection. The doors and windows were opened for 48 h after the disinfection.
The same tested paper was added 3 days before disinfection. After disinfection for 30 days, the paper was sealed in a sealed bag and sent to the laboratory for testing. The reference standard for breakage resistance detection was the Determination of paper breakage resistance (GB/T 454-2002).
For the XPS survey spectrum ( Figure S1), besides O 1s and Ti 2p peaks, the C 1s peak at 288.3 eV is detected, due to the adventitious hydrocarbon from the air [18]. For the Ti 2p spectrum (Figure 2a), the two main peaks of Ti2p 1/2 at 464.4 eV and Ti2p 3/2 at 458.6 eV are ascribed to Ti 4+ of TiO 2 [19]. Furthermore, an associated satellite peak located at 458.2 eV is detected [20], corresponding to Ti 3+ . The O 1s spectrum of TiO 2 (Figure 2b) can be fitted with three peaks at 529.9, 531.2, and 532.0 eV, ascribing to O 2− (O-Ti-O bonds), O − (Ti 3+ induced oxygen vacancies), and Ti-OH groups, respectively [21]. Furthermore, the ratio of Ti 4+ and Ti 3+ in the TiO 2 -750 sample is 91.5:8.5, based on their characteristic peaks in XPS spectra. Based on the above-discussed analysis, the XPS results confirm the existence of oxygen vacancies in TiO 2 -750. Calcination is the most common approach for forming TiO 2 defects, such as oxygen vacancies [21]. Thermal annealing of the amorphous TiO 2 at 750 • C for 4 h led to the production of oxygen vacancies in TiO 2 -750. For the XPS survey spectrum ( Figure S1), besides O 1s and Ti 2p peaks, the C 1s p at 288.3 eV is detected, due to the adventitious hydrocarbon from the air [18]. For th 2p spectrum (Figure 2a), the two main peaks of Ti2p1/2 at 464.4 eV and Ti2p3/2 at 458.6 are ascribed to Ti 4+ of TiO2 [19]. Furthermore, an associated satellite peak located at 45 eV is detected [20], corresponding to Ti 3+ . The O 1s spectrum of TiO2 ( Figure 2b) can fitted with three peaks at 529.9, 531.2, and 532.0 eV, ascribing to O 2− (O-Ti-O bonds) (Ti 3+ induced oxygen vacancies), and Ti-OH groups, respectively [21]. Furthermore, ratio of Ti 4+ and Ti 3+ in the TiO2-750 sample is 91.5:8.5, based on their characteristic pe in XPS spectra. Based on the above-discussed analysis, the XPS results confirm the ex ence of oxygen vacancies in TiO2-750. Calcination is the most common approach for fo ing TiO2 defects, such as oxygen vacancies [21]. Thermal annealing of the amorphous T at 750 °C for 4 h led to the production of oxygen vacancies in TiO2-750. The microstructure of TiO2 synthesized with and without annealing was analyzed SEM and TEM (Figures 3 and 4).   For the XPS survey spectrum ( Figure S1), besides O 1s and Ti 2p peaks, the C 1s peak at 288.3 eV is detected, due to the adventitious hydrocarbon from the air [18]. For the Ti 2p spectrum (Figure 2a), the two main peaks of Ti2p1/2 at 464.4 eV and Ti2p3/2 at 458.6 eV are ascribed to Ti 4+ of TiO2 [19]. Furthermore, an associated satellite peak located at 458.2 eV is detected [20], corresponding to Ti 3+ . The O 1s spectrum of TiO2 (Figure 2b) can be fitted with three peaks at 529.9, 531.2, and 532.0 eV, ascribing to O 2− (O-Ti-O bonds), O − (Ti 3+ induced oxygen vacancies), and Ti-OH groups, respectively [21]. Furthermore, the ratio of Ti 4+ and Ti 3+ in the TiO2-750 sample is 91.5:8.5, based on their characteristic peaks in XPS spectra. Based on the above-discussed analysis, the XPS results confirm the existence of oxygen vacancies in TiO2-750. Calcination is the most common approach for forming TiO2 defects, such as oxygen vacancies [21]. Thermal annealing of the amorphous TiO2 at 750 °C for 4 h led to the production of oxygen vacancies in TiO2-750.  The microstructure of TiO 2 synthesized with and without annealing was analyzed by SEM and TEM (Figures 3 and 4).  nanosheets with the diameter of 50-100 nm (Figure 4a) and the thickness of 10-20 nm (Figure 4b). The HRTEM image in Figure 4b presents that TiO2 nanospheres are extremely crystallized. The crystal lattice of 0.357 and 0.320 nm indicate anatase TiO2 (101) facets and rutile TiO2 (110) facets, respectively [10]. The anatase and rutile phases in TiO2 nanospheres can form the heterophase junctions, which will efficiently enhance the separation of photogenerated carriers. The EDS elemental mapping images ( Figure S2) reveal the existence of Ti and O elements, further confirming the formation of TiO2. The specific surface areas were tested by nitrogen adsorption-desorption isotherms. The BET surface area of TiO2 nanospheres is significantly increased after annealing (Table  S1). The annealing temperature does not affect the specific surface area of TiO2 nanospheres. TiO2-750 still presents a relatively large specific surface area (297 m 2 /g). Interestingly, TiO2-750 (Table S1) presents the largest pore diameter (4.2 nm), which is beneficial for the absorption of bacteria.
As shown in Figure 5a, the TiO2-A only shows the absorption of UV light and exhibits absorption edges at about 375 nm. Interestingly, after annealing at 750 °C , the absorption edge of TiO2-750 is expanded to 442 nm, indicating that the formation of the rutile/anatase The specific surface areas were tested by nitrogen adsorption-desorption isotherms. The BET surface area of TiO 2 nanospheres is significantly increased after annealing (Table S1). The annealing temperature does not affect the specific surface area of TiO 2 nanospheres. TiO 2 -750 still presents a relatively large specific surface area (297 m 2 /g). Interestingly, TiO 2 -750 (Table S1) presents the largest pore diameter (4.2 nm), which is beneficial for the absorption of bacteria.
As shown in Figure 5a, the TiO 2 -A only shows the absorption of UV light and exhibits absorption edges at about 375 nm. Interestingly, after annealing at 750 • C, the absorption edge of TiO 2 -750 is expanded to 442 nm, indicating that the formation of the rutile/anatase junction and oxygen vacancies in TiO 2 -750 result in a longer-wavelength (visible) absorption edge and have significant effects on the optical property. Figure 5b shows the calculated bandgap (E g ) for TiO 2 nanospheres. The calculated E g of TiO 2 -A, TiO 2 -250, TiO 2 -350, TiO 2 -550, and TiO 2 -750 is estimated to be 3.30, 3.10, 3.05, 3.00, and 2.80 eV, respectively. Interestingly, TiO 2 -750 exhibits the narrowest bandgap, implying the best light absorption.
To prove the photocatalytic antibacterial performance of TiO 2 nanospheres, the sterilization of E. coli was measured under simulated solar light irradiation (Figure 6a). In the light control experiment (Figure S3), the cell density of E. coli shows a limited decrease without photocatalysts under simulated solar light irradiation, suggesting the negligible effect of the simulated solar light on the bacterial cells. The TiO 2 nanosphere without annealing (TiO 2 -A) is not a good photocatalyst (Figure 6a). The sterilization rate of TiO 2 -A for 120 min is only 12%. After annealing, the photocatalytic antibacterial activity of TiO 2 nanospheres is significantly improved. Compared with other annealing temperature samples (TiO 2 -250, TiO 2 -350, and TiO 2 -550), TiO 2 nanosphere annealing at 750 • C (TiO 2 -750) presents the best photocatalytic antibacterial activity. After irradiation for 120 min, the E. coli sterilization rate of TiO 2 -750 is 98%. This is ascribed to the formed rutile/anatase heterojunctions, which effectively promote the separation of carriers. In addition, the relatively larger specific surface area (297 m 2 g −1 ) and the largest pore diameter (4.2 nm) of TiO 2 -750 are beneficial for the absorption of bacteria. Moreover, TiO 2 -750 has the narrowest bandgap and can efficiently utilize more light. Moreover, there is no significant loss of E. coli sterilization rate over TiO 2 -750 after three cycles ( Figure S4), suggesting excellent sterilization stability. Nanomaterials 2021, 11, x FOR PEER REVIEW 6 of 10 junction and oxygen vacancies in TiO2-750 result in a longer-wavelength (visible) absorption edge and have significant effects on the optical property. Figure 5b shows the calculated bandgap (Eg) for TiO2 nanospheres. The calculated Eg of TiO2-A, TiO2-250, TiO2-350, TiO2-550, and TiO2-750 is estimated to be 3.30, 3.10, 3.05, 3.00, and 2.80 eV, respectively. Interestingly, TiO2-750 exhibits the narrowest bandgap, implying the best light absorption. To prove the photocatalytic antibacterial performance of TiO2 nanospheres, the sterilization of E. coli was measured under simulated solar light irradiation (Figure 6a). In the light control experiment (Figure S3), the cell density of E. coli shows a limited decrease without photocatalysts under simulated solar light irradiation, suggesting the negligible effect of the simulated solar light on the bacterial cells. The TiO2 nanosphere without annealing (TiO2-A) is not a good photocatalyst (Figure 6a). The sterilization rate of TiO2-A for 120 min is only 12%. After annealing, the photocatalytic antibacterial activity of TiO2 nanospheres is significantly improved. Compared with other annealing temperature samples (TiO2-250, TiO2-350, and TiO2-550), TiO2 nanosphere annealing at 750 °C (TiO2-750) presents the best photocatalytic antibacterial activity. After irradiation for 120 min, the E. coli sterilization rate of TiO2-750 is 98%. This is ascribed to the formed rutile/anatase heterojunctions, which effectively promote the separation of carriers. In addition, the relatively larger specific surface area (297 m 2 g −1 ) and the largest pore diameter (4.2 nm) of TiO2-750 are beneficial for the absorption of bacteria. Moreover, TiO2-750 has the narrowest bandgap and can efficiently utilize more light. Moreover, there is no significant loss of E. coli sterilization rate over TiO2-750 after three cycles ( Figure S4), suggesting excellent sterilization stability. To confirm the photocatalytic antibacterial mechanism of TiO2-750, three main reactive species (•O2 − , •OH, and h + ) in the photocatalytic sterilization were measured using the radical scavenger experiments (Figure 6b). Benzoquinone (1 mM), methanol (1:15/V:V), and EDTA (10 mM) were used as •O2 − , •OH, and h + radical scavengers, respectively. After adding EDTA, the photocatalytic efficiency is slightly decreased compared with no trapping agent, indicating that h + has a slight effect on photocatalytic sterilization. The addition of methanol and benzoquinone significantly reduces photocatalytic sterilization, indicating that •O2 − and •OH are the main active substances for the sterilization of E. coli in TiO2-750.
The photocatalytic antibacterial mechanism is proposed in Figure 7. The rutile phase has upper positions of both VB and CB than anatase [22]. For TiO2-750 with rutile/anatase heterojunctions (Figure 7a), during the photocatalytic sterilization, the electrons and holes of both anatase and rutile are excited (Equation (1)). The photoinduced electrons in the CB of rutile are migrated to the anatase and react with dissolved O2 to produce •O2 -(Equation (2)). In the meantime, the holes in the VB of anatase are transferred to rutile and captured by H2O to produce •OH (Equation (3)), finally inactivating E. coli cells.
The strong oxidizing reactive oxygen species (ROS), such as •O2 -and •OH, are generated in the TiO2-750 with rutile/anatase heterojunctions, confirmed by the reactive spe- To confirm the photocatalytic antibacterial mechanism of TiO 2 -750, three main reactive species (•O 2 − , •OH, and h + ) in the photocatalytic sterilization were measured using the radical scavenger experiments (Figure 6b). Benzoquinone (1 mM), methanol (1:15/V:V), and EDTA (10 mM) were used as •O 2 − , •OH, and h + radical scavengers, respectively. After adding EDTA, the photocatalytic efficiency is slightly decreased compared with no trapping agent, indicating that h + has a slight effect on photocatalytic sterilization. The addition of methanol and benzoquinone significantly reduces photocatalytic sterilization, indicating that •O 2 − and •OH are the main active substances for the sterilization of E. coli in TiO 2 -750.
The photocatalytic antibacterial mechanism is proposed in Figure 7. The rutile phase has upper positions of both VB and CB than anatase [22]. For TiO 2 -750 with rutile/anatase heterojunctions (Figure 7a), during the photocatalytic sterilization, the electrons and holes of both anatase and rutile are excited (Equation (1)). The photoinduced electrons in the CB of rutile are migrated to the anatase and react with dissolved O 2 to produce •O 2 − (Equation (2)). In the meantime, the holes in the VB of anatase are transferred to rutile and captured by H 2 O to produce •OH (Equation (3)), finally inactivating E. coli cells.  Inspired by the excellent photocatalytic antibacterial activity of TiO2-750 with rutile/anatase heterojunctions, we designed a photocatalytic bactericidal spray (water:TiO2-750:epichlorohydrin = 1000:1:5) with TiO2-750 with rutile/anatase heterojunctions as the photocatalysts to protect the file paper. As shown in Figure 8, the photocatalytic bactericidal spray method has a positive effect on the paper breakage resistance, and the breakage resistance decreases 7.5% after photocatalytic bactericidal spraying after 30 days, which is better than the paper without photocatalytic bactericidal spraying (decreasing 24.6% after 30 days) and the paper fumigated with potassium permanganate and formaldehyde (decreasing 18.7% after 30 days). Our designed photocatalytic bactericidal spray using TiO2-750 with rutile/anatase heterojunctions as the photocatalysts can maintain the durability of paper in the process of archival protection.

Conclusions
In conclusion, TiO2 nanospheres with rutile/anatase heterojunctions are successfully fabricated. With the merits of improved carrier transfer by rutile/anatase heterojunctions, and good adsorption performance for bacteria by large specific surface area and pore size, the photocatalytic antibacterial efficiency of TiO2 nanospheres with rutile/anatase hetero-  (Figure 6b), triggering oxidative stress and cell damage in bacteria [23].
However, for the pure anatase phase (TiO 2 -350 and TiO 2 -550) (Figure 7b), under light irradiation, the holes and electrons are generated (Equation (1)). The holes and electrons can be quickly recombined, which induces the low photocatalytic performance.
Inspired by the excellent photocatalytic antibacterial activity of TiO 2 -750 with rutile/anatase heterojunctions, we designed a photocatalytic bactericidal spray (water:TiO 2 -750:epichlorohydrin = 1000:1:5) with TiO 2 -750 with rutile/anatase heterojunctions as the photocatalysts to protect the file paper. As shown in Figure 8, the photocatalytic bactericidal spray method has a positive effect on the paper breakage resistance, and the breakage resistance decreases 7.5% after photocatalytic bactericidal spraying after 30 days, which is better than the paper without photocatalytic bactericidal spraying (decreasing 24.6% after 30 days) and the paper fumigated with potassium permanganate and formaldehyde (decreasing 18.7% after 30 days). Our designed photocatalytic bactericidal spray using TiO 2 -750 with rutile/anatase heterojunctions as the photocatalysts can maintain the durability of paper in the process of archival protection. Inspired by the excellent photocatalytic antibacterial activity of TiO2-750 with rutile/anatase heterojunctions, we designed a photocatalytic bactericidal spray (water:TiO2-750:epichlorohydrin = 1000:1:5) with TiO2-750 with rutile/anatase heterojunctions as the photocatalysts to protect the file paper. As shown in Figure 8, the photocatalytic bactericidal spray method has a positive effect on the paper breakage resistance, and the breakage resistance decreases 7.5% after photocatalytic bactericidal spraying after 30 days, which is better than the paper without photocatalytic bactericidal spraying (decreasing 24.6% after 30 days) and the paper fumigated with potassium permanganate and formaldehyde (decreasing 18.7% after 30 days). Our designed photocatalytic bactericidal spray using TiO2-750 with rutile/anatase heterojunctions as the photocatalysts can maintain the durability of paper in the process of archival protection.

Conclusions
In conclusion, TiO2 nanospheres with rutile/anatase heterojunctions are successfully fabricated. With the merits of improved carrier transfer by rutile/anatase heterojunctions, and good adsorption performance for bacteria by large specific surface area and pore size,

Conclusions
In conclusion, TiO 2 nanospheres with rutile/anatase heterojunctions are successfully fabricated. With the merits of improved carrier transfer by rutile/anatase heterojunctions, and good adsorption performance for bacteria by large specific surface area and pore size, the photocatalytic antibacterial efficiency of TiO 2 nanospheres with rutile/anatase heterojunctions towards E. coli is significantly improved. Under light illumination, the photoinduced electrons in the CB of rutile are immigrated to the anatase through rutile/anatase heterojunctions. Meanwhile, the holes in the anatase's VB are transferred to rutile. These photo-excited electrons and holes can react with O 2 and H 2 O to produce a great quantity of ROS (e.g., •O 2 − and •OH), which can induce serious damage to bacterial membranes, thus resulting in bacterial death. Inspired by the excellent photocatalytic antibacterial activity of TiO 2 nanospheres with rutile/anatase heterojunctions as the photocatalysts to protect the file paper, the paper breakage resistance is significantly improved after spraying with the photocatalysts. Therefore, it is a potential candidate for maintaining the durability of paper in the process of archival protection.