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Biocompatible Eu-doped TiO2 nanodot film with in situ protein adsorption characterization property
TSF-34009; No of Pages 4 Thin Solid Films xxx (2015) xxx–xxx
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Biocompatible Eu-doped TiO2 nanodot film with in situ protein adsorption characterization property Kui Cheng a, Yifei Zhu a, Wenjian Weng a,b,⁎, Jun Lin c, Huiming Wang c a Department of Materials Science and Engineering, State Key Laboratory of Silicon Materials, Cyrus Tang Center for Sensor Materials and Applications, Zhejiang University, Hangzhou 310027, China b The Shanghai Institute of Ceramics, Chinese Academy of Sciences, 1295 Dingxi Road, Shanghai, 200050, China c The First Affiliated Hospital of Medical College, Zhejiang University, Hangzhou, 310003, China
a r t i c l e
i n f o
Available online xxxx Keywords: Europium Doped oxides Titanium dioxide Nanoparticles Thin films Photoluminescence Protein
a b s t r a c t Europium (Eu) doped TiO2 nanodot films were prepared through a phase-separation-induced self-assembly method. Eu was doped to impart the nanodots with luminescence property so that the protein adsorption could be in situ characterized quantitatively. Bovine serum albumin (BSA) was adsorbed on the surface of Eudoped titanium nanodot films. It was found that the photo luminescence intensity at 616 nm decreased with the increase of BSA adsorption time. Also, Eu-doped TiO2 nanodot films showed good biocompatibility. These results suggested that Eu-doped TiO2 nanodot films could provide a feasible in situ way to evaluate protein adsorption if prepared on the surface of bioimplants. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Implantation of bioimplants usually initiates surface interactions between the materials and the host. Cells adhere, proliferate and differentiate on the implants' surface. In most cases, cells can only survive when attached to the culture surface mediated by pre-adsorbed extracellular matrix proteins [1,2]. The adsorption behavior and amount of the protein greatly influence the subsequent cellular responses. Therefore, it is of great importance if a facile method could be applied to evaluate the amount of protein adsorption. Many methods have been proposed to evaluate the amount of protein adsorption, such as BCA (bicinchoninic acid protein assay) [3–5], quartz crystal microbalance [6,7], and enzyme linked immunosorbent assay [8,9]. However, most of them are not in situ, which means protein removal from the surface is inevitable. That may bring some deviations between the data obtained and real situation. E.g., the prevalent BCA method needs a thorough elution process to remove the adsorbed protein. The residual protein molecules on the surface may greatly affect the accuracy of the result. On the other hand, cells need an appropriate microenvironment for growth [10,11]; an excellent biomaterial surface with great nanoscale topography was developed and reported to be favorable for the proper functioning of the attached cells [12,13]. It was found that microscale or ⁎ Corresponding author at: Department of Materials Science and Engineering, State Key Laboratory of Silicon Materials, Cyrus Tang Center for Sensor Materials and Applications, Zhejiang University, Hangzhou 310027, China. Tel./fax: +86 571 87953787. E-mail address: [email protected] (W. Weng).
nanoscale topography, such as nanodots, nanofibers, nanorods and nanotubes can promote cell growth [14–17]. According to our previous work, europium doped TiO2 nanodot films could be easily prepared. Such nanodot films showed good luminescence property. Also, it is found that mouse pre-osteoblastic MC3T3-E1 cells can grow and spread well on TiO2 nanodot films [18]. Therefore, it is interesting to explore if the luminescence property brought by Eu doping could be utilized to in situ evaluate the protein adsorption without removing it from the surface. In this work, Eu-doped TiO2 nanodot films were prepared and characterized. The photoluminescence (PL) property of such nanodot films was investigated as a simple in situ indicator to evaluate the amount of protein adsorbed. 2. Experimental 2.1. Synthesis and characterization of Eu-doped TiO2 nanodot films Phase-separation-induced self-assembly method was used to prepare Eu doped TiO2 nanodots on quartz (silica) substrate [19]. Briefly, Eu(NO 3) 3 ·6H2 O (0.01 mol/l) or Tris(2,2,6,6-tetramethyl3,5-heptanedionato) europium (Eu(TMHD) 3, 0.01 mol/l) was dissolved in ethanol (Sinopharm Chemical Reagent, AR, N99.7%) and stirred for 60 min to be clear and stable ethanol solution (about 5 ml), acetylacetone (AcAc, Lingfeng Chemical Reagent, AR, N99%), deionized water, tetrabutyl titanate (TBOT, Sinopharm Chemical Reagent, CP, N 98% 0.1 mol/l) and polyvinyl pyrrolidone (40 g/l) with an average molecular weight of 30,000 (K30, Sinopharm Chemical
http://dx.doi.org/10.1016/j.tsf.2015.01.003 0040-6090/© 2015 Elsevier B.V. All rights reserved.
Please cite this article as: K. Cheng, et al., Biocompatible Eu-doped TiO2 nanodot film with in situ protein adsorption characterization property, Thin Solid Films (2015), http://dx.doi.org/10.1016/j.tsf.2015.01.003
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K. Cheng et al. / Thin Solid Films xxx (2015) xxx–xxx
Reagent, AR, N99%) were added to the solution to obtain a precursor solution. The concentration of TBOT was 0.1 mol/l, and the molar ratio of AcAc: H2O: TBOT was fixed at 0.3:1:1. Then, 15 μl solutions were dropped on the high speed (8000 rpm) rotating substrate to coat for 40 s, and the sample was put in a muffle furnace at 500 °C and fired for 2 h to be Eu doped TiO2 nanodot films. The morphology of Eu doped TiO2 nanodot films was evaluated using scanning electron microscopy (SEM, SU-70, Hitachi). The chemical composition of Eu doped TiO2 nanodot films was investigated by energy dispersive spectrometer (EDS) attached to SEM.
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3. Biocompatibility test 3.1. Cell culture Mouse pre-osteoblastic MC3T3-E1 cells (CRL-2594, ATCC) were cultured in alpha minimum essential media (Gibco) supplemented with 10% fetal bovine serum (Hyclone), 1% minimum essential medium non-essential amino acids (Gibco), 1% antibiotic solution containing 10,000 mg/ml streptomycin (Gibco) and 10,000 units/ml penicillin, and 1% sodium pyruvate (Gibco). The cells were incubated under standard cell culture conditions at 37 °C in an atmosphere of 5% CO2. Cultured cells were harvested with 0.25% trypsin/ethylene diamine tetraacetic acid (Gibco), suspended in fresh culture media for the experiments.
(b)
3.2. Cell adhesion and proliferation Cell adhesion and proliferation were measured by cell counting kit-8 (CCK-8) assay [20]. Briefly, cell cultured at a density of 5 × 104 cells/cm2 were seeded on TiO2 nanodot films with and without Eu doping in 24 wells. CCK-8 was added in the wells as the concentration ratio of 1:10 with culture solution. After reacted for 3 h in the incubator, the optical density (OD) of supernatant liquid was measured at 450 nm with a microplate reader (Multiskan MK3) after cultured for different times (6 h, 24 h, 72 h).
Fig. 1. SEM images of Eu-doped TiO2 nanodot films with different Eu(NO3)2·6H2O addition: (a) 10% mol and (b) 5% mol. The scale bar in the inset image is 200 nm.
4. Protein adsorption and characterization Bovine serum albumin (BSA) was dissolved into phosphate buffered saline to get 1 mg/ml solution. The samples were soaked in the solution for different times to simulate different amounts of protein adsorption. The fluorescence spectrometer (FLS920, Edinburgh Instruments) was used to measure the luminescence property of Eu doped TiO2 nanodot film with different amounts of protein adsorption. The excitation wavelength and slit width were set at 300 nm and 5 nm respectively. 5. Results and discussions The morphology of Eu-doped TiO 2 nanodot films with Eu(NO3) as the Eu source was shown in Fig. 1. Clearly, with increasing Eu(NO3)3·6H2O content, films with more dense nanodots were observed. However, as shown in the inset, some parts of the films showed different morphology. That actually means that the incorporation of Eu(NO3)3·6H2O affects the formation of TiO2 nanodot films. It is assumed that the water molecules brought may influence the hydrolysis of TBOT and eventually the morphology of nanodot films. The unevenness of nanodot films may affect cellular responses, which are not suitable for further investigation. In Fig. 2, the morphology of Eu-doped TiO2 nanodot films with Eu(TMHD)3 as the Eu source was shown in Fig. 2. Spherical, well dispersed Eu-doped TiO2 nanodots formed nanodot films on the substrate. Evaluated from diameters of the nanodots, the average thickness of the films is about 90–110 nm. The densities of nanodots in the films are about 84/μm2, 70/μm2 and 67/μm2 respectively with different Eu(TMHD)3 amounts. Clearly, the morphology and size of nanodots do 3·6H2O
not change much when the amount of Eu doping increases. That indicates that Eu(TMHD)3 is suitable for incorporation of Eu. EDS was used in order to verify the existence of Eu element under the voltage of 20 kV; the working distance is 15 mm. However, almost no Eu signal was observed. The reason was ascribed to that the amount of Eu was too low, and fell out of range of the detection limit of EDS. In order to study the luminescence property of Eu doped TiO2 nanodot films, PL spectra were measured at room temperature (25 °C). According to data in the literature, excitation light with a wavelength of 300 nm was used in this experiment in order to get stronger luminescence intensity. Fig. 3 showed the PL spectra of undoped and Eu doped TiO2 nanodot films. According to reference, there are 5 emission peaks at 579 nm, 592 nm, 614 nm, 654 nm and 702 nm in PL spectra, which correspond to 5D0 → 7 F0, 5D0 → 7 F1, 5D0 → 7 F2, 5D0 → 7 F3 and 5D0 → 7 F4 transitions of Eu3+ ion respectively [21]. In this work, the peak at 579 nm can hardly be found; only a very small shoulder peak was observed on that with 15% Eu (Eu/Ti molar ratio). As for the peak at 702 nm, it overlapped with the peaks from the substrate. Only three obvious peaks of 592 nm, 616 nm and 656 nm were observed. The slight shift of the latter two could be ascribed to that different excitation wavelength was used. The peak located at 465 nm was ascribed to quartz (silica) substrate [22,23]. As seen from the characteristic peak at 616 nm, the emission intensity increased with the increasing Eu concentration. 15% doping led to almost twice luminescence intensity that that of 5% doping. Obviously, the doping concentration of Eu has a great impact on the luminescence property of Eu-doped TiO2 nanodot films.
Please cite this article as: K. Cheng, et al., Biocompatible Eu-doped TiO2 nanodot film with in situ protein adsorption characterization property, Thin Solid Films (2015), http://dx.doi.org/10.1016/j.tsf.2015.01.003
K. Cheng et al. / Thin Solid Films xxx (2015) xxx–xxx
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PL intensity (arb.units)
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5%-Eu-TiO2 10%-Eu-TiO2 15%-Eu-TiO2
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Wavelength (nm) Fig. 3. PL intensities of Eu-doped TiO2 nanodot films with different amounts of Eu.
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observed. The OD value on Eu doped TiO2 nanodot film was slightly smaller, but no statistic difference was found. Basically, Eu doped TiO2 nanodot film showed good biocompatibility and no adverse effect on cells was observed. Cells could grow and proliferate well on Eu-doped TiO2 nanodot films. Eu doped TiO2 nanodot film with 10% doping was used to characterize the protein adsorption, since the luminescence intensity is high enough. The samples were soaked in the BSA solution for different times and then the PL intensity was measured. As shown in Fig. 5, the peak intensity at 616 nm decreased obviously with increasing soaking time initially, and then kept almost unchanged with elongated soaking time from 2 h to 16 h. That means after 2 h, BSA adsorption gets saturated. According to our previous work, BSA adsorption increases with increasing soaking time in the initial hours. The results in this work actually means that the luminescence property of Eu could be utilized to evaluate the amount of such adsorption through doping in TiO 2 nanodot films. Owing to the excellent biocompatibility, such Eu-doped TiO2 nanodot film could be widely applied on the surface of many bioimplants and impart them with a feasible and easy way to evaluate the protein adsorption.
3.0 Pure TiO2
Fig. 2. SEM images of Eu-doped TiO2 nanodot films with different Eu(TMHD)3: (a) 5% mol, (b)10% mol, and (c)15% mol. The scale bar in the inset image is 200 nm.
CCK-8 kit was used to detect the biocompatibility of Eu doped TiO 2 nanodot films. A TiO2 nanodot film without Eu doping was used as a reference. As shown in Fig. 4, the OD value, which indicates the number of attached cells, showed almost the same value for the first 6 h for both films. With longer culture time of 24 h, the OD value of Eu-doped TiO 2 nanodot film was a little bit higher than that of the undoped. Such results indicate that the initial cell adhesions of the two films are basically similar. When the culture time increased to about 72 h, fibroblasts proliferated and more cells were
OD value
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2.0 1.5 1.0 0.5 0.0 6h
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Time (h) Fig. 4. CCK-8 results of NIH 3T3 cells cultured on nanodot films with and without Eu doping.
Please cite this article as: K. Cheng, et al., Biocompatible Eu-doped TiO2 nanodot film with in situ protein adsorption characterization property, Thin Solid Films (2015), http://dx.doi.org/10.1016/j.tsf.2015.01.003
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PL Intensity (arb. units)
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Wavelength (nm) Fig. 5. PL intensities of Eu-doped TiO2 nanodots with different soaking times in BSA solution.
6. Conclusions Eu doped TiO 2 nanodot films can be easily prepared by sol–gel and phase-separation-induced self-assembly method, and they showed good biocompatibility. The PL intensity of Eu decreased gradually in the first few hours with increasing soaking time in protein solution. Such films on bio-implant could provide an easy in situ way to evaluate surface protein adsorption without detrimental effect on biocompatibility. Acknowledgments This work is financially supported by the National Basic Research Program of China (973 Program, 2012CB933600), the National Natural Science Foundation of China (51372217, 51472216, 51272228, 81171003, 81371120), the Fundamental Research Funds for the Central Universities (2013QNA4010, 2014XZZX005) and the Medical Technology and Education of Zhejiang Province of China (wkj-zj-11). References [1] L. Bacakova, V. Mares, V. Lisa, V. Svorcik, Molecular mechanisms of improved adhesion and growth of an endothelial cell line cultured on polystyrene implanted with fluorine ions, Biomaterials 21 (11) (2000) 1173. [2] A. Kikuchi, H. Taira, T. Tsuruta, M. Hayashi, K. Kataoka, Adsorbed serum protein mediated adhesion and growth behavior of bovine aortic endothelial cells on polyamine graft copolymer surfaces, J. Biomater. Sci. Polym. Ed. 8 (2) (1996) 77.
[3] Y. Takami, S. Yamane, K. Makinouchi, G. Otsuka, J. Glueck, R. Benkowski, Y. Nose, Protein adsorption onto ceramic surfaces, J. Biomed. Mater. Res. 40 (1) (1998) 24. [4] L. Chen, Z. Yu, Y.J. Lee, X. Wang, B. Zhao, Y.M. Jung, Quantitative evaluation of proteins with bicinchoninic acid (BCA): resonance Raman and surface-enhanced resonance Raman scattering-based methods, Analyst 137 (24) (2012) 5834. [5] F. Kratz, C. Muller, N. Korber, N. Umanskaya, M. Hannig, C. Ziegler, Characterization of protein films on dental materials: bicinchoninic acid assay (BCA) studies on loosely and firmly adsorbed protein layers, Phys. Status Solidi A 210 (5) (2013) 964. [6] N. Chandrasekaran, S. Dimartino, C.J. Fee, Study of the adsorption of proteins on stainless steel surfaces using QCM-D, Chem. Eng. Res. Des. 91 (9) (2013) 1674. [7] T. Yoshioka, H. Yonekura, T. Ikoma, M. Tagaya, J. Tanaka, Investigation of multilayered protein adsorption on carbonate apatite with a QCM technique, in: K. Ishikawa, Y. Iwamoto (Eds.),24th Symposium and Annual Meeting of International Society for Ceramics in Medicine, Fukuoka, JAPAN, October 21–24, 2012, Bioceramics, 529-530, 2013, p. 74. [8] M. Balcells, D. Klee, H. Hocker, Quantitative description of protein adsorption processes at polymer surfaces by means of ELISA, Mater. Werks. 30 (12) (1999) 846. [9] Y. Zhang, C. Tan, J.Q. Zhang, W. Sheng, S. Wang, A sensitive sandwich ELISA for the rapid detection of mung bean protein: development and evaluation of the effect of thermal processing on detection, Food Anal. Methods 7 (6) (2014) 1305. [10] M.P. Lutolf, H.M. Blau, Artificial stem cell niches, Adv. Mater. 21 (32–33) (2009) 3255. [11] E. Martínez, A. Lagunas, C.A. Mills, S. Rodríguez-Seguí, M. Estévez, S. Oberhansl, J. Comelles, J. Samitier, Stem cell differentiation by functionalized micro- and nanostructured surfaces, Nanomedicine 4 (1) (2009) 65. [12] M.J. Dalby, N. Gadegaard, R. Tare, A. Andar, M.O. Riehle, P. Herzyk, C.D.W. Wilkinson, R.O.C. Oreffo, The control of human mesenchymal cell differentiation using nanoscale symmetry and disorder, Nat. Mater. 6 (12) (2007) 997. [13] F. Iwasa, N. Hori, T. Ueno, H. Minamikawa, M. Yamada, T. Ogawa, Enhancement of osteoblast adhesion to UV-photofunctionalized titanium via an electrostatic mechanism, Biomaterials 31 (10) (2010) 2717. [14] K. Vasilev, Z. Poh, K. Kant, J. Chan, A. Michelmore, D. Losic, Tailoring the surface functionalities of titania nanotube arrays, Biomaterials 31 (3) (2010) 532. [15] L. Zhao, S. Mei, P.K. Chu, Y. Zhang, Z. Wu, The influence of hierarchical hybrid micro/ nano-textured titanium surface with titania nanotubes on osteoblast functions, Biomaterials 31 (19) (2010) 5072. [16] K. Kubo, N. Tsukimura, F. Iwasa, T. Ueno, L. Saruwatari, H. Aita, W.-A. Chiou, T. Ogawa, Cellular behavior on TiO2 nanonodular structures in a micro-to-nanoscale hierarchy model, Biomaterials 30 (29) (2009) 5319. [17] V.V. Divya Rani, L. Vinoth-Kumar, V.C. Anitha, K. Manzoor, M. Deepthy, V.N. Shantikumar, Osteointegration of titanium implant is sensitive to specific nanostructure morphology, Acta Biomater. 8 (5) (2012) 1976. [18] Y. Hong, M.F. Yu, W.J. Weng, K. Cheng, H.M. Wang, J. Lin, Light-induced cell detachment for cell sheet technology, Biomaterials 34 (1) (2013) 11. [19] M. Luo, K. Cheng, W.J. Weng, C.L. Song, P.Y. Du, G. Shen, G. Xu, G.R. Han, Size- and density-controlled synthesis of TiO2 nanodots on a substrate by phase-separationinduced self-assembly, Nanotechnology 20 (2009) 215605. [20] V. Guarani, G. Deflorian, C.A. Franco, M. Krüger, L.K. Phng, K. Bentley, L. Toussaint, F. Dequiedt, R. Mostoslavsky, M.H.H. Schmid, B. Simmermann, R.P. Brandes, M. Mione, C.H. Westphal, T. Braun, A.M. Zeiher, H. Gerhardt, S. Dimmeler, M. Potente, Acetylation-dependent regulation of endothelial notch signalling by the SIRT1 deacetylase, Nature 473/7346 (2011) 234–238. [21] L. Li, C.K. Tsung, Z. Yang, G.D. Stucky, L.D. Sun, J.F. Wang, C.H. Yan, Rare-earth-doped nanocrystalline titania microspheres emitting luminescence via energy transfer, Adv. Mater. 20 (5) (2008) 903. [22] C. Leostean, M. Stefan, O. Pana, A.I. Cadis, R.C. Suciu, T.D. Silipas, E. Gautron, Properties of Eu doped TiO2 nanoparticles prepared by using organic additives, J. Alloys Compd. 579 (2013) 29. [23] F. Gao, Y. Sheng, Y.H. Song, Facile synthesis and luminescence properties of europium(III) -doped silica nanotubes, J. Sol-Gel Sci. Technol. 71 (2) (2014) 313.
Please cite this article as: K. Cheng, et al., Biocompatible Eu-doped TiO2 nanodot film with in situ protein adsorption characterization property, Thin Solid Films (2015), http://dx.doi.org/10.1016/j.tsf.2015.01.003
Contents lists available at ScienceDirect
Thin Solid Films journal homepage: www.elsevier.com/locate/tsf
Biocompatible Eu-doped TiO2 nanodot film with in situ protein adsorption characterization property Kui Cheng a, Yifei Zhu a, Wenjian Weng a,b,⁎, Jun Lin c, Huiming Wang c a Department of Materials Science and Engineering, State Key Laboratory of Silicon Materials, Cyrus Tang Center for Sensor Materials and Applications, Zhejiang University, Hangzhou 310027, China b The Shanghai Institute of Ceramics, Chinese Academy of Sciences, 1295 Dingxi Road, Shanghai, 200050, China c The First Affiliated Hospital of Medical College, Zhejiang University, Hangzhou, 310003, China
a r t i c l e
i n f o
Available online xxxx Keywords: Europium Doped oxides Titanium dioxide Nanoparticles Thin films Photoluminescence Protein
a b s t r a c t Europium (Eu) doped TiO2 nanodot films were prepared through a phase-separation-induced self-assembly method. Eu was doped to impart the nanodots with luminescence property so that the protein adsorption could be in situ characterized quantitatively. Bovine serum albumin (BSA) was adsorbed on the surface of Eudoped titanium nanodot films. It was found that the photo luminescence intensity at 616 nm decreased with the increase of BSA adsorption time. Also, Eu-doped TiO2 nanodot films showed good biocompatibility. These results suggested that Eu-doped TiO2 nanodot films could provide a feasible in situ way to evaluate protein adsorption if prepared on the surface of bioimplants. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Implantation of bioimplants usually initiates surface interactions between the materials and the host. Cells adhere, proliferate and differentiate on the implants' surface. In most cases, cells can only survive when attached to the culture surface mediated by pre-adsorbed extracellular matrix proteins [1,2]. The adsorption behavior and amount of the protein greatly influence the subsequent cellular responses. Therefore, it is of great importance if a facile method could be applied to evaluate the amount of protein adsorption. Many methods have been proposed to evaluate the amount of protein adsorption, such as BCA (bicinchoninic acid protein assay) [3–5], quartz crystal microbalance [6,7], and enzyme linked immunosorbent assay [8,9]. However, most of them are not in situ, which means protein removal from the surface is inevitable. That may bring some deviations between the data obtained and real situation. E.g., the prevalent BCA method needs a thorough elution process to remove the adsorbed protein. The residual protein molecules on the surface may greatly affect the accuracy of the result. On the other hand, cells need an appropriate microenvironment for growth [10,11]; an excellent biomaterial surface with great nanoscale topography was developed and reported to be favorable for the proper functioning of the attached cells [12,13]. It was found that microscale or ⁎ Corresponding author at: Department of Materials Science and Engineering, State Key Laboratory of Silicon Materials, Cyrus Tang Center for Sensor Materials and Applications, Zhejiang University, Hangzhou 310027, China. Tel./fax: +86 571 87953787. E-mail address: [email protected] (W. Weng).
nanoscale topography, such as nanodots, nanofibers, nanorods and nanotubes can promote cell growth [14–17]. According to our previous work, europium doped TiO2 nanodot films could be easily prepared. Such nanodot films showed good luminescence property. Also, it is found that mouse pre-osteoblastic MC3T3-E1 cells can grow and spread well on TiO2 nanodot films [18]. Therefore, it is interesting to explore if the luminescence property brought by Eu doping could be utilized to in situ evaluate the protein adsorption without removing it from the surface. In this work, Eu-doped TiO2 nanodot films were prepared and characterized. The photoluminescence (PL) property of such nanodot films was investigated as a simple in situ indicator to evaluate the amount of protein adsorbed. 2. Experimental 2.1. Synthesis and characterization of Eu-doped TiO2 nanodot films Phase-separation-induced self-assembly method was used to prepare Eu doped TiO2 nanodots on quartz (silica) substrate [19]. Briefly, Eu(NO 3) 3 ·6H2 O (0.01 mol/l) or Tris(2,2,6,6-tetramethyl3,5-heptanedionato) europium (Eu(TMHD) 3, 0.01 mol/l) was dissolved in ethanol (Sinopharm Chemical Reagent, AR, N99.7%) and stirred for 60 min to be clear and stable ethanol solution (about 5 ml), acetylacetone (AcAc, Lingfeng Chemical Reagent, AR, N99%), deionized water, tetrabutyl titanate (TBOT, Sinopharm Chemical Reagent, CP, N 98% 0.1 mol/l) and polyvinyl pyrrolidone (40 g/l) with an average molecular weight of 30,000 (K30, Sinopharm Chemical
http://dx.doi.org/10.1016/j.tsf.2015.01.003 0040-6090/© 2015 Elsevier B.V. All rights reserved.
Please cite this article as: K. Cheng, et al., Biocompatible Eu-doped TiO2 nanodot film with in situ protein adsorption characterization property, Thin Solid Films (2015), http://dx.doi.org/10.1016/j.tsf.2015.01.003
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K. Cheng et al. / Thin Solid Films xxx (2015) xxx–xxx
Reagent, AR, N99%) were added to the solution to obtain a precursor solution. The concentration of TBOT was 0.1 mol/l, and the molar ratio of AcAc: H2O: TBOT was fixed at 0.3:1:1. Then, 15 μl solutions were dropped on the high speed (8000 rpm) rotating substrate to coat for 40 s, and the sample was put in a muffle furnace at 500 °C and fired for 2 h to be Eu doped TiO2 nanodot films. The morphology of Eu doped TiO2 nanodot films was evaluated using scanning electron microscopy (SEM, SU-70, Hitachi). The chemical composition of Eu doped TiO2 nanodot films was investigated by energy dispersive spectrometer (EDS) attached to SEM.
(a)
3. Biocompatibility test 3.1. Cell culture Mouse pre-osteoblastic MC3T3-E1 cells (CRL-2594, ATCC) were cultured in alpha minimum essential media (Gibco) supplemented with 10% fetal bovine serum (Hyclone), 1% minimum essential medium non-essential amino acids (Gibco), 1% antibiotic solution containing 10,000 mg/ml streptomycin (Gibco) and 10,000 units/ml penicillin, and 1% sodium pyruvate (Gibco). The cells were incubated under standard cell culture conditions at 37 °C in an atmosphere of 5% CO2. Cultured cells were harvested with 0.25% trypsin/ethylene diamine tetraacetic acid (Gibco), suspended in fresh culture media for the experiments.
(b)
3.2. Cell adhesion and proliferation Cell adhesion and proliferation were measured by cell counting kit-8 (CCK-8) assay [20]. Briefly, cell cultured at a density of 5 × 104 cells/cm2 were seeded on TiO2 nanodot films with and without Eu doping in 24 wells. CCK-8 was added in the wells as the concentration ratio of 1:10 with culture solution. After reacted for 3 h in the incubator, the optical density (OD) of supernatant liquid was measured at 450 nm with a microplate reader (Multiskan MK3) after cultured for different times (6 h, 24 h, 72 h).
Fig. 1. SEM images of Eu-doped TiO2 nanodot films with different Eu(NO3)2·6H2O addition: (a) 10% mol and (b) 5% mol. The scale bar in the inset image is 200 nm.
4. Protein adsorption and characterization Bovine serum albumin (BSA) was dissolved into phosphate buffered saline to get 1 mg/ml solution. The samples were soaked in the solution for different times to simulate different amounts of protein adsorption. The fluorescence spectrometer (FLS920, Edinburgh Instruments) was used to measure the luminescence property of Eu doped TiO2 nanodot film with different amounts of protein adsorption. The excitation wavelength and slit width were set at 300 nm and 5 nm respectively. 5. Results and discussions The morphology of Eu-doped TiO 2 nanodot films with Eu(NO3) as the Eu source was shown in Fig. 1. Clearly, with increasing Eu(NO3)3·6H2O content, films with more dense nanodots were observed. However, as shown in the inset, some parts of the films showed different morphology. That actually means that the incorporation of Eu(NO3)3·6H2O affects the formation of TiO2 nanodot films. It is assumed that the water molecules brought may influence the hydrolysis of TBOT and eventually the morphology of nanodot films. The unevenness of nanodot films may affect cellular responses, which are not suitable for further investigation. In Fig. 2, the morphology of Eu-doped TiO2 nanodot films with Eu(TMHD)3 as the Eu source was shown in Fig. 2. Spherical, well dispersed Eu-doped TiO2 nanodots formed nanodot films on the substrate. Evaluated from diameters of the nanodots, the average thickness of the films is about 90–110 nm. The densities of nanodots in the films are about 84/μm2, 70/μm2 and 67/μm2 respectively with different Eu(TMHD)3 amounts. Clearly, the morphology and size of nanodots do 3·6H2O
not change much when the amount of Eu doping increases. That indicates that Eu(TMHD)3 is suitable for incorporation of Eu. EDS was used in order to verify the existence of Eu element under the voltage of 20 kV; the working distance is 15 mm. However, almost no Eu signal was observed. The reason was ascribed to that the amount of Eu was too low, and fell out of range of the detection limit of EDS. In order to study the luminescence property of Eu doped TiO2 nanodot films, PL spectra were measured at room temperature (25 °C). According to data in the literature, excitation light with a wavelength of 300 nm was used in this experiment in order to get stronger luminescence intensity. Fig. 3 showed the PL spectra of undoped and Eu doped TiO2 nanodot films. According to reference, there are 5 emission peaks at 579 nm, 592 nm, 614 nm, 654 nm and 702 nm in PL spectra, which correspond to 5D0 → 7 F0, 5D0 → 7 F1, 5D0 → 7 F2, 5D0 → 7 F3 and 5D0 → 7 F4 transitions of Eu3+ ion respectively [21]. In this work, the peak at 579 nm can hardly be found; only a very small shoulder peak was observed on that with 15% Eu (Eu/Ti molar ratio). As for the peak at 702 nm, it overlapped with the peaks from the substrate. Only three obvious peaks of 592 nm, 616 nm and 656 nm were observed. The slight shift of the latter two could be ascribed to that different excitation wavelength was used. The peak located at 465 nm was ascribed to quartz (silica) substrate [22,23]. As seen from the characteristic peak at 616 nm, the emission intensity increased with the increasing Eu concentration. 15% doping led to almost twice luminescence intensity that that of 5% doping. Obviously, the doping concentration of Eu has a great impact on the luminescence property of Eu-doped TiO2 nanodot films.
Please cite this article as: K. Cheng, et al., Biocompatible Eu-doped TiO2 nanodot film with in situ protein adsorption characterization property, Thin Solid Films (2015), http://dx.doi.org/10.1016/j.tsf.2015.01.003
K. Cheng et al. / Thin Solid Films xxx (2015) xxx–xxx
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Wavelength (nm) Fig. 3. PL intensities of Eu-doped TiO2 nanodot films with different amounts of Eu.
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observed. The OD value on Eu doped TiO2 nanodot film was slightly smaller, but no statistic difference was found. Basically, Eu doped TiO2 nanodot film showed good biocompatibility and no adverse effect on cells was observed. Cells could grow and proliferate well on Eu-doped TiO2 nanodot films. Eu doped TiO2 nanodot film with 10% doping was used to characterize the protein adsorption, since the luminescence intensity is high enough. The samples were soaked in the BSA solution for different times and then the PL intensity was measured. As shown in Fig. 5, the peak intensity at 616 nm decreased obviously with increasing soaking time initially, and then kept almost unchanged with elongated soaking time from 2 h to 16 h. That means after 2 h, BSA adsorption gets saturated. According to our previous work, BSA adsorption increases with increasing soaking time in the initial hours. The results in this work actually means that the luminescence property of Eu could be utilized to evaluate the amount of such adsorption through doping in TiO 2 nanodot films. Owing to the excellent biocompatibility, such Eu-doped TiO2 nanodot film could be widely applied on the surface of many bioimplants and impart them with a feasible and easy way to evaluate the protein adsorption.
3.0 Pure TiO2
Fig. 2. SEM images of Eu-doped TiO2 nanodot films with different Eu(TMHD)3: (a) 5% mol, (b)10% mol, and (c)15% mol. The scale bar in the inset image is 200 nm.
CCK-8 kit was used to detect the biocompatibility of Eu doped TiO 2 nanodot films. A TiO2 nanodot film without Eu doping was used as a reference. As shown in Fig. 4, the OD value, which indicates the number of attached cells, showed almost the same value for the first 6 h for both films. With longer culture time of 24 h, the OD value of Eu-doped TiO 2 nanodot film was a little bit higher than that of the undoped. Such results indicate that the initial cell adhesions of the two films are basically similar. When the culture time increased to about 72 h, fibroblasts proliferated and more cells were
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Time (h) Fig. 4. CCK-8 results of NIH 3T3 cells cultured on nanodot films with and without Eu doping.
Please cite this article as: K. Cheng, et al., Biocompatible Eu-doped TiO2 nanodot film with in situ protein adsorption characterization property, Thin Solid Films (2015), http://dx.doi.org/10.1016/j.tsf.2015.01.003
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K. Cheng et al. / Thin Solid Films xxx (2015) xxx–xxx
PL Intensity (arb. units)
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Wavelength (nm) Fig. 5. PL intensities of Eu-doped TiO2 nanodots with different soaking times in BSA solution.
6. Conclusions Eu doped TiO 2 nanodot films can be easily prepared by sol–gel and phase-separation-induced self-assembly method, and they showed good biocompatibility. The PL intensity of Eu decreased gradually in the first few hours with increasing soaking time in protein solution. Such films on bio-implant could provide an easy in situ way to evaluate surface protein adsorption without detrimental effect on biocompatibility. Acknowledgments This work is financially supported by the National Basic Research Program of China (973 Program, 2012CB933600), the National Natural Science Foundation of China (51372217, 51472216, 51272228, 81171003, 81371120), the Fundamental Research Funds for the Central Universities (2013QNA4010, 2014XZZX005) and the Medical Technology and Education of Zhejiang Province of China (wkj-zj-11). References [1] L. Bacakova, V. Mares, V. Lisa, V. Svorcik, Molecular mechanisms of improved adhesion and growth of an endothelial cell line cultured on polystyrene implanted with fluorine ions, Biomaterials 21 (11) (2000) 1173. [2] A. Kikuchi, H. Taira, T. Tsuruta, M. Hayashi, K. Kataoka, Adsorbed serum protein mediated adhesion and growth behavior of bovine aortic endothelial cells on polyamine graft copolymer surfaces, J. Biomater. Sci. Polym. Ed. 8 (2) (1996) 77.
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Please cite this article as: K. Cheng, et al., Biocompatible Eu-doped TiO2 nanodot film with in situ protein adsorption characterization property, Thin Solid Films (2015), http://dx.doi.org/10.1016/j.tsf.2015.01.003