Nitrogen and Fluorine Codoped, Colloidal TiO2 Nanoparticle: Tunable

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Nitrogen and Fluorine Codoped, Colloidal TiO2 Nanoparticle: Tunable Doping, Large Red Shifted Band Edge, Visible Light Induced Photocatalysis and Cell Death Aritra Biswas, Atanu Chakraborty, and Nikhil R. Jana ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b14025 • Publication Date (Web): 19 Dec 2017 Downloaded from http://pubs.acs.org on December 20, 2017

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ACS Applied Materials & Interfaces

Nitrogen and Fluorine Codoped, Colloidal TiO2 Nanoparticle: Tunable Doping, Large Red Shifted Band Edge, Visible Light Induced Photocatalysis and Cell Death

Aritra Biswas, Atanu Chakraborty and Nikhil R. Jana* Centre for Advanced Materials, Indian Association for the Cultivation of Science, Kolkata700032, India *Corresponding authors E-mail: [email protected]

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ACS Applied Materials & Interfaces 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Abstract: Visible light photocatalysis by TiO2 requires efficient doping of other elements with red shifted band edge to visible region. However, preparation of such TiO2 with tunable doping is challenging. Here we report a method of making nitrogen (N) and fluorine (F) codoped TiO2 nanoparticle with tunable doping between 1-7 atomic %. The preparation of N, F codoped TiO2 nanoparticle involves reaction of colloidal TiO2 nanorods with ammonium fluoride-urea mixture at 300 °C and the extent of N/F doping is tuned by varying the amount of ammonium fluorideurea and reaction time. Resultant colloidal N, F codoped TiO2 nanoparticle show doping dependent shifting of band edge from UV to NIR region, visible light induced generation of reactive oxygen species (ROS) and visible light photodegradation of bisphenol A. Colloidal form of doped TiO2 nanoparticle offers labeling of cells, visible light induced ROS generation inside cell and successive cell death. This work shows the potential advantage of anisotropic nanoparticle precursor for tunable doping and colloidal form of N, F codoped TiO2 nanoparticle as visible light photocatalyst.

KEYWORDS: nanoparticle, doping, band edge shifting, visible light photocatalysis, reactive oxygen species, bisphenol A, cytotoxicity


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Introduction Titanium dioxide (TiO2) is most popularly used photocatalyst due to its chemical stability, low toxicity and low cost.1-6 It occurs mainly in rutile, anatase and brookite form. Among which rutile form is thermodynamically more stable in bulk phase, anatase form is more stable in nanoscale structure7 and brookite form is relatively less studied.8 However, TiO2 nanoparticle has wide band gap of 3.2 eV that restricts the use only under UV light (λ < 400 nm) and a small portion of the solar spectrum (2-3 %) can be utilized for such catalysis.5 Thus there are undergoing researches for the development of visible light active TiO2 and other photocatalyst in order to utilize most of the solar spectrum.9,10 These approaches include doping with metal/nonmetal,6,11 introducing crystal defect12 and making hybrids nanostructures with heterojunction structures.13 However, most of these approaches are not efficient enough for complete capturing of sunlight and for any practical application. Origin of such failure is the rapid electron-hole pair recombination that occurs just after light absorption and thus various strategies are introduced for effective charge separation, hole trapping and electron trapping.5 Although cation or anion doped TiO2 nanoparticle are successfully used for photocatalysis, anion doping has more substantial effect in extending the absorption spectra toward visible region.5,6 In particular nitrogen (N) doped TiO2 nanoparticle with the extended visible light absorption has been widely used for photocatalysis.14-23 However, N doping leads to oxygen vacancy sites in the TiO2 lattice and produces unstable particle along with increased recombination kinetics of the electron-hole pair.6,21-23 In contrast codoping of N with fluorine (F) in the TiO2 nanoparticle can eliminate this defective crystal structure.24 This is because O2-/N3-/F- are isoelectric, comparable in size and two O2- ions are equivalent to one N3- ion and one F- ion.24 It is shown that codoping of N and F in the TiO2 nanoparticle can be achieved as high as 15 atomic % and band edge of



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TiO2 nanoparticle can be red shifted as high as 550 nm.24 These type N, F codoped TiO2 nanoparticle have been studied recently by various groups with the hope to improve the photocatalytic performance under visible light.24-32 Most of the synthesis use solid phase approach that involves heating of powder TiO2 sample with N/F precursors followed by annealing at > 500 °C.29-32 In addition substrate TiO2 particles are > 100 nm in size and non-dispersible.25-32 Thus resultant doped nanoparticles sinter with each other, non-dispersible or poorly dispersible and doping is generally inefficient. Additionally the extent of N/F doping is difficult to control in most of these approaches. Considering the fact that doping can be enhanced by > 4 times if micron size TiO2 substrate particles are replaced by 6-10 nm particles,15 we presume that use of colloidal anisotropic nanoparticle as doping substrate may improve doping performance. Here we report N and F codoping approach using colloidal TiO2 nanorod (2-3 nm diameter and 25-35 nm length) as substrate. We found that efficient doping can be achieved at 300 °C using colloidal/dispersed nanorod substrate with the formation of N and F codoped TiO2 nanoparticles of 70-225 nm size. The presented doping approach and resultant N, F codoped TiO2 nanoparticles have five specific advantages over reported doping approaches/doped materials. (see Supporting Information, Table S1 for details) First, extent of N and F doping can be tuned between 1-7 atomic % by simple variation of doping precursor and doping time. Second, N, F codoped nanoparticles are colloidal in nature and they can be further functionalized for specific application. Third, band edge of colloidal doped nanoparticles is red shifted by > 600 nm and they absorb visible to NIR light. Fourth, colloidal doped nanoparticles show N/F doping dependent visible light photocatalytic activity. Fifth, colloidal doped nanoparticles can be used to produce reactive oxygen species (ROS) inside cell under visible light irradiation and to induce cell death.


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Experimental Section Materials. Ammonium fluoride was purchased from Loba Chemie Pvt. Ltd. Urea was purchased from Merck. Methylthiazolyl diphenyl tetrazolium bromide (MTT) purchased from Himedia. Oleic acid, oleylamine, titanium(IV) isopropoxide, poly(ethyleneglycol) methacrylate, 3sulfopropyl

methacrylate

potassium

(methyacryloyloxy)ethyl]phosphate,

Igepal

salt,

ammonium CO-520,

persulfate,

cyclohexane,

bis[2 N,N,N,N

tetramethylethylenediamine, N-(3-aminopropyl) methacrylamide, dialysis tube (MWCO ~ 12,000-14,000 Da), Dulbecco’s Modified Eagle Medium (DMEM), 2´, 7´-dichlorofluorescin diacetate (DCFDA), propidium iodide (PI) and terephthalic acid were purchased from Sigma Aldrich. Instrumentation. Transmission electron microscopy (TEM) images, energy dispersive X-ray spectra (EDS) and selected area electron diffraction (SAED) patterns were measured using ultra high resolution field emission gun transmission electron microscope (UHRFEG-TEM), JEOL, JEM 2100 F field-emission electron microscope. Wide-angle X-ray diffraction (XRD) was measured with Bruker D8 Advance powder diffractometer using Cu Kα (λ = 1.5406 Å) as the incident radiation. X-ray photoelectron spectroscopy (XPS) measurement was performed using Omicron (serial number: 0571) X-ray photoelectron spectrometer. Raman spectra were recorded by J-Y Horiba Confocal Triple Raman Spectrometer (Model:T64000) fitted with gratings of 1800 groove/mm, and a TE cooled Synapse CCD detector from J-Y Horiba. The samples were excited using a 532 nm Nd:YAG laser (Spectra Physics). The scattered signals were collected at 180° scattering angle to the excitations from an Olympus open Stage microscope of 50× objective. The detector and the data acquisition were controlled by LabSpec 5 software of Horiba. HPLC (Waters) analysis was performed using C18 column (5 µm 4.6×250 mm).



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UV−visible absorption spectra of samples were collected using Shimadzu UV-2550 UV−visible spectrophotometer. Emission spectra were measured using a Synergy Mx Multi-Mode Microplate Reader. CHN analysis was performed with Model No-2400 SERIES II CHNS/O ANALYZER (Perkin Elmer). Visible light intensity was measured using visible light meter (model HTC LX101). Fourier transform infrared (FTIR) spectra were measured on a PerkinElmer Spectrum 100 FTIR spectrometer using a KBr powder pressed pellet of solid samples. EPR spectra were recorded on a JEOL JES-FA 200 instrument. The hydrodynamic sizes and ζ potentials were measured via dynamic light scattering (DLS) using NanoZS (Malvern) instrument. The differential interference contrast (DIC) and fluorescence (F) images of cells were measured by Olympus IX81 microscope using Image-Pro Plus version 7.0 software. Synthesis of N and F codoped TiO2 nanoparticle from TiO2 nanorod. About 6.0 mL oleic acid was loaded in a three-necked round-bottomed flask and purged with nitrogen for 15-20 min under 70-80 °C. Next, 0.2 mL titanium isopropoxide was injected at the stirring condition and temperature was increased in the range of 300 °C. Then 0.7 mL oleylamine was injected under stirring conditions and temperature was maintained for 2 h. Next, heating was stopped and solution was cooled to room temperature and mixed with 180-360 mg ammonium fluoride (4.869.72 mmole) and 180-360 mg urea (3.0-6.0 mmole). Next, temperature was raised to 300 °C under magnetically stirring condition in open air, maintained for 1-2 h and then heating was stopped. As the temperature increased, the bubbles were observed from solution that increases with time and then decrease within 15-30 mins. In addition there was a gradual change of solution color from white to green within 15-30 mins. Nanoparticles were precipitated from solution by adding acetone and isolated precipitates were dissolved in chloroform. Next, second


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round of purification of nanoparticles was performed by ethanol-based precipitation and chloroform/cyclohexane/toluene-based redispersion. Finally, purified solution was prepared in chloroform/cyclohexane/toluene and used as a stock solution. In order to characterize TiO2 nanorods produced, a part of solution was collected before adding ammonium fluoride-urea, precipitated from the solution by adding acetone and isolated precipitate was dissolved in chloroform. Nanorods were further purified via ethanol-based precipitation and chloroform/cyclohexane/toluene-based redispersion for two times. Finally, purified nanorods were dissolved in chloroform and used for characterization studies. Synthesis of polyacrylate-coated doped TiO2 nanoparticle. About 3 mg doped TiO2 nanoparticle was dissolved in 2 mL of Igepal-cyclohexane reverse micelle, mixed with reverse micelle solution of different acrylate monomers to make a total volume of 10-12 mL and was taken in a 50 mL three necked round-bottom flask. Next, 100 µL of N,N,N,N-tetramethyl ethylenediamine base was added and the whole solution was degassed under an argon atmosphere for 15 min. Next, 3 mg of ammonium persulfate, dissolved in 100 µL water, was introduced and reaction was continued for 30 min under stirring conditions. Next, ethanol was added to precipitate the particles and repeatedly washed with chloroform/ethanol. Finally, particles were dissolved in water and purified by dialysis against distilled water using dialysis membrane (MWCO ∼12,000−14,000 Da). Specifically, we have used poly(ethylene glycol) methacrylate, 3-sulfopropyl methacrylate and N-(3-aminopropyl) methacrylamide to introduce for polyethyleneglycol, anionic SO3- and primary amine groups on the nanoparticle surface, respectively. Additionally, 5 mole % bis[2(methyacryloyloxy)ethyl]phosphate was used as a cross-linker. In selected cases we have used



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fluorescein o-methacrylate as one of the monomer for preparing FITC functionalized nanoparticles. Photocatalytic experiment. Colloidal doped nanoparticle was washed 4-5 times by precipitation and redispersion method described above, dried well and then 3 mg of nanoparticle was dispersed in 50 mL bisphenol A solution with a concentration of 100 mg/L. The suspension was stirred in the dark for 90 min to establish adsorption−desorption equilibrium and then irradiated with visible light source, which was a 250 W Hg vapor lamp (>380 nm wavelength) with light intensity ∼5 mW/cm2. A certain volume of aliquot was withdrawn at different time intervals, nanoparticles were separated by high speed centrifuge, the aqueous solution was filtered with 0.2 micron centrifugal filter and supernatant was passed through HPLC column to verify the degradation of bisphenol A. UV detector was used with the absorbance at 275 nm as detection signal. In order to estimate the reactive oxygen species (ROS) generated by nanoparticles, 3 mg of as synthesized doped nanoparticle or polyacrylate coated doped nanoparticle was suspended in a 50 mL aqueous terephthalic acid solution with a concentration of 5 × 10−4 M, and the pH was adjusted to basic using NaOH solution. Next, the solution was stirred for 90 min to reach homogeneity at room temperature and then irradiated with a 250 W Hg vapor lamp with light intensity ∼5 mW/cm2. At different time intervals a part of the solution was collected, nanoparticles were separated by centrifuge and fluorescence of the supernatant was measured by exciting at 315 nm. Investigation of nanoparticle-induced reactive oxygen species (ROS) generation inside HeLa cells under visible light exposure. HeLa cells were cultured in 24-well plate under 37 °C with 5 % CO2 using DMEM media with 10 % heat activated fetal bovine serum (FBS) and 1%


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penicillin streptomycin. After 24 h cells were taken in fresh media and mixed with 50 µL polyacrylate coated nanoparticle solution. After 60 min of incubation cells were washed with PBS buffer solution (pH=7.4) to remove unbound nanoparticles. Then cells were taken in fresh media and irradiated with a 250 W Hg vapor lamp for 30 min with light intensity ∼5 mW/cm2. For reactive oxygen species (ROS) detection, 5 µL DCF solution (10 mM) was added to the media and incubated for 20-30 min under 37 °C. Next, cells were washed with PBS (pH=7.4) and fresh DMEM media was added. After that, the cells were imaged under fluorescence microscope. In order to determine the membrane damage, visible light irradiated cells were treated with PI staining solution (10 µg/mL), incubated in dark at 37 °C for 10 min and washed cells were imaged under green excitation. MTT assay. HeLa cells were seeded into 24-well tissue culture plates in the presence of 500 µL of DMEM medium supplemented with 10 % fetal bovine serum and 1 % penicillin/ streptomycin at 37 °C and 5 % CO2. After 24 h cells were taken in fresh media, and then cells were incubated with polyacrylate coated doped nanoparticles for 60 min. Next, cells were irradiated with visible light (250 W Hg vapor lamp with light intensity ∼5 mW/cm2) for 30 min followed by 24 h incubation under dark. Next, cells were washed with PBS buffer, and 500 µL of fresh DMEM medium was added. Then, cells were incubated with 50 µL of MTT (5 mg/mL) solution for 4 h, violet formazan was dissolved in a DMF−water solution of sodium dodecyl sulfate and absorbance of the solution was measured at 570 nm in a microplate reader. The relative cell viability was measured by assuming 100 % cell viability for control cells without nanoparticles. Control cytotoxicity experiments were performed under the same conditions, except that cells were not exposed under visible light.



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Results and Discussion Transformation of TiO2 nanorod to N, F codoped TiO2 nanoparticle. Synthesis approach of N and F codoped TiO2 nanoparticle involves reaction of colloidal TiO2 nanorod with a mixture of ammonium fluoride and urea under high temperature. (Scheme 1) At first, TiO2 nanorod is prepared at 300 °C by hydrolysis of titanium isopropoxide in oleic acid medium under nitrogen atmosphere.33 Next, colloidal nanorods are mixed with solid ammonium fluoride and urea and heated in air at 300 °C under magnetically stirring condition. Under this condition, intermediates (HF and NH3) produced from ammonium fluoride-urea reacts with nanorod and produces N, F codoped TiO2 nanoparticle. The formation of doped TiO2 nanoparticle is observed by gradual change of solution color from white to green within 15-30 mins. Extent of N/F doping is varied by changing amount of ammonium fluoride/urea and reaction time. (Table 1) After reaction, doped nanoparticles are purified by conventional precipitation-redispersion method and dispersed in chloroform/cyclohexane/toluene. Figure 1a and Supporting Information, Figure S1 show the TEM image of TiO2 nanorod substrate and N, F codoped TiO2 nanoparticle, prepared under three different conditions. Table 1 summarizes their preparation condition, size and extent of doping. While nanorods are 2-3 nm in diameter and 25-35 nm in length, doped nanoparticles are 70-225 nm in size. Depending on the increased extent of doping, they are termed as d-TiO2 (1), d-TiO2 (2) and d-TiO2 (3) where the doping of N and F are in the range of 1-7 atomic %. The atomic % of N and F are 1.4, 1.2 for dTiO2 (1), 3.0, 6.6 for d-TiO2 (2) and 6.8, 3.5 for d-TiO2 (3) (see below for details) and these variations are achieved by using different amounts of ammonium fluoride-urea and changing reaction time. (see Table 1 for details)


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Crystalline phases of TiO2 nanoparticle is characterized by wide angle X-ray diffraction study. (Figure 1b) Both the doped and undoped TiO2 nanoparticles are crystalline with anatase structure. The crystal planes of the synthesized nanoparticles are characteristics of tetragonal phase according to the standard data (JCPDS no. 84-1286). No additional reflections are observed for the doped TiO2 nanoparticles which indicate that either doping of N/F occurs inside crystal lattice or homogenously distributed over nanoparticle surface. However, some of the broad reflections of undoped TiO2 become sharp in doped TiO2, and this may be due to increased particle size. Fluorescence spectral study of doped TiO2 nanoparticles show no emission band around 600 nm, suggesting that oxygen vacancies are absent in the crystal structure. (Supporting Information, Figure S2) Raman analysis shows the signature of anatase TiO2 phase and doping leads to the shifting of low frequency 144 cm-1 (Eg) band to ~155 cm-1 because of nonstochiometric TiO2-x phase and appearance of a new band at 204 cm-1 corresponding to the firstorder scatterings of non-stoichiometric titanium nitride (Ti-N).19 (Figure 1b) Core level X-ray photoelectron spectroscopy (XPS) has been performed in order to determine the nature of N and F doping. (Figure 2) In the case of undoped TiO2 there is no F 1s signal in XPS, but doped TiO2 shows characteristic signals of F1s. In particular d-TiO2 (1) shows signal at ~688 eV for directly substituted F atom in the TiO2 lattice and d-TiO2 (3) shows F 1s signals at ~685 eV that can be assigned to the interstitially or surface doped F atoms. However, d-TiO2 (2) shows signals at 688 eV and ~ 685.8 eV due to both directly substituted F and interstitial/surface doped F. F doping is further confirmed from FTIR analysis that shows a Ti-F stretching vibration at 944-1030 cm-1 region. The effect of F doping leads to the shifting of Ti 2p signals. Ti 2p3/2 and Ti p1/2 signals of undoped TiO2 nanorod appears at ~458.6 eV and ~464.2 eV, respectively. However, in d-TiO2



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(1) they are shifted to higher binding energies of ~461.2 eV and ~467.1 eV, respectively. These shifting are relatively less for d-TiO2 (2) (values are ~459.2 eV and ~464.9 eV, respectively) and shifting is almost insignificant for d-TiO2 (3) (values are ~458.1 eV and ~463.7 eV, respectively). Deconvoluted N 1s signal for the undoped TiO2 appears at ~ 399.9 eV which can be assigned as surface adsorbed nitrogen atoms coming from oleylamine precursor. In contrast, d-TiO2 (1) shows N 1s signals at ~ 400.7 eV; d-TiO2 (2) shows signals at ~402.0 eV, ~400.4 eV and ~399.6 eV and d-TiO2 (3) shows signals at ~400.6 eV, ~399.3 eV and ~398 eV. These bands may arise due to surface adsorbed, interstitial or substitutional doping of the N atoms. Control experiments show that high temperature annealing at 400-500 °C or surface cleaning by Argon ion sputtering before XPS cannot remove the N 1s signal below ~400.0 eV, suggesting that N 1s signals below ~400.0 eV are due to interstitially doped or directly substituted N into the host lattice. (Supporting Information, Figure S3) The extent of N and F dopants are determined from XPS. In addition the nitrogen amount has been determined via elemental analysis and correlated with XPS data. The atomic % of N and F are 1.4, 1.2 for d-TiO2 (1), 3.0, 6.6 for d-TiO2 (2) and 6.8, 3.5 for d-TiO2 (3). In order to further understand the origin of unusual color we have performed EPR study and observed very weak signal of Ti3+ (g ~ 1.98) and associated with paramagnetic oxygen (g ~ 2.06) signals. (Supporting Information, Figure S4) This result indicates partially reduced TiO2 is present in a small percent in the doped nanoparticle. Formation of mechanism of N, F codoped TiO2 nanoparticle. We found that anisotropic nanorod structure, 300 °C temperature and colloidal form are three important criteria for efficient N and F doping. Under the similar doping condition, if shorter nanorods or near spherical nanoparticles are used, the doping becomes inefficient. (Supporting Information, Figure S5)


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Similarly, longer nanowires are less effective substrate for doping. (Supporting Information, Figure S5) Moreover, if thin film of TiO2 nanorods is mixed with drop casted solution of ureaammonium fluoride and dried film is then heated at 300 °C doping is less efficient. (Supporting Information, Figure S6) However, if solid nanorods are mixed with solid urea-ammonium fluoride and then heated at 300 °C, doping can be achieved but the doping tunability is less effective via changing conditions. (Supporting Information, Figure S7) If < 300 °C is used, the conversion of nanorods to doped nanoparticle becomes insignificant. (Supporting Information, Figure S8) In order to further understand the doping mechanism we have investigated time dependent evaluation of doped nanocrystals. (Figure 3) UV-visible spectra show gradual increase of absorption band covering the entire visible region. Isolation of particle at different time interval shows that blue-green particles appear typically after 10-15 min of reaction and becomes more intense in color as time progress. TEM image shows aggregated TiO2 nanorods at 5 min, partially dissolved particles along with the nucleated cubic/tetragonal shaped particles at 10-20 min time and cubic/tetragonal shaped particles of larger size as the time progress. Based on these observations we propose a tentative mechanism of formation of N, F codoped TiO2 nanoparticle. (Supporting Information, Scheme S1) At the first step the capping ligands around TiO2 nanorods are partially removed and nanorods become unstable. Next, intermediate HF and NH3 (which are produced from ammonium fluoride and urea, respectively) starts reacting with nanorod, induces partial dissolution of nanorod (by reaction with HF) and nucleation of doped TiO2 nanoparticle which eventually lead to N, F codoping. Finally, well developed nanocube/nanorhombohedra are produced depending upon the amount of doping precursor and reaction time. It is reported earlier that addition of hydrochloric acid to a titania suspension lead to rapid development of anatase



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particles.34 It is also known that impurity incorporation into TiO2 nanoparticle is easier under the conditions where nanoparticle acts as unit (rather than atom) and the growth occurs via nanoparticle-nanoparticle attachment.35 Thus acidic condition and high temperature offers structural nonequillibrium of nanorod crystal phase, high reactivity of HF offers rapid F doping and induced N doping and nucleation-growth of N, F codoped TiO2 occur from partially dissolved

nanorods.

We

have

also

replaced

ammonium

fluoride-urea

mixture

by

hydrazine/urea/5-fluorouracil and found that TiO2 nanorods remain intact and doping becomes insignificant, which further support the proposed mechanism (Supporting Information, Figure S9). Doping dependent large (> 600 nm) red shifting of band gap. The most interesting aspect of N, F codoped TiO2 nanoparticle is their intense and stable blue-green color. While undoped solid TiO2 nanoparticle is white in color, the solid samples or colloidal solution of all N, F codoped TiO2 nanoparticles are blue-green in color. Figure 4 shows the solid samples and UV-visible absorption spectra of their dilute dispersions. All the three doped TiO2 nanoparticles shows strong absorption in visible region covering from 350 nm to 800 nm and the colour becomes stable to more than month. While undoped TiO2 nanorod does not have any band in visible region, the d-TiO2 (1) shows broad absorption band with a peak at 350 nm, d-TiO2 (2) shows broad absorption band with a peak at 450 nm and d-TiO2 (3) shows broad absorption band with peak at 500 nm. In addition the band edges are 340 nm, 800 nm, 900 nm and 950 nm for undoped TiO2 nanorod, d-TiO2 (1), d-TiO2 (2) and d-TiO2 (3), respectively. This suggest that the absorption band edge is red shifted typically by > 600 nm. N, F codoped TiO2 nanoparticles are highly dispersible in organic solvents such as chloroform, toluene and dispersion remain stable for weeks/months. (Figure 4) However, doped TiO2


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nanoparticle are insoluble in water or polar organic solvents such as ethanol, methanol. This is because nanoparticles are capped with long chain fatty acid/amine. In order to extend the application, doped TiO2 nanoparticles are transformed into water soluble nanoparticle via polyacrylate coating.36 Polyacrylate coating is widely used for preparation of nanoparticle with modular surface charge and surface chemistry that can be used in tuning the interaction with bioenvironment.36,37 Here we have used polyacrylate coating to introduce polyethylene glycol and zwitterionic surface charge. (Supporting Information, Scheme S2 and Figure S10, S11) The coated nanoparticles are 140-350 nm in hydrodynamic size with good colloidal stability at physiological pH and with overall surface charge of near zero values. Doping dependent reactive oxygen species (ROS) generation and photocatalytic activity by TiO2 nanoparticle under visible light. We have investigated visible light induced generation of reactive oxygen species (ROS) by N, F codoped TiO2 nanoparticle. This determination is important as ROS is actually responsible for photocatalytic activity. The ROS is generated after the absorption of visible light by doped TiO2 nanoparticle followed by formation of electron-hole pair and reaction of the excited state electrons/holes with solvent/dissolved oxygen.6 ROS is determined by estimating the free radicals produced by doped TiO2 nanoparticle under visible light. Typically, colloidal doped TiO2 nanoparticle is mixed with terephthalic acid and irradiated under visible light. The photogenerated ROS is trapped by terephthalic acid with the formation of fluorescent products which is then used for estimation of ROS.38 Results are summarized in Figure 5a and Supporting Figure S12. It shows that emission of terephthalic acid gradually increases with increasing irradiation time of visible light, suggesting the increased ROS generation with time. However, the rate of ROS generation varies for different doped samples. Figure 5a shows the extent ROS generation against irradiation time under the similar



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experimental condition, except that nanoparticle is varied. Results show that undopped TiO2 nanoparticle cannot produce ROS even after 6 h of visible light irradiation, d-TiO2 (1) produces very low amount of ROS, d-TiO2 (2) produces ROS that increases with time and most interestingly d-TiO2 (3) produces highest amount of ROS with the highest production rate within 60-80 min. This result suggests that d-TiO2 (3) would be the most effective photocatalyst among the three nanoparticles. In next experiment we have studied the visible light photodegradation of bisphenol A using N, F codoped TiO2 nanoparticle as photocatalyst. Bisphenol A is a well known endocrine disrupting chemical and its degradation by sunlight is important scientific research aspect.39 Typically, colloidal doped TiO2 nanoparticle is mixed with bisphenol A and irradiated under visible light. Next, degradation of bisphenol A is monitored via HPLC. (Figure 5b and Supporting Information, Figure S13) Results show that bisphenol A degradation kinetics follows trends similar to ROS generation trend as shown in Figure 5a. Degradation rate of bisphenol A is highest by d-TiO2 (3), lowest by d-TiO2 (1), intermediate by d-TiO2 (2) and insignificant by undopped TiO2 nanoparticle. This result clearly suggests that extent of N, F doping dictates the photocatalytic performance and heavily doped TiO2 nanoparticle is preferred for better photocatalysis. Visible light induced cell death by N, F codoped TiO2 nanoparticle via ROS generation. Next, we have investigated visible light induced cytotoxicity of doped TiO2 nanoparticle. We have selected d-TiO2 (2) and d-TiO2 (3) for this study as they produces significant amount of ROS within 30 min, in comparison to d-TiO2 (1). Doped TiO2 nanoparticle is transformed into polyacrylate coated nanoparticle of 140-350 nm hydrodynamic size and with zwitterionic surface charge. The zwitterionic surface charge of nanoparticle is introduced for efficient cell labelling


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without appreciable cytotoxicity.36,37 In order to study the cell labelling; these nanoparticles are further conjugated with FITC so that they can be seen under fluorescence microscope. When HeLa cells are incubated with colloidal solution of doped TiO2 nanoparticle, the cells get labelled by nanoparticles and distinct green emission is observed from HeLa cells. (Figure 6 and Supporting Information, Figure S14) In next experiment, cells are labelled with FITC non-conjugated, doped nanoparticle and then exposed under visible light for 30 min. Next, generation of ROS inside cell is investigated via fluorescent imaging using dichlorofluorescein diacetate as probe.40 Results show that HeLa cells labeled with doped TiO2 nanoparticle and exposed with visible light shows green emission due to the formation of ROS. In contrast, undoped TiO2 nanoparticle cannot produce ROS under visible light and doped TiO2 nanoparticle cannot produce ROS if they are not exposed with visible light. (Figure 7 and Supporting Information, Figure S15) Propidium ioide (PI) staining of nanoparticle labeled and visible light exposed cells show strong red emission, indicating the formation of membrane pores that induce permeation of PI to intercalate with the DNA of cell nucleus.41 Clearly such red emission is absent for control samples, suggesting that doping of TiO2 nanoparticle and visible light exposure are essential for membrane pore formation. ROS producing materials are widely used for antifungal42and antibacterial43-45activity. It is known that ROS generation inside cell leads to cell death via apoptosis and necrosis.46 So we have estimated the ROS mediated cytotoxicity using MTT-based cell viability assay. Typically, doped TiO2 nanoparticle labeled cells are exposed with visible light for 30 min and after 24 hrs of incubation in culture media, MTT assay has been performed. Results show that cell viability of doped TiO2 nanoparticle labeled and visible light exposed cells greatly decreased and the effect becomes more prominent at higher nanoparticle concentration. (Figure 8) In contrast,



Page 18 of 37

undoped TiO2 nanoparticle labeled cells exposed with visible light or doped TiO2 nanoparticle labeled cells without visible light exposure are less toxic as they do not produce ROS. N, F codoped colloidal TiO2 nanoparticle: Advantage of TiO2 nanorod-based doping approach. Presented doping approach has three unique features and advantages as compared to reported N, F codoping approaches. First, the doping requires relatively less drastic condition. While we have used 300 °C for doping, most of the earlier works use > 300 °C. Second, nanometer size and dispersible TiO2 nanorods are used for doping in their colloidal form, while most of the earlier works use either micron size particle or non-dispersible particles. Third, the extent of N and F doping can be tuned between 1-7 atomic %. There are only few earlier reports that can tune N, F doping typically between 0.1-0.4 atomic %.25 The reasons for this unique doping are the use of colloidal TiO2 nanorods as doping substrate. The small size, anisotropic shape and dispersed form offer maximum exposure of surface atoms for reaction with dopant precursors at less drastic conditions. In addition dispersible substrate allows controlled doping by simple variation of doping precursor concentration and reaction time. Control experiments show that if colloidal nanorods are replaced by nanorod films or physical mixtures in the powder form, the doping becomes inefficient and non-tunable. Resultant N, F codoped TiO2 nanoparticles have four specific advantages over reported N, F codoped TiO2 nanoparticles. First, colloidal doped nanoparticles have red shifted band edge upto 900 nm and they can absorb visible to NIR light. Considering the maximum red shift upto 550 nm for reported N, F codoped TiO2 nanoparticles, this is a significantly large shift. Second, doped nanoparticle is stable under long preservation and the colour becomes stable for more than month, either before or after use as catalyst (Supporting Information, Figure S16). Third, N, F codoped TiO2 nanoparticles show visible light photocatalysis depending on the extent of N/F


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doping. Clearly this type of doping dependent photocatalysis is not reported. Fourth, doped nanoparticles are colloidal in nature and they can be transformed into polymer coated and functional nanoparticle for specific applications. For example, they can be used for cell targeting and visible light induced cell death via ROS generation. Considering the unusually large red shift of band edge and strong absorption in visible region, further studies are necessary to understand the property of this material. There are earlier reports of N, F codoped green colored TiO2 or N doped green colored TiO2 and a small percent of Ti3+ is responsible for such color.28,47 HF doped blue TiO2 and NHF doped brown TiO2 are also reported.48 In our case, we are unable to detect Ti3+ species by XPS. This is either due to low concentration or they are deep inside lattice. However, we observed a very weak EPR signal associated with Ti3+ which may be one of the reasons for blue-green color and large red shift of band edge. In addition unique doping approach that involves dissolution of TiO2 nanorod followed by nucleation-growth of doped nanoparticle, may lead to efficient doping both in the lattice and at surface. In particular directly substituted F (Ti-F) is confirmed from XPS and FTIR, directly substituted N (Ti-N) is confirmed from XPS and Raman spectra. It is known that N, F codoping leads to the formation of isolated N 2p band at the top of the O 2p valence band and possibly high doping and nature of doping (substituted and interstitial/surface doping) further influence red shifting of band edge.24 In addition good durability and color stability of the doped material indicate the high application potential of this material.



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Conclusion Nitrogen and fluorine codoped TiO2 nanoparticle has been synthesized from colloidal TiO2 nanorods. The approach produces colloidal doped TiO2 nanoparticle and doping of nitrogen/fluorine can be tuned between 1-7 atomic %. Doped TiO2 nanoparticles have doping dependent heavily red shifted band gap (> 600 nm) and their colloidal dispersion absorbs from visible to NIR light. They produce reactive oxygen species under visible light exposure that can be used for photocatalytic degradation of toxic chemicals, cells and pathogens. Colloidal form of doped TiO2 nanoparticle offer labeling of cells followed by visible light induced cell death via reactive oxygen species generation inside cell. This work shows the potential advantage of nitrogen and fluorine codoping of TiO2 nanoparticle for efficient utilization of sunlight. ASSOCIATED CONTENT Supporting Information Summary of earlier N/F doped TiO2 nanoparticle with their properties, details of the characterization of N, F codoped TiO2 nanoparticle, control doping experimental data at different conditions, characterization of polyacrylated coated doped TiO2 nanoparticle, details of photocatalysis data, cell labeling and ROS imaging data of doped nanoparticles. This material is available free of charge via the Internet at http://pubs.acs.org. Notes The authors declare no competing financial interests. Acknowledgement. The authors acknowledge DST and CSIR, Government of India for financial assistance. (Grant numbers DST/TM/WTI/2K16/02(G), SR/NM/NS-1143/2016 and 02(0249)/15/EMR-II) A.B. and A.C. acknowledge CSIR, India for providing research fellowship. 20 ACS Paragon Plus Environment

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Table 1. Synthetic conditions and property of N, F codoped TiO2 nanoparticle. Sample

TiO

2

nanorod d-TiO2 (1)

Doping conditions (NH4F, urea, time)@ ---

Size$

Atomic % of F, N#

Band edge (absorption maxima)

2-3 nm x 25-35 nm (30-100 nm)

0.0, 1.1 (1.1)

--(340 nm)

Visible light induced ROS generation, photocatalysis, cytotoxicity no, no, no

180 mg, 180 mg, 70-100 nm 1.2, 1.4 800 nm poor, poor, poor 120 min (140-230 nm) (2.3) (350 nm) d-TiO2 (2) 360 mg, 360 mg, 150-220 nm 6.6, 3.0 900 nm good, good, good 120 min (180-350 nm) (3.1) (450 nm) TiO2 (3) 360 mg, 360 mg, 175-225 nm 3.5, 6.8 950 nm good, good, good 60 min (180-350 nm) (6.5) (500 nm) @ All other conditions are mentioned in experimental section. $ Values are derived from TEM. Values in parenthesis are polyacrylate coated particles measured by DLS. # Values are derived from XPS. Values in parenthesis are measured by elemental analysis.



N2 gas

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N 2 gas Titanium isopropoxide, oleylamine

NH 4 F, (NH 2 )2 CO 300 °C, 1-2 hrs

300 °C, 2 hrs Oleic acid

TiO 2 nanorods

N,F codoped TiO 2

Scheme 1. Synthetic approach for N, F codoped TiO2 nanoparticle. In first step TiO2 nanorod is prepared at 300 °C by hydrolysis of titanium isopropoxide in oleic acid medium. Next, colloidal TiO2 nanorod is reacted with ammonium fluoride and urea at 300 °C. Extent of N/F doping is varied by changing amount of ammonium fluoride/urea and reaction time.


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Figure 1. a) Transmission electron microscopic (TEM) image of TiO2 nanorod and three different N, F codoped TiO2 nanoparticles at low and high resolution. Selected area electron diffraction pattern is shown in the inset of high resolution images. b. i) X-ray diffraction pattern of TiO2 nanorod and three different N, F codoped TiO2 nanoparticles with respect to the bulk anatase TiO2. ii) Raman analysis of TiO2 nanorod and three different N, F codoped TiO2 nanoparticles. Signals at 144 cm-1 (Eg), 197 cm-1 (Eg), 399 cm1

(B1g), 519 cm-1 (A1g, B1g), 639 cm-1 (Eg) correspond to anatase phase of undoped TiO2 nanorod.

However, in doped nanoparticles a new band at 204 cm-1 appears (shown as red arrow) that corresponds to the first-order scatterings of non-stoichiometric titanium nitride (Ti-N). In 29 ACS Paragon Plus Environment


addition the low frequency 144 cm-1 (Eg) band is slightly shifted to ~155 cm-1 which is attributed to non-stochiometric TiO2-x phase.

b)

Ti 2p

d-TiO2 (3)

470

465

460

455 450 d-TiO2 (2)

470

465

460

455

450

d-TiO2(1) 470

465

460

455

450

Intensity (CPS)

Intensity (CPS)

a)

N 1s

d-TiO 2 (3)

408

404

400

396 392 d-TiO2 (2)

408

404

400

396 392 d-TiO 2 (1)

408

404

400

396 TiO 2

392

408

404

400

396

392

TiO2 465

460

455

450

Binding Energy (eV)

c)

F 1s

Binding Energy (eV)

d)

d-TiO 2 (3)

692

688

684

680 d-TiO2 (2)

692

688

684

680 d-TiO2 (1)

692

688

684

680 TiO2

692

688

684

680

Binding Energy (eV)

d-TiO2 (3)

%Transmittance

470

Intensity (CPS)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 37

d-TiO2 (2)

d-TiO2 (1)

Ti-F str.

Ti-F str. Ti-O-Ti str.

TiO2

3200

2400 1600 Wavenumber (cm-1)

800

Figure 2. Evidence of N, F codoping via XPS and FTIR analysis of doped TiO2 nanoparticle. a) Deconvoluted Ti 2p3/2 and Ti p1/2 signals that shift to higher binding energies after doping. While undoped TiO2 shows their signals at ~458.6 eV and ~464.2 eV; in d-TiO2 (1) they are shifted to ~461.2 eV and ~467.1 eV, respectively. These shifting are relatively less for d-TiO2 (2) and almost insignificant for d-TiO2 (3). b) Deconvoluted N 1s signal at ~ 400.7 eV for d-TiO2 (1); at ~402.0 eV, ~400.4 eV, ~399.6 eV for d-TiO2 (2); at ~400.6 eV, ~399.3 eV, ~398 eV for d-TiO2 (3). In undoped TiO2 it appears at ~ 399.9 eV due to surface adsorbed oleylamine. The N1s bands below ~400.0 eV are attributed as interstitially doped or directly substituted N into the host 30 ACS Paragon Plus Environment

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lattice as they are present even after surface cleaning by argon ion sputtering. c) Characteristic signals of F1s in doped TiO2 nanoparticle, which is absent in undoped TiO2 nanorods. The dTiO2 (1) shows signal at ~688 eV for directly substituted fluorine atom in the TiO2 lattice and dTiO2 (3) shows F1s signals at ~685 eV that can be assigned to the interstitially or surface doped fluorine atoms. However, d-TiO2 (2) shows signals at 688 eV and ~ 685.8 eV due to both directly substituted fluorine and interstitial/surface doped fluorine. d) FTIR spectra of doped TiO2 nanoparticles, showing Ti-F stretching vibration at 944-1030 cm-1.

Figure 3. a) Time dependent evaluation of UV-visible absorption spectra of doped TiO2 nanoparticle from colloidal TiO2 nanorods at their early stages. b) Acetone solution of isolated doped TiO2 nanoparticle at different time point of reaction, showing that blue-green particles appear typically after 10-20 min of reaction and color becomes more intense as the time progress. c) TEM image of starting TiO2 nanorods used with length distribution in the inset. d) TEM image TiO2 nanoparticles at different time points, showing that nanorods aggregate after 5 min (with length distribution in the inset), partially dissolve at 10-20 min (with size distribution in the inset), nucleate to cubic/tetragonal shaped particles at 20-30 min and then grow into larger size with time (with size distribution in the inset).



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Figure 4. a) UV-visible absorption spectra of dilute dispersions of N, F codoped TiO2 nanoparticles in chloroform showing their intense absorption in visible region covering from 350 nm to 900 nm. While undoped TiO2 nanoparticle does not have any band in visible region and band edge at 340 nm, the d-TiO2 (1) shows broad band having peak at 350 nm with band edge at 800 nm, d-TiO2 (2) shows broad band having peak at 450 nm with band edge at 900 nm and dTiO2 (3) shows broad band having peak at 500 nm with band edge at 950 nm. b) Digital image of solid doped TiO2 nanoparticles and their respective colloidal dispersion in chloroform. While undoped solid TiO2 nanorods are white in color, the solid samples and colloidal dispersions are blue-green in color.


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b) 260

1.0

d-TiO2 (3)

208

d-TiO2 (2)

156

d-TiO2 (1) TiO2

104

C / C0

a)

I / I0

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0.8

TiO2 d-TiO2 (1)

0.6

d-TiO2 (2)

0.4

d-TiO2 (3)

52

0.2

0

0.0

0

60 120 180 240 300 360 420 Time (min)

0

60 120 180 240 300 360 420 Time (min)

Figure 5. a) Visible light induced reactive oxygen species (ROS) generation by N, F codoped TiO2 nanoparticle. Typically, same concentrations of colloidal doped TiO2 nanoparticles and terephthalic acid are mixed together, irradiated by visible light under similar condition and then photogenerated ROS (ratio of emission at 420 nm after and before light irradiation, I/I0) is measured at different time of irradiation. Results show increased ROS generation with irradiation time and varied rate of ROS generation for different doped samples which are in the order of dTiO2 (3) > d-TiO2 (2) > d-TiO2 (1) > undopped TiO2. b) Visible light photodegradation of bisphenol A using N, F codoped TiO2 nanoparticle as photocatalyst showing that bisphenol A degradation kinetics by nanoparticles is in the order of dTiO2 (3) > d-TiO2 (2) > d-TiO2 (1) > undopped TiO2. Colloidal doped TiO2 nanoparticle is mixed with bisphenol A, irradiated by visible light and then degradation of bisphenol A (ratio of final to initial bisphenol A concentration, C/C0) is monitored via HPLC.



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Figure 6. Labeling of HeLa cells by N, F codoped TiO2 nanoparticle. Typically, d-TiO2 (3) is transformed into polyacrylate coated nanoparticle of 180-350 nm hydrodynamic size with zwitterionic surface charge and conjugated with FITC. Next, cells are incubated with colloidal solution of nanoparticle for 3 hrs and washed cells are imaged under bright field (BF) or fluorescence (F) mode. Strong green emission from cell indicates that cells are labelled with nanoparticle.


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Figure 7. a) Evidence of visible light induced ROS generation inside cell by doped TiO2 nanoparticle. HeLa cells are labeled with N, F codoped TiO2 nanoparticle (FITC non-conjugated) and then exposed under visible light for 30 min. Next, cells are incubated with DCF followed by fluorescence imaging under bright field (BF) or fluorescence (F) mode. Strong green fluorescence indicates ROS generation inside cells. b) ROS induced cell membrane rupture by doped TiO2 nanoparticle. Nanoparticle labeled HeLa cells are exposed with visible light for 30 min and then incubated with propidium iodide (PI) followed by fluorescence imaging under bright field (BF) or fluorescence (F) mode. Damaged membrane allows PI entry into cell, intercalate with the DNA of the nucleus and shows red fluorescence. Strong red fluorescence from cell nucleus indicates membrane rupture. i) d-TiO2 (3) with visible light exposure for 30 min ii) d-TiO2 (3) without any visible light exposure iii) undoped TiO2 nanorod exposed under visible light for 30 min.



120 100 Cell Viability (%)

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TiO2 + light

d-TiO2 (3)

d-TiO2 (3) + light

80 60 40 20 0

0.00

0.05 0.10 0.02 Concentration (µg/mL)

Figure 8. Visible light induced toxicity of N, F codoped TiO2 nanoparticle labelled cells. Typically, nanoparticle labeled cells are exposed with visible light for 30 min and after 24 hrs of incubation in culture media, MTT assay has been performed. Results show that cell viability greatly decreased due to visible light exposure and the effect becomes more prominent at higher dose of nanoparticle. In contrast, undoped TiO2 nanorod exposed with visible light or doped TiO2 nanoparticle without visible light exposure is less toxic. The mean ± SD of three determinations are represented in bars.


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Nitrogen and Fluorine Codoped, Colloidal TiO2 Nanoparticle: Tunable

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