A General Method for Fabrication of Biocompatible Surfaces by

Surface analysis and biocorrosion properties of nanostructured surface sol–gel coatings on Ti6Al4V titanium alloy implants. Maria C. Advincula , Don...
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A General Method for Fabrication of Biocompatible Surfaces by Modification with Titania Layer Ghanashyam Acharya and Toyoki Kunitake* Frontier Research System, The Institute of Physical and Chemical Research (RIKEN), 2-1 Hirosawa, Wako, Saitama 351-0198, Japan Received October 21, 2002 A new methodology for conversion of inert surfaces to biocompatible surfaces was proposed. A gold plate, a quartz plate, a silicon wafer, and a Teflon sheet were selected as substrates. The surfaces of the first three substrates were hydroxylated by chemical methods, and the Teflon surface was activated by oxygen plasma treatment. A titania layer was adsorbed on these substrates to provide active surfaces for adsorption of biomolecules. The adsorption process of four representative biomolecules (collagen, fibrinogen, insulin, and heparin) was monitored by quartz crystal microbalance and IR and X-ray photoelectron spectroscopies. The adsorbed proteins were stably bound up to 60 °C and at a wide pH range. The biological activity of adsorbed fibrinogen was confirmed by treatment with thrombin to produce a fibrin network on the surface, as evidenced by scanning electron microscopy.

1. Introduction Proteins have an inherent tendency to deposit on surfaces of artificial materials as tightly bound adsorbates, thus exerting influence on subsequent cellular interactions with the surfaces.1-4 When an external surface comes into contact with biological fluids, the protein adsorption event occurs well before cells arrive at the surface. Therefore, cells see primarily a protein layer rather than the actual artificial surface of the biomaterial. Since cells respond specifically to proteins, formation of this interfacial protein film may be the event that controls subsequent bioreactions to implants. The biological performance of artificial materials largely depends on their surface properties, and biological responses to biomaterials, on the other hand, are dominated by their surface chemistry, structure, and morphology; hence, if surface modification is properly effected, their biological performance will be improved. These surface modification strategies have been used to engineer biomaterials to optimize specific surface properties such as lubricity, protein resistance, and enhanced protein retention. Modification of solid surfaces by the chemical attachment of biomolecules has proven to be an effective and important method for altering the interaction of solids with biological environments.5-7 A cursory look at the literature revealed the existence of various surface modification methods to activate solid surfaces upon which biological molecules are adsorbed. Selected examples of the important surface modification methods are as follows: (a) chemical vapor deposition (CVD) polymerization, wherein 4-amino[2,2]paracyclo* Corresponding author. E-mail: [email protected]. Fax: +81-48-464-6391. (1) Biomaterials Science, An Introduction to Materials in Medicine; Ratner, B. D., Hoffman, A. S., Schoen, F. J., Lemons, J. E., Eds.; Academic Press: San Diego, 1996. (2) Castner, D. G.; Ratner, B. D. Surf. Sci. 2002, 500, 28. (3) Kasemo, B. Surf. Sci. 2002, 500, 656. (4) Mohwald, H.; Lichtenfeld, H.; Moya, S.; Voigt, A.; Sukhorukov, G.; Leporatti, S.; Dahne, L.; Antipov, A.; Gao, C. Y.; Donath, E. Stud. Surf. Sci. Catal. 2001, 132, 485. (5) Lvov, Y.; Price, R.; Bruce, G.; Ichinose, I. Colloids Surf., A 2002, 198-200, 375. (6) Bentaleb, A.; Ball, V.; Haikel, Y.; Voegel, J. C.; Schaaf, P. Langmuir 1997, 13, 729. (7) Cacciafesta, P.; Humphris, A. D. L.; Jandt, K. D.; Miles, M. J. Langmuir 2000, 16, 8167.

phane is used to coat a copper surface, generating bioactive coatings for medical implant materials;8 (b) surfaceinitiated polymerization of L-lactide for coating solid surfaces with biodegradable polymers;9,10 (c) surface functionalization of silicon with terminal amine groups that are used for preparation of surface-grafted polymer layers;11 (d) thermal and anodic oxidation of a titanium surface to generate titania for biomedical applications;12 (e) oxidation of a titanium surface under corrosive conditions, such as H2O2/HCl solutions, to generate a titania layer;13 (f) hydrothermal modification of titanium metal in calcium hydroxide solutions for forming a calciumion-containing surface layer on titanium;14 (g) use of a polystyrene surface for culturing human monocytes to study apoptosis and cytokine release.15 All of these processes are substrate specific and involve multistep synthetic procedures; hence individual protocols have to be developed for each substrate. In addition, inert metal alloys and polymers often suffer from oxide defects and reactive zones that will differently influence adsorption and chemical interactions at their surfaces. For example, the electrospray deposition method is useful to fabricate functionally active protein films, wherein the protein is sprayed from a nondenaturing solvent onto a conducting electrode resulting in the formation of an ultrathin protein film.16 This method requires an expensive instrumentation setup including a high-voltage power supply and a vacuum chamber. Adsorption on polymer films such as polystyrene is useful but involves spin coating of the polymer on solid surfaces (8) Lahann, J.; Hocker; H.; Langer, R. Angew. Chem., Int. Ed. 2001, 40, 726. (9) Choi, I. S.; Langer, R. Macromolecules 2001, 34, 5361. (10) Kim, B. S.; Harkach, J. S.; Langer, R. Biomaterials 2000, 21, 259. (11) Schmidmaier, G.; Wildemann, B.; Stemberger, A.; Haas, N. P.; Raschke, M. J. Biomed. Mater. Res. 2001, 58, 449. (12) Sieval, A. B.; Linke, R.; Heij, G.; Meijer, G.; Zuilhof, H.; Sudholter, E. J. R. Langmuir 2001, 17, 7554. (13) Velten, D.; Biehl, V.; Aubertin, F.; Valeske, B.; Possart, W.; Breme, J. J. Biomed. Mater. Res. 2002, 59, 18. (14) Wang, X, X.; Hayakawa, S.; Tsuru, K.; Osaka, A. Biomaterials 2002, 23, 1353. (15) Hamada, K.; Kon, M.; Hanawa, T.; Yokoyama, K.; Miyamoto, Y.; Asaoka, K. Biomaterials 2002, 23, 2265. (16) Morozov, V. N.; Morozova, T. Y. Anal. Chem. 1999, 71, 1415.

10.1021/la0267239 CCC: $25.00 © 2003 American Chemical Society Published on Web 02/08/2003

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prior to protein adsorption. It also suffers from longer processing times and weak adsorption leading to up to a 38% loss of protein upon washing, as the proteins are adsorbed on polymers via hydrophobic interactions.17-19 Preparation of a titanium oxide substrate by the template stripping method involves thermal evaporation of a 200 nm thick layer of titanium on a freshly cleaved mica surface, which upon exposure to oxygen forms a titanium oxide surface.20 The multistep, cumbersome procedures of the existing methods prompted us to develop a simple and more efficient method for such surfaces. Our methodology presents a simple, efficient, and commercially viable protocol to fabricate ultrathin protein films. This method gives flexibility to coat most of the inert surfaces with biocompatible titania, upon which various proteins and peptides can be adsorbed. 2. Experimental Section 2.1. General Procedures. All of the solvents and reagents were used as received. Silicon wafers (N100 type) and quartz substrates were cut into approximately 1 × 3 cm squares. Fibrinogen, insulin, collagen, and heparin were purchased from Sigma Chemical Co. and used as received. The quartz crystal microbalance (QCM) experiments were conducted on a USI system, Fukuoka, Japan. Scanning electron microscopic (SEM) studies were conducted on a Hitachi S-900 instrument. X-ray photoelectron spectroscopy (XPS) experiments were carried out on an ESCALAB 250 (VG) using Al KR (1486.6 eV) radiation. The applied power was operated at 15 kV and 20 mA. The base pressure in the analysis chamber was less than 10-8 Pa. Smoothening, background removal, and curve fitting were performed with VG ECLIPS analysis software. Peak positions were normalized to the carbon peak at 285 eV. Attenuated total reflection infrared (ATR-IR) measurements were carried out on a Nicolet AVATAR 320 FT-IR instrument. 2.2. Surface Activation. 2.2.1. Silicon.7 A silicon wafer was cut into squares approximately 1 cm × 3 cm. Each piece was then individually soaked in EtOH (30 min) followed by sonication in a bath sonicator (10 min) and rinsing thrice with deionized water. The substrates were then immersed in 20 mL of piranha solution (3 parts 30% H2O2/7 parts concd H2SO4) for 5 h. At the end of this period, the substrates were rinsed twice with deionized water and thrice with MeOH and dried under a N2 stream. The substrates thus activated contain free -OH groups on the surface. 2.2.2. Quartz. The substrate was cut into squares of approximately 1 cm × 3 cm. Each piece was then individually soaked in EtOH (30 min) followed by sonication in a bath sonicator (10 min) and rinsing thrice with deionized water. The substrates were then immersed in concentrated H2SO4 (20 mL, 5 h). At the end of this period, the substrates were rinsed twice with deionized water and thrice with MeOH and dried under a N2 stream. The quartz substrate thus activated contains free -OH groups on the surface. 2.2.3. Teflon.26 A Teflon sheet was cut into squares of approximately 1 cm × 3 cm. Each piece was then individually soaked in EtOH (30 min) followed by sonication in a bath sonicator (10 min) and rinsing thrice with deionized water. The substrates were then exposed to O2 plasma (35 W, 45 min). Exposure of Teflon to O2 plasma generates peroxide groups on the surface. At the end of this period, the substrates were rinsed with deionized water and soaked in 20 mL of 1 N hydrochloric acid for 30 min, to decompose the peroxide groups into free hydroxyl groups. Finally, the substrates were rinsed twice with deionized water and thrice with MeOH and dried under a N2 stream. 2.2.4. Mica. Freshly cleaved mica was immersed in poly(ethyleneimine) (PEI) for 30 min, rinsed with water, dried, and (17) Brynda, E.; Houska, M.; Wikerstal, A.; Pientka, Z.; Dyr, E. J.; Brandenburg, A. Langmuir 2000, 16, 4352. (18) Lee, E. J.; Saavedra, S. S. Langmuir 1996, 12, 4025. (19) Baty, A. M.; Leavitt, P. K.; Siedlecki, A. C.; Tyler, B. J.; Suci, A. P.; Marchant, R. E.; Geesey, G. G. Langmuir 1997, 13, 5702. (20) Cacciafesta, P.; Humphris, A. D. L.; Jandt, K. D.; Miles, M. J. Langmuir 2000, 16, 8167.

Langmuir, Vol. 19, No. 6, 2003 2261 then immersed in poly(acrylic acid) (PAA) (30 min). At the end of this period, the mica substrate was rinsed with EtOH, dried, and then immersed in Ti(OBu)4 solution (3 min), rinsed with EtOH, dipped in water (3 min), and dried. The PAA-titania cycle was repeated thrice to obtain a uniform titania film. 2.3. FTIR-ATR Measurements. The adsorption of protein on the titania surface was monitored by ATR-IR. The spectra were recorded using a Nicolet AVATAR 320 FT-IR instrument equipped with a broad-band mercury cadmium telluride detector. The spectra were collected with 20 cm-1 resolution, and the number of averaged scans was 200. Neat titania-coated substrate was used as a background correction. 2.4. QCM Measurements. QCM measurements were performed on 9 MHz gold QCM electrodes. The gold QCM resonator was dipped in thioethanol (in EtOH) for 12 h, and it was rinsed with EtOH and dried. The thioethanol-coated gold QCM was dipped in Ti(OBu)4 solution (3 min) and after rinsing with EtOH was dipped in water (3 min) to hydrolyze the Ti(OBu)4 to Ti(OH)4, followed by drying under a N2 stream. The decrease in frequency was measured. This titania-coated QCM was dipped in PAA for 30 min; at the end of this period, it was rinsed with EtOH and dried under a N2 stream. This process was repeated thrice in order to obtain a uniform titania surface. The QCM with a uniform titania surface thus prepared was dipped in protein solutions for 30 min, rinsed with water, and dried under a stream of N2. At the end of every adsorption step, the decrease in frequency was recorded. The decrease with respect to the frequency of the thioethanol-coated QCM resonator was plotted against the cycle of adsorption. 2.5. pH Measurements. The pH experiments were conducted on an IQ Scientific Instruments model IQ 240 pH meter. A 1 M HCl solution (pH 0.8) was carefully diluted with deionized water and dilute sodium hydroxide solutions to obtain the required acidic pH. The required alkaline pH was obtained by diluting a 1 M NaOH solution with deionized water and dilute hydrochloric acid.

3. Results and Discussion 3.1. General Strategy. A simple, general methodology to generate robust, biocompatible surfaces is highly desired in biomaterials and biomedical research to construct safe implants. As mentioned above, all the surface activation procedures that have been reported in the past are substrate specific and there is no general protocol to prepare ultrathin biocompatible films on the substrate surface for protein adsorption. A key element in substrate-independent fabrication of biocompatible surfaces is to develop a general way to activate biologically inert surfaces of different kinds. We have been interested in the formation of metal oxide ultrathin films by the surface sol-gel process, wherein adsorption of metal alkoxide precursors on hydroxylated surfaces and the subsequent activation of the adsorbed layer are repeated regularly to give uniform multilayers. It was also demonstrated that water-soluble globular proteins such as cytochrome c and myoglobin smoothly underwent alternate layer-by-layer adsorption with Zr(OPr)4.21 It was clear that adsorbed metal oxide layers acted as efficient protein binding sites. This observation prompted us to develop a general method to convert inert surfaces to biocompatible ones, since adsorption of metal alkoxides is readily achieved on oxygenated surfaces. Oxygenated surfaces may be formed from inert surfaces such as Teflon, silicon wafers, and gold by chemical treatments. They are then readily modified with metal alkoxides to give activated metal oxide surfaces. Organic and biological molecules, if properly selected, would be readily adsorbed on such surfaces to make them biocompatible. (21) Ichinose, I.; Kunitake, T. Unpublished results.

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Figure 1. Schematic diagram of metal oxide treatment of a hydroxylated surface followed by biomolecular adsorption.

Figure 2. Partial molecular structure of heparin.

To accomplish this goal, we chose titania (titanium hydroxide polymer) as an intermediate layer that can react with both biological molecules and underlying solid substrates (Figure 1). Titania layers have a strong affinity for positively charged species and bind strongly to organic molecules with carboxy and hydroxy functionalities to form ester and ether linkages, respectively.22 As representative biological molecules, we chose collagen and fibrinogen as proteins, insulin as a peptide, and heparin as a polysaccharide.23 Collagen is a hydrophilic, triple-helical protein with three polypeptide chains. It is abundantly present in skin, bone, and cartilage, acting as the stress-bearing component of connective tissue, and is often used as a biocompatible surface layer. It contains Arg and Lys residues with positive charges, Ser and Thr residues containing hydroxyl groups, and free carboxylic acid groups. Fibrinogen is a blood clotting protein present in blood and promotes wound healing. The molecule consists of three pairs of homologous polypeptide chains. Fibrinogen has free carboxylic acid groups and is positively charged. In addition, it is reactive toward thrombin, leading to the formation of a polymeric fibrin; hence its bioactivity can be examined by fibrin formation. Insulin is a polypeptide containing 51 amino acid residues and is neutral. It binds to the titania surface through free carboxylic acid groups of the four glutamic acid (Glu) residues and hydroxyl groups of the three each serine (Ser) and tyrosine (Tyr) residues. Heparin is a negatively charged polysaccharide containing sulfate and carboxylate groups, present in the arterial walls (Figure 2). It is an anticoagulant and is used as a surface layer to prevent blood clotting. These biomolecules have specific functional groups and electrostatic charges to interact with titania and are useful in estimating the extent of adsorption on negatively charged titana. In addition to accomplishing adsorption of the biological molecules, we are interested in understanding their retention of biological activity after adsorption on the metal oxide surface. For example, fibrinogen is reactive to the blood clotting enzyme thrombin to form polymeric fibrin, and we can test its bioactivity by exposure to thrombin. 3.2. Surface Activation. The first step of the general procedure is surface activation of solid substrates. The surface activation involves two major steps: (i) surface

oxidation and (ii) formation of the active titania layer. For a substrate to be reactive toward Ti(OBu)4, its surface should be oxygenated or contain free hydroxyl groups. Surface hydroxyl groups can be generated on the majority of materials rather readily. We confirmed this by selecting quartz, silicon, Teflon, and gold as representative inert surfaces. The period of activation required for generating hydroxyl groups was determined by monitoring the appearance of sufficiently strong intensities of IR signals (-OH and -NH groups at 3500-3200 cm-1 and amide bonds at 1640 cm-1) after formation of the titania layer and protein adsorption. Examination of the individual steps was not possible since the surface hydroxyl signals were too weak to be monitored by IR. It was also confirmed that protein molecules did not adsorb directly on hydroxylated Teflon and silicon surfaces. In the second step of surface activation, we chose titania as the active metal oxide layer. As mentioned above, the titania layer is reactive toward carboxylic and hydroxyl groups on a Teflon sheet and a silicon wafer. The gold substrate was activated by soaking in mercaptoethanol (10 mM, 12 h). This process yielded a uniformly organized monolayer with hydroxyl end groups, which can react with Ti(OBu)4 to form a titania thin film upon hydrolysis.22 The quartz substrate was activated by thorough rinsing with EtOH and sonication, followed by soaking in concentrated H2SO4 (10 h), washing with deionized water and MeOH, and drying. This process generated on the quartz surface free hydroxyl groups that are suitable for titania adsorption.24,25 The titania coating of a quartz plate was characterized by titania absorption at 250 nm since a neat quartz plate was silent in this region. The silicon wafer was activated by thorough rinsing with EtOH and sonication, followed by soaking in piranha solution (5 h). At the end of this period, the substrate was rinsed with deionized water and MeOH and dried under a stream of N2. The surface thus activated contains free -OH groups, which are sufficiently reactive toward Ti(OBu)4.26 The Teflon surface was activated by exposing it to an oxygen plasma discharge (35 W, 60 min) after rinsing and sonication in EtOH.26 The substrate was then soaked in water (1 h) and dried under a stream of N2. It was sufficiently reactive toward Ti(OBu)4 to form a titania film by the surface sol-gel process.

(22) Ichinose, I.; Kawakami, T.; Kunitake, T. Adv. Mater. 1998, 10, 535. (23) Voet, D.; Voet, J. G. Biochemistry, 2nd ed.; John Wiley & Sons: New York, 1995.

(24) Ichinose, I.; Senzu, H.; Kunitake, T. Chem. Mater. 1997, 12, 1296. (25) Lee, S.-W.; Ichinose, I.; Kunitake, T. Langmuir 1998, 14, 2857. (26) Ichinose, I.; Kunitake, T. Adv. Mater. 1999, 11, 413.

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Figure 3. QCM profiles of precursor layers and biomolecules: (A) collagen, (B) fibrinogen, (C) insulin, and (D) heparin.

3.3. Adsorption of Biomolecules on a TitaniaCoated Gold Surface. QCM resonators are most convenient for semiquantitative determination of biomolecules adsorbed on the titania-modified gold surface.27 The modified gold-coated 9 MHz QCM resonator was allowed to react with Ti(OBu)4.23,24 Titania and PAA were alternately adsorbed in the present case. We chose the titania/ PAA combination rather than titania layer alone, since the PAA component gives a more uniform, smoother layer without structural defects.20 Adsorption experiments of biomolecules were performed on this uppermost titania surface. A frequency decrease of 1 Hz corresponds to 0.9 ng of mass adsorbed, and the film thickness can be calculated from the frequency change using the following equation:

2d ) -∆F/1.83F

(1)

wherein d is thickness of the film (Å); ∆F is the frequency change (Hz); and F is the density of protein (g cm-3), which is approximately 1.3 g/cm-3.30 A linear decrease in frequency with each cycle of adsorption indicates a uniform and regular growth of the film. The following four different biomolecules were subjected to QCM experiments: positively charged fibrinogen and collagen, neutral insulin, and the negatively charged polysaccharide heparin. Figure 3 describes adsorption processes of precursor layers and biomolecules, to indicate that these biomolecules are efficiently adsorbed on the (27) Okahata, Y.; Ebara, Y.; Sato, T. Protein Architecture; Lvov, Y., Mo¨hwald, H., Eds.; Marcel Dekker: New York, 2000; p 55. (28) Ingall, M. D. K.; Honeyman, C. H.; Mercure, J. V.; Bianconi, P. A.; Kunz, R. R. J. Am. Chem. Soc. 1999, 121, 3607. (29) Kang, E. T.; Zhang, Y. Adv. Mater. 2000, 12, 1481. (30) Lvov, Y.; Ariga, K.; Ichinose, I.; Kunitake, T. J. Am. Chem. Soc. 1995, 117, 6117.

Table 1. Adsorption of Biomolecules on Titania-Coated QCM Resonators

no. biomolecule 1 2 3 4

collagen fibrinogen insulin heparin

charge (+)ve (+)ve no charge (-)ve

frequency mass decrease increase functionality (Hz) (ng) -COOH -COOH -COOH -OH

630 383 215 227

567 344 193 204

titania surface. In the case of collagen, the frequency decrease was 573 Hz and the second dip resulted in a decrease of 57 Hz, suggesting that the surface was almost saturated in one dip. With fibrinogen, the first frequency decrease was 361 Hz and the second decrease was 22 Hz, again indicating saturation of adsorption. In the case of insulin, the frequency decrease was 215 Hz and the next dip resulted in a 56 Hz decrease. Heparin decreased the frequency by 205 Hz in the first dip and 12 Hz in the second dip. From the frequency change, the thickness of the protein films can be estimated by using eq 1. The thickness of the collagen film was estimated to be 12 nm by assuming complete coverage. In the case of fibrinogen, the film thickness was estimated as 7.6 nm. Neutral insulin and negatively charged heparin were not adsorbed as much as collagen and fibrinogen. These results can be explained on the basis of the nature of the functional groups involved. Collagen and fibrinogen contain protonated amino side chains of lysine and free carboxylic acid groups of glutamic acid. These functional groups imbue them with a high affinity for the titania surface, and collagen and fibrinogen adsorb quite strongly on the titania surface, 567 and 344 ng, respectively, as is evident from Table 1. Heparin, a sulfated polysaccharide, presents an interesting situation, as it has a high content of negatively charged sulfonate and carboxylate groups

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Figure 4. IR spectra of biomolecules adsorbed on a titania-coated Teflon substrate: (A) collagen, (B) fibrinogen, (C) insulin, and (D) heparin.

which could suppress its adsorption onto a negatively charged titania surface. However, the adsorption was still possible, presumably through free -OH groups of the sugar backbone. 3.4. Protein Adsorption on Other Activated Surfaces. It is demonstrated from the above experiments that a titania-coated gold surface effectively binds representative biomolecules. We subsequently tested the adsorption on other representative surfaces: a quartz plate, a Teflon film, and a silicon wafer. Since the QCM measurements cannot be performed directly on these surfaces, the adsorption process was monitored by ATRIR and XPS techniques. The substrate surfaces were activated via hydroxylation protocols as described in section 3.2. The titania-coated substrates were immersed in solutions of the biomolecules for a stipulated time (30 min), followed by thorough rinsing with water and drying under a stream of N2, and were subjected to ATR-IR examination. A minimum adsorption time of 30 min was required to saturate the titania surface with the biomolecules to obtain strong ATR signals. There was no increase in the signal intensity for adsorption times greater than 30 min. An adsorption time of less than 30 min resulted in a reduced ATR signal intensity due to incomplete coverage of the surface with biomolecules. In all these IR experiments, neat substrate surfaces coated with titania were used as a background, to cancel out the ATR absorption peaks of the substrate. As can be seen from ATR spectra obtained by the biomolecular adsorption on the Teflon substrate (Figure 4), all the characteristic peaks, broad O-H and N-H stretching vibrations between 3500 and 2700 cm-1, and a sharp amide stretching vibration at 1640 cm-1 were present. In the case of collagen adsorbed on titania-coated Teflon, the characteristic ATR signal contains a broad peak arising from the stretching vibrations of -NH and -OH groups from 3400 to 2700 cm-1. A sharp amide I peak at 1650 cm-1 is accompanied by a weaker amide II band arising from the N-H bending vibrations at 1620 cm-1. Fibrinogen adsorbed on titania-coated Teflon exhibited a broad peak arising from the stretching vibrations of -NH and -OH groups from 3400 to 2700 cm-1. The amide CdO stretching vibrations (amide I band) appeared as a sharp peak at 1650 cm-1, and a weaker amide II band arising from the N-H bending vibrations at 1620 cm-1 was overlapped by the amide I band. Insulin exhibited a broad peak at 3400-2700 cm-1. The CdO stretching

Figure 5. XPS survey spectrum of fibrinogen adsorbed on a titania-coated quartz plate (insets: narrow scans of the carbon, nitrogen, and oxygen signals).

vibrations of the amide groups (amide I band) appeared as a sharp peak at 1650 cm-1, immediately followed by a separate amide II band arising from the N-H bending vibrations at 1620 cm-1. Heparin adsorbed on Teflon also exhibited a broad stretching band from 3400 to 2700 cm-1 and a strong CdO stretching band at 1640 cm-1 from carboxylate anions, followed by the sulfate stretching frequency at 1415 cm-1. Virtually identical IR spectra were obtained when these biomolecules were adsorbed on titania-coated surfaces of quartz and silicon. An XPS survey spectrum of fibrinogen on a titaniacoated quartz substrate (Figure 5) reveals peaks of oxygen (O1s), carbon (C1s), and nitrogen (N1s) arising from the protein overlayer as well as those of titanium (Ti2p) and silicon (Si2p) from the substrate.31 In the oxygen signals, the binding energy component at ∼535 eV corresponds to metal oxide substrate (titania) and oxygen of the amide bonds of fibrinogen. The carbon signal can be resolved into two different contributions. The higher energy component has a binding energy of 288 eV and is assigned to the aliphatic carbons of fibrinogen. The next contribution at 292 eV corresponds to the C-N bond in the amide and amino groups of fibrinogen. Finally, the nitrogen (31) Ruiz-Taylor, L. A.; Martin, T. L.; Wagner, P. Langmuir 2001, 17, 7313.

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Figure 6. Effect of temperature on biomolecular films adsorbed on titania surfaces.

signal shows a single contribution at 404 eV corresponding to the peptide bond and the protonated amine groups of fibrinogen. In the case of collagen as well, the main peaks are oxygen (O1s), carbon (C1s), and nitrogen (N1s) from the collagen overlayer and titanium (Ti2p) and silicon (Si2p) from the substrate. In the oxygen signals, the binding energy component at ∼536.0 eV corresponds to metal oxide substrate (titania) and oxygen of the amide bond. The carbon signal can be resolved into two different contributions. The component with the highest intensity has a binding energy of 289 eV and is assigned to the aliphatic carbons of collagen. The next contribution at 292 eV corresponds to the C-N bond in the amide groups and the C-N of amino groups in collagen. Finally, the nitrogen signal shows a single contribution at 404 eV corresponding to the peptide bond and the protonated amino groups of collagen. The nitrogen peaks are ascribed to the biomolecule film alone, confirming the adsorption of proteins on the titania surface. 3.5. Stability of Adsorbed Biomolecules. 3.5.1. Temperature Stability. The effect of temperature on the stability of biological molecules adsorbed on titaniacoated QCM resonators was examined. The biomolecules adsorbed on QCM resonators were incubated (5 min) in deionized water at every 10 °C from 20 to 60 °C. The QCM frequencies were measured at room temeprature at the end of each 5 min of incubation. As can be seen from the QCM profiles of Figure 6, collagen did not show a mass decrease up to 50 °C,

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suggesting very strong interaction with the titania surface. A similar trend was observed in the case of fibrinogen. But insulin and heparin started to desorb at 30 °C. We separately confirmed that a mass decrease was not observed even at 70 °C, when a titania-coated QCM electrode was not subjected to protein adsorption. It is clear that collagen and fibrinogen are stably bound to the titania surface probably through their multiple binding sites and electrostatic interactions. Neutral insulin is less strongly bound to the surface and began to drop off at 30 °C. The negatively charged heparin was rather weakly bound, probably because of electrostatic repulsions. 3.5.2. pH Stability. The adsorbed biomolecules remain stably attached to the surface under a wide range of moderate pHs. However, they are desorbed under highly acidic or alkaline conditions. The pH of the solutions was carefully adjusted by addition of aqueous alkali or hydrochloric acid in small portions. Under acidic pH conditions (Figure 7A), collagen and fibrinogen were stably bound to titania up to pH 1.3 and underwent 10% (52 ng) and 20% (62 ng) desorption at pH 1.2, respectively. Rapid and complete desorption of these two proteins occurred only at pH 1.1. Insulin was stably bound to titania up to pH 1.7 and underwent 15% (23 ng) desorption at pH 1.5 and total desorption at pH 1.3. Heparin was stable up to pH 2.1 and underwent 25% (43 ng) and 84% desorption at pH 2 and 1.7, respectively, and complete desorption at pH 1.5. When sulfuric acid and nitric acid were used to adjust pH, erratic results were obtained, probably due to undesirable chemical reactions on the surface. Under alkaline conditions, it is evident from Figure 7B that collagen and fibrinogen are stably bound to titania up to pH 12.3 and are desorbed by 48% (252 ng) and 59% (188 ng), respectively, at pH 12.7. Both proteins were completely desorbed at pH 13. Insulin and heparin were stably bound up to pH 12.3 and underwent 62% (73 and 72 ng, respectively) desorption at pH 12.6 and complete desorption at pH 12.7. Collagen and fibrinogen have pI values of 6.6 and 5.6, respectively. The fact that these proteins are stably bound at pH 1-12 suggests that the role of the surface charge is not particularly significant in the current study. Thus, these proteins can be stably attached to the activated inert surfaces under physiological conditions. The titania layer is an ideal candidate as a surface modifier on biomedical implants. 3.6. Bioactivity of Adsorbed Fibrinogen. It is important to see if biomolecules adsorbed on titania-coated substrates retain their native morphologies and activities. As a typical example, we examined the biological activity

Figure 7. Effect of acidic and alkaline media on biomolecular layers adsorbed on titania surfaces: (A) acidic media and (B) alkaline media.

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Even after adsorption on a solid surface, fibrinogen still retained its chemical reactivity toward thrombin to undergo polymerization. This suggests that fibrinogen, after adsorption on titania, is not denatured and still retains its native conformation and reactivity. 4. Conclusions

Figure 8. Scanning electron micrographs of (A) neat fibrinogen on a titania layer before thrombin treatment and (B) the formation of a fibrin network after thrombin treatment. The rectangle in (B) indicates the region where the fiber width was measured.

of adsorbed fibrinogen. Thrombin in vivo polymerizes fibrinogen to fibrin via a series of proteolytic reactions, resulting in the formation of blood clots. Thus, we treated fibrinogen adsorbed on titania-coated quartz with aqueous thrombin. As can be seen from the SEM picture in Figure 8A, the fibrinogen-coated surface was smooth with no microstructures on it. In contrast, the thrombin-treated fibrinogen surface revealed the presence of a highly crosslinked, intricate network of the protein molecules with an average fiber width of 270 nm (Figure 8B). It is known that the fibrinogen molecule is converted to a fine, long, intertwined network in solution.32 The structural morphology of Figure 8B is clearly different, probably due to tight binding of fibrinogen to the titania surface.

On the basis of surface sol-gel synthesis of a titania overlayer, we have successfully established a general methodology to make biocompatible surfaces. Inert surfaces such as a Teflon sheet, a silicon wafer, and a quartz plate are converted to activated surfaces for biomolecular adsorption by taking advantage of the titania overlayer. Proteins, peptides, and polysaccharides are strongly bound to the ultrathin titania layer. It is important to note that the adsorbed fibrinogen protein is receptive to the enzymatic activity of thrombin. Apparently, the titania layer does not suppress the biological reactivity of the adsorbed protein. Since the surface activation by adsorption of the titania layer can be conducted for a wide variety of inert surfaces, the current methodology makes it possible to convert such inert surfaces to biocompatible surfaces. In addition, the ultrathin titania layer can be produced on curved surfaces. These unique features are particularly useful for fabrication of biocompatible surfaces on implant devices. Acknowledgment. S.N.G.A. thanks the Japan Science and Technology Corporation (JST) for an STA fellowship. LA0267239 (32) Veklich, Y.; Francis, C. W.; White, J.; Weisel, J. W. Blood 1998, 92, 4721.