Tartrazine - American Chemical Society

Nov 13, 2014 - We examined the ROS production and degradation of a widely used azo dye, FD&C Yellow No. 5 (tartrazine), triggered by photoactivated Ti...
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FD&C Yellow No. 5 (Tartrazine) Degradation via Reactive Oxygen Species Triggered by TiO2 and Au/TiO2 Nanoparticles Exposed to Simulated Sunlight Meng Li,†,§ Weiwei He,† Yi Liu,† Haohao Wu,†,§ Wayne G. Wamer,‡ Y. Martin Lo,§ and Jun-Jie Yin*,† †

Division of Analytical Chemistry and ‡Division of Bioanalytical Chemistry, Office of Regulatory Science, Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration, College Park, Maryland 20740, United States § Department of Nutrition and Food Science, University of Maryland, College Park, Maryland 20742, United States S Supporting Information *

ABSTRACT: When exposed to light, TiO2 nanoparticles (NPs) become photoactivated and create electron/hole pairs as well as reactive oxygen species (ROS). We examined the ROS production and degradation of a widely used azo dye, FD&C Yellow No. 5 (tartrazine), triggered by photoactivated TiO2 NPs. Degradation was found to follow pseudo-first order reaction kinetics where the rate constant increased with TiO2 NP concentration. Depositing Au on the surface of TiO2 largely enhanced electron transfer and ROS generation, which consequently accelerated dye degradation. Alkaline conditions promoted ROS generation and dye degradation. Results from electron spin resonance spin-trap spectroscopy suggested that at pH 7.4, both hydroxyl radical (•OH) and singlet oxygen (1O2) were responsible for dye discoloration, whereas at pH 5, the consumption of 1O2 became dominant. Implications for dye degradation in foods and other consumer products that contain both TiO2 and FD&C Yellow No. 5 as ingredients are discussed. KEYWORDS: TiO2 nanoparticles, ROS, FD&C Yellow No. 5, ESR, color degradation, tartrazine



INTRODUCTION Titanium dioxide (TiO2) is used as an ingredient in a wide range of consumer products. Important examples include its use as a UV protectant in sunscreens, as pigment in paints, and as coloring and anticaking agents in drugs and food products.1 Additionally, TiO2, particularly in its anatase form, is an efficient photocatalyst. For this reason, TiO2 has also been extensively used as a photocatalyst for wastewater treatment to detoxify biocides or other hazardous pollutants.2,3 To date, in the food industry, TiO2 is primarily found in soft drinks, yogurts, ice cream, chips, pickles, honey, mustard, gum, puddings and gelatins, confectionary products, and baked goods.4 While use of TiO2 in food is generally considered safe in the United States, there is a 1% by weight maximum allowance in food,5 unlike Japan where it is permitted without limitations, except in specific types of food.6 On the other hand, a tighter control over the use of TiO2 is seen in different parts of the world. The European Union authorizes its use at 100 mg/L (individually or in combination with other dyes) in nonalcoholic flavored drinks, 0.015% in desserts including flavored milk products, and 0.02% in candied fruits and vegetables.7 In India, the use of TiO2 is restricted to chewing gum and bubble gum at no more than 1% and to powdered concentrate mixes for fruit beverage drinks not to exceed 0.01% by weight.8,9 Recent studies revealed that the size of a significant amount of food-grade TiO2 falls into nanorange.10−12 Since nanoparticles (NPs) can exhibit increased chemical and biological activity, which result from a reduction in particle size and increased surface area, the behavior of nanosized TiO2 in foods may differ from that of larger-sized TiO2. When irradiated, TiO2 NPs absorb photon energy equal to or higher than their © XXXX American Chemical Society

band gap (3.0 eV for the rutile and 3.2 eV for the anatase phase) and excite electrons in the valence band to the conduction band, which results in conduction band electrons and leaves valence band holes that can extract electrons from water or hydroxyl ions while hydroxyl radicals (•OH) are generated.13,14 This process also involves formation of other reactive oxygen species (ROS), including superoxide radicals (O2−•) and singlet oxygen (1O2),15 as well as intermediate species such as H2O2 or O2.13 By taking advantage of their light-tunable ability to generate ROS, TiO2 NPs have been used in food package materials as an antimicrobial agent16,17 as well as in intelligent ink for oxygen detection.18 ROS generated by photoexcited TiO2 NPs may lead to oxidative damage in biological systems.19,20 Of particular interest are food matrices because they are complex and contain many reactive compounds. ROS have been observed to cause degradation of the azo dye tartrazine,21,22 which is certifiable as FD&C Yellow No. 5 by the U.S. Food and Drug Administration (FDA) for use in coloring foods and other FDA-regulated products. Therefore, FD&C Yellow No. 5 was selected as a model for the current study to evaluate the decomposition of this type of dyes when exposed to simulated sunlight in the presence of TiO2 NPs. Received: September 18, 2014 Revised: November 4, 2014 Accepted: November 13, 2014

A

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Figure 1. TEM images of (a) TiO2 NPs and (b) Au/TiO2 hybrid nanostructures; (c) dark field STEM of Au/TiO2 hybrid nanostructures, inset shows EDS analysis; and (d) HRTEM of Au/TiO2 (Au:TiO2 ratio = 1:100 by weight) hybrid nanostructures formation after photodeposition. All scale bars are 10 nm.



water, and then dried at 45 °C overnight in oven and used for further experiments. Characterization of TiO2 NPs and Au/TiO2 Nanostructures. UV−vis absorption spectra were obtained using a Varian Cary 300 spectrophotometer. Transmission electron microscopy (TEM) and high resolution TEM (HRTEM) images were captured on a JEM 2100 FEG (JEOL) transmission electron microscope (accelerating voltage of 200 kV) located at the NanoCenter, University of Maryland, College Park, MD. Energy-dispersive X-ray spectroscopy analysis (EDS) was conducted using the same microscope. The samples for TEM analysis were prepared by adding drops of the redispersed colloidal solutions onto standard holey carbon-coated copper grids, which were then air-dried at room temperature. Dye Degradation. For examining the degradation of Y5, 10 mL of an aqueous solution containing 25 mg/L MY5 or Y5 and selected concentrations of TiO2 or Au/TiO2 NPs was continuously stirred in the dark for 30 min to establish an adsorption−desorption equilibrium between the NPs and dyes. The TiO2 and Au/TiO2 NPs were dispersed as suspensions for all experiments. Each suspension was transferred to a 50 mL quartz Erlenmeyer flask, sealed, and irradiated using a light source to deliver simulated sunlight. The light resource consisted of a Universal Arc Lamp Power Supply (69920 Universal Supply, Newport, Irvine, CA) and an Oriel Xenon Arc Lamp (880 W) solar simulator. During irradiation, the suspension was continuously stirred and kept in ice water bath. The degradation process was monitored using a Varian Cary 300 spectrophotometer. Before UV− vis analysis, the reaction mixture was centrifuged to remove the TiO2 or Au/TiO2 particles. The photocatalytic degradation of MY5 was observed to follow pseudo-first-order kinetics. When the dye concentration was in the millimolar range, the apparent first-order rate constant, kapp, was determined from the slope of ln (C/C0) versus irradiation time according to the following relation:

MATERIALS AND METHODS

Materials. The sample of FD&C Yellow No. 5 (Y5) used in this study originated from a batch submitted to the FDA for certification. The purified main component of FD&C Yellow No. 5 (MY5) was separated by countercurrent chromatography from a portion of Y5 and was generously provided by Dr. Adrian Weisz of the FDA.23 Methanol (Fisher Scientific, Fair Lawn, NJ, USA), ammonium acetate (NH4OAc, Fisher), and water were of chromatography grade. The mixed-phase titanium dioxide Deggussa Aeroxide P25 (75% anatase and 25% rutile phase; surface area, 50 ± 15 m2/g) was purchased from Degussa Corporation (Parsippany, NJ). The spin-trap, 5-tert-butoxycarbonyl 5methyl-1-pyrroline N-oxide (BMPO), was purchased from Applied Bioanalytical Laboratories (Sarasota, FL). The 2,2,6,6-tetramethyl-4piperidone (TEMP) was purchased from Wako Pure Chemical Industry, Ltd. Japan; and 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) was purchased from Alexis, Enzo Life Sciences, Inc. (Farmingdale, NY). The 15N-labeled 4-oxo-2,2,6,6-tetramethylpiperidine-d16−1-oxyl (15N-PDT) was purchased from Cambridge Isotope Laboratories (Andover, MA). Superoxide dismutase (SOD) and standard buffer solutions were purchased from Sigma Chemical Co. (St. Louis, MO). Before use, each buffer stock solution (pH 3.9 HAc-NaAc, pH 5.0 PBS, pH 7.4 PBS, all at 0.1 M) was treated with Chelex100 molecular biology-grade resin from Bio-Rad Laboratories (Hercules, CA) to remove trace metal ions. Distilled deionized water (18.2 MΩ cm) from a Milli-Q water purification system was used in all experiments. Preparation of Au/TiO2 Hybrid Nanostructures. Au/TiO2 hybrid nanostructures were prepared according to a previously reported photoreduction method with modifications.24,25 To obtain a 1.00 wt % Au/TiO2 sample, 10 mL of 2.0 mg/mL TiO2 suspension in methanol (50% by volume) was mixed with 0.1 mL of an aqueous solution of HAuCl4 (2 mg/mL Au) in a 50 mL quartz Erlenmeyer flask and sealed. The above mixture was sonicated for 5 min and then irradiated for 30 min under simulated sunlight with magnetic stirring in an ice water bath. The light source was an Oriel Xenon Arc Lamp Solar Simulator. A clearly evident color change from white to greyish pink was observed, which indicated the formation of Au/TiO2 hybrid nanostructures. After centrifugation, UV spectral analysis of the supernatant after each photodeposition revealed that the Au had been almost completely (>99%) deposited as Au on the TiO2 particles. The precipitate was collected, washed three times with double distilled

− ln

C = kappt C0

(1)

where t is the irradiation time, C0 is the initial concentration of dye, and C is the concentration of dye (mg/L) at time t.26 The concentration was determined by spectrophotometry. The UV−vis spectrum of MY5 consists of two characteristic peaks at 425 and 258 nm. The λmax value of the dye (425 nm) was chosen for quantitative degradation studies. B

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Electron Spin Resonance. ESR spin-trap spectroscopy is a powerful tool for evaluating the capability of NPs to generate reactive oxygen species and free radicals.27 It was employed to detect the generation of ROS and occurrence of electron transfer during irradiation of samples with simulated sunlight. The spin-trap, BMPO, was used to detect the generation of superoxide radicals (O2−•) or hydroxyl radicals (•OH). TEMP was used to investigate the generation of singlet oxygen (1O2). TEMPO, a stable radical that exhibits an ESR signal with three identical peaks, was used as spin probe to study the electrons generated in the TiO2 aqueous suspension. Oxygen consumption during dye degradation photocatalyzed by TiO2 NPs was monitored by ESR oximetry. A watersoluble spin label, 15N-PDT, was used to detect the oxygen content in sample solutions. The shape of the 15N-PDT ESR spectrum is dependent on the amount of O2 that interacts with it, where higher peak-to-peak amplitude (A) and narrower peak-to-peak line width (W) are found when the levels of O2 in solution are reduced.28 The O2 concentration was calculated by a calibration standard curve described by

[O2 ] = 419.72 × W − 44.12

presence of nano-TiO2 was carried out under simulated sunlight. Figure 2, panel a illustrates the effect of the initial

(2)

where [O2] is the concentration in solution (μM). The consumption of oxygen was expressed as [O2‑time]/[O2‑initial]. Fifty microliter aliquots of control or sample solutions were put in glass capillary tubes with internal diameters of 1 mm and sealed using Critoseal capillary tube sealant, then placed into the microwave cavity of a Bruker EMX ESR Spectrometer (Billerica, MA) to obtain ESR spectra. All ESR measurements were carried out in situ using the following settings for detection of the spin adducts: 10 mW microwave power for detection of spin adducts using spin-traps and 1 mW microwave power for ESR oximetry using 15N-PDT, 100 G scan range, and 1 G field modulation. A Schoeffel xenon arc (450 W) solar simulator was used to irradiate samples during ESR measurements. ESR spectra were collected during irradiation at different time intervals. All measurements were performed in replicate at ambient temperature. Control groups, either without catalysts or without irradiation, were also recorded for comparison. The final concentration of each component is described in each figure caption. All experiments were repeated three times. Data are expressed as mean ± standard deviation.



RESULTS AND DISCUSSION

Characterization of TiO2 NPs and Au/TiO2 Hybrid Nanostructures. The morphology of TiO2 NPs and Au/TiO2 hybrid nanostructures was characterized by TEM. Figure 1, panel a shows the original TiO2 P25 agglomerates with the size range of 22−48 nm. Figure 1, panel b shows the Au NPs (dark dots) located on the surface of the individual TiO2 NPs. The size of the photodeposited Au NPs was estimated to be 4−10 nm. After Au was doped onto TiO2 surface, the surface characteristics and size of the TiO2 NPs remained unchanged. EDS analysis was performed to further confirm the formation of Au/TiO2 nanostructure. The EDS spectrum in Figure 1, panel c shows a strong signal for Ti and a weak signal for Au. The mass ratio of Au/TiO2 for the selected area was estimated to be approximately 5% based on this spectrum. Elemental copper and carbon were also detected by EDS, which was associated with the supporting carbon-coated copper grids. The HRTEM image (Figure 1d) taken from the interfacial region of Au and TiO2 shows the lattice image with a lattice spacing of 0.353 nm that corresponds to the {001} facets of anatase phase. Lattice fringes with interplanar distance of 0.202 nm were observed on the dark dots in Figure 1, panel d, which is similar to the planar distance for Au {200} (d = 0.204 nm). Degradation of FD&C Yellow No. 5. Effect of Initial TiO2 Concentration. The degradation of Y5 and MY5 in the

Figure 2. Decomposition of purified MY5 under simulated sunlight. Yaxis (C/C0) shows the concentration of remaining dye at irradiation time divided by the initial dye concentration. Reactions were carried out in (a) water solutions of MY5 and 0 (control), 0.01, 0.02, 0.05, 0.1, and 0.2 mg/mL TiO2 NPs irradiated by simulated sunlight for different time intervals or without sunlight exposure (control); (b) 10 mM PBS buffer (pH 5.0, 6.0, and 7.4) or 10 mM acetic acid buffer (pH 3.9) solutions containing MY5 and 0.1 mg/mL TiO2 NPs; (c) water solutions of MY5 or batch-certified Y5 containing 0.1 mg/mL TiO2 NPs or Au/TiO2 hybrid nanostructures. The initial dye concentration in all samples was fixed at 25 mg/mL.

TiO2 NPs concentration on degradation kinetics. The initial concentration of MY5 was fixed at 25 mg/L, while the concentration of TiO2 NPs was adjusted to 0.01, 0.02, 0.05, 0.1, and 0.2 mg/mL. Figure 2, panel a demonstrates that MY5 remains unchanged after 120 min for the control groups, either without TiO2 NPs but exposed to simulated sunlight or added with TiO2 NPS but without exposure to simulated sunlight. Dos C

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purified main component according to data obtained by spectrophotometric analysis. This result points to a higher stability of the batch-certified Y5 compared to MY5, which may be attributable to the more complex mixture of dye components in Y5. Effect of Deposition of Au onto TiO2 Surface. Noble metals have been widely used to enhance the photoactivity of TiO2. Au NPs deposited on TiO2 act as electron traps and enhance the electron−hole separation and the subsequent transfer of the trapped electron to the adsorbed O2 that acts as an electron acceptor. Therefore, to investigate the relationship between dye degradation and electron/hole pair as well as ROS generation, we synthesized Au/TiO2 hybrid nanostructure by depositing Au onto the surface of TiO2. Figure 2, panel c shows that, for both MY5 and Y5, Au/TiO2 exhibited enhanced photoactivity and accelerated color degradation, again with a higher degradation rate for MY5. TiO2-Generated Electrons and ROS. Detection of Electron Reactivity during Photoexcitation of TiO2 NPs. ESR was employed to investigate the generation of ROS and occurrence of electron transfer during photoexcitation of TiO2 in aqueous suspension. The reactive electrons generated in 0.1 mg/mL TiO2 NPs were detected using a stable spin probe, TEMPO. TEMPO is a stable radical that has been widely used as a spin label in ESR spectroscopy, and it exhibits a triplet ESR spectrum, whereas its reduced form, hydroxyl amine (TEMPOH), is ESR silent.37 As shown Figure 3, panel a, the

Santos et al. reported a significant reduction of tartrazine in aqueous solution with 15 min of UV irradiation.21 The difference between this study and our results may be attributed to the different light sources and intensities used. Figure 2, panel a shows that an increase in the concentration of TiO2 resulted in a higher rate of degradation for MY5. At TiO2 NP concentration of 0.1 mg/mL, the dye was not spectrophotometrically detectable after 30 min of exposure to simulated sunlight. The inset of Figure 1, panel a shows the calculated reaction constant, kapp, at different concentrations of TiO2. The highest rate constant (kapp = 0.133 min−1) was observed at 0.2 mg/mL TiO2 NPs. Effect of pH. The pH value of the reaction mixture plays multiple roles in the dye degradation process by affecting the ionization state of the TiO2 surface and hydroxyl radical generation in the solution26 as well as the structure of tartrazine.23 In this study, we investigated pH values of 3.9, 5.0, 6.0, and 7.4 to mimic the pH range of particular food products. Figure 2, panel b shows that under slightly alkaline conditions (pH = 7.4), the degradation rate was significantly promoted (kapp = 0.084 min−1), whereas the rate constant kapp was calculated to be 0.028, 0.035, and 0.039 min−1 for pH values 3.9, 5.0, and 6.0, respectively. The effect of pH value plays multiple roles in dye decomposition, including influences on •OH generation and adsorption as well as the adsorption of dye. Higher pH value could provide higher concentration of hydroxyl ions to react with valence band holes (h+) to form •OH.29 However, under acidic conditions (pH < 6.8), the TiO2 surface is positively charged, whereas it is negatively charged for pH > 6.8.30 When TiO2 is negatively charged, Coulombic repulsion takes place, which repels the hydroxyl ion and oxygen molecule from adsorbing on its surface and thus decreases the availability of hydroxyl and superoxide radical for dye degradation.31 On the other hand, in acidic media, a strong adsorption of the dye onto the TiO2 particle has been observed, which results from the significantly high electrostatic attraction force between the positively-charged TiO2 and the anionic dye molecule, which causes an increase in adsorption and accelerates the degradation process.32,33 Therefore, with regard to the influence of pH on azo dye degradation, some reported a bell-shaped figure in which the dye degradation reaches its maximum around neutral pH.34,35 For Y5 (tartrazine) decomposition triggered by TiO2, the rate constant kapp was found to increase with pH in the pH range of 2.2−11.36 Effect of Dye Composition. Y5 is batch certified by the FDA to ensure compliance with specifications in the Code of Federal Regulations (CFR).23 Among the specifications is a limit of 1% for each of two polysulfonated subsidiary colors: the tetrasodium salt of 4-((4,5-disulfo(1,1-biphenyl)-2-yl) hydrazono)-4,5-dihydro-5-oxo-1-(4-sulfophenyl)-1H-pyrazole3-carboxylic acid and the trisodium salt of 4,4′-(4,5-dihydro-5oxo-4-((4-sulfophenyl)hydrazono)-1H-pyrazol-1,3-diyl)bis(benzenesulfonic acid), which have been assigned the abbreviations Pk5 and Pk7, respectively.23 To our best knowledge, most of the Y5 samples in previously published studies were obtained from commercially available tartrazine samples that contain around 85% MY5. It is noteworthy that the purity and composition of those samples may lead to different results. Therefore, we studied a certified batch of dye (Y5, ∼86% purity) used for coloring foods, drugs, and cosmetics as well as its purified main component (MY5, ∼99% purity). Under the same treatment conditions, the FDAcertified dye showed a lower degradation rate compared to the

Figure 3. Detection of photogenerated electrons and ROS in solutions containing 0.1 mg/mL TiO2 NPs or Au/TiO2 hybrid nanostructures. ESR spectra obtained from samples mixed with different spin probes: (a) 0.02 mM TEMPO; (b) 25 mM BMPO; and (c) 2 mM TEMP. First row of each column presents both of the control groups, which contained the spin probe either without TiO2 NPs or without simulated sunlight irradiation. All the spectra were recorded after 3 min of irradiation with simulated sunlight.

triplet-peak ESR signal of TEMPO remained unchanged for both control groups, either without TiO2 NPs but exposed to simulated sunlight or added with TiO2 NPS but without exposure to simulated sunlight. When TiO2 NPs were added (0.1 mg/mL) and simulated sunlight irradiation was applied for 3 min, the TEMPO signal intensity was reduced. Further flattening of ESR spectrum was observed for samples containing the same amount of Au/TiO2 NPs, which indicates a higher fraction of TEMPO being reduced to TEMPOH. Since our results indicated that TEMPO does not react with other species in the solution, such as hydroxyl radical or superoxide D

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Figure 4. ESR detection of •OH during decomposition of dye upon simulated sunlight irradiance: (a) ESR spectra recorded after 1 min of irradiation by simulated sunlight; and (b) time-dependent ESR signal intensity obtained by measuring the peak-to-peak height of the second line of the ESR spectrum of BMPO/•OH. Reactions were conducted in 10 mM pH 5.0 or 7.4 PBS buffer solutions containing 25 mM BMPO spin-trap. All sample solutions contained 0.1 mg/mL TiO2 NPs with (or without) 25 mg/mL MY5.

We used the spin probe TEMP to study singlet oxygen (1O2) generation during irradiation of TiO2 NPs. TEMP is ESR silent. When it reacts with singlet oxygen, the reaction product, a nitroxide radical, 2, 2, 6, 6-tetramethylpiperidine-N-oxyl (TEMPONE), is formed and shows a 1:1:1 peak ESR spectrum.41 No ESR signal was observed for both control groups containing the spin probe TEMP, either without TiO2 NPs but exposed to simulated sunlight or added with TiO2 NPS but without exposure to simulated sunlight (Figure 3c). ESR spectra with three lines with equal intensities (aN = 16.0 G), typical of nitroxide radicals, were found for irradiated suspensions containing TEMP and TiO2 (Figure 3c). The hyperfine splitting constant and g factor of the observed signal were identical to those of commercial TEMPONE, which indicates the generation of singlet oxygen during irradiation. A similar spectrum with higher intensity was observed for Au/ TiO2. Previous studies found evidence that singlet oxygen was formed through the following pathway:42

radical generated by H2O2 (Figure S1, Supporting Information), the reduction of TEMPO necessarily resulted from the electrons generated during photoexcitation of TiO2 NPs. Generation of Hydroxyl and Superoxide Radicals. The generation of hydroxyl radical by photoexcited TiO2 NPs in aqueous suspension was confirmed using the spin-trap BMPO. Being ESR silent itself, BMPO can trap hydroxyl radical or superoxide radical and become ESR detectable. The spinadduct BMPO/•OH exhibits a 1:2:2:1 ratio in peak ESR signal intensity, with hyperfine coupling constant aN = 13.5 G, aHβ = 15.3 G, and aHγ = 0.62 G, while the spin-adduct of BMPO and superoxide radical (BMPO/O2−•) has a typical four-line ESR spectrum with hyperfine splitting structure (aN = 13.4 G, aHβ = 12.1 G).38 Figure 3, panel b shows an ESR spectrum identical to the BMPO/•OH spin-adduct signal for TiO2 exposed to simulated sunlight. A similar ESR pattern with higher intensity was observed for Au/TiO2 NPs under the same experimental conditions. Previous studies have reported difficulties when using BMPO as a spin-trap for a system containing both •OH and O2−• because of possible overlap of BMPO/•OH and BMPO/O2−• signal.14 For irradiated TiO2 solutions, the formation of •OH has been widely recognized, while several studies have reported observation of O2−• detected by ESR spectroscopy.39 To determine whether superoxide radical is involved in this process, superoxide dismutase (SOD) was employed to scavenge superoxide radicals in order to eliminate any BMPO/O2−• signal (Figure S2, Supporting Information). With the addition of SOD (0.2 U/ml) to the above TiO2 NP suspension, the 1:2:2:1 peak ESR signal remained the same pattern that is identical to BMPO/•OH spin-adduct. However, the intensity of the above signal was reduced to half. The reduction of this ESR signal suggests that that superoxide radical is involved in ROS generation in photoexcited TiO2 NP suspensions. Generation of Singlet Oxygen. In addition to hydroxyl and superoxide radicals, singlet oxygen is another important ROS that has been implicated in the degradation of dye mixtures.40

O2−• + h+ vb → 1O2

(3)

To test this theory, SOD (0.2 U/mL) was added to the above mixture. This resulted in a reduction of this triplet ESR signal (Figure S2, Supporting Information). TEMPO signal significantly reduced upon the addition of SOD. Again, because of the fact that SOD is a specific enzyme that catalyzes the dismutation of superoxide (O2−•), this result confirms that during photoexcitation of TiO2 NP aqueous suspension, superoxide radicals play a role in the formation of singlet oxygen. Interaction between ROS and FD&C Yellow No. 5. Hydroxyl Radical Consumption. A time and pH dependence study was carried out to further investigate the role of hydroxyl radical in the decomposition of Y5 (Figure 4). Figure 4, panel a shows the ESR spectra of the BMPO/•OH spin-adduct recorded for photoexcited TiO2 NP suspensions with or without MY5 at pH 5.0 or 7.4 after 1 min of simulated sunlight irradiation. The time-dependent ESR signal intensity shown in E

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Figure 5. ESR detection of 1O2 during decomposition of dye upon simulated sunlight irradiance: (a) ESR spectra recorded after 10 min of irradiation by simulated sunlight; (b) time-dependent ESR signal intensity obtained by measuring the peak-to-peak height of the first line of the ESR spectrum of TEMP/1O2 adduct. Reactions were conducted in 10 mM pH 5.0 or 7.4 PBS buffer solutions containing 5 mM TEMP spin probe. All sample solutions contained 0.1 mg/mL TiO2 NPs with (or without) 25 mg/mL MY5.

Figure 6. O2 consumption detection using ESR oximetry during decomposition of dye upon simulated sunlight irradiation: (a) ESR spectra of spinprobe 15N-PDT in sample solutions containing 0.1 mg/mL TiO2 and 25 mg/mL MY5 recorded before (0 min) or after 3 min of irradiation by simulated sunlight; and (b) O2 consumption kinetics. O2 concentration was calculated based on the peak-to-peak line width of spin-probe 15N-PDT using a calibrated standard curve described in the Material and Methods section. Reactions were conducted in 10 mM pH 5.0 or 7.4 PBS buffer solutions containing 0.2 mM 15N-PDT spin-probe. All sample solutions contained 0.1 mg/mL TiO2 NPs with (or without) 25 mg/mL MY5.

only, which indicates that under the simulated sunlight, hydroxyl radicals generated on photoexcited TiO2 NPs were consumed by the azo dye MY5, which results in decomposition. Interestingly, at pH 5.0, the addition of MY5 did not result in a significant change in ESR intensity for •OH spin-adduct, which implies that little or no •OH was used for dye degradation. The results from this ESR time- and pH-dependent study agree with our previous observations shown in Figure 2, panel b, where a lower reaction rate constant was found for acidic solution.

Figure 4, panel b was obtained by measuring the peak-to-peak height of the second line of the ESR spectra. For suspensions containing only TiO2 NPs, the signal intensities of BMPO/ •OH adduct tested at pH 5.0 were significantly lower than those of samples tested at pH 7.4. This is most likely due to the lower availability of hydroxide ions in acidic media. It has been stated that in alkaline solution, •OH generation is promoted due to oxidation of more hydroxide ions available on TiO2 surface.26 At pH 7.4, TiO2 solutions with MY5 show significantly lowered ESR intensity compared to TiO2 NPs F

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Singlet Oxygen Consumption. Figure 5 shows the time dependence of ESR signal for the formation of TEMPONE from TEMP due to the generation of 1O2. Figure 5, panel a exhibits the ESR spectra of TEMPONE observed after 10 min irradiation of TiO2 NP suspensions with or without MY5 at pH 5.0 or 7.4. The time dependence of the ESR signal intensity (Figure 5b) was obtained by measuring the peak-to-peak height of the first line of this ESR spectrum. ESR intensity detected at pH 7.4 was significantly higher compared to that at pH 5. This observation agrees with a previous study by Daimon et al. in which the generation of 1O2 was suppressed when pH < 5 and reaches its maximum between pH 6−11.15 The decrease in 1O2 at pH 5 compared to pH 7.4 is attributable to the suppression of O2−• at the TiO2 surface by taking consideration that the 1O2 originated from the oxidation of O2−• by the valence band holes h+ (eq 1). Unlike hydroxyl radical, the consumption of 1O2 by the azo dye MY5 was found for both acidic and alkaline conditions. At pH 5, the ESR signal for 1O2 was largely reduced when MY5 was added into the reaction mixture; thus, we did not observe any ESR signal for 1O2. However, when BMPO is used in the above solution, a six-peak ESR signal was recorded by ESR spectroscopy (data not shown), which indicates the formation of carbon center radical28 that was associated the oxidation of dye MY5. The decrease in 1O2 may take place by two mechanisms: one is by direct reaction with the Y5 dye anion, and the other is due to the decrease of available O2−• that has been consumed by dye oxidation. Oxygen Consumption. It has been well established that the O2−• in aqueous solutions containing TiO2 NPs results from the reduction of oxygen by conduction band electrons (e−cb): O2 + e

− cb

→ O2

−•

when exposed to simulated sunlight, TiO2 NPs at low concentration can trigger decomposition of the azo dye Y5, which is widely used in food, drug, and cosmetic products. By using ESR spin trapping, the only technique that directly monitors the existence of free radicals and identifies them,43 we demonstrated that dye degradation proceeds via reaction with ROS generated on the surface of photoexcited TiO2, and a higher degradation rate was associated with increased ROS generation. At pH 7.4, a significant reduction in both hydroxyl radical and singlet oxygen was observed after the dye was added into the suspension, while singlet oxygen became the main reactive species at pH 5.0. Our study characterized the decomposition mechanism and directly monitored the involvement of ROS that may occur in food, drugs, cosmetics, and other consumer products that contain both TiO2 and Y5 as ingredients. The findings in this study may provide important insights for the design of new applications or evaluation of the safety of nano-TiO2 in these products. Our results demonstrate that the interaction between nanomaterials and other ingredients should be investigated in addition to studies on the nanomaterials as independent components. Our results were obtained in a simplified chemical system. Further studies are needed to determine the effects of complex food matrices on factors such as aggregation, ROS quenching, and light transmission, which might moderate the photocatalytic activity of TiO2.



ASSOCIATED CONTENT

S Supporting Information *

Effect of hydroxyl radical on BMPO and TEMPO spin probes. Effect of SOD on ESR signal generated from TiO2 NP solutions exposed to simulated sunlight. This material is available free of charge via the Internet at http://pubs.acs.org.

(4)

−•

Although O2 was indirectly detected by adding SOD to the suspension containing both TiO2 NPs and BMPO spin-trap as discussed above, it was unclear how O2−• was involved in the decomposition process of MY5. Since difficulties arise when O2−• is detected by ESR due to the overlap of BMPO/O2−• and BMPO/•OH spin adducts, a time- and pH-dependent study was conducted for O2 consumption using ESR oximetry. The inset in Figure 6, panel a depicts the ESR spectrum of a commonly used water-soluble spin-label 15N-PDT. Figure 6, panel a shows that after 3 min of light irradiation, higher peak intensity with broader line width was observed for the sample containing TiO2 and food color, which is associated with the reduction of oxygen in the solution. Figure 6, panel b demonstrates that for suspensions only containing TiO2 NPs, higher O2 consumption was observed at pH 7.4 compared to that at pH 5. The addition of the dye MY5 led to a further decrease in O2 level in both acidic (pH 5) and alkaline (pH 7.4) media, and the degree of this further consumption appeared to be similar for both pHs. On the basis of the observation that upon sunlight irradiation, O2 did not directly react with the azo dye without TiO2 or that the reaction was too slow to be observed and thus negligible (control groups in Figure 2a,b), the main reason for oxygen consumption is very likely to be the reduction of O2 by e− (eq 4). Therefore, by determination of oxygen consumption, we observed the involvement of O2−• in the decomposition process. Our findings suggest it is very possible that pH primarily affects the initial rate of O2−• generation rather than the interaction between the azo dye and O2−•. Nanosized TiO2 particles may be found in a broad range of consumer products. In conclusion, our results confirmed that



AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: 240-402-1991. Fax: 201-436-2624. Funding

This work was supported by a regulatory science grant under the FDA Nanotechnology CORES Program and by the Office of Cosmetics and Colors, CFSAN/FDA. W.H. appreciates the support from the National Natural Science Foundation of China (Grant No. 21303153) and the Program for Science and Technology Innovation Talents in Universities of Henan Province (Grant No. 14HASTIT008). Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS The authors thank Dr. Adrian Weisz (CFSAN/FDA) for his valuable help and comments. We acknowledge the support of the Maryland NanoCenter and its NispLab. This article is not an official U.S. FDA guidance or policy statement. No official support or endorsement by the U.S. FDA is intended or should be inferred.



ABBREVIATIONS USED Y5, FD&C Yellow No. 5; MY5, purified main component of FD&C Yellow No. 5; NPs, nanoparticles; ESR, electron spin resonance; ROS, reactive oxygen species; H2O2, hydrogen peroxide; •OH, hydroxyl radical; O2•‑, superoxide radical; 1O2, G

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(18) Mills, A. Oxygen indicators and intelligent inks for packaging food. Chem. Soc. Rev. 2005, 34 (12), 1003−1011. (19) Wamer, W. G.; Yin, J.-J.; Wei, R. R. Oxidative damage to nucleic acids photosensitized by titanium dioxide. Free Radical Biol. Med. 1997, 23 (6), 851−858. (20) Liu, S.; Xu, L.; Zhang, T.; Ren, G.; Yang, Z. Oxidative stress and apoptosis induced by nanosized titanium dioxide in PC12 cells. Toxicology 2012, 267, 1−3. (21) Dos Santos, T. C.; Zocolo, G. J.; Zanoni, M. V. B.; Morales, D. A.; Umbuzeiro, G. D. A. Assessment of the breakdown products of solar/UV-induced photolytic degradation of food dye tartrazine. Food Chem. Toxicol. 2014, 68, 307−315. (22) Oancea, P.; Meltzer, V. Photo-Fenton process for the degradation of tartrazine (E102) in aqueous medium. J. Taiwan Inst. Chem. Eng. 2013, 44, 990−994. (23) Weisz, A.; Ridge, C. D.; Roque, J. A.; Mazzola, E. P.; Ito, Y. Preparative separation of two subsidiary colors of FD&C Yellow No. 5 (tartrazine) using spiral high-speed counter-current chromatography. J. Chromatogr., A 2014, 1343, 91−100. (24) He, W.; Kim, H.-K.; Wamer, W. G.; Melka, D.; Callahan, J. H.; Yin, J.-J. Photogenerated reactive oxygen species and charge carriers in ZnO/Au hybrid nanostructures are correlated with enhanced photocatalytic and antibacterial activity. J. Am. Chem. Soc. 2014, 136 (2), 750−757. (25) Tanaka, A.; Ogino, A.; Iwaki, M.; Hashimoto, K.; Ohnuma, A.; Amano, F.; Ohtani, B.; Kominami, H. Gold−titanium(IV) oxide plasmonic photocatalysts prepared by a colloid-photodeposition method: Correlation between physical properties and photocatalytic activities. Langmuir 2012, 28 (36), 13105−13111. (26) Konstantinou, I. K.; Albanis, T. A. TiO2-assisted photocatalytic degradation of azo dyes in aqueous solution: Kinetic and mechanistic investigationsA review. Appl. Catal., B 2004, 49 (1), 1−14. (27) He, W.; Liu, Y.; Wamer, W. G.; Yin, J. J. Electron spin resonance spectroscopy for the study of nanomaterial-mediated generation of reactive oxygen species. J. Food Drug Anal. 2014, 22 (1), 49−63. (28) Yin, J. J.; Zhao, B.; Xia, Q.; Fu, P. P. Electron Spin Resonance Spectroscopy for Studying the Generation and Scavenging of Reactive Oxygen Species by Nanomaterials in Nanopharmaceutics: The Potential Application of Nanomaterials; World Scientific Pub. Co.: Singapore, 2012; pp 375−400. (29) Zhang, Y.; Wong, J. W.; Liu, P.; Yuan, M. Heterogeneous photocatalytic degradation of phenanthrene in surfactant solution containing TiO2 particles. J. Hazard Mater. 2011, 191 (1−3), 136− 143. (30) Poulios, I.; Tsachpinis, I. Photodegradation of the textile dye Reactive Black 5 in the presence of semiconducting oxides. J. Chem. Technol. Biotechnol. 1999, 74 (4), 349−357. (31) Choudhury, B.; Borah, B.; Choudhury, A. Extending photocatalytic activity of TiO2 nanoparticles to visible region of illumination by doping of cerium. Photochem. Photobiol. 2012, 88 (2), 257−264. (32) Banerjee, S.; Chattopadhyaya, M. C. Adsorption characteristics for the removal of a toxic dye, tartrazine, from aqueous solutions by a low-cost agricultural by-product. Arabian J. Chem. 2013, DOI: 10.1016/j.arabjc.2013.06.005. (33) Rauf, M. A.; Meetani, M. A.; Hisaindee, S. An overview on the photocatalytic degradation of azo dyes in the presence of TiO2 doped with selective transition metals. Desalination 2011, 276 (1−3), 13−27. (34) Meetani, M. A.; Rauf, M. A.; Hisaindee, S.; Khaleel, A.; Al Zamly, A.; Ahmad, A. Mechanistic studies of photoinduced degradation of Orange G using LC/MS. RSC Adv. 2011, 1 (3), 490−497. (35) Soutsas, K.; Karayannis, V.; Poulios, I.; Riga, A.; Ntampegliotis, K.; Spiliotis, X.; Papapolymerou, G. Decolorization and degradation of reactive azo dyes via heterogeneous photocatalytic processes. Desalination 2010, 250 (1), 345−350. (36) Gupta, V. K.; Jain, R.; Nayak, A.; Agarwal, S.; Shrivastava, M. Removal of the hazardous dyeTartrazine by photodegradation on titanium dioxide surface. Mater. Sci. Eng., C 2011, 31 (5), 1062−1067.

singlet oxygen; BMPO, 5-tert-butoxycarbonyl 5-methyl-1pyrroline N-oxide; TEMP, 2,2,6,6-tetramethyl-4-piperidone; TEMPO, 2,2,6,6 -tetramethylpiperidine-1-oxyl; 15N-PDT, 4oxo-2,2,6,6-tetramethylpiperidine-d16-1-oxyl; SOD, superoxide dismutase



REFERENCES

(1) Carlotti, M. E.; Ugazio, E.; Gastaldi, L.; Sapino, S.; Vione, D.; Fenoglio, I.; Fubini, B. Specific effects of single antioxidants in the lipid peroxidation caused by nanotitania used in sunscreen lotions. J. Photochem. Photobiol., B 2009, 96 (2), 130−135. (2) Hamal, D. B.; Haggstrom, J. A.; Marchin, G. L.; Ikenberry, M. A.; Hohn, K.; Klabunde, K. J. A multifunctional biocide/sporocide and photocatalyst based on titanium dioxide (TiO2) codoped with silver, carbon, and sulfur. Langmuir 2010, 26 (4), 2805−2810. (3) Ochiai, T.; Tago, S.; Nakano, R.; Nakata, K.; Murakami, T.; Fujishima, A.; Ikekita, M.; Morito, Y.; Hara, M.; Nojima, Y.; Masuko, K.; Suzuki, T.; Niitsu, Y.; Kobayashi, G.; Kurano, M.; Serizawa, I.; Horio, K. Development of a hybrid environmental purification unit by using of excimer VUV lamps with TiO2-coated titanium mesh filter. Chem. Eng. J. 2013, 218, 327−332. (4) Gomez, M.; Arancibia, V.; Rojas, C.; Nagles, E. Adsorptive stripping voltammetric determination of tartrazine and sunset yellow in gelatins and soft drink powder in the presence of cetylpyridinium bromide. Int. J. Electrochem. Sci. 2012, 7 (8), 7493−7502. (5) Code of Federal Regulations, Title 21, Section 73.575; U.S. Food and Drug Administration: Washington, DC, 2014. (6) Japan’s Specifications and Standards for Food Additives, 7th ed.; Ministry of Health and Welfare: Tokyo, Japan, 2000. (7) EU European Parliament and Council Directive, 94/36/EC of 30 June 1994 on Colours for Use in Foodstuffs. Off. J. Eur. Communities 1994, L237, 13−29. (8) Chewing gum and bubble gum. In The Prevention of Food Adulteration Act and Rules, 1955, Appendix B, Section A.25.02.01, Directorate General of Health Services; Ministry of Health: New Delhi, India, 2004. (9) Powdered concentrate mixes for fruit beverage drinks. In The Prevention of Food Adulteration Act and Rules, 1955, Appendix C, Table 3, Directorate General of Health Services; Ministry of Health: New Delhi, India, 2004. (10) Yang, Y.; Doudrick, K.; Bi, X.; Hristovski, K.; Herckes, P.; Westerhoff, P.; Kaegi, R. Characterization of food-grade titanium dioxide: The presence of nanosized particles. Environ. Sci. Technol. 2014, 48 (11), 6391−400. (11) Weir, A.; Westerhoff, P.; Fabricius, L.; Hristovski, K.; Von, G. N. Titanium dioxide nanoparticles in food and personal care products. Environ. Sci. Technol. 2012, 46 (4), 2242−50. (12) Chen, X. X.; Cheng, B.; Yang, Y. X.; Cao, A.; Liu, J. H.; Du, L. J.; Liu, Y.; Zhao, Y.; Wang, H. Characterization and preliminary toxicity assay of nano-titanium dioxide additive in sugar-coated chewing gum. Small 2013, 9, 9−10. (13) Mao, S. S.; Chen, X. Selected nanotechnologies for renewable energy applications. Int. J. Energy Res. 2007, 31 (6−7), 619−636. (14) Yin, J. J.; Liu, J.; Ehrenshaft, M.; Roberts, J. E.; Fu, P. P.; Mason, R. P.; Zhao, B. Phototoxicity of nano-titanium dioxides in HaCaT keratinocytesGeneration of reactive oxygen species and cell damage. Toxicol. Appl. Pharmacol. 2012, 263 (1), 81−88. (15) Daimon, T.; Hirakawa, T.; Kitazawa, M.; Suetake, J.; Nosaka, Y. Formation of singlet molecular oxygen associated with the formation of superoxide radicals in aqueous suspensions of TiO2 photocatalysts. Appl. Catal., A 2008, 340 (2), 169−175. (16) Yu, K. P.; Huang, Y. T.; Yang, S. C. The antifungal efficacy of nanometals supported TiO2 and ozone on the resistant Aspergillus niger spore. J. Hazard Mater. 2013, 261, 155−162. (17) Wu, P.; Xie, R.; Imlay, K.; Shang, J. K. Visible-light-induced bactericidal activity of titanium dioxide codoped with nitrogen and silver. Environ. Sci. Technol. 2010, 44 (18), 6992−6997. H

dx.doi.org/10.1021/jf5045052 | J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry

Article

(37) Lavi, R.; Sinyakov, M.; Samuni, A.; Shatz, S.; Friedmann, H.; Shainberg, A.; Breitbart, H.; Lubart, R. ESR detection of 1O2 reveals enhanced redox activity in illuminated cell cultures. Free Radical Res. 2004, 38 (9), 893−902. (38) Zhao, H.; Joseph, J.; Zhang, H.; Karoui, H.; Kalyanaraman, B. Synthesis and biochemical applications of a solid cyclic nitrone spin trap: A relatively superior trap for detecting superoxide anions and glutathiyl radicals. Free Radical Biol. Med. 2001, 31 (5), 599−606. (39) Li, M.; Yin, J. J.; Wamer, W. G.; Lo, Y. M. Mechanistic characterization of titanium dioxide nanoparticle-induced toxicity using electron spin resonance. J. Food Drug Anal. 2014, 22 (1), 76−85. (40) Rembold, M.; Kramer, H. Singlet oxygen as an intermediate in the catalytic fading of dye mixtures. J. Soc. Dyers Colour. 1978, 94 (1), 12−17. (41) Lion, Y.; Delmelle, M.; Van, V. A. New method of detecting singlet oxygen production. Nature 1976, 263 (5576), 442−443. (42) Daimon, T.; Nosaka, Y. Formation and behavior of singlet molecular oxygen in TiO2 photocatalysis studied by detection of nearinfrared phosphorescence. J. Phys. Chem. C 2007, 111 (11), 4420− 4424. (43) Fu, P. P.; Xia, Q.; Hwang, H. M.; Ray, P. C.; Yu, H. Mechanisms of nanotoxicity: Generation of reactive oxygen species. J. Food Drug Anal. 2014, 22 (1), 64−75.

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