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Titanium dioxide nanotube films
Materials Science and Engineering C 37 (2014) 374–382
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Titanium dioxide nanotube films Preparation, characterization and electrochemical biosensitivity towards alkaline phosphatase Ioan Roman a,⁎, Roxana Doina Trusca a, Maria-Laura Soare a, Corneliu Fratila b, Elzbieta Krasicka-Cydzik c, Miruna-Silvia Stan d, Anca Dinischiotu d a
S.C. METAV-Research and Development S.R.L., Bucharest, 31C. A. Rosetti, 020011, Romania Research and Development National Institute for Nonferrous and Rare Metals, Pantelimon, 102 Biruintei, 077145, Romania c University of Zielona Gora, Department of Biomedical Engineering Division, 9 Licealna, 65-417, Poland d University of Bucharest, Department of Biochemistry and Molecular Biology, 36-46 Mihail Kogalniceanu, 050107, Romania b
a r t i c l e
i n f o
Article history: Received 27 June 2013 Received in revised form 26 November 2013 Accepted 8 January 2014 Available online 24 January 2014 Keywords: TiO2 nanotube Anodization Osteoblast Alkaline phosphatase Electrochemical impedance spectroscopy
a b s t r a c t Titania nanotubes (TNTs) were prepared by anodization on different substrates (titanium, Ti6Al4V and Ti6Al7Nb alloys) in ethylene glycol and glycerol. The influence of the applied potential and processing time on the nanotube diameter and length is analyzed. The as-formed nanotube layers are amorphous but they become crystalline when subjected to subsequent thermal treatment in air at 550 °C; TNT layers grown on titanium and Ti6Al4V alloy substrates consist of anatase and rutile, while those grown on Ti6Al7Nb alloy consist only of anatase. The nanotube layers grown on Ti6Al7Nb alloy are less homogeneous, with supplementary islands of smaller diameter nanotubes, spread across the surface. Better adhesion and proliferation of osteoblasts was found for the nanotubes grown on all three substrates by comparison to an unprocessed titanium plate. The sensitivity towards bovine alkaline phosphatase was investigated mainly by electrochemical impedance spectroscopy in relation to the crystallinity, the diameter and the nature of the anodization electrolyte of the TNT/Ti samples. The measuring capacity of the annealed nanotubes of 50 nm diameter grown in glycerol was demonstrated and the corresponding calibration curve was built for the concentration range of 0.005–0.1 mg/mL. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Although electrochemical growth of titanium oxide nanotubes (TNTs) on titanium and titanium alloys has a history of 14 years [1], the electrolytes, processing conditions and morphology of these nanostructured films are still an open discussion [2–7]. Also, several functionalization procedures of these films were proposed for various applications [8–12]. In the field of medicine, these functionalized surfaces can be used as biosensors for protein measurement in diagnostics of diseases. Alkaline phosphatase (ALP) is an enzyme present mainly in the liver, osteoblasts and placenta, and its activity and expression are clinical references in the diagnosis of hepatic or bone diseases. The bone ALP represents a biochemical marker of bone formation and general osteoblast activity. This enzyme is also involved in bone mineralization process [13]. The paper defines controlled electrochemical growth conditions in order to obtain predetermined sizes of the nanotubes films, in organic ⁎ Corresponding author. Tel.: +40 214055013; fax: +40 214055012. E-mail address: [email protected] (I. Roman). 0928-4931/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.msec.2014.01.036
electrolytes, both glycerol and ethylene glycol, for which growth equations are proposed, for exposure times of practical interest (short exposure). The ALP adhesion at the oxide layer interface is favoured by the specific nature, diameter and length of TNT [14]. TNT films grown on three of the most common titanium base materials, titanium and TiAl6V4 and Ti6Al7Nb alloys are interdisciplinary and comparatively investigated, as their mechanical strength, biocompatibility, non-toxicity, corrosion resistance and easy processing [15,16] are well known. The purpose of this analysis is to select the best support and preparation conditions in order to maximize the nanotube film biosensitivity towards ALP. Therefore, specific morphology, crystallinity and biocompatibility of these films were pursued. Also, among the selection criteria of TNT films, the degree of electrochemical homogeneity and their wetting ability were also considered. All these features are important in the surface response to the presence of protein markers such as ALP enzyme, for direct electrochemical evaluation. Under polarization, specific oxidation and/or reduction processes can occur at the interface or the electrochemical double layer characteristics may be altered and these changes, related to the marker, can be measured by cyclic voltammetry or by electrochemical impedance spectroscopy.
I. Roman et al. / Materials Science and Engineering C 37 (2014) 374–382 Table 1 Preparation and features of experimental samples. Sample Substrate Conditions 1 2 3 4 5 6
Ti Ti Ti Ti Ti Ti
Ethylene glycola, 40 V, 300 minb Ethylene glycola, 10 V, 120 minb Ethylene glycola, 15 V, 30 minb Ethylene glycola, 15 V, 60 minb Ethylene glycola, 15 V, 120 minb Ethylene glycola, 20 V, 30 minb
7
Ti
Ethylene glycola, 20 V, 60 minb
8
Ti
Ethylene glycola, 20 V, 120 minb
9 10
Ti Ti
Ethylene glycola, 30 V, 30 minb Ethylene glycola, 30 V, 60 minb
11
Ti
Ethylene glycola, 30 V, 120 minb
12 13 14 15 16 17 18
Ti Ti Ti Ti Ti Ti Ti
21
Glycerolc, 2.5 V, 120 minb Glycerolc, 5 V, 120 minb Glycerolc, 10 V, 120 minb Glycerolc, 15 V,120 minb Glycerolc, 20 V,120 minb Glycerolc, 30 V, 120 minb Ethylene glycola, 22 V, 10 min; annealedd Ti6Al4V Ethylene glycola, 22 V, 15 min; annealedd Ti6Al7Nb Ethylene glycola, 15 V, 30 min, annealedd Ti6Al7Nb Ethylene glycola, 15 V, 30 minb
22
Ti
19 20
23 24 25 26 27 28 29 a b c d
Glycerolc, 10 V, 120 min, annealedd Ti6Al4V Glycerolc, 10 V, 120 min, annealedd Ti6Al7Nb Glycerolc, 10 V, 120 min, annealedd Ti Polished titanium plate Ti Ethylene glycola, 10 V, 120 min, annealedd Ti Ethylene glycola, 30 V, 60 min, annealedd Ti Glycerolc, 5 V, 120 min, annealedd Ti Glycerolc, 30 V, 120 min, annealedd
Features Cavities, Ø = 85 nm Ø = 25 nm; l = 1400 nm Ø = 50 nm; l = 850 nm Ø = 50 nm; l = 1100 nm Ø = 50 nm; l = 1990 nm Ø = 55 ± 5 nm; l = 1000 nm Ø = 65 ± 5 nm; l = 1300 nm Ø = 65 ± 5 nm; l = 2200 nm Ø = 90 nm; l = 1600 nm Ø = 95 ± 5 nm; l = 1900 nm Ø = 90 ± 10 nm; l = 3000 nm Ø = 11.5 nm; l = 700 nm Ø = 17.5 nm; l = 1500 nm Ø = 45 nm; l = 425 nm Ø = 65 nm; l = 550 nm Ø = 80 nm; l = 560 nm Ø = 110 nm; l = 650 nm Ø = 50 ± 10 nm, l = 500 ± 50 nm Ø = 50 ± 5 nm, l = 500 ± 50 nm Ø = 52 ± 4 nm, l = 800 ± 100 nm Ø = 47 ± 5 nm, l = 750 ± 50 nm Ø = 50 ± 10 nm; l = 500 ± 25 nm Ø = 50 nm; l = 500 nm Ø = 45 ± 5 nm; l = 475 ± 25 nm – Ø = 20 nm; l = 1400 nm Ø = 100 nm; l = 1900 nm Ø = 20 nm; l = 1500 nm Ø = 100 nm; l = 650 nm
98.45% ethylene glycol, 0.55% NH4F, 1% H2O. As-formed. 90% glycerol, 9.3% H2O, 0.7% NH4F. 550°C, 1 h.
2. Experimental The samples, Table 1, were prepared on 10 × 10 mm titanium plates (Al = 0.30; Cd = 0.003; Cr = 0.010; Cu = 0.020; Fe = 0.040; Mg = 0.05; Mn = 0.005; Mo = 0.005; Ni = 0.009; Pb = 0.40; Sb = 0.020; Si = 0.05; Zn = 0.005), Ti6Al4V alloy plates (N = 0.0051; C = 0.030; Al = 5.53; V = 3.90; Fe = 0.13; Si = 0.05–0.1; Ni = 0.01–0.05; Cr = 0.005–0.01; Co b 0.005; Cu ≅ 0.001; Pb b 0.005), and Ti6Al7Nb alloy plates (C b 0.08; N b 0.05; Fe b 0.25; H b 0.009; O b 0.20; Ta b 0.5; Al 5.5–6.5; Nb 6.5–7.5). Glycerol and ethylene glycol-based electrolytes (90% glycerol, 9.3% H2O, 0.7% NH4F and 98.45% ethylene glycol, 1% H2O, 0.55% NH4F, respectively, Sigma-Aldrich pro analysis reagents) were used, due to their high viscosity which influences the diffusion of ionic species, the kinetics of nanotubes formation and their morphology. Prior to anodization, metallic plates were initially polished employing emery paper in successive grits of 320, 400 and 600 and finally with diamond paste, degreased
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by sonication in acetone followed by rinsing with deionised water, and drying in hot air stream. Anodization was performed in a standard two-electrode bath with circular platinum mesh cathode and MLW DC power source, of 150 V and 10 A. The anodization temperature was fixed to lab temperature, 25 °C. Afterwards, the resulting TiO2 nanotube films were rinsed with distilled water. Some samples were subsequently heat-treated in a VULCAN 3-350 furnace, in air, at 550 °C for 1 h [17,18]. The nanotube films were investigated by scanning electron microscopy (SEM, QUANTA INSPECT F) equipped with energy dispersive Xray spectroscopy analyzer (EDAX), high resolution transmission electron microscopy (HRTEM, TECNAI F30 G2) with a line resolution of 1 Å (selected area electron diffraction (SAED) images were also generated) and X-ray diffraction (XRD, PANalytical X' PERT MPD), in order to define film morphology and crystallinity, as well as the diameter and length of nanotubes. The length was typically measured in a section of the film of nanotubes [19]. The contact angle was measured using a PG-3 goniometer (Klimatest), using double distilled water. Due to the high porosity of the tested samples, the evaluation of the contact angles was carried out in the dynamic sessile drop mode. Biocompatibility tests were carried out using G292 osteoblastic cells (ATCC CRL-1423) cultured in McCoy's 5a medium (Gibco, USA) supplemented with 10% foetal bovine serum (Gibco, USA), 100 U/mL penicillin and 100 μg/mL streptomycin, in a humidified atmosphere (5% CO2) at 37 °C. The culture medium was changed every 2 days until cells reached confluence and then were trypsinized with 0.25% trypsin–0.03% EDTA (Sigma-Aldrich). The cells were seeded onto titanium samples or cultured directly on tissue culture polystyrene (TCPS) in a six-well plate at a density of 2 × 104 cells/well, for 24 h. The samples were sterilized at 180 °C for 30 min prior to biological experiments. In order to detect alkaline phosphatase, the cells were fixed in 4% paraformaldehyde for 20 min at 4 °C and permeabilized with 0.5% Triton X-100 in phosphate buffered saline (PBS, Sigma-Aldrich) for 10 min. Cells were then blocked with 2% BSA in PBS for 30 min. Primary antibody against human alkaline phosphatase (Santa Cruz Biotechnology) was added on cells for 2 h at room temperature. After three washes with PBS, cells were incubated with TRITC-conjugated goat anti-mouse secondary antibody (Santa Cruz Biotechnology) for 30 min. The nuclei were counterstained with 4′,6′-diamidino-2-phenylindole (DAPI) and the cells were observed using an Olympus IX71 fluorescent microscope and fluorescence intensity was quantified using ImageJ 1.43 software. The data were expressed as average ± SD (three independent experiments) and analyzed for statistical significance using Student's test. A value of P b 0.05 was considered significant. Electrochemical measurements were performed using a VoltaLab PGZ 301 Radiometer potentiostat connected to a classical threeelectrode cell. TNT samples were used as working electrodes (electrode surface at 0.2826 cm2), a platinum plate was used as a counter electrode and a saturated calomel electrode (SCE) was used as reference electrode. Time evolution of the open circuit potential (OCP) was monitored for 30 min in PBS and several simulated body fluids (SBF): Carter– Brugirard artificial saliva (prepared according to AFNOR/NF (French Association of Normalization) 591-141), synthetic human plasma and synthetic human blood, both supplied by the “Babes-Bolyai” University of Cluj Napoca [20–22]. Cyclic voltammetry measurements were performed in the range of −500–1500 mV vs SCE, with 25 mV/s, without ohmic drop compensation. Impedance spectra were acquired in the frequency range of 100 kHz to 50 mHz. The applied amplitude of the AC potential was 25 mV. The DC potential was 0 mV vs SCE. The analyses and interpretations were based on the Nyquist curves and on a specific parameter denoted L, which represents half of the rough loop diameter measured on the real part axis of impedance, Fig. 18a. Solutions of bovine intestinal alkaline phosphatase (ALP, SigmaAldrich, pro analysis reagent) were prepared with double distilled water just before use, in the range of 0.005–0.1 mg/mL.
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Fig. 1. Dependence of the TNT (a) length on the anodization time in ethylene glycol and (b) diameter on the applied potential in ethylene glycol and glycerol.
3. Results and discussion
resulted from the oxidation of water present in the electrolyte) triggered by an increased applied potential [24].
3.1. TNT preparation In order to improve the anodization, the influence of the processing time and anodizing voltage in two conventional electrolytes, ethylene glycol [23] and glycerol [5] was investigated for the nanotubes grown on titanium substrate, samples 2–17. The TNT length increases with the processing time and the applied potential, showing an ideal second order polynomial dependence, Fig. 1a. However, a processing time as high as 300 min is critical, resulting in the dissolution of the formed nanotubes, a SEM image of the surface of sample 1 showing an array of hexagonal cavities originating from the initial arrangement in which the TNTs have grown, Fig. 2. The nanotube diameter increases linearly with the anodizing voltage, showing a similar trend in both solvents, Fig. 1b. Length and diameter increase of nanotubes is the result of the competition between the potential-induced oxide formation and fluoride-induced oxide dissolution [19], in the used electrolytes. In the case of sample 1, the dissolution rate of the oxidic structures greatly exceeded their growth rates, a long exposure time leading to their disappearance. The increase of the nanotubes diameter may also be explained by a stronger oxygen release (oxygen bubbles at the anode
Fig. 2. SEM image of sample 1 surface, subjected to extended processing time.
3.2. Surface and phase composition analysis of TNT samples Several series of samples produced both on titanium and on Ti6Al4V and Ti6Al7Nb alloys were investigated. A comparative analysis of the nanotubes produced in similar conditions by anodization and subsequent annealing on the three different substrates was carried out (samples 18–20 in Table 1). In order to observe the effect of annealing, samples 11 and 21, which were not subjected to further thermal treatment, were used for comparison. SEM, HRTEM and XRD analyses of samples 11 and 18–21 were performed, Figs. 3–7. Fig. 3a shows the TNT specific morphology, where a bottom layer (formed by the close ends of the tubes) is followed by a second one, constituted by the nanotube film, as reported in literature [12,25]. The nanotubes grow as hexagonal rings (Figs. 2 and 3b) with a wall thickness of 7 nm, which overlap, as evidenced by SEM (Fig. 3a) and HRTEM (Fig. 4b). The SEM images show that TNTs grown on titanium and Ti6Al4V are homogeneous, Figs. 3a and 4a, unlike those grown on Ti6Al7Nb which present a gofer arrangement upon which islands of nanotubes of smaller diameters (zone 1) are spread over the whole sample surface (zone 2), Figs. 5a and 6a. EDAX analysis of sample 20 revealed that in zone 1 there is a higher niobium content than in zone 2, Table 2, corresponding to the presence of β phase in the substrate [26,27]. HRTEM images of the nanotube wall and SAED images showed that sample 20 (annealed) is crystalline, Fig. 5b and c, while sample 21 is amorphous, Fig. 6b and c. Hence, annealing does not affect the TNT morphology (Figs. 5a and 6a) but it induces crystallinity [12,28], Fig. 7. Both HRTEM and XRD analyses (Fig. 7) showed that anatase is formed by thermal treatment of the TNTs grown on Ti6Al7Nb substrate (Fig. 5c) whereas both anatase and rutile are characteristic for the annealed TNTs grown on titanium and Ti6Al4V, Figs. 3c and 4c. This demonstrates that crystallization process of nanotubes (grown in glycerol or ethylene glycol) is influenced by the specific chemical composition of the base metal, by local diffusion of alloying elements. Niobium increases the conversion temperature of anatase to rutile [29,30], hence rutile is not formed when annealing the TNT grown on Ti6Al7Nb substrate at 550 °C. Fig. 8 shows the comparative profile of two TNT films of roughly 50 nm grown on titanium in ethylene glycol and glycerol, respectively. It can be observed that the morphology is not significantly influenced by the nature of the anodization electrolyte.
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Fig. 3. (a) SEM image of TNTs for sample 11 and (b) HRTEM and (c) SAED images of TNTs for sample 18.
Fig. 4. (a) SEM, top view, outer TNT diameters, (b) HRTEM and (c) SAED images of TNT, sample 19.
Fig. 5. (a) SEM, top view, outer (white) and inner (black) TNT diameters, (b) HRTEM and (c) SAED images of TNT, sample 20.
Fig. 6. (a) SEM, top view, inner TNT diameters, (b) HRTEM and (c) SAED images of TNT, sample 21.
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SEM images; therefore, Ti6Al7Nb might not be an ideal substrate for the targeted biosensor. Generally, the nanotubes grown in glycerol show a more pronounced passivity state, probably due to a better uniformity in terms of nanotube length. 3.4. Contact angle measurement The measurement of the contact angle on samples 18 and 19 revealed that both types of annealed TNT films, obtained on titanium and Ti6Al4V substrates, have a pronounced hydrophilic character. The contact angle reached 31° in both cases, but this occurred 10 times faster in the case of the TNT film on titanium substrate than in the case of the TNT film with Ti6Al4V substrate, Fig. 10. 3.5. Osteoblastic cell culture
Fig. 7. XRD analysis of (a) sample 21; (b) sample 20; (c) sample 18 and (d) sample 19.
Table 2 EDAX analysis of zones 1 and 2 in Fig. 6a, sample 20. EDAX
Wt.%
Element
Zone 1
Zone 2
OK AlK TiK NbK Total
20.53 4.18 63.20 12.09 100.00
21.93 4.23 65.40 8.43 100.00
In vitro biocompatibility was tested for polished titanium sample 25 and for three TNT samples (22, 23 and 24) using G-292 osteosarcoma cells which were found to be a valid experimental model for primary human osteoblasts expressing alkaline phosphatase [31]. Taking into account that surface topography has a major influence on osteoblast attachment and proliferation [32], it seems that only the samples with nanotubes can provide appropriate bioactive surfaces that promote a three dimensional growth of cells and the activation of alkaline phosphatase biosynthesis [14]. Indeed, as shown in Fig. 11, sample 25 did not manage to maintain a proper level of ALP which had decreased by 44% compared to TCPS control cells. A high level of ALP can be explained by the fast growth of osteoblasts, because this protein represents a major product of these cells. On the other hand, the samples with nanotube films induced a good cell proliferation reflected in ALP expression. The levels were above the positive control for the cells cultured on these TNT samples.
3.3. Monitoring of the OCP 3.6. Testing of electrochemical sensitivity towards ALP Time evolution of the OCP of the nanotubes grown on the three types of substrates, both in ethylene glycol (samples 18, 19 and 20 in Table 1) and in glycerol (samples 22, 23 and 24 in Table 1) was monitored in various SBFs. The behaviour of the samples in Carter–Brugirard artificial saliva is given in Fig. 9. The TNT/Ti samples show a passivity behaviour in all media, while the TNT/Ti6Al4V samples show an initial activation of the nanotubes surface, followed by a passivity state after approx. 500 s. This different behaviour indicates that in case of the TNT/Ti6Al4V samples there is a less compact film which allows for the electrolyte to penetrate the surface; therefore, the interface equilibrium is reached in a longer time. For the TNT/Ti6Al7Nb samples, the evolution of the potential presents a characteristic noise in all cases, which is consistent with an inhomogeneous surface, as previously observed in the
Based on the results presented so far, titanium was chosen as optimal substrate for the TNT films. Hence, electrochemical sensitivity towards different concentrations of bovine ALP (0.005, 0.5 and 0.1 mg/mL) was tested on such nanotubes (samples 14, 18, 22, 25–29 in Table 1). The behaviour of sample 18 (annealed nanotubes) was compared to that of samples 25 and 14 (as-formed nanotubes). No quantitative correlations were observed in case of samples 25 and 14 (Figs. 12 and 13); moreover, while the polished titanium (sample 25) only shows a qualitative dependence on the bovine ALP concentrations, sample 14 shows no rational dependence. Conversely, for sample 18, both CV (Fig. 14) and EIS (Fig. 15) show clear signal changes related to the different bovine ALP content. In CV, the anodic current increases with potential and ALP content in the
Fig. 8. SEM images of (a) sample 18 and (b) sample 22.
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Fig. 9. Time evolution of the OCP in Carter–Brugirard artificial saliva of TNT samples grown in (a) ethylene glycol and (b) glycerol.
Fig. 10. Water contact angle evolution on (a) sample 18 for 50 s; (b) sample 19 for 575 s.
solution; an anodic oxidation occurs in the entire potential range, along with the anodic oxidation of water. The influence of the nanotube diameter on the electrochemical sensitivity towards the bovine ALP content is stronger in case of the TNTs grown in glycerol than for those in ethylene glycol, Figs. 16 and 17, respectively. The sensitivity increases with the nanotube diameter, in both cases, probably due to a better dimensional and morphological compatibility of ALP macromolecules with larger diameter nanotubes.
These results support the use of the nanotubes prepared in glycerol and subsequently annealed as an electrochemical sensor for bovine ALP. Calibration tests were performed for the EIS results obtained for bovine ALP in distilled water, by using sample 22 as electrode. Calibration curve of the impedimetric response measured in aqueous solutions of bovine ALP of different concentrations (corresponding to enzyme activities of 1, 2, 3.5, 15, 18.5 and 25 U/L) is presented in Fig. 18. A very good linear correlation was obtained in the range of concentrations of 0.005–
Fig. 11. (a) Immunofluorescence detection of ALP in human osteoblasts (G292 cells) cultured on samples 22–25. White scale bar: 50 μm. (b) Fluorescence intensity quantification of ALP expression in cells grown on TCPS and samples 22–25 *** indicates a statistically significance compared to TCPS (P b 0.001).
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Fig. 12. Nyquist curves for aqueous solutions of different concentrations of bovine ALP, at 28 °C, recorded on sample 25.
Fig. 14. CV curves, with 25 mV/s, for aqueous solutions of different concentrations of bovine ALP, at 28 °C, recorded on sample 18.
TNT with controllable dimensional features can be prepared by anodic exposure in organic electrolytes at lab temperature, under a DC voltage driven on a specific route. Structural and morphological analyses revealed that nanotube growth occurs by the overlap of hexagonal rings, with a cross section of about 7 nm diameter. This diameter also represents the nanotube wall thickness. XRD analysis showed that the annealed nanotubes grown on titanium and TiAl6V4 alloy substrates annealed at 550 °C consist of a mixture of rutile and anatase, while TNT crystallinity on TiAl6Nb7 alloy, annealed at the same temperature, is simpler, as only anatase is present. These samples also show islands of nanotubes with smaller diameters (on β phase) with higher niobium content.
Fluorescence microscopy studies showed that TNT films support osteoblast attachment and proliferation, being able to induce ALP biosynthesis. On contrary, polished titanium sample did not provide the right surface for cell adhesion that was reflected by the lower ALP content. On annealed TNT/Ti samples, both cyclic voltammetry and electrochemical impedance data show clear quantitative dependence on the bovine ALP concentration. This aspect proves that the TNT interface is able to capture the enzyme, affecting the double layer. Both when prepared in glycerol and ethylene glycol, the TNT films present electrochemical sensitivity to the variation of ALP content in distilled water. Sensitivity increases with increasing nanotubes diameter, in both cases. However, the influence of the diameter on the TNT/ Ti electrochemical sensitivity towards ALP is stronger for the nanotubes grown in glycerol than in ethylene glycol. Therefore, the nanotubes of 50 nm diameter grown in glycerol are a new credible candidate impedimetric sensor for bovine ALP. The results of calibration tests gave a good correlation in the range of 0.005–0.1 mg/mL.
Fig. 13. Nyquist curves for aqueous solutions of different concentrations of bovine ALP, at 28 °C, recorded on sample 14.
Fig. 15. Nyquist curves for aqueous solutions of different concentrations of bovine ALP, at 28 °C, recorded on sample 18.
0.1 mg/mL, with standard errors below 10%, both for the slope and for the intercept. The experimental point corresponding to 0.001 mg/mL bovine ALP content is outside the linear range. 4. Conclusions
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Acknowledgements We would like to express our gratitude to Prof. Radu SilaghiDumitrescu from “Babes-Bolyai” University of Cluj Napoca for supplying the synthetic human plasma and synthetic human blood. The financial support from MNT-ERA.NET Transnational Call 2010 — TNTBIOSENS project is gratefully acknowledged.
References
Fig. 16. Nyquist curves for aqueous solutions of 0.05 mg/mL bovine ALP, at 28 °C, recorded on samples with various nanotube diameters obtained in glycerol.
Fig. 17. Nyquist curves for aqueous solutions of 0.05 mg/mL bovine ALP, at 28 °C, recorded on samples with various nanotube diameters obtained in ethylene glycol.
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Fig. 18. Nyquist curves recorded at 28 °C (a) and the corresponding calibration curve (b) for sample 22 in bovine ALP aqueous solutions.
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[18] A. Tighineanu, T. Ruff, S. Albu, R. Hahn, P. Schmuki, Conductivity of TiO2 nanotubes: influence of annealing time and temperature, Chem. Phys. Lett. 494 (2010) 260–263. [19] A. Ghicov, H. Tsuchiya, J.M. Macak, P. Schmuki, Titanium oxide nanotubes prepared in phosphate electrolytes, Electrochem. Commun. 7 (2005) 505–509. [20] E. Fischer-Fodor, A. Mot, F. Deac, M. Arkosi, R. Silaghi-Dumitrescu, Towards hemerythrin-based blood substitutes: comparative performance to hemoglobin on human leukocytes and umbilical vein endothelial cells, J. Biosci. 36 (2011) 215–221. [21] F. Deac, B. Iacob, E. Fischer-Fodor, G. Damian, R. Silaghi-Dumitrescu, Derivatization of hemoglobin with periodate-generated reticulation agents: evaluation of oxidative reactivity for potential blood substitutes, J. Biochem. 149 (2011) 75–82. [22] B.J. Reeder, M. Grey, R. Silaghi-Dumitrescu, D.A. Svistunenko, L. Bülow, C.E. Cooper, M.T. Wilson, Tyrosine residues as redox cofactors in human hemoglobin: implications for engineering non toxic blood substitutes, J. Biol. Chem. 283 (2008) 30780–30787. [23] C.J. Frandsen, K. Noh, K.S. Brammer, G. Johnston, S. Jin, Hybrid micro/nano-topography of a TiO2 nanotube-coated commercial zirconia femoral knee implant promotes bone cell adhesion in vitro, Mater. Sci. Eng. C 33 (2013) 2752–2756. [24] D. Regonini, A. Satka, A. Jaroenworaluck, D.W.E. Allsopp, C.R. Bowen, R. Stevens, Factors influencing surface morphology of anodized TiO2 nanotubes, Electrochim. Acta 74 (2012) 244–253. [25] D. Gong, C.A. Grimes, O.K. Varghese, W. Hu, R.S. Singh, Z. Chen, E.C. Dickey, Titanium oxide nanotube arrays prepared by anodic oxidation, J. Mater. Res. 16 (2001) 3331–3334. [26] E.-D. Stoica, F. Fedorov, M. Nicolae, M. Uhlemann, A. Gebert, L. Schultz, Ti6Al7Nb surface modification by anodization in electrolytes containing HF, U. P. B. Sci. Bull. Ser. B 74 (2012) 277–288. [27] E. Krasicka-Cydzik, Anodic Layer Formation on Titanium and Its Alloys for Biomedical Applications, Chapter 8 in Titanium Alloys — Towards Achieving Enhanced Properties for Diversified Applications, InTech, Rijeka2012. [28] L. Zhang, Y. Han, Enhanced bioactivity of self-organized ZrO2 nanotube layer by annealing and UV irradiation, Mater. Sci. Eng. C 31 (2011) 1104–1110. [29] J.M. Macak, H. Tsuchiya, A. Ghicov, K. Yasuda, R. Hahn, S. Bauer, P. Schmuki, TiO2 nanotubes: self-organized electrochemical formation, properties and applications, Curr. Opin. Solid State Mater. Sci. 11 (2007) 3–18. [30] J. Arbiol, J. Cerdà, G. Dezanneau, A. Cirera, F. Peiró, A. Cornet, J.R. Morante, Effects of Nb doping on the TiO2 anatase-to-rutile phase transition, J. Appl. Phys. 92 (2002) 853–861. [31] P.G. Bradford, J.M. Maglich, A.S. Ponticelli, K.L. Kirkwood, The effect of bone morphogenetic protein-7 on the expression of type I inositol 1,4,5-trisphosphate receptor in G-292 osteosarcoma cells and primary osteoblast cultures, Arch. Oral Biol. 45 (2000) 159–166. [32] W. Singhatanadgit, Biological responses to new advanced surface modifications of endosseous medical implants, Bone Tissue Regen. Insights 2 (2009) 1–11.
Ioan Roman, BSc in Inorganic Chemistry Engineering at “POLITEHNICA” University Timisoara in 1980 and Doctor in Physical Chemistry at “POLITEHNICA” University Bucharest in 1999, Senior researcher and Project Manager at METAVResearch Development Centre Bucharest, Romania. Interests: Electrochemical techniques applied on electrochemical waste water treatment and materials, nanomaterials and thin layers (titania nanotubes, hydroxyapatite), biomaterials (including titanium, nickel and cobalt-base superalloys), corrosion and sensors for medicine. Publications: over 40 papers (19 in ISI journals), 6 patents and 2 books. Others: Member of the Romanian Academy Corrosion Commission and of the Romanian Biomaterials Society, reviewer for Materials Science and Engineering B.
Roxana Doina Trusca. Education: Bachelor's Degree in Materials Science at POLITEHNICA University Bucharest in 1984. Interests: Structural and microstructural investigation of materials (ceramics, composites, polymers, metallic materials, etc.) by X-ray diffraction analysis, scanning electron microscopy (HRSEM) and X-ray spectrometry (EDAX). Publications: 25 ISI papers (most of them in nanomaterials field) and one book–co-author. Current position: Senior Researcher in METAV R&D Centre, Bucharest. Fellow worker in over 25 research projects and team leader of 4 research projects.
Maria-Laura Soare, PhD in Chemical Engineering (2013), Early Stage Researcher at METAV-Research Development, Bucharest, Romania (since 2007). Interests: electrochemistry of titania nanostructured films and nanotubes and biomaterials (titanium, nickel and cobalt-base superalloys), design of electrochemical sensors, electrocatalysis of CO and CO2 on Cu-based surfaces, electrochemical degradation of water pollutants, drug delivery systems. Author of 12 papers (8 in ISI journals). Fellow worker in 6 national and 2 international research projects (EU FP7 PEOPLE-2007-1-1-ITN Marie Curie project — ELCAT, no. 214936 and MNT-ERA.NET Transnational Call 2010 — TNTBIOSENS). Member of ACS. Responsible in several Technical Committees of the Romanian Standards Association.
Corneliu Fratila, BSc of University POLITEHNICA of Bucharest, Faculty of Materials Science in 1977, Senior Researcher at the National Institute of Nonferrous and Rare Metals. Interests: nonferrous metals metallurgy, electrochemical technology, surface coating of metallic and ceramic supports, bio-materials (including titanium and titanium alloys base), electrochemical surface processing of titanium for nano-bio-sensors, design of composite materials with metallic matrix. Author of 7 ISI papers and 1 patent. Managerial experience in more than 17 research projects.
Elzbieta Krasicka-Cydzik, PhD, DSc, Professor of University of Zielona Gora, Poland. Interests: titanium and its alloys, bioactivity of materials, electrochemistry of surface layer, TiO2 based biosensors, corrosion. Author of 2 monographs, 3 book charters, 75 papers and 4 patents, with 3 invited lectures at international conferences. Life Member of Clare Hall Cambridge University (UK), member of Polish Acad. Sci, Mat. Science, ISE, Polish and European Biomaterials Soc, Polish Material Soc., American Chem Soc., member of the Editorial Board the Open Corr Journal, and the co-founder of biomedical engineering studies at her university in 2007. http://www.ibem.uz. zgora.pl/zib/ekc/index.html.
Miruna-Silvia Stan is a PhD student in the first year at the Faculty of Biology, University of Bucharest. She began her research activity in 2009 in the Department of Biochemistry and Molecular Biology. Her PhD thesis involves the study of cytotoxicity of various synthesized nanomaterials (TiO2 nanotubes and silicon quantum dots) in order to provide new biochemical and molecular mechanisms involved in regulating redox homeostasis and cell proliferation. In addition, her interest in the field of nanotube biocompatibility was well appreciated at International Congresses.
Anca Dinischiotu is a full professor at the Faculty of Biology from the University of Bucharest, a Senior Researcher in the Department of Biochemistry and Molecular Biology and director of numerous national and international grants. She attended post-doc fellowships at Catholic University in Leuven, Belgium, and at Paris XI University in France. She led numerous research projects in molecular toxicology and tissue biocompatibility, and her vast experience in these fields comprises over 50 papers in ISI rated journals, books and patents.
Contents lists available at ScienceDirect
Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Titanium dioxide nanotube films Preparation, characterization and electrochemical biosensitivity towards alkaline phosphatase Ioan Roman a,⁎, Roxana Doina Trusca a, Maria-Laura Soare a, Corneliu Fratila b, Elzbieta Krasicka-Cydzik c, Miruna-Silvia Stan d, Anca Dinischiotu d a
S.C. METAV-Research and Development S.R.L., Bucharest, 31C. A. Rosetti, 020011, Romania Research and Development National Institute for Nonferrous and Rare Metals, Pantelimon, 102 Biruintei, 077145, Romania c University of Zielona Gora, Department of Biomedical Engineering Division, 9 Licealna, 65-417, Poland d University of Bucharest, Department of Biochemistry and Molecular Biology, 36-46 Mihail Kogalniceanu, 050107, Romania b
a r t i c l e
i n f o
Article history: Received 27 June 2013 Received in revised form 26 November 2013 Accepted 8 January 2014 Available online 24 January 2014 Keywords: TiO2 nanotube Anodization Osteoblast Alkaline phosphatase Electrochemical impedance spectroscopy
a b s t r a c t Titania nanotubes (TNTs) were prepared by anodization on different substrates (titanium, Ti6Al4V and Ti6Al7Nb alloys) in ethylene glycol and glycerol. The influence of the applied potential and processing time on the nanotube diameter and length is analyzed. The as-formed nanotube layers are amorphous but they become crystalline when subjected to subsequent thermal treatment in air at 550 °C; TNT layers grown on titanium and Ti6Al4V alloy substrates consist of anatase and rutile, while those grown on Ti6Al7Nb alloy consist only of anatase. The nanotube layers grown on Ti6Al7Nb alloy are less homogeneous, with supplementary islands of smaller diameter nanotubes, spread across the surface. Better adhesion and proliferation of osteoblasts was found for the nanotubes grown on all three substrates by comparison to an unprocessed titanium plate. The sensitivity towards bovine alkaline phosphatase was investigated mainly by electrochemical impedance spectroscopy in relation to the crystallinity, the diameter and the nature of the anodization electrolyte of the TNT/Ti samples. The measuring capacity of the annealed nanotubes of 50 nm diameter grown in glycerol was demonstrated and the corresponding calibration curve was built for the concentration range of 0.005–0.1 mg/mL. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Although electrochemical growth of titanium oxide nanotubes (TNTs) on titanium and titanium alloys has a history of 14 years [1], the electrolytes, processing conditions and morphology of these nanostructured films are still an open discussion [2–7]. Also, several functionalization procedures of these films were proposed for various applications [8–12]. In the field of medicine, these functionalized surfaces can be used as biosensors for protein measurement in diagnostics of diseases. Alkaline phosphatase (ALP) is an enzyme present mainly in the liver, osteoblasts and placenta, and its activity and expression are clinical references in the diagnosis of hepatic or bone diseases. The bone ALP represents a biochemical marker of bone formation and general osteoblast activity. This enzyme is also involved in bone mineralization process [13]. The paper defines controlled electrochemical growth conditions in order to obtain predetermined sizes of the nanotubes films, in organic ⁎ Corresponding author. Tel.: +40 214055013; fax: +40 214055012. E-mail address: [email protected] (I. Roman). 0928-4931/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.msec.2014.01.036
electrolytes, both glycerol and ethylene glycol, for which growth equations are proposed, for exposure times of practical interest (short exposure). The ALP adhesion at the oxide layer interface is favoured by the specific nature, diameter and length of TNT [14]. TNT films grown on three of the most common titanium base materials, titanium and TiAl6V4 and Ti6Al7Nb alloys are interdisciplinary and comparatively investigated, as their mechanical strength, biocompatibility, non-toxicity, corrosion resistance and easy processing [15,16] are well known. The purpose of this analysis is to select the best support and preparation conditions in order to maximize the nanotube film biosensitivity towards ALP. Therefore, specific morphology, crystallinity and biocompatibility of these films were pursued. Also, among the selection criteria of TNT films, the degree of electrochemical homogeneity and their wetting ability were also considered. All these features are important in the surface response to the presence of protein markers such as ALP enzyme, for direct electrochemical evaluation. Under polarization, specific oxidation and/or reduction processes can occur at the interface or the electrochemical double layer characteristics may be altered and these changes, related to the marker, can be measured by cyclic voltammetry or by electrochemical impedance spectroscopy.
I. Roman et al. / Materials Science and Engineering C 37 (2014) 374–382 Table 1 Preparation and features of experimental samples. Sample Substrate Conditions 1 2 3 4 5 6
Ti Ti Ti Ti Ti Ti
Ethylene glycola, 40 V, 300 minb Ethylene glycola, 10 V, 120 minb Ethylene glycola, 15 V, 30 minb Ethylene glycola, 15 V, 60 minb Ethylene glycola, 15 V, 120 minb Ethylene glycola, 20 V, 30 minb
7
Ti
Ethylene glycola, 20 V, 60 minb
8
Ti
Ethylene glycola, 20 V, 120 minb
9 10
Ti Ti
Ethylene glycola, 30 V, 30 minb Ethylene glycola, 30 V, 60 minb
11
Ti
Ethylene glycola, 30 V, 120 minb
12 13 14 15 16 17 18
Ti Ti Ti Ti Ti Ti Ti
21
Glycerolc, 2.5 V, 120 minb Glycerolc, 5 V, 120 minb Glycerolc, 10 V, 120 minb Glycerolc, 15 V,120 minb Glycerolc, 20 V,120 minb Glycerolc, 30 V, 120 minb Ethylene glycola, 22 V, 10 min; annealedd Ti6Al4V Ethylene glycola, 22 V, 15 min; annealedd Ti6Al7Nb Ethylene glycola, 15 V, 30 min, annealedd Ti6Al7Nb Ethylene glycola, 15 V, 30 minb
22
Ti
19 20
23 24 25 26 27 28 29 a b c d
Glycerolc, 10 V, 120 min, annealedd Ti6Al4V Glycerolc, 10 V, 120 min, annealedd Ti6Al7Nb Glycerolc, 10 V, 120 min, annealedd Ti Polished titanium plate Ti Ethylene glycola, 10 V, 120 min, annealedd Ti Ethylene glycola, 30 V, 60 min, annealedd Ti Glycerolc, 5 V, 120 min, annealedd Ti Glycerolc, 30 V, 120 min, annealedd
Features Cavities, Ø = 85 nm Ø = 25 nm; l = 1400 nm Ø = 50 nm; l = 850 nm Ø = 50 nm; l = 1100 nm Ø = 50 nm; l = 1990 nm Ø = 55 ± 5 nm; l = 1000 nm Ø = 65 ± 5 nm; l = 1300 nm Ø = 65 ± 5 nm; l = 2200 nm Ø = 90 nm; l = 1600 nm Ø = 95 ± 5 nm; l = 1900 nm Ø = 90 ± 10 nm; l = 3000 nm Ø = 11.5 nm; l = 700 nm Ø = 17.5 nm; l = 1500 nm Ø = 45 nm; l = 425 nm Ø = 65 nm; l = 550 nm Ø = 80 nm; l = 560 nm Ø = 110 nm; l = 650 nm Ø = 50 ± 10 nm, l = 500 ± 50 nm Ø = 50 ± 5 nm, l = 500 ± 50 nm Ø = 52 ± 4 nm, l = 800 ± 100 nm Ø = 47 ± 5 nm, l = 750 ± 50 nm Ø = 50 ± 10 nm; l = 500 ± 25 nm Ø = 50 nm; l = 500 nm Ø = 45 ± 5 nm; l = 475 ± 25 nm – Ø = 20 nm; l = 1400 nm Ø = 100 nm; l = 1900 nm Ø = 20 nm; l = 1500 nm Ø = 100 nm; l = 650 nm
98.45% ethylene glycol, 0.55% NH4F, 1% H2O. As-formed. 90% glycerol, 9.3% H2O, 0.7% NH4F. 550°C, 1 h.
2. Experimental The samples, Table 1, were prepared on 10 × 10 mm titanium plates (Al = 0.30; Cd = 0.003; Cr = 0.010; Cu = 0.020; Fe = 0.040; Mg = 0.05; Mn = 0.005; Mo = 0.005; Ni = 0.009; Pb = 0.40; Sb = 0.020; Si = 0.05; Zn = 0.005), Ti6Al4V alloy plates (N = 0.0051; C = 0.030; Al = 5.53; V = 3.90; Fe = 0.13; Si = 0.05–0.1; Ni = 0.01–0.05; Cr = 0.005–0.01; Co b 0.005; Cu ≅ 0.001; Pb b 0.005), and Ti6Al7Nb alloy plates (C b 0.08; N b 0.05; Fe b 0.25; H b 0.009; O b 0.20; Ta b 0.5; Al 5.5–6.5; Nb 6.5–7.5). Glycerol and ethylene glycol-based electrolytes (90% glycerol, 9.3% H2O, 0.7% NH4F and 98.45% ethylene glycol, 1% H2O, 0.55% NH4F, respectively, Sigma-Aldrich pro analysis reagents) were used, due to their high viscosity which influences the diffusion of ionic species, the kinetics of nanotubes formation and their morphology. Prior to anodization, metallic plates were initially polished employing emery paper in successive grits of 320, 400 and 600 and finally with diamond paste, degreased
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by sonication in acetone followed by rinsing with deionised water, and drying in hot air stream. Anodization was performed in a standard two-electrode bath with circular platinum mesh cathode and MLW DC power source, of 150 V and 10 A. The anodization temperature was fixed to lab temperature, 25 °C. Afterwards, the resulting TiO2 nanotube films were rinsed with distilled water. Some samples were subsequently heat-treated in a VULCAN 3-350 furnace, in air, at 550 °C for 1 h [17,18]. The nanotube films were investigated by scanning electron microscopy (SEM, QUANTA INSPECT F) equipped with energy dispersive Xray spectroscopy analyzer (EDAX), high resolution transmission electron microscopy (HRTEM, TECNAI F30 G2) with a line resolution of 1 Å (selected area electron diffraction (SAED) images were also generated) and X-ray diffraction (XRD, PANalytical X' PERT MPD), in order to define film morphology and crystallinity, as well as the diameter and length of nanotubes. The length was typically measured in a section of the film of nanotubes [19]. The contact angle was measured using a PG-3 goniometer (Klimatest), using double distilled water. Due to the high porosity of the tested samples, the evaluation of the contact angles was carried out in the dynamic sessile drop mode. Biocompatibility tests were carried out using G292 osteoblastic cells (ATCC CRL-1423) cultured in McCoy's 5a medium (Gibco, USA) supplemented with 10% foetal bovine serum (Gibco, USA), 100 U/mL penicillin and 100 μg/mL streptomycin, in a humidified atmosphere (5% CO2) at 37 °C. The culture medium was changed every 2 days until cells reached confluence and then were trypsinized with 0.25% trypsin–0.03% EDTA (Sigma-Aldrich). The cells were seeded onto titanium samples or cultured directly on tissue culture polystyrene (TCPS) in a six-well plate at a density of 2 × 104 cells/well, for 24 h. The samples were sterilized at 180 °C for 30 min prior to biological experiments. In order to detect alkaline phosphatase, the cells were fixed in 4% paraformaldehyde for 20 min at 4 °C and permeabilized with 0.5% Triton X-100 in phosphate buffered saline (PBS, Sigma-Aldrich) for 10 min. Cells were then blocked with 2% BSA in PBS for 30 min. Primary antibody against human alkaline phosphatase (Santa Cruz Biotechnology) was added on cells for 2 h at room temperature. After three washes with PBS, cells were incubated with TRITC-conjugated goat anti-mouse secondary antibody (Santa Cruz Biotechnology) for 30 min. The nuclei were counterstained with 4′,6′-diamidino-2-phenylindole (DAPI) and the cells were observed using an Olympus IX71 fluorescent microscope and fluorescence intensity was quantified using ImageJ 1.43 software. The data were expressed as average ± SD (three independent experiments) and analyzed for statistical significance using Student's test. A value of P b 0.05 was considered significant. Electrochemical measurements were performed using a VoltaLab PGZ 301 Radiometer potentiostat connected to a classical threeelectrode cell. TNT samples were used as working electrodes (electrode surface at 0.2826 cm2), a platinum plate was used as a counter electrode and a saturated calomel electrode (SCE) was used as reference electrode. Time evolution of the open circuit potential (OCP) was monitored for 30 min in PBS and several simulated body fluids (SBF): Carter– Brugirard artificial saliva (prepared according to AFNOR/NF (French Association of Normalization) 591-141), synthetic human plasma and synthetic human blood, both supplied by the “Babes-Bolyai” University of Cluj Napoca [20–22]. Cyclic voltammetry measurements were performed in the range of −500–1500 mV vs SCE, with 25 mV/s, without ohmic drop compensation. Impedance spectra were acquired in the frequency range of 100 kHz to 50 mHz. The applied amplitude of the AC potential was 25 mV. The DC potential was 0 mV vs SCE. The analyses and interpretations were based on the Nyquist curves and on a specific parameter denoted L, which represents half of the rough loop diameter measured on the real part axis of impedance, Fig. 18a. Solutions of bovine intestinal alkaline phosphatase (ALP, SigmaAldrich, pro analysis reagent) were prepared with double distilled water just before use, in the range of 0.005–0.1 mg/mL.
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Fig. 1. Dependence of the TNT (a) length on the anodization time in ethylene glycol and (b) diameter on the applied potential in ethylene glycol and glycerol.
3. Results and discussion
resulted from the oxidation of water present in the electrolyte) triggered by an increased applied potential [24].
3.1. TNT preparation In order to improve the anodization, the influence of the processing time and anodizing voltage in two conventional electrolytes, ethylene glycol [23] and glycerol [5] was investigated for the nanotubes grown on titanium substrate, samples 2–17. The TNT length increases with the processing time and the applied potential, showing an ideal second order polynomial dependence, Fig. 1a. However, a processing time as high as 300 min is critical, resulting in the dissolution of the formed nanotubes, a SEM image of the surface of sample 1 showing an array of hexagonal cavities originating from the initial arrangement in which the TNTs have grown, Fig. 2. The nanotube diameter increases linearly with the anodizing voltage, showing a similar trend in both solvents, Fig. 1b. Length and diameter increase of nanotubes is the result of the competition between the potential-induced oxide formation and fluoride-induced oxide dissolution [19], in the used electrolytes. In the case of sample 1, the dissolution rate of the oxidic structures greatly exceeded their growth rates, a long exposure time leading to their disappearance. The increase of the nanotubes diameter may also be explained by a stronger oxygen release (oxygen bubbles at the anode
Fig. 2. SEM image of sample 1 surface, subjected to extended processing time.
3.2. Surface and phase composition analysis of TNT samples Several series of samples produced both on titanium and on Ti6Al4V and Ti6Al7Nb alloys were investigated. A comparative analysis of the nanotubes produced in similar conditions by anodization and subsequent annealing on the three different substrates was carried out (samples 18–20 in Table 1). In order to observe the effect of annealing, samples 11 and 21, which were not subjected to further thermal treatment, were used for comparison. SEM, HRTEM and XRD analyses of samples 11 and 18–21 were performed, Figs. 3–7. Fig. 3a shows the TNT specific morphology, where a bottom layer (formed by the close ends of the tubes) is followed by a second one, constituted by the nanotube film, as reported in literature [12,25]. The nanotubes grow as hexagonal rings (Figs. 2 and 3b) with a wall thickness of 7 nm, which overlap, as evidenced by SEM (Fig. 3a) and HRTEM (Fig. 4b). The SEM images show that TNTs grown on titanium and Ti6Al4V are homogeneous, Figs. 3a and 4a, unlike those grown on Ti6Al7Nb which present a gofer arrangement upon which islands of nanotubes of smaller diameters (zone 1) are spread over the whole sample surface (zone 2), Figs. 5a and 6a. EDAX analysis of sample 20 revealed that in zone 1 there is a higher niobium content than in zone 2, Table 2, corresponding to the presence of β phase in the substrate [26,27]. HRTEM images of the nanotube wall and SAED images showed that sample 20 (annealed) is crystalline, Fig. 5b and c, while sample 21 is amorphous, Fig. 6b and c. Hence, annealing does not affect the TNT morphology (Figs. 5a and 6a) but it induces crystallinity [12,28], Fig. 7. Both HRTEM and XRD analyses (Fig. 7) showed that anatase is formed by thermal treatment of the TNTs grown on Ti6Al7Nb substrate (Fig. 5c) whereas both anatase and rutile are characteristic for the annealed TNTs grown on titanium and Ti6Al4V, Figs. 3c and 4c. This demonstrates that crystallization process of nanotubes (grown in glycerol or ethylene glycol) is influenced by the specific chemical composition of the base metal, by local diffusion of alloying elements. Niobium increases the conversion temperature of anatase to rutile [29,30], hence rutile is not formed when annealing the TNT grown on Ti6Al7Nb substrate at 550 °C. Fig. 8 shows the comparative profile of two TNT films of roughly 50 nm grown on titanium in ethylene glycol and glycerol, respectively. It can be observed that the morphology is not significantly influenced by the nature of the anodization electrolyte.
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Fig. 3. (a) SEM image of TNTs for sample 11 and (b) HRTEM and (c) SAED images of TNTs for sample 18.
Fig. 4. (a) SEM, top view, outer TNT diameters, (b) HRTEM and (c) SAED images of TNT, sample 19.
Fig. 5. (a) SEM, top view, outer (white) and inner (black) TNT diameters, (b) HRTEM and (c) SAED images of TNT, sample 20.
Fig. 6. (a) SEM, top view, inner TNT diameters, (b) HRTEM and (c) SAED images of TNT, sample 21.
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SEM images; therefore, Ti6Al7Nb might not be an ideal substrate for the targeted biosensor. Generally, the nanotubes grown in glycerol show a more pronounced passivity state, probably due to a better uniformity in terms of nanotube length. 3.4. Contact angle measurement The measurement of the contact angle on samples 18 and 19 revealed that both types of annealed TNT films, obtained on titanium and Ti6Al4V substrates, have a pronounced hydrophilic character. The contact angle reached 31° in both cases, but this occurred 10 times faster in the case of the TNT film on titanium substrate than in the case of the TNT film with Ti6Al4V substrate, Fig. 10. 3.5. Osteoblastic cell culture
Fig. 7. XRD analysis of (a) sample 21; (b) sample 20; (c) sample 18 and (d) sample 19.
Table 2 EDAX analysis of zones 1 and 2 in Fig. 6a, sample 20. EDAX
Wt.%
Element
Zone 1
Zone 2
OK AlK TiK NbK Total
20.53 4.18 63.20 12.09 100.00
21.93 4.23 65.40 8.43 100.00
In vitro biocompatibility was tested for polished titanium sample 25 and for three TNT samples (22, 23 and 24) using G-292 osteosarcoma cells which were found to be a valid experimental model for primary human osteoblasts expressing alkaline phosphatase [31]. Taking into account that surface topography has a major influence on osteoblast attachment and proliferation [32], it seems that only the samples with nanotubes can provide appropriate bioactive surfaces that promote a three dimensional growth of cells and the activation of alkaline phosphatase biosynthesis [14]. Indeed, as shown in Fig. 11, sample 25 did not manage to maintain a proper level of ALP which had decreased by 44% compared to TCPS control cells. A high level of ALP can be explained by the fast growth of osteoblasts, because this protein represents a major product of these cells. On the other hand, the samples with nanotube films induced a good cell proliferation reflected in ALP expression. The levels were above the positive control for the cells cultured on these TNT samples.
3.3. Monitoring of the OCP 3.6. Testing of electrochemical sensitivity towards ALP Time evolution of the OCP of the nanotubes grown on the three types of substrates, both in ethylene glycol (samples 18, 19 and 20 in Table 1) and in glycerol (samples 22, 23 and 24 in Table 1) was monitored in various SBFs. The behaviour of the samples in Carter–Brugirard artificial saliva is given in Fig. 9. The TNT/Ti samples show a passivity behaviour in all media, while the TNT/Ti6Al4V samples show an initial activation of the nanotubes surface, followed by a passivity state after approx. 500 s. This different behaviour indicates that in case of the TNT/Ti6Al4V samples there is a less compact film which allows for the electrolyte to penetrate the surface; therefore, the interface equilibrium is reached in a longer time. For the TNT/Ti6Al7Nb samples, the evolution of the potential presents a characteristic noise in all cases, which is consistent with an inhomogeneous surface, as previously observed in the
Based on the results presented so far, titanium was chosen as optimal substrate for the TNT films. Hence, electrochemical sensitivity towards different concentrations of bovine ALP (0.005, 0.5 and 0.1 mg/mL) was tested on such nanotubes (samples 14, 18, 22, 25–29 in Table 1). The behaviour of sample 18 (annealed nanotubes) was compared to that of samples 25 and 14 (as-formed nanotubes). No quantitative correlations were observed in case of samples 25 and 14 (Figs. 12 and 13); moreover, while the polished titanium (sample 25) only shows a qualitative dependence on the bovine ALP concentrations, sample 14 shows no rational dependence. Conversely, for sample 18, both CV (Fig. 14) and EIS (Fig. 15) show clear signal changes related to the different bovine ALP content. In CV, the anodic current increases with potential and ALP content in the
Fig. 8. SEM images of (a) sample 18 and (b) sample 22.
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Fig. 9. Time evolution of the OCP in Carter–Brugirard artificial saliva of TNT samples grown in (a) ethylene glycol and (b) glycerol.
Fig. 10. Water contact angle evolution on (a) sample 18 for 50 s; (b) sample 19 for 575 s.
solution; an anodic oxidation occurs in the entire potential range, along with the anodic oxidation of water. The influence of the nanotube diameter on the electrochemical sensitivity towards the bovine ALP content is stronger in case of the TNTs grown in glycerol than for those in ethylene glycol, Figs. 16 and 17, respectively. The sensitivity increases with the nanotube diameter, in both cases, probably due to a better dimensional and morphological compatibility of ALP macromolecules with larger diameter nanotubes.
These results support the use of the nanotubes prepared in glycerol and subsequently annealed as an electrochemical sensor for bovine ALP. Calibration tests were performed for the EIS results obtained for bovine ALP in distilled water, by using sample 22 as electrode. Calibration curve of the impedimetric response measured in aqueous solutions of bovine ALP of different concentrations (corresponding to enzyme activities of 1, 2, 3.5, 15, 18.5 and 25 U/L) is presented in Fig. 18. A very good linear correlation was obtained in the range of concentrations of 0.005–
Fig. 11. (a) Immunofluorescence detection of ALP in human osteoblasts (G292 cells) cultured on samples 22–25. White scale bar: 50 μm. (b) Fluorescence intensity quantification of ALP expression in cells grown on TCPS and samples 22–25 *** indicates a statistically significance compared to TCPS (P b 0.001).
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Fig. 12. Nyquist curves for aqueous solutions of different concentrations of bovine ALP, at 28 °C, recorded on sample 25.
Fig. 14. CV curves, with 25 mV/s, for aqueous solutions of different concentrations of bovine ALP, at 28 °C, recorded on sample 18.
TNT with controllable dimensional features can be prepared by anodic exposure in organic electrolytes at lab temperature, under a DC voltage driven on a specific route. Structural and morphological analyses revealed that nanotube growth occurs by the overlap of hexagonal rings, with a cross section of about 7 nm diameter. This diameter also represents the nanotube wall thickness. XRD analysis showed that the annealed nanotubes grown on titanium and TiAl6V4 alloy substrates annealed at 550 °C consist of a mixture of rutile and anatase, while TNT crystallinity on TiAl6Nb7 alloy, annealed at the same temperature, is simpler, as only anatase is present. These samples also show islands of nanotubes with smaller diameters (on β phase) with higher niobium content.
Fluorescence microscopy studies showed that TNT films support osteoblast attachment and proliferation, being able to induce ALP biosynthesis. On contrary, polished titanium sample did not provide the right surface for cell adhesion that was reflected by the lower ALP content. On annealed TNT/Ti samples, both cyclic voltammetry and electrochemical impedance data show clear quantitative dependence on the bovine ALP concentration. This aspect proves that the TNT interface is able to capture the enzyme, affecting the double layer. Both when prepared in glycerol and ethylene glycol, the TNT films present electrochemical sensitivity to the variation of ALP content in distilled water. Sensitivity increases with increasing nanotubes diameter, in both cases. However, the influence of the diameter on the TNT/ Ti electrochemical sensitivity towards ALP is stronger for the nanotubes grown in glycerol than in ethylene glycol. Therefore, the nanotubes of 50 nm diameter grown in glycerol are a new credible candidate impedimetric sensor for bovine ALP. The results of calibration tests gave a good correlation in the range of 0.005–0.1 mg/mL.
Fig. 13. Nyquist curves for aqueous solutions of different concentrations of bovine ALP, at 28 °C, recorded on sample 14.
Fig. 15. Nyquist curves for aqueous solutions of different concentrations of bovine ALP, at 28 °C, recorded on sample 18.
0.1 mg/mL, with standard errors below 10%, both for the slope and for the intercept. The experimental point corresponding to 0.001 mg/mL bovine ALP content is outside the linear range. 4. Conclusions
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Acknowledgements We would like to express our gratitude to Prof. Radu SilaghiDumitrescu from “Babes-Bolyai” University of Cluj Napoca for supplying the synthetic human plasma and synthetic human blood. The financial support from MNT-ERA.NET Transnational Call 2010 — TNTBIOSENS project is gratefully acknowledged.
References
Fig. 16. Nyquist curves for aqueous solutions of 0.05 mg/mL bovine ALP, at 28 °C, recorded on samples with various nanotube diameters obtained in glycerol.
Fig. 17. Nyquist curves for aqueous solutions of 0.05 mg/mL bovine ALP, at 28 °C, recorded on samples with various nanotube diameters obtained in ethylene glycol.
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Ioan Roman, BSc in Inorganic Chemistry Engineering at “POLITEHNICA” University Timisoara in 1980 and Doctor in Physical Chemistry at “POLITEHNICA” University Bucharest in 1999, Senior researcher and Project Manager at METAVResearch Development Centre Bucharest, Romania. Interests: Electrochemical techniques applied on electrochemical waste water treatment and materials, nanomaterials and thin layers (titania nanotubes, hydroxyapatite), biomaterials (including titanium, nickel and cobalt-base superalloys), corrosion and sensors for medicine. Publications: over 40 papers (19 in ISI journals), 6 patents and 2 books. Others: Member of the Romanian Academy Corrosion Commission and of the Romanian Biomaterials Society, reviewer for Materials Science and Engineering B.
Roxana Doina Trusca. Education: Bachelor's Degree in Materials Science at POLITEHNICA University Bucharest in 1984. Interests: Structural and microstructural investigation of materials (ceramics, composites, polymers, metallic materials, etc.) by X-ray diffraction analysis, scanning electron microscopy (HRSEM) and X-ray spectrometry (EDAX). Publications: 25 ISI papers (most of them in nanomaterials field) and one book–co-author. Current position: Senior Researcher in METAV R&D Centre, Bucharest. Fellow worker in over 25 research projects and team leader of 4 research projects.
Maria-Laura Soare, PhD in Chemical Engineering (2013), Early Stage Researcher at METAV-Research Development, Bucharest, Romania (since 2007). Interests: electrochemistry of titania nanostructured films and nanotubes and biomaterials (titanium, nickel and cobalt-base superalloys), design of electrochemical sensors, electrocatalysis of CO and CO2 on Cu-based surfaces, electrochemical degradation of water pollutants, drug delivery systems. Author of 12 papers (8 in ISI journals). Fellow worker in 6 national and 2 international research projects (EU FP7 PEOPLE-2007-1-1-ITN Marie Curie project — ELCAT, no. 214936 and MNT-ERA.NET Transnational Call 2010 — TNTBIOSENS). Member of ACS. Responsible in several Technical Committees of the Romanian Standards Association.
Corneliu Fratila, BSc of University POLITEHNICA of Bucharest, Faculty of Materials Science in 1977, Senior Researcher at the National Institute of Nonferrous and Rare Metals. Interests: nonferrous metals metallurgy, electrochemical technology, surface coating of metallic and ceramic supports, bio-materials (including titanium and titanium alloys base), electrochemical surface processing of titanium for nano-bio-sensors, design of composite materials with metallic matrix. Author of 7 ISI papers and 1 patent. Managerial experience in more than 17 research projects.
Elzbieta Krasicka-Cydzik, PhD, DSc, Professor of University of Zielona Gora, Poland. Interests: titanium and its alloys, bioactivity of materials, electrochemistry of surface layer, TiO2 based biosensors, corrosion. Author of 2 monographs, 3 book charters, 75 papers and 4 patents, with 3 invited lectures at international conferences. Life Member of Clare Hall Cambridge University (UK), member of Polish Acad. Sci, Mat. Science, ISE, Polish and European Biomaterials Soc, Polish Material Soc., American Chem Soc., member of the Editorial Board the Open Corr Journal, and the co-founder of biomedical engineering studies at her university in 2007. http://www.ibem.uz. zgora.pl/zib/ekc/index.html.
Miruna-Silvia Stan is a PhD student in the first year at the Faculty of Biology, University of Bucharest. She began her research activity in 2009 in the Department of Biochemistry and Molecular Biology. Her PhD thesis involves the study of cytotoxicity of various synthesized nanomaterials (TiO2 nanotubes and silicon quantum dots) in order to provide new biochemical and molecular mechanisms involved in regulating redox homeostasis and cell proliferation. In addition, her interest in the field of nanotube biocompatibility was well appreciated at International Congresses.
Anca Dinischiotu is a full professor at the Faculty of Biology from the University of Bucharest, a Senior Researcher in the Department of Biochemistry and Molecular Biology and director of numerous national and international grants. She attended post-doc fellowships at Catholic University in Leuven, Belgium, and at Paris XI University in France. She led numerous research projects in molecular toxicology and tissue biocompatibility, and her vast experience in these fields comprises over 50 papers in ISI rated journals, books and patents.