MSA  Vol.12 No.2 , February 2021
Preparation of Anatase Titanium Dioxide Nanoparticle Powders Submitting Reactive Oxygen Species (ROS) under Dark Conditions
Abstract: Recently, under the circumstances of pandemic of COVID-19 much attention has been paid to titanium dioxide TiO2 as bactericidal agent; however, conventional TiO2 requires ultraviolet radiation or visible light to exercise its photocatalytic properties and its induced antimicrobial activity. In order to expand its applications directed at wide civil life, antibacterial TiO2 being usable under dark conditions has been demanded. The present paper describes the powder characterization of newly developed potassium K and phosphorous P co-doped nanometer-size anatase TiO2 powders using X-ray diffraction (XRD), scanning and transmission electron microscopies (SEM & TEM), Brunauer-Emmett-Teller method (BET), fourier-transform infrared spectroscopy (FT-IR), X-ray absorption fine structure (XAFS), electron spin resonance (ESR) and chemiluminescence (CL). It was found for the first time that thus prepared anatase TiO2 could submit much reactive oxygen species (ROS) even in the dark, which has close relation with bactericidal activity in light interception.

1. Introduction

Three crystal structures of titanium dioxide are mainly reported: low-temperature form tetragonal anatase (a-TiO2), high-temperature stable tetragonal rutile (r-TiO2) and middle temperature orthorhombic brookite (b-TiO2) [1]. The first a-TiO2 and third b-TiO2 transform easily into r-TiO2 at higher temperatures than 1188 K and 923 K, respectively [2]; these temperatures depend on their particle and crystallite sizes, purity and synthetic process conditions [3]. Among them, r-TiO2 has been widely used in the industry that is textiles, electronics, wastewater treatment, and catalysis [4] [5] [6]. Recently, TiO2 nanoparticles (TiO2 NPS), have been attracting much attention due to their functional and physicochemical properties, such as, a white pigment and a personal skin care product (due to its brightness and high refractive index with high safety margin), and bactericidal agents (photocatalytic properties and its induced antimicrobial activity under ultraviolet (UV) radiation or visible light) [7] [8] [9]. Up to now, many papers and reviews concerning about toxicity mechanism have been published from the viewpoints of reactive oxygen species (ROS) [10] - [17], that is hydroxyl radical ·OH and superoxide anion O 2 which are generated by hole-electron pairs in the valence and conduction bands of TiO2, respectively. Their bactericidal mechanism has been explained by introducing bio-cell wall damage and lipid peroxidation of membrane, and TiO2 NPS’s adherence to intercellular organelles and biological macro molecules [17] [18] [19] [20] [21].

The authors have been studying metal oxide powders which can reveal strong antimicrobial activity in the shade for long time. Biocompatible zinc oxide ZnO also has been interested due to its microbial toxicity. However, its activity is a little lower than TiO2 NPS. Recently, ZnO powders have been prepared by hydrothermal treatment in zinc nitrate aqueous solution with a concentration of 3 mol·L−1 at 443 K for 2.52 × 104 s, followed by re-oxidation heating at 873 K for 3.6 × 103 s in air. And then it has been cleared that thus obtained ZnO powders can reveal strong antibacterial activity even under dark conditions [22] [23] [24]. During investigation of antibacterial ZnO, the authors found that 1) among commercially available TiO2 powders, some a-TiO2 could submit a small amount of ROS in the dark, however, r-TiO2 did not, and furthermore, 2) among a-TiO2, ROS were submitted from a-TiO2 which contained a trace amount of potassium K and phosphorus P as impurities.

The effects of potassium K doping on physicochemical properties of TiO2, such as their crystallinity, surface areas SA, solubility, photocatalytic activity, and band gap Eg, were studied by several researchers [25] [26] [27] [28]. Their results were summarized as follows; K-solubility was a very low around 0.2 at%, therefore, Eg was not changed and photocatalytic activity was decreased under visible light conditions. Hao et al. [29] studied the antibacterial activity of K-doped TiO2 powders, which were prepared using molten potassium nitrate KNO3 and metal Ti under various calcination temperatures. The obtained samples consisted of a-TiO2 as well as metal Ti. Doped (2.6 - 10.6 at% K) TiO2 samples showed a photocatalytic activity under visible light conditions, despite having a small change in the band gap (3.14 eV), contradicting a previous study [27].They assumed that K+ ions decreased Eg of a-TiO2 and the doped samples killed bacteria under visible light conditions. Under dark conditions, the samples also showed some antibacterial activity.

Yu et al. [30] studied phosphorus-doped TiO2 powders and reported their microstructures; phosphate P doping inhibited the grain growth during calcination, significantly increasing SA, and decreased the pore size. About the values of Eg, opposite results were reported, Eg became larger [30] and smaller [31] [32] than those of undoped one. This might be explained in terms of a different source of P and way of synthesis method. Shi et al. [32] investigated the bonding of P in the crystal structure and reported that P was only present as P5+, and only Ti-O-P bonds were detected. This substitution consequently led to a charge imbalance, which needs to be compensated. The compensation was proved through the decrease in oxygen vacancies. It is known that decreasing oxygen vacancies improved photocatalytic activity, because oxygen vacancies acted as recombination centres for electron-hole pairs [33] [34]. In addition, they also observed an increase in the photocatalytic activity with increasing the P content. Jin et al. [35] found that P doping increased hydroxyl radical emission from the surface, which was also favourable in terms of photocatalytic activity.

Up to now, there is no report on the generation of ROS from a-TiO2 NPS under dark conditions. Then, we started to investigate the relationship between the amount of ROS and the contents of K, P, and their combined doping, from the viewpoint of the microstructure and physicochemical properties. Finally, we found that anatase (a-TiO2) powders which could submit a lot of ROS even in the dark; the amounts of ROS were much higher than our previous antibacterial ZnO, in addition, this powder could be prepared with much simpler process. The present paper treats the physicochemical properties in relation with the microbial toxicity of thus prepared anatase (a-TiO2) as a function of impurity contents and its doping method.

2. Experimental Procedure

2.1 Preparation of Doped TiO2 Powders

As shown in Figure 1, fine anatase TiO2 powder (W-4038, 99.92% of purity, Sakai Chemical Industry Co., Ltd., Osaka, Japan) with a BET SA of 55.1 m2/g, i.e., particle size Ps of 0.0280 µm, which was calculated from the values of SA and theoretical density Dx of 3.895 Mg·m−3 (Powder Diffraction File PDF: #21-1272), was used as the starting material. 0.1 mol of this powder and a certain amount of KHCO3 (99.7%, Sigma Aldrich, Japanese agency Nacalai Tesque Chemicals, Kyoto, Japan) and (NH4)2HPO4 (≥98%, Sigma Aldrich) with various inner atomic ratios (0.01 - 10.0 at%) of K and P for single or double additions (co-doping), as shown in Table 1, were mixed together in 1.5 × 10−5 m3 (15 mL) ethanol for 9.0 × 102 s at room temperature. The mixtures were subsequently dried at 393 K for 4.32 × 104 s in air, and then heated at 973 K for 3.6 × 103 s in air or oxygen atmosphere. These doped TiO2 powders were pulverized in ethanol with a planetary

Figure 1. Flowchart of preparation and evaluation of K and P doped anatase TiO2. SEM photo shows the starting material of a-TiO2 W-4037, BET surface area of SA = 55.1 m2·g−1, theoretical density Dx = 3.895 Mg·m−3, particle size of Ps = 0.0280 µm.

Table 1. The sample numbers of TiO2 doped with various amount of K and P.

ball milling apparatus (P-7, Fritsch Japan, Yokohama, Japan) for 1.8 × 103 s (30 min) using yttria-stabilized tetragonal ZrO2 (YTZ) balls with a diameter of 2.0 × 10−3 m (2.0 mmf), at a rotating speed of 6.67 round per sec (400 round per minute rpm; centrifugal force about 11 G, gravitation acceleration unit) under the ratio of the powder, YTZ balls, and ethanol were 1.0 × 10−3 kg: 1.0 × 10−2 kg: 1.0 × 10−5 m3 (10 mL).

2.2. Evaluation

2.2.1. Physicochemical Property

X-ray diffraction (XRD; Smartlab, Rigaku, Tokyo, Japan) analysis using CuKα radiation (wavelength of 0.15418 nm) with a graphite monochromator was utilized for the crystalline phases and lattice parameter determinations. Microstructural observation with a field emission-type scanning electron microscope (FE-SEM; SU8020, Hitachi High-Technologies Corporation, Tokyo, Japan) and a transmission electron microscope (TEM, JEM-2100F, JEOL, Tokyo, Japan) equipped with energy-dispersive X-ray spectroscopy (EDS, JED-2300/F, JEOL). BET surface areas SA of powders were measured using an automatic surface area and porosimetry analyzer (Tristar II, Micromeritics, Japanese agency Shimadzu, Kyoto, Japan) at room temperature. X-ray photoelectron spectroscopy (XPS) or electron spectroscopy for chemical analysis (ESCA) (ESCA3057, ULVAC-PHI. Inc., Chigasaki, Kanagawa, Japan) was utilized to analyze the surface chemical state of K and P in TiO2 powders. A monochromatic AlKα X-ray source (1486.6 eV) was used and operated at 150 W in a pressure of 1.333 × 10−12 Pa. All the binding energies were referenced to the Au4f7/2 peak located at 84.2 eV attributed to the sputtered-Au on the surface. Fourier transform infrared spectroscopy (FT-IR, IRAffinity, Shimadzu) was performed under the following conditions; measuring method was attenuated total reflection (ATR, MIRacle 10, Shimadzu) with the resolution capacity of 4.0 cm−1, cumulated number is 20, measuring wavenumber domain was 600 - 4000 cm−1 at room temperature. The local chemical structure around metal ions in anatase TiO2 crystalline structure had also been investigated by extended X-ray absorption fine structure (EXAFS) and X-ray absorption near edge structure (XANES) both of which were performed at Aichi synchrotron radiation research center. The storage ring was operated at the ring-energy of 1.2 GeV and a stored current of 300 mA. Data were collected in transmission mode with two flat Si (311) crystals as a monochromator with a RH-coated double mirror (0.8 mrad) to reject the higher harmonics. The EXAFS data were processed using the computer program “Athena”. The EXAFS functions were Fourier-transformed (FT) in the region 0.4 - 0.9 nm−1 to obtain the radial structure function (RSF). These were powerful tools for studying interatomic bonding- and electronic-states, and can provide information on the structure of a metal and an oxide ion, and/or a reactant. However, the local structure around K and P ions in TiO2 crystal were not well known. Instrument and attachment, integrating sphere: ISR-2200 and UV-Vis spectroscopy: UV-2400PC, were used for the band gap Eg measurement. The values of Eg of a-TiO2 powders were estimated by measuring reflectance spectrum intensity and transformed into absorbing spectrum intensity using Kubelka-Munk transformation [36].

The pH dependence of the emission of reactive oxygen species (ROS) which is emitted from the surface of a-TiO2 immersed in various PH buffer solutions was analyzed using an electron spin resonance apparatus (ESR, JES FA-100, JEOL) with the maximum magnetic field of 0.65 T. Among all ROS, as hydroxyl radical OH· has the highest redox potential (2.8 V/SHE) and the longest life-time [37], this ROS is the most powerful to kill bacteria. At ESR measurement, [power: 4 mW, central field: 328.3 mT, sweeping width: 1 ± 10 mT, sweeping time: 30 s, modular width: 0.3 × 1 mT, time constant: 0.03 s, at 293 K], 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) was used as a scavenger in order to trap OH·. As DMPO traps both hydroxyl radical and superoxide oxygen anion O2·, the reaction of O2· with DMPO is very slow when compared with OH· and the collected radical is less stable than the one formed with OH·. Moreover, a small fraction is subsequently decomposed into DMPO-OH.

2.2.2. Evaluation of Reactive Oxygen Species (ROS)

Chemiluminescence (CL) of TiO2 powders (10 μmol) in a 2.5 × 10−7 m3 (0.25 mL) aqueous luminol solution with a concentration of 5.0 × 10−9 mol·m−3 (5.0 μmol·L−1) mixed with 3.0 × 10−6 m3 (3.0 mL) carbonic acid buffer solution (NaOH/ NaHCO3, pH = 10.8) was observed under dark conditions using a CL detector (CLD-100FC, Tohoku Electronic Industrial Co., Ltd., Sendai, Japan). After dropping the luminol solution in a 1.2 × 102 s’ warming up of the detector, the intensity of CL was integrated between 1.2 - 6.0 × 102 s. Chemiluminescence (CL) with scavengers such as, 2-5 dimethyl furan (for singlet oxygen, 1O2, Fuji-film Wako Pure Chemical Co., Ltd., Osaka, Japan), nitro blue tetrazolium (for superoxide, · O 2 , Nacalai Tesque Chemicals), 2-propanol (for hydroxyl radical, OH·, Nacalai Tesque Chemicals), and riboflavin (for hydrogen per oxide, H2O2, Fuji-film Wako Pure Chemical Co., Ltd.) measurements were performed at room temperature in the dark to identify the reactive oxygen species (ROS) of TiO2 powders.

3. Results and Discussion

Figure 2 shows SEM photographs of a) No. 18: TiO2 doped with 5.0 at% P heated at 973 K for 3.6×103 s in air with a surface area of SA = 35.6 m2·g−1, here No.18 is the sample number displayed in Table 1, afterward the same manner, b) No. 23: 5.0 at% K doped TiO2 with SA = 29.0 m2·g−1, c) No. 35: K:P = 0.75:2.25 at% (K/P = 1/3 atomic ratio) doped TiO2 with SA = 28.3.0 m2·g−1, and d) No. 38: K:P = 1.5:4.5 at% (K/P = 1/3 atomic ratio) TiO2 doped with SA = 21.0 m2·g−1g. From these SA values, as they showed also anatase phase confirmed by XRD as will be described later, their particle sizes Ps were calculated to be Ps (No. 2)1 = 0.103 µm,Ps (No. 18) = 0.0433 µm, Ps (No. 23) = 0.0531 µm, Ps (No. 35) = 0.0544 µm andPs (No. 35) = 0.0734 µm using the same theoretical density of anatase Dx = 3.895 Mg·m−3 (PDF#21-1272). They were nanoparticle powders (NPS). It is interesting that after heating, a small amount of P doped sample gave high SA value (35.6 m2·g−1) and decreased gradually with increasing the content of K or/and P, from 35.6 to 21.0 m2·g−1. It is easily noticed that P addition has much effect to suppress the particle growth at 973 K.

The effect of P-doping on the decrement of Ps and increment of SA agreed well with the previous results reported by Yu et al. [30] and Shi et al. [32] . About the other K-doping effects have been described by Chen et al. [25] and Yan et al. [27]; the former pointed out that decrease in SA with more than 4.6 at% K, on the other hand the latter increase in SA with a 3 at% K addition.

Figure 2. SEM photographs of K and P doped anatase TiO2, No. of samples are shown in Table 1: (a) No. 18, K:P = 0:5, SA = 35.6 m2·g−1, Ps = 0.0433 µm, (b) No. 23, K:P = 5:0 at%, SA = 29.0 m2·g−1, Ps = 0.0531 µm, (c) No. 35, K:P = 0.75:2.25, SA = 28.3 m2·g−1, Ps = 0.0544 µm, and (d) No. 38, K:P = 1.5:4.5, SA = 21.0 m2·g−1, Ps = 0.0734 µm.

Figure 3 shows XRD patterns of the representative TiO2 powders without and with a small amount of K and P; a) No. 18 (5.0 at% P), b) No. 23 (5.0 at% K), c) No. 35 (K:P = 0.75:2.25 at%), and d) No. 38 (K:P = 1.5:4.5 at%). There were no other phases except anatase, even though the diffraction intensity of anatase was much scattered. This X-ray diffraction peak broadening caused by decreasing both crystallinity and crystallite sizes is the same result as Chen et al. [25] and Yan et al. [27].

Then the lattice parameters a and c of anatase phase were estimated by the method of least squares refinement using Si as an internal standard. As the amount of P is much higher than that of K, the lattice parameter changes in both a and c were evaluated as a function of input P. Figure 4 shows, (i) a of the combined (K/P = 1/3 atomic ratio) added a-TiO2 powders, (ii)a of the single addition of P, (iii) c of the single addition of P, and (iv) c of the combined (1·K+3·P; K/P = 1/3 atomic ratio) addition are displayed. It is clear that the single addition gives the decrease in both a and c values. This might be explained due to the small ionic radius r of P5+ with the coordination IV, r(P5+)VI = 0.038 nm, in comparison with r of Ti4+,r(Ti4+)VI = 0.0605 nm. However, (i) a and (iv) c values of the combined addition of (1·K+3·P) are almost constant; this could be explained by that the average r of (1·K+3·P), calculated from r(K+)VI = 0.138 nm, and r(P5+)VI = 0.038 nm, [(0.138 + 3.0.038)/4 = 0.063 nm], is almost the same as Ti4+. These ionic radii values were reported by Shannon [38]. Furthermore, the combined addition could make it easy to dope into a-TiO2 lattice, because its average electric valence is +4, the same as titanium. In addition, single addition of

Figure 3. XRD patterns of (a) No. 18 (K:P = 0:5), (b) No. 23 (K:P = 5:0), (c) No. 35 (K:P = 0.75:2.25), and (d) No. 38 (K:P = 1.5:4.5).

Figure 4. Lattice parameters of a-TiO2 doped with various contents of P (at%).

K+ and P5+ introduced the formation of oxygen excess and deficiency, respectively. These results are accordance with the previous studies that the solubility limit of K was around 0.2 at% by impregnation method (Yildizhan et al., [28] ), and a charge imbalance induced by P5+ resulted in the reduction of oxygen deficiency (Shi et al., [32] ).

As the amount of chemiluminescence CL corresponding to that of ROS which can disinfect bacteria, the CL emitted from the surface of a-TiO2 was observed under dark conditions as described before.

At first as preliminary experiment, ROS from various co-doped powders prepared by heating in air was investigated; their results were displayed in Figure 5. From the CL curves of No. 28 (K/P = 0.1/0.3 at%) heated at 973, 1073, and 1173 K, it was confirmed that 973 K heating gave the highest CL. Then, the CL values of other co-doped powders such as No. 35 (K/P = 0.75/2.25 at%), No.38 (1.5/4.5 at%), and No. 41 (3.0/9.0 at%), were investigated using CL-scavenger combined system; this system indicated which ROS was submitted from a-TiO2 powders. For example, the much reduction in CL intensity from No-scavenger to 2-popanol (scavenger for OH·) added samples, suggests that the ROS is hydroxyl radical OH·.

Then, the effect of the heat-treatment atmosphere during solid state reaction for doping was investigated. Figure 6 shows the CL values of various a-TiO2 as a function of input P content. After heating at 973 K for 3.6 × 103 s in oxygen, co-doped (1·K + 3·P) a-TiO2 showed significantly high CL values comparing with those of other K or P single added samples heated in air at same conditions.

In order to research the inner structure of doped TiO2, XPS measurement has been performed. Figure 7 shows the spectra of (i) TiO2, Ti 2P1/2, Ti4+ 2P3/2, (ii) P2p,

Figure 5. CL values of various a-TiO2 co-doped with K and P; their atomic ratio is a constant of 1:3, varying K/P = 0.1/0.3 (sample No. 28), 0.75/2.25 (No. 35), 1.5:4.5 (No. 38), and 3.0:9.0 (No. 41).

Figure 6. CL values of various single or co-doped a-TiO2 as a function of P, which materials were heated at 973 K in air or O2 atmosphere.

Figure 7. XPS spectra of a-TiO2 doped with various K and P prepared by heating in air, corresponding to (i) TiO2, (ii) P, (iii) K, and (iv) O. Here, No. 1, starting material, No. 2, non doped anatase, No. 18, K:P = 0:5, No. 23, K:P = 5:0, No. 35, K:P = 0.75:2.25, No.38, K:P = 1.5:4.5, No.41, K:P = 3:9 at%.

(iii) K2s, and (iv) O1s of various a-TiO2, such as No. 1 (as-received a-TiO2), No. 2 (973 K-heated No. 1), and No. 18, 23, 35, 38 and 41 samples which are denoted in Table 1. From these XPS spectra, there is not so much difference among all, except for O1s spectra in (iv). In these spectra, the peaks of 529.9 eV @ TiO2 lattice oxygen, 530.3 eV @ Ti2O3, and 531.6 eV @ non-lattice are mixed at around 530 eV. However, the weak shoulder peaks of P doped samples (No. 18, 35, 38, 41) are observed around 532 eV; these could be attributed to O in O-H or P-O.

Then, we measured FT-IR spectra of the same doped anatase samples to check the presence of O-H and P-O. As shown in Figure 8, it was clearly detected phosphate group ( PO 4 3 ) around 1000 cm−1. However, OH group (OH) was not found. Furthermore, the inner structure of doped TiO2 was studied by using XAFS [39]; the results are presented in Figure 9; (i) local chemical environment around K+ (XANES), all the spectra except for K2SO4 (the reference substance) were measured by partial fluorescence yield (PFY) mode. From upper the 2nd to

Figure 8. FT-IR spectra of a-TiO2 doped with various K and P prepared by heating in air; No. corresponding to the samples shown in Table 1.

Figure 9. EXAFS of (1.5 at%·K + 4.5 at%·P) co-doped a-TiO2 prepared by heating in air.

5th curves, 5 at% K, 1·K + 2·P, 1·K + 3·P, 1·K + 4·P (here, K = 0.75 at%) doped samples; small differences are seen for the peak at ~3614 eV and the shoulder structure in the higher energy side of the peak. And (ii) shows local chemical environment around P5+ (XANES), as the same as (i) in which all the spectra except for Ca3(PO4)2 (the reference substance) were measured by PFY mode. K-edge XANES spectrum of P elements in P-doped a-TiO2 was very similar to single addition of 5 at% P and compound addition. Therefore, the local chemical environment around P5+ is almost the same. EXAFS results of P K-edge spectrum of (1·K + 3·P, K = 0.75 at%)-doped a-TiO2 are shown in Figure 10, Fourier transformation of K-range had been done from 0.3 to 1.25 nm (3.0 - 12.5 Å) (upper right side), then radial structural function (lower right side) was obtained. The distance between P-O ions was considered to be shorter than the distance of P-O in PO 4 3 i.e., from 0.145 to 0.15 nm (1.45 to 1.50 Å). However, the coupling distance, calculated based on the radial structure function obtained through Fourier transformation of EXAFS oscillation depends on a factor which called as phase factor. The details are still unknown because it is several orders of magnitude shorter than the actual coupling distance. Therefore, O was present in the nearest neighbor of P, the same results reported by Shi et al. [32], and there was almost no ordering beyond that.

Then, the band structure of undoped and doped a-TiO2 were evaluated; their results are shown in Figure 11. These figures contain the function expressed as F(R) * hν2 on the ordinate, here, F(R) is reflectance spectrum intensity, hν (eV) (h: Planck constant, ν: wave number), and eV on the abscissa. Band gap Eg values were determined at the point of intersection between the line of tangency of each F(R) * hν2 curve and horizontal axis [40]. The Eg values of No. 38 (K:P =

Figure 10. (i) K of K-edge XANES and (ii) P of K-edge XANES of (1.5 at%·K + 4.5 at%·P) co-doped a-TiO2 prepared by heating in air.

Figure 11. Band gap energy spectra of various a-TiO2; No. corresponding to the samples shown in Table 1.

1.5:4.5 at%) 3.43 eV and 41 (K:P = 3:9 at%) 3.46 eV are almost the same. However, their energy levels were smaller than those (3.61 and 3.64 eV) of No. 1 and 2, respectively; this might be due to the co-doping (K and P) effect (this phenomenon is usually called as red shift [26] [27] [41]. In addition, these values of pure anatase (No.1 and No. 2) are much higher than those (3.2 - 3.3 eV) reported for the previous papers on anatase; this could be originated from the high purity more than 99.92% and fine particle (Ps of 0.028 µm: 28 nm). As it is very difficult to synthesize high purity/single-phase/fine anatase powder, for example, a very popular nano titanium dioxide powder (Ps ~ 30.3 nm, BET: 50 m2/g), “P25” AEROXIDE®, consists 80% anatase and 20% rutile phases. Therefore, the smaller band gap ~3.2 to ~3.3 eV for the anatase powder could be explained due to a small amount of impurities or double phases; because there are a few descriptions about their purity in the previous papers.

In the present study, pure a-TiO2 (No. 1 and heated 2) reveal only one Eg value, however, the doped samples have one or more energy gaps. Based on the viewpoints of Eg, it is difficult to explain the reason why these anatase can produce ROS even under dark conditions, because if we presume that the photocatalytic property of TiO2, i) generation of electrons and holes in the conduction and valence bands, respectively, should be occurred by absorbing excitation energy which is greater than Eg (3.43 - 3.64 eV). ii) It has been explained that these excited electrons and holes can produce ROS, such as, O 2 from O2 and ·OH from OH, respectively [42]. iii) However, as visible light energy, around 1.59 - 3.10 eV, is lower than Eg, visible light cannot excite the electrons in valence band of K&P doped anatase TiO2. iv) Therefore, the present a-TiO2 cannot submit much ROS under dark conditions. By the way, ultra violet wave (UV: λ < 387 nm) in solar light is only 5%, then effect of UV excitation could be ignored.

TEM images were taken to investigate the particle sizes of co-doped a-TiO2. As shown in Figure 12, it can be seen that the prepared powders calcined in O2 atmosphere possess a spherical shape and nanoparticle sizes irrespective of ball-milling. The crystallinity of the powders may be confirmed from crisp pictures. Ball-milling effect on the CL amount is described as follows.

Figure 12. TEM images of 1·K + n·P (n = 2, 3, 4, here K = 1.5 at%), co-doped a-TiO2 prepared by heating in O2 followed by ball milling.

Figure 13. Integrated CL values of 1·K+ n·P (n = 2, 3, 4, here K = 1.5 at%) co-doped a-TiO2 prepared by heating in O2 (non BM), and followed by ball milling (BM).

The integrated CL emission from the K and P co-doped a-TiO2 powders calcined in O2 at 973 K without and with ball-milling (BM) are represented in Figure 13. It appears that the TiO2doped with 1.5 at% K and 4.5 at% P, i.e., (1·K + 3·P) shows the highest CL value, however, after BM, 1·K + 4·P doped a-TiO2 revealed much higher CL value, that is, the peak-top of CL amount shifted to the higher P content. Here, the surface area SA values of these powders are also indicated at the top of each bar graph. At first we thought that this increase might be originated from the increment in SA introduced by BM. However, on the contrary to this expectation, the change in SA is not so much. Therefore, a small amount of strain, which was introduced in the anatase lattice by ball-milling (BM), might have some effect on the submission of CL. Based on our preliminary experience to check the effect of ball-milling time on CL value, it was cleared that 1.8 × 103 s (30 min) BM gave the best results and after much longer than it, the integrated CL values decreased, suggesting that too much strain could reduce the CL emission.

Then, different scavengers corresponding to each ROS were added to the highly efficient powder (No. 39, 1·K + 4·P with BM) during CL measurement in order to determine which ROS was emitted. All CL values with scavengers were compared to those without scavenger. It can be seen in Figure 14 that all ROS— hydroxyl radical OH· (2-propanol, PrOH, as scavenger [43] [44] [45] ), superoxide radical · O 2 (nitroblue tetrazolium, NBT [46] [47] and singlet oxygen 1O2 (dimethylfluran, DMF [48] )—are emitted. By comparing i) (No. 39) with iv) (No. 39 + PrOH), and v) (No. 39 + BM) with viii) (No. 39 + BM + PrOH) CL curves, hydroxyl radical OH· seems to be the least emitted ROS, whereas superoxide radical · O 2 is emitted the most by comparing i) (No. 39) with iii) (No. 39 + NBT), and v) (No. 39 + BM) with vii) (No. 39 + BM + NBT) CL curves. Regarding singlet oxygen 1O2, its emission was proved by comparing i) (No. 39) with ii) (No. 39+DMF), and v) (No. 39 + BM) with vi) (No. 39 + BM + DMF) CL curves, an interesting comment should be made. As a matter of fact, according to previous study [32], the emission of singlet oxygen requires a hole. Therefore, it is a photo-induced ROS as it requires an electron-hole pair to be formed as well. However, obtained results suggest that there is also presence of singlet oxygen 1O2 even in the dark. Hence, further study must be made on the exact mechanisms of formation of singlet oxygen under dark conditions. Table 2 shows

Figure 14. CL values of 1·K + 4·P (K = 1.5 at%) co-doped a-TiO2 (No. 39) with various scavengers.

Table 2. ROS of TiO2 doped with various amounts of K and P.

Both single doped and co-doped anatase powder heated in O2 submitted ROS under dark conditions.

the summary of what kinds of ROS were emitted from K/P doped a-TiO2. It should be noted that hydroxyl radical OH· which is much harmful to many kinds of bacteria was produced by K/P doped a-TiO2 with the atomic ratios of 1·K+ n·P, where n was 2 and 4. This means that a little deviated valance from +4, i.e., 1·K + 2·P à 3.66 (−0.34) and 1·K + 4·P à 4.2 (+0.2) could be close relation to form ·OH.

In order to evaluate the amount of ROS which were produced in various pH buffer solutions, ESR measurements [49] were performed without protection from light on the most efficient powder above mentioned, i.e., 1.5 at% K and 6 at% P co-doped, calcined in O2 and ball milled powder (No.39+ BM). Applying a spin trapping method with a spin-trap agent of 2,2-oxo-5,5-dimethyl-1-pyrroline1yloxyl (DMPO), thus obtained ESR results are presented on Figure 15. In Figure 15(a), ESR spectra measured at various pH values from 7 to 10 are shown; it appears that from the detected signal which is originated from the DMPO-OH radical, the ROS emitted from K/P doped a-TiO2 is mainly hydroxyl radical, indicating the opposite result shown in Figure 14. This could be explained that the difference in measuring conditions between CL and ESR; the former and the latter were performed under dark and light conditions, respectively.

In order to investigate the pH dependence of the ROS emission, an integral ESR signal was calculated. The result is shown in Figure 15(b). To this purpose, noise was eliminated and the integral was performed on the characteristic peaks of DMPO-OH radical only. From this figure, it was clear that ROS emission was the highest around pH = 9.5.

The ROS generation mechanism in doped anatase could be explained based on their non-stoichiometry as shown in Figure 16, metal-rich structure Ti 1 + x O 2 ( x > 0 ) [29], x in the formula represents the amount of interstitial titanium Ti i which is the main source to produce ROS [23] [24]. After doping of K or/and P and heating at high temperatures, Ti i will be created and increased. These Tii will react with oxygen as following reaction: Ti i + 2O Ti i 4 + 4e + O 2 . Then, ROS could be formed on the surface of TiO2 by the equations below.

Figure 15. (a) ESR spectrum of co-doped a-TiO2 (No. 39) measured under various pH values. (b) Peak integral intensity of the ESR spectra at different pH values.

Figure 16. Proposed antibacterial mechanism for doped a-TiO2 under dark conditions.

Ti i Ti i 4 + 4e (1)

Ti i 4 + 4H 2 O 4 OH + Ti i + 4H + (2)

4H + + 4e + 2O 2 2H 2 O 2 (3)

Eventually 4H 2 O + 2O 2 4 OH + 2H 2 O 2 .

Thus, one of reactive oxygen species, hydroxyl radical ·OH could be produced from the surface of a-TiO2.

4. Conclusion

Anatase titanium dioxide (a-TiO2) nanoparticle powders (NPS) have been prepared by combined doping of K and P with the atomic ratio of K/P = 1/3 into high purity (≥99.92%) a-TiO2 powder, followed by heating at 973 K for 3.6 × 103 s in oxygen atmosphere and ball-milled. These powders can submit much reactive oxygen species (ROS) under dark conditions. The microstructure and physicochemical properties such as lattice parameters, particle sizes Ps, and oxygen deficiency, reactive oxygen species (ROS) of thus prepared powders were examined using XRD, SEM, BET, XPS, FT-IR, XAFS, ESR and chemiluminescence (CL). The amount of ROS submitted from a-TiO2 NPS in the dark reached about 10 times higher than pure a-TiO2. Addition to this much hydroxyl radical OH·, one of ROS, has been recognized in 1·K + 2·P or 1·K + 4·P doped a-TiO2 NPS in light interception. The mechanism of generation of ROS from the doped anatase powder has been also proposed. The present results will open new academic and industrial fields in which metal oxides can sterilize both bacteria and virus even under dark condition for better civil life in a future.


The authors express their appreciation to Dr. H. OJI (NUSR/AichiSR, Japan), for his technical assistance and advice for the EXAFS measurement of doped a-TiO2. The authors thank Ms. M. Toda of the Doshisha University Research Centre for Interfacial Phenomena, for FE-SEM and TEM observations of the samples.


1No. 2 sample was the powder without K and P addition, which was heated at 973 K for 3.6 × 103 s in air as a reference.

Cite this paper: Nguyen, T. , Lemaitre, P. , Kato, M. , Hirota, K. , Tsukagoshi, K. , Yamada, H. , Terabe, A. , Mizutani, H. and Kanehira, S. (2021) Preparation of Anatase Titanium Dioxide Nanoparticle Powders Submitting Reactive Oxygen Species (ROS) under Dark Conditions. Materials Sciences and Applications, 12, 89-110. doi: 10.4236/msa.2021.122006.

[1]   Fischer, K., Gawel, A., Rosen, D., Krause, M., Latif, A.A., Griebe, J., et al. (2017) Low-Temperature Synthesis of Anatase/Rutile/Brookite TiO2 Nanoparticles on a Polymer Membrane for Photocatalysis. Catalyst, 7, 209-222.

[2]   Catauro, M., Tranquillo, E., Poggetto, G.D., Pasquali, M., Dell’Era, A. and Ciprioti, S.V. (2018) Influence of the Heat Treatment on the Particles Size and on the Crystalline Phase of TiO2 Synthesized by the Sol-Gel Method. Materials, 11, 2364-2374.

[3]   Pena, M., Meng, X., Korfiatis, G.P. and Jing, C. (2006) Adsorption Mechanism of Arsenic on Nanocrystalline Titanium Dioxide. Environmental Science & Technology, 40, 1257-1262.

[4]   Kuo, C.Y., Wu, C.H., Wu, J.T. and Chen, Y.R. (2015) Synthesis and Characterization of a Phosphorus-Doped TiO2 Immobilized Bed for the Photodegradation of Bisphenol A under UV and Sunlight Irradiation. Reaction Kinetics, Mechanisms and Catalysis, 114, 753-766.

[5]   Liou, J.W. and Chang, H.H. (2012) Bactericidal Effects and Mechanisms of Visible Light-Responsive Titanium Dioxide Photocatalysts on Pathogenic Bacteria. Archivum Immunologiae et Therapiae Experimentalis, 60, 267-275.

[6]   Akpan, U.G. and Hameed, B.H. (2009) Parameters Affecting the Photocatalytic Degradation of Dyes Using TiO2-Based Photocatalysts: A Review. Journal of Hazardous Materials, 17, 520-529.

[7]   Tachikawa, T., Tojo, S., Fujitsuka, M. and Majima, T. (2004) Influences of Adsorption on TiO2 Photocatalytic One-Electron Oxidation of Aromatic Sulfides Studied by Time-Resolved Diffuse Reflectance Spectroscopy. The Journal of Physical Chemistry B, 108, 5859-5866.

[8]   Carp, O., Huisma, C.L. and Reller, A. (2004) Photoinduced Reactivity of Titanium Dioxide. Progress in Solid State Chemistry, 32, 133-177.

[9]   Sunada, K., Watanabe, T. and Hashimoto, K. (2003) Studies on Photokilling of Bacteria on TiO2 Thin Film. Journal of Photochemistry and Photobiology A: Chemistry, 156, 227-233.

[10]   Akhavan, O. (2009) Lasting Antibacterial Activities of Ag-TiO2/Ag/a-TiO2 Nanocomposite Thin Film Photocatalysts under Solar Light Irradiation. Journal of Colloid and Interface Science, 336, 117-124.

[11]   Ashkarran, A.A., Aghigh, S.M., Kavianipour, M. and Farahani, N.J. (2011) Visible Light Photo- and Bioactivity of Ag/TiO2 Nanocomposite with Various Silver Contents. Current Applied Physics, 11, 1048-1055.

[12]   Sun, C., Li, Q., Gao, S., Cao, L. and Shang, J.K. (2010) Enhanced Photocatalytic Disinfection of Escherichia coli Bacteria by Silver and Nickel Comodification of a Nitrogen-Doped Titanium Oxide Nanoparticle Photocatalyst under Visible-Light Illumination. Journal of the American Ceramic Society, 93, 531-535.

[13]   Yu, J.C., Ho, W.K., Lin, J., Yip, H. and Wong, P.K. (2003) Photocatalytic Activity, Antibacterial Effect, and Photoinduced Hydrophilicity of TiO2 Films Coated on a Stainless Steel Substrate. Environmental Science & Technology, 37, 2296-2301.

[14]   Sunada, K., Kikuchi, Y., Hashimoto, K. and Fujishima, A. (1998) Bactericidal and Detoxification Effects of TiO2 Thin Film Photocatalysts. Environmental Science & Technology, 32, 726-728.

[15]   Salih, F.M. (2002) Enhancement of Solar Inactivation of Escherichia coli by Titanium Dioxide Photocatalytic Oxidation. Journal of Applied Microbiology, 92, 920- 926.

[16]   Zaleska, A. (2008) Doped-TiO2: A Review. Recent Patents on Engineering, 2, 157- 164.

[17]   Le, P.H., Le, T.H., Lam, T.N., Nguyen, T.N.H., Nguyen, V.T., Le, T.C.T., et al. (2018) Enhanced Photocatalytic Performance of Nitrogen-Doped TiO2 Nanotube Arrays Using a Simple Annealing Process. Micromachines, 9, 618-630.

[18]   Hou, J., Wang, L., Wang, C., Zhang, S., Liu, H., Li, S., et al. (2019) Toxicity and Mechanisms of Action of Titanium Dioxide Nanoparticles in Living Organisms. Jour- nal of Environmental Science, 75, 40-53.

[19]   Venkatasubbu, D., Baskarb, R., Anusuya, T., Seshan, C.A. and Chelliah, R. (2016) Toxicity Mechanism of Titanium Dioxide and Zinc Oxide Nanoparticles against Food Pathogens. Colloids and Surfaces B: Biointerfaces, 148, 600-606.

[20]   Iavicoli, I., Leso, V., Fontana, L. and Berganaschi, A. (2011) Toxicological Effects of Titanium Dioxide Nanoparticles: A Review of in Vitro Mammalian Studies. European Review for Medical and Pharmacological Sciences, 15, 481-508.

[21]   Santhoshkumar, T., Rahuman, A.A., Jayaseelan, C., Rajakumar, G., Marimuthu, S., Kirthi, A.V., et al. (2014) Green Synthesis of Titanium Dioxide Nanoparticles Using Psidium guajava Extract and Its Antibacterial and Antioxidant Properties. Asian Pacific Journal of Tropical Medicine, 7, 968-976.

[22]   Hirota, K., Sugimoto, M., Kato, M., Tsukagoshi, K., Tanigawa, T. and Sugimoto, H. (2010) Preparation of Zinc Oxide Ceramics with a Sustainable Antibacterial Activity under Dark Conditions. Ceramics International, 36, 497-506.

[23]   Nguyen, T.M.P., Hirota, S., Suzuki, Y., Kato, M., Hirota, K., Taniguchi, H., et al. (2018) Preparation of ZnO Powders with Strong Antibacterial Activity under Dark Conditions. Journal of the Japan Society of Powder and Powder Metallurgy, 65, 316-324.

[24]   Nguyen, T.M.P., Hirota, K., Kato, M., Tsukagoshi, K., Yamada, H., Terabe, A., et al. (2019) Dependence of Antibacterial Activity of ZnO Powders on Their Physicochemical Properties. Journal of the Japan Society of Powder and Powder Metallurgy, 66, 435-441.

[25]   Chen, L.C., Huang, C.M. and Tsai, F.R. (2007) Characterization and Photocatalytic Activity of K+-Doped TiO2 Photocatalysts. Journal of Molecular Catalysis A: Chemical, 265, 133-140.

[26]   Bessekhouad, Y., Robert, D., Weber, J.V. and Chaoui, N. (2004) Effect of Alkaline-Doped TiO2 on Photocatalytic Efficiency. Journal of Photochemistry and Photobiology A: Chemistry, 167, 49-57.

[27]   Yang, G., Yan, Z., Xiao, T. and Yang, B. (2013) Low-Temperature Synthesis of Alkalis Doped TiO2 Photocatalysts and Their Photocatalytic Performance for Degradation of Methyl Orange. Journal of Alloys and Compounds, 580, 15-22.

[28]   Yildizhan, M.M., Sturm, S. and Gulgun, M.A. (2016) Structural and Electronic Modifications on TiO2 Anatase by Li, K or Nb Doping below and above the Solubility Limit. Journal of Materials Science, 51, 5912-5923.

[29]   Hao, L., Guan, S., Takaya, S., Yoshida, H., Tochihara, M. and Lu, Y. (2017) Preparation of Visible-Light-Responsive TiO2 Coatings Using Molten KNO3 Treatment and Their Photocatalytic Activity. Applied Surface Science, 407, 276-281.

[30]   Yu, J.C., Zhang, L., Zheng, Z. and Zhao, J. (2003) Synthesis and Characterization of Phosphated Mesoporous Titanium Dioxide with High Photocatalytic Activity. Chemistry of Materials, 15, 2280-2286.

[31]   Lin, L., Lin, W., Zhu, Y., Zhao, B. and Xie, Y. (2005) Phosphor-Doped Titania—A Novel Photocatalyst Active in Visible Light. Chemistry Letters, 34, 284-285.

[32]   Shi, Q., Yang, D., Jiang, Z. and Li, J. (2006) Visible-Light Photocatalytic Regeneration of NADH Using P-Doped TiO2 Nanoparticles. Journal of Molecular Catalysis B: Enzymatic, 43, 44-48.

[33]   Pan, X., Yang, M.Q., Fu, X., Zhang, N. and Xu, Y.J. (2013) Defective TiO2 with Oxygen Vacancies: Synthesis, Properties and Photocatalytic Applications. Nanoscale, 5, 3601.

[34]   Lin, L., Lin, W., Xie, J., Zhu, Y., Zhao, B. and Xie, Y. (2007) Photocatalytic Properties of Phosphor-Doped Titania Nanoparticles. Applied Catalysis B: Environmental, 75, 52-58.

[35]   Jin, C., Zheng, R.Y., Guo, Y., Xie, J.L., Zhu, Y.X. and Xie, Y.C. (2009) Hydrothermal Synthesis and Characterization of Phosphorous-Doped TiO2 with High Photocatalytic Activity for Methylene Blue Degradation. Journal of Molecular Catalysis A: Chemical, 313, 44-48.

[36]   Alfred, A.C., Olav, M.K. and Rance, A.V. (1995) Quantitative Analysis in Diffuse Reflectance Spectrometry: A Modified Kubelka-Munk Equation. Vibrational Spectroscopy, 9, 19-27.

[37]   Mousset, E., Oturan, N. and Oturan, M.A. (2018) An Unprecedented Route of OH Radical Reactivity Evidenced by an Electrocatalytical Process: Ipso-Substitution with Perhalogenocarbon Compounds. Applied Catalysis B: Environmental, 226, 135-146.

[38]   Shannon, R.D. (1976) Revised Effective Ionic Radii and Systematic Studies of Interatomic Distances in Halides and Chalcogenides. Acta Crystallographica Section A, 32, 751-767.

[39]   Lin, X.C., Tijana, R., Zhiyu, W. and Marion, C.T. (1997) XAFS Studies of Surface Structures of TiO2 Nanoparticles and Photocatalytic Reduction of Metal Ions. The Journal of Physical Chemistry B, 101, 10688-10697.

[40]   Bandna, B., Santosh, K., Heung, N.L. and Rajesh, K. (2016) Formation of Oxygen Vacancies and Ti3+ State in TiO2 Thin Film and Enhanced Optical Properties by Air Plasma Treatment. Scientific Reports, 6, Article No. 32355.

[41]   Ma, J., Li, W., Le, N.T., Díaz-Real, J.A., Body, M., Legein, C., et al. (2019) Red-Shifted Absorptions of Cation-Defective and Surface Functionalized Anatase with Enhanced Photoelectrochemical Properties. ACS Omega, 4, 10929-10938.

[42]   Fujishima, A. and Zhang, C.R. (2006) Titanium Dioxide Photocatalysis: Present Situation and Future Approaches. Chimie, 9, 750-760.

[43]   Smith, B.A., Teel, A.L. and Watts, R.J. (2004) Identification of the Reactive Oxygen Species Responsible for Carbon Tetrachloride Degradation in Modified Fenton’s Systems. Environmental Science & Technology, 38, 5465-5469.

[44]   Kozmér, Z., Takács, E., Wojnárovits, L., Alapi, T., Hernádi, K. and Dombi, A. (2016) The Influence of Radical Transfer and Scavenger Materials in Various Concentrations on the Gamma Radiolysis of Phenol. Radiation Physics and Chemistry, 124, 52-57.

[45]   Kormali, P., Triantis, T., Dimotikali, D., Hiskia, A. and Papaconstantinou, E. (2006) On the Photooxidative Behavior of TiO2 and PW12O403-: OH Radicals versus Holes. Applied Catalysis B: Environmental, 68, 139-146.

[46]   Nishibori, S. and Namiki, K. (1998) Superoxide Anion Radical-Scavenging Ability of Fresh and Heated Vegetable Juices. Nippon Shokuhin Kagaku Kogaku Kaishi, 45, 144-148.

[47]   Wang, L., Liu, S., Zheng, Z., Pi, Z., Song, F. and Liu, Z. (2015) Rapid Assay for Testing Superoxide Anion Radical Scavenging Activities to Natural Pigments by Ultra-High Performance Liquid Chromatography-Diode-Array Detection Method. Analytical Methods, 7, 1535-1542.

[48]   Ao, Y., Satoh, K., Shibano, K., Kawahito, Y. and Shioda, S. (2008) Singlet Oxygen Scavenging Activity and Cytotoxicity of Essential Oils from Rutaceae. Journal of Clinical Biochemistry and Nutrition, 43, 6-12.

[49]   Noda, H., Oikawa, K., Ogata, T., Matsuki, K. and Kamada, H. (1986) Preparation Method and Characterization of Titanium Oxide (IV). Journal of the Chemical Society of Japan, 8, 1084-1090. (In Japanese)