Stability of Self-Assembled Monolayers on Titanium and Gold

May 30, 2008 - Fei Hua Li , Jason D. Fabbri , Raisa I. Yurchenko , Alexander N. Mileshkin , J. Nathan Hohman , Hao Yan , Hongyuan Yuan , Ich C. Tran ...
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Stability of Self-Assembled Monolayers on Titanium and Gold Gopinath Mani,† Dave M. Johnson,*,†,‡ Denes Marton,†,§ Victoria L. Dougherty,‡ Marc D. Feldman,†,| Devang Patel,| Arturo A. Ayon,† and C. Mauli Agrawal*,† Departments of Biomedical Engineering and of Chemistry, The UniVersity of Texas at San Antonio, One UTSA Circle, San Antonio, Texas 78249, and Department of Radiology and DiVision of Cardiology, The UniVersity of Texas Health Science Center at San Antonio, 7703 Floyd Curl DriVe, San Antonio, Texas 78229 ReceiVed June 23, 2007. ReVised Manuscript ReceiVed March 10, 2008 Methyl- and hydroxyl-terminated phosphonic acid self-assembled monolayers (SAMs) were coated on Ti from aqueous solution. Dodecyl phosphate and dodecyltrichlorosilane SAMs were also coated on Ti using solution-phase deposition. The stability of SAMs on Ti was investigated in Tris-buffered saline (TBS) at 37 °C using X-ray photoelectron spectroscopy, contact angle goniometry, and atomic force microscopy. For comparison purposes, a hydroxyl-terminated thiol SAM was coated on Au, and its stability was also investigated under similar conditions. In TBS, a significant proportion of phosphonic acid or phosphate molecules were desorbed from the Ti surface within 1 day, while the trichlorosilane SAM on Ti or thiol SAM on Au was stable for up to 7 days under similar conditions. The stability of hydroxyl-terminated phosphonic acid SAM coated Ti and thiol SAM coated Au was investigated in ambient air and ultraviolet (UV) light. In ambient air, the phosphonic acid SAM on Ti was stable for up to 14 days, while the thiol SAM on Au was not stable for 1 day. Under UV-radiation exposure, the alkyl chains of the phosphonic acid SAM were decomposed, leaving only the phosphonate groups on the Ti surface after 12 h. Under similar conditions, decomposition of alkyl chains of the thiol SAM was observed on the Au surface accompanied by oxidation of thiolates.

1. Introduction Self-assembled monolayers (SAMs) are single-layered organic coatings that are deposited on metal or metal oxide surfaces by the adsorption of organic molecules from a solution.1,2 A variety of biomolecules involving proteins,3 peptides,4 DNA,5 carbohydrates,4 antibodies,6 and therapeutics7–9 have been attached to SAMs for biomedical applications. Au is the most commonly used metal for studying the science and bioengineering aspects of SAM coatings because of the well-behaved thiol on Au system. However, the stability of SAMs on metals is a concern especially for long-term biomedical applications. Numerous studies have investigated the stability of SAMs on Au in phosphate-buffered * To whom correspondence should be addressed. E-mail: Dave.Johnson@ utsa.edu (D.M.J.). † Department of Biomedical Engineering, The University of Texas at San Antonio. ‡ Department of Chemistry, The University of Texas at San Antonio. § Department of Radiology, The University of Texas Health Science Center at San Antonio. | Division of Cardiology, The University of Texas Health Science Center at San Antonio.

(1) Ulman, A. Chem. ReV. 1996, 96, 1533–1554. (2) Love, J. C.; Estroff, L. A.; Kriebel, J. K.; Nuzzo, R. G.; Whitesides, G. M. Chem. ReV. 2005, 105, 1103–1169. (3) Spinke, J.; Liley, M.; Guder, H. J.; Angermaier, L.; Knoll, W. Langmuir 1993, 9, 1821–1825. (4) Houseman, B. T.; Gawalt, E. S.; Mrksich, M. Langmuir 2003, 19, 1522– 1531. (5) Huang, E.; Zhou, F.; Deng, L. Langmuir 2000, 16, 3272–3280. (6) Herrwerth, S.; Rosendahl, T.; Feng, C.; Fick, J.; Eck, W.; Himmelhaus, M.; Dahint, R.; Grunze, M. Langmuir 2003, 19, 1880–1887. (7) Mani, G.; Mahapatro, A.; Johnson, D. M.; Patel, D. N.; Feldman, M. D.; Ayon, A. A.; Agrawal, C. M. Therapeutic self-assembled monolayers. BMES Proceedings, Baltimore, MD, Sept 28 to Oct 1, 2005; Biomedical Engineering Society: Landover, MD, 2005; Abstract No. 652. (8) Mani, G.; Johnson, D. M.; Marton, D.; Mahapatro, A.; Feldman, M.; Patel, D.; Ayon, A.; Agrawal, C. M. Drug elution using self-assembled monolayers. Transactions of the 31st Annual Meeting of the Society for Biomaterials, Pittsburgh, PA, April 26-29, 2006; Society for Biomaterials: Mt. Laurel, NJ, 2006; Abstract No. 307. (9) Mahapatro, A.; Johnson, D. M.; Patel, D. N.; Feldman, M. D.; Ayon, A. A.; Agrawal, C. M. Langmuir 2006, 22, 901–905.

saline,10 calf serum,10 ambient laboratory air,11–15 and ultraviolet light.16–20 In general, these studies have proposed that oxidation of thiolates to sulfonates (sulfonates do not have strong affinity toward Au as thiolate does) precedes desorption of SAMs. Compared to SAMs on Au, there are comparatively fewer reports of SAMs on biomaterials such as Ti,21–28 stainless steel,29–32 tantalum,24,33–35 and nitinol.36 The stability of SAMs under physiological conditions is crucial for successfully using this technique to coat medical implants. Also, the shelf life of SAMs and their stability under sterilization conditions are also important for biomedical applications. The stability of SAMs on Ti mainly depends on two factors: (a) the type of head groups (10) Flynn, N. T.; Tran, T. N. T.; Cima, M. J.; Langer, R. Langmuir 2003, 19, 10909–10915. (11) Li, Y.; Huang, J.; Mciver, R. T.; Hemminger, J. C. J. Am. Chem. Soc. 1992, 114, 2428–2432. (12) Tarlov, M. J.; Newman, J. G. Langmuir 1992, 8, 1398–1405. (13) Schoenfisch, M. H.; Pemberton, J. E. J. Am. Chem. Soc. 1998, 120, 4502– 4513. (14) Norrod, K. L.; Rowlen, K. L. J. Am. Chem. Soc. 1998, 120, 2656–2657. (15) Lee, M. T.; Hsueh, C. C.; Freund, M. S.; Ferguson, G. S. Langmuir 1998, 14, 6419–6423. (16) Huang, J.; Hemminger, J. C. J. Am. Chem. Soc. 1993, 115, 3342–3343. (17) Lewis, M.; Tarlov, M. J. Am. Chem. Soc. 1995, 117, 9574–9575. (18) Hutt, D. A.; Leggett, G. J. J. Phys. Chem. 1996, 100, 6657–6662. (19) Brewer, N. J.; Rawsterne, R. E.; Kothari, S.; Leggett, G. J. J. Am. Chem. Soc. 2001, 123, 4089–4090. (20) Ishida, T.; Sano, M.; Fukushima, H.; Ishida, M.; Sasaki, S. Langmuir 2002, 18, 10496–10499. (21) Gawalt, E. S.; Avaltroni, M. J.; Koch, N.; Schwartz, J. Langmuir 2001, 17, 5736–5738. (22) Helmy, R.; Fadeev, A. Y. Langmuir 2002, 18, 8924–8928. (23) Gawalt, E. S.; Avaltroni, M. J.; Danahy, M. P.; Silverman, B. M.; Hanson, E. L.; Midwood, K. S.; Schwarzbauer, J. E.; Schwartz, J. Langmuir 2003, 19, 200–204. (24) Hofer, R.; Textor, M.; Spencer, N. D. Langmuir 2001, 17, 4014–4020. (25) Tosatti, S.; Michel, R.; Textor, M.; Spencer, N. D. Langmuir 2002, 18, 3537–3548. (26) Zwahlen, M.; Tosatti, S.; Textor, M.; Hahner, G. Langmuir 2002, 18, 3957–3962. (27) Silverman, B. M.; Wieghaus, K. A.; Schwartz, J. Langmuir 2005, 21, 225–228. (28) Adden, N.; Gamble, L. J.; Castner, D. G.; Hoffmann, A.; Gross, G.; Menzel, H. Langmuir 2006, 22, 8197–8204.

10.1021/la8003646 CCC: $40.75  2008 American Chemical Society Published on Web 05/30/2008

Stability of SAMs on Titanium and Gold

and their affinity toward metal oxide; (b) coating methods used to deposit SAMs. SAMs with head groups such as phosphonic acid,21–23 phosphate,24–26 and trichlorosilane22 have been coated on Ti. A phosphonic acid SAM is coated on Ti using aerosol spray/ annealing,21 T-BAG/annealing,27 and gas plasma pretreatment/ solution-phase deposition.37 Since these coating methods involve the use of organic solvents, a novel method was developed to coat SAMs (alkyl phosphates) on Ti from aqueous solution.24 Aqueous solution-based coating systems are highly preferred for biomedical applications.24–26 The presence of surface hydroxyl groups is crucial for the formation of trichlorosilane SAMs on Ti or any other metal oxides.1,22 The stability of silane SAMs on metal oxides is determined by one or more of the following chemical bonds: (a) covalent bonds between silane molecules and the underlying metal; (b) cross-links between neighboring silane molecules; (c) hydrogen bonds between the silanol groups of silane molecules; (d) van der Waals interactions between alkyl chains.1,38 The standard procedure for coating thiols on Au is by solutionphase deposition.2 Unlike SAMs on Au, it is not common in the literature to find an optimized procedure for coating SAMs on Ti. The two methods that are commonly employed to coat SAMs on Ti are (a) pretreating Ti using gas plasma followed by solutionphase deposition24,37 and (b) annealing-based deposition.21,27 Oxygen plasma was used to create pure and stoichiometric surface oxide (TiO2) on Ti in a controlled manner.39 Also, the oxide created by oxygen plasma has less affinity for hydrocarbonbased contaminations.39 Oxygen plasma treatment is also found to introduce hydroxyl groups on a Ti surface,40 which is useful for the covalent attachment of SAMs. Gas plasma was extensively used in the literature to pretreat metal oxides before the deposition of SAMs.24,30,41,42 The stability of octadecyltriethoxysilane and octadecyltrichlorosilane SAMs on mica was significantly improved by the gas plasma pretreatment process.43,44 The other novel method was developed by Schwartz and co-workers for coating phosphonic acid SAMs on Ti.21,27 This method involves heating Ti specimens (after depositing phosphonic acid) for a period of time to make the coating more stable on Ti. Arginine-glycine-aspartic acid (RGD) peptides23 and bone morphogenic proteins28 were attached to phosphonic acid SAMs to enhance the cell attachment on Ti surfaces. We have previously shown attachment of therapeutics to phosphonic acid SAMs on (29) Shustak, G.; Domb, A. J.; Mandler, D. Langmuir 2004, 20, 7499–7506. (30) Mahapatro, A.; Johnson, D. M.; Patel, D. N.; Feldman, M. D.; Ayon, A. A.; Agrawal, C. M. Langmuir 2006, 22, 901–905. (31) Raman, A.; Dubey, M.; Gouzman, I.; Gawalt, E. S. Langmuir 2006, 22, 6469–6472. (32) Raman, A.; Gawalt, E. S. Langmuir 2007, 23, 2284–2288. (33) Brovelli, D.; Hahner, G.; Ruiz, L.; Hofer, R.; Kraus, G.; Waldner, A.; Schlosser, J.; Oroszlan, P.; Ehrat, M.; Spencer, N. D. Langmuir 1999, 15, 4324– 4327. (34) Textor, M.; Ruiz, L.; Hofer, R.; Rossi, A.; Feldman, K.; Hahner, G.; Spencer, N. D. Langmuir 2000, 16, 3257–3271. (35) De Palma, R.; Laureyn, W.; Frederix, F.; Bonroy, K.; Pireaux, J.-J.; Borghs, G.; Maes, G. Langmuir 2007, 23, 443–451. (36) Quinones, R.; Gawalt, E. S. Langmuir 2007, 23, 10123–10130. (37) Kanta, A.; Sedev, R.; Ralston, J. Colloids Surf., A 2006, 291, 51–58. (38) Marcinko, S.; Fadeev, A. Y. Langmuir 2004, 20, 2270–2273. (39) Aronsson, B. O.; Lausmaa, J.; Kasemo, B. J. Biomed. Mater. Res. 1997, 35, 49–73. (40) Yoshinari, M.; Hayakawa, T.; Matsuzaka, K.; Inoue, T.; Oda, Y.; Shimono, M.; Ide, T.; Tanaka, T. Biomed. Res. 2006, 27, 29–36. (41) Mahapatro, A.; Johnson, D. M.; Patel, D.; Feldman, M.; Ayon, A.; Agrawal, M. Nanomedicine: Nanotechnol., Biol., Med. 2006, 2, 182–190. (42) Zschieschang, U.; Halik, M.; Klauk, H. Langmuir, in press. (43) Kim, S.; Christenson, H. K.; Curry, J. E. Langmuir 2002, 18, 2125–2129. (44) Kim, S.; Sohn, H.; Boo, J.-H.; Lee, J. Thin Solid Films 2008, 516, 940– 947.

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Ti for drug delivery applications.45 Although the use of SAMs on Ti in biomedical applications is increasing rapidly, the literature available on its stability under physiological conditions is very limited.27 Also, the stability of SAMs on Ti in ambient air and ultraviolet (UV) irradiation is not clearly known.37 In this study, first, we investigated the formation of methyland hydroxyl-terminated phosphonic acid SAMs on Ti from aqueous solution. Second, the stability of the phosphonic acid, phosphate, and trichlorosilane SAMs on Ti (deposited by the solution-phase method) was investigated under physiological conditions. Third, the stability of phosphonic acid SAMs in ambient air and UV irradiation was investigated. For comparison purposes, the stability of thiol SAMs on Au was investigated under the same conditions. Since the SAMs on Au are well studied and reported to be highly stable, this system was used as a benchmark.

2. Materials and Methods Acetone, absolute ethanol (200 proof), and hexane were purchased from Pharmco-AAPER and used as received. Microscope glass slides, nitric acid, anhydrous toluene, and 11-mercapto-1-undecanol were all purchased from Sigma-Aldrich and used as received. Au (99.99% pure) and Ti (99.998% pure) sputtering targets were purchased from Atomic Spectroscopy Instruments, Inc. Dodecyl phosphate was purchased from Alfa Aesar. Dodecyltrichlorosilane was purchased from Gelest, Inc. Dodecylphosphonic acid,46 (11-hydroxyundecyl)phosphonic acid,46 and the ammonium salt of dodecyl phosphate24 were synthesized according to previously described methods. 2.1. Preparation of Ti and Au Specimens. The Ti substrates were prepared by sputter coating (Denton Vacuum Desk-II turbo sputter coater) the glass slides with a 40 nm thick layer of Ti at a deposition rate of ∼0.04 nm/s. The Au substrates were prepared by sputter coating the glass slides first with a 20 nm thick layer of Ti (to improve adhesion of Au on glass) followed by a 150 nm thick layer of Au. The Au deposition was carried out at a rate of about 1.0 nm/s. 2.2. Coating of -CH3-Terminated Dodecylphosphonic Acid, Dodecyl Phosphate, and Dodecyltrichlorosilane SAMs on Ti Surfaces. The Ti-coated glass substrates (Ti specimens) were oxygen gas plasma treated for 150 s using a radio frequency glow discharge system (Harrick Scientific Corp.). Then the specimens were immediately immersed in 3 mL of 1 mM dodecylphosphonic acid in doubly distilled water (dd-H2O),47 3 mL of the 1 mM ammonium salt of dodecyl phosphate in dd-H2O,24 and 3 mL of 1 mM dodecyltrichlorosilane in anhydrous toluene. After 48 h, the Ti specimens coated with phosphonic acid or phosphate SAMs were rinsed in copious amount of running dd-H2O, and the specimens coated with trichlorosilane SAMs were rinsed in toluene and ddH2O. The dodecylphosphonic acid, dodecyl phosphate, and dodecyltrichlorosilane SAM coated Ti specimens are referred here as DDPA/Ti, DDPO4/Ti, and DDTS/Ti, respectively. 2.3. Coating of -OH-Terminated Phosphonic Acid SAMs on Ti and Thiol SAMs on Gold. The Ti specimens were rinsed in 100% absolute ethanol (EtOH), acetone, 40% HNO3, and dd-H2O. Finally, before being coated with SAMs, the specimens were dried and treated with oxygen gas plasma for 150 s. The cleaned Ti specimens were immersed in 5 mL of 2 mM (11-hydroxyunde(45) Mani, G.; Johnson, D. M.; Marton, D.; Dougherty, V.; Feldman, M.; Patel, D.; Ayon, A.; Agrawal, C. M. Immobilization of therapeutics on metal surfaces using self-assembled monolayers. Transactions of the 32nd Annual Meeting of the Society for Biomaterials, Chicago, IL, April 18-21, 2007; Society for Biomaterials: Mt. Laurel, NJ, 2007; Abstract No. 4. (46) Dougherty, V.; Johnson, D. M. Synthesis and characterization of ω-functional alkylphosphonic acids to be used as self-assembling monolayers on titanium dioxide. Abstracts of Papers, 233rd ACS National Meeting, Chicago, IL, March 25-29, 2007; American Chemical Society: Washington, DC, 2007. (47) Phosphonic acid compound was dissolved in dd-H2O by heating the solution to 50 °C. Then the solution was sonicated for 5 min. If the compound still did not dissolve in dd-H2O, then the heating (to 50 °C) and sonication steps were repeated until the compound dissolved.

6776 Langmuir, Vol. 24, No. 13, 2008 cyl)phosphonic acid solution in dd-H2O. After 48 h, the Ti specimens coated with -OH-terminated phosphonic acid SAMs (OH-DDPA/ Ti) were rinsed in dd-H2O and dried using N2 gas. The Au-coated glass substrates (Au specimens) were rinsed in hexane and EtOH before SAM deposition. Then the Au specimens were immersed in 5 mL of a 1 mM solution of 11-mercapto-1undecanol in ethanol. After 48 h, the thiol SAM coated Au specimens (thiol/Au) were rinsed in EtOH for 60 s followed by N2 gas drying. 2.4. Immersion in Tris-Buffered Saline (TBS) Solution. DDPA/ Ti, DDPO4/Ti, DDTS/Ti, OH-DDPA/Ti, and thiol/Au were immersed in 3 mL of 10 mM TBS and incubated at 37 °C for up to 7 days. After removal from the solution, the Ti and Au specimens were rinsed in dd-H2O. The Au specimens were additionally rinsed in EtOH. After rinsing, all specimens were dried using N2 gas. 2.5. Immersion in dd-H2O. OH-DDPA/Ti and thiol/Au were immersed in dd-H2O at room temperature and removed after 1 and 7 days. The specimens were rinsed and dried as described above. 2.6. Exposure to Ambient Laboratory Conditions. OH-DDPA/ Ti and thiol/Au were stored under ambient laboratory conditions48 in air for up to 14 days. Upon removal, the Ti and Au specimens were rinsed in dd-H2O and EtOH, respectively, followed by N2 gas drying. 2.7. Exposure to UV Light. OH-DDPA/Ti and thiol/Au were placed inside a biological safety cabinet, and a Sylvania germicidal 30 W Hg tube (G30T8) was used as the UV source. The wavelength and intensity of the UV light were 254 nm and 100 µW cm-2, respectively. The specimens were placed 680 mm from the source and exposed to the UV light for 1 and 12 h. After the UV exposure, the specimens were rinsed (Ti in dd-H2O; Au in EtOH) and N2 dried. 2.8. X-ray Photoelectron Spectroscopy (XPS). XPS measurements were carried out using a Kratos Axis-Ultra instrument equipped with a monochromatic Al KR X-ray source (E ) 1486.7 eV, 225 W), a dual-anode Al/Mg X-ray gun, a hemispherical electron energy analyzer, and a channeltron detector array. A base pressure of 110° (Table 2). This significant increase in the contact angle suggests the formation of ordered monolayer films with -CH3-terminal groups. This value is consistent with several other studies on the formation of well-ordered SAMs.24,32,36 OH-DDPA/Ti showed a hydrophilic surface with a contact angle of 44.2 ( 2°. (61) Graubner, V.-M.; Jordan, R.; Nuyken, O.; Schnyder, B.; Lippert, T.; Kotz, R.; Wokaun, A. Macromolecules 2004, 37, 5936–5943. (62) Wang, D.; Oleschuk, R. D.; Horton, J. H. Langmuir 2008, 24, 1080–1086. (63) Spanos, C. G.; Ebbens, S. J.; Badyal, J. P. S.; Goodwin, A. J.; Merlin, P. J. Macromolecules 2001, 34, 8149–8155.

Stability of SAMs on Titanium and Gold

Figure 2. XPS-determined O 1s components of (a) control Ti, (b) DDPA/ Ti, (c) DDPO4/Ti, (d) DDTS/Ti, (e) OH-DDPA/Ti.

In the literature, AFM imaging was extensively used to determine the uniformity of SAMs on metal substrates.31,32,36,64 Figure 3 shows the AFM contact mode images of Ti specimens obtained before and after the formation of SAMs. The rms roughness value of control Ti was measured as 0.30 ( 0.04 nm. The rms roughness values of DDPA/Ti, DDPO4/Ti, DDTS/Ti, and OH-DDPA/Ti were 0.22 ( 0.003, 0.34 ( 0.12, 0.34 ( 0.08, and 0.28 ( 0.05 nm, respectively. No significant differences were observed between the rms roughness values of control and SAM coated Ti specimens. This indicates the formation of complete and uniform monolayer films on Ti surfaces.31,32,36,64 3.2. Formation of SAMs on Au Specimens. In the literature, the BE for S 2p3/2 in unbound (physically adsorbed) thiols is between 163.5 and 164 eV and decreases to 162 eV after the thiol groups are chemically attached to Au.65 The decrease in BE is due to the formation of a Au-thiolate chemical bond.65 High-resolution Au 4f, C 1s, O 1s, and S 2p XPS spectra were obtained for all the Au specimens that were involved in this study. The S 2p3/2 and S 2p1/2 peaks for unbound thiols are at 164.1 and 165.2 ( 0.2 eV, respectively (see Supporting Information (SI) Figure 1a). Also, the presence of S 2p3/2 and S 2p1/2 peaks at 162 and 163.2 ( 0.1 eV confirmed the presence of thiolates which are chemically bound to Au. This implies that, after the formation of the SAM, some thiol molecules were physically present at the top of the SAM. An effective cleaning procedure was required to remove physically adsorbed molecules but to keep the SAM coating intact. Here, two simple cleaning procedures were employed and compared: (1) manual rinsing (20 mL of solvent was added to a Petri dish, and the specimens (64) Quinones, R.; Raman, A.; Gawalt, E. Surf. Interface Anal. 2007, 39, 593–600. (65) Castner, D. G.; Hinds, K.; Grainger, D. W. Langmuir 1996, 12, 5083– 5086.

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were manually shaken in the solvent for 60 s using a tweezer), (2) sonication for 2 min. The absence of S 2p3/2 and S 2p1/2 peaks at 164.1 and 165.2 eV showed that both the cleaning procedures were efficient in removing the physically adsorbed molecules (SI Figure 1b,c). However, we preferred manual rinsing over sonication for the following reason: though the intermediate Ti coating improved the adhesion of Au on glass substrates, delamination of the Au coating occurred at the edges of some of the sonicated specimens. Hence, all the SAM coated Au specimens involved in the study were cleaned by manual rinsing. High-resolution Au 4f XPS spectra of the cleaned specimens showed a doublet at 84 eV (4f7/2) and 87.7 eV (4f5/2) (data not shown). The C 1s spectra showed two peaks: the major peak at 284.7 eV was assigned to C-C/C-H species, and the minor peak at 286.5 eV was assigned to the carbon atom which is attached to the terminal -OH group. The O 1s peak at 532.5 eV was assigned to the oxygen atom in the terminal -OH group. The contact angle measured for the Au specimens coated with -OH-terminated thiol SAMs was 27.6 ( 4.3°. Thus, the attachment of SAMs on Ti and Au was confirmed, and thereafter, the stability of SAMs was investigated under different conditions. 3.3. Investigation of SAM Stability on Ti in TBS and dd-H2O. Parts b-e of Table 1 show the change in the elemental concentration of SAM coated Ti specimens immersed in TBS at 37 °C for 1, 3, and 7 days. In the case of DDPA/Ti and DDPO4/ Ti, a significant decrease in the concentrations of P and C was observed after day 1 (Table 1b). Concurrently, the concentrations of Ti and O increased during this period. This suggests that a significant amount of DDPA and DDPO4 SAM molecules desorb from the Ti surfaces within 1 day. In the case of DDTS/Ti, no significant differences were observed in the concentrations of any elements for up to 7 days (Table 1b-d). This suggests that the DDTS SAM is highly stable on Ti surfaces under physiological conditions. Wang et al.66 investigated the stability of silane SAMs on silicon surfaces in saline solution at 37 °C for up to 10 days. In this study, XPS elemental concentrations (before and after saline immersion) were used to determine the percentage of SAM molecules desorbed or remaining on the metal surface. Here, we used such an approach to calculate the percentage of SAM molecules desorbed from the Ti surface. The ratios of P 2p to Ti 2p and C 1s to Ti 2p were calculated on the basis of the changes in the elemental concentrations (Figure 4). The degradation rate of the SAM was calculated as {(P 2p:Ti 2p)i - (P 2p:Ti 2p)t}/(P 2p:Ti 2p)i, where “i” and “t” refer to ratios of P 2p to Ti 2p before and after immersion in TBS for a predetermined time, respectively. On the basis of this calculation, we estimated 85% and 82% of DDPA and DDPO4 SAM molecules were desorbed in 1 day, respectively. However, in the case of the DDTS SAM, desorption was less than 10% even after 7 days of immersion in TBS. Similarly, the C 1s:Ti 2p ratio of DDPA/Ti decreased from 5.5 ( 0.4 to 1.8 ( 0.04 in 1 day (Figure 4b). For DDPO4/Ti, the ratio decreased from 3.2 ( 0.1 to 1.6 ( 0.2 in 1 day. However, the ratio obtained for DDTS/Ti remained constant for a period of 7 days. Parts e and f of Table 1 show the changes in the elemental concentrations of OH-DDPA/Ti immersed in TBS at 37 °C and dd-H2O at room temperature. A significant decrease in the concentration of P was observed from day 0 to day 1 and from day 1 to day 7 in TBS immersion. In the case of dd-H2O immersion, a significant decrease in the concentration of P was observed from day 0 to (66) Wang, A.; Tang, H.; Cao, T.; Salley, S. O.; Ng, K. Y. S. J. Colloid Interface Sci. 2005, 291, 438–447.

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Figure 3. AFM contact mode images of (a) control Ti, (b) DDPA/Ti, (c) DDPO4/Ti, and (d) DDTS/Ti.

day 1 but not from day 1 to day 7. The P 2p:Ti 2p and C 1s:Ti 2p ratios were also calculated (Figure 5a,b). On the basis of the P 2p:Ti 2p ratio, approximately 55% and 75% of the OH-DDPA SAM molecules were desorbed in TBS in 1 and 7 days, respectively (Figure 5a). In the case of dd-H2O immersion, approximately 30% of the SAM molecules were desorbed in 7 days (Figure 5a). Similar behavior was also observed in the case of the C 1s:Ti 2p ratio (Figure 5b). These observations suggest that the rate and amount of SAM molecules desorbed in dd-H2O are lower when compared to those in TBS. In the previous studies, where SAM coated metal substrates were incubated in saline solution, the contact angle values changed with respect to the original value due to disorder and/or desorption of alkyl chain molecules.10,66 When SAMs terminated with hydrophobic groups (-CH3) were desorbed, a sharp decrease in contact angle values was observed.10 When SAMs terminated with -OH or -NH2 groups were desorbed, an increase in the contact angle values was observed.10,66 Thus, the changes in the surface wettability observed in these studies have been attributed to the loss of SAM integrity and desorption.10,66 The contact angles of Ti specimens were measured during the degradation studies (Table 2). In the case of both DDPA/Ti and DDPO4/Ti, there is an abrupt drop in contact angle from >110° to ∼63° after day 1. This strongly suggests desorption of SAM

molecules from the Ti surface within 1 day. A significant change in the contact angle was also observed for OH-DDPA/Ti as the contact angle increased from 44° to 52° within day 1. However, in the case of DDTS/Ti, the contact angle remains ∼110° even after 7 days of immersion in TBS. These results are in excellent agreement with our XPS data that suggest the DDTS SAM is highly stable on the Ti surface under physiological conditions while the DDPA and DDPO4 SAMs are not stable on the Ti surface under the same conditions. AFM images were captured during the degradation studies (Figure 6), and the rms roughness values were measured (Figure 7). For DDPA/Ti, an increase in the surface roughness was observed from 0.22 ( 0.003 nm (before immersion in TBS) to 0.41 ( 0.18 nm (after 3 days of immersion in TBS). Gawalt and co-workers36,64 have previously showed that an increase in surface roughness values occurs when the SAM is disordered and the molecules are not uniformly distributed on the metal. In our study, we would attribute this increase in surface roughness values to the monolayer that was disordered during immersion in TBS. Interestingly, after 7 days of immersion in TBS, the surface roughness value of DDPA/Ti decreased to 0.35 ( 0.08 nm. This might indicate that when the molecules were completely desorbed from the Ti, the surface was free of irregularities and the roughness values were closer to that of control Ti (0.30 ( 0.04 nm). Similar

Stability of SAMs on Titanium and Gold

Figure 4. (a) P 2p:Ti 2p peak area ratios and (b) C 1s:Ti 2p peak area ratios of DDPA/Ti, DDPO4/Ti, and DDTS/Ti immersed in TBS at 37 °C.

behavior was also observed for DDPO4/Ti and OH-DDPA/Ti. However, the substrates immersed in TBS showed roughness values with large standard deviations when compared to control Ti or SAM coated Ti specimens. This relatively large scatter might be because of the presence of disordered SAM molecules on the Ti surface after the TBS immersion. There is always a possibility that the surface roughness value increases because of the adsorption of impurities from the TBS and/or atmosphere during sample preparation and characterization.67 However, the chances of contamination are less likely, since both XPS and contact angle data showed disruption and desorption of phosphonic acid or phosphate molecules on the Ti surfaces during immersion in TBS. In the case of DDTS/Ti, we did not observe an increase in surface roughness values for up to 7 days of TBS immersion. This is also in agreement with our XPS and contact angle data that suggest the uniformity of DDTS is undisturbed in TBS. Desorption of phosphonic acid and phosphate SAM molecules in TBS could be attributed to the hydrolytic instability of phosphonates or phosphates on Ti. Kanta et al.37 showed that the phosphonic acid SAM was not removed from the Ti even after 18 h of immersion in cyclohexane, toluene, or acetone. However, the stability of the monolayers was significantly affected during immersion in water, and it was proposed that low molecular volume liquids, such as water, could penetrate the SAM coating and affect the stability.37 In our study, the higher degree of desorption of phosphonic acid SAM molecules in TBS than ddH2O could be attributed to the presence of Na+ and Cl- ions in TBS, and perhaps the higher temperature (37 °C vs room temperature) may play a role as well. Also, the stability of -OHterminated phosphonic acid SAMs (55% desorption in 1 day) is comparatively better than than that of -CH3-terminated phosphonic acid SAMs (85% desorption in 1 day). The role of terminal (67) Petri, D. F. S.; Wenz, G.; Schunk, P.; Schimmel, T. Langmuir 1999, 15, 4520–4523.

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groups in the stability of SAMs has been previously investigated.68,69 Hydroxyl and carboxylic acid terminal groups provide better stability to SAMs because of the hydrogen bonding between them.68,69 Fadeev and co-workers22 compared the reactivity of silane SAMs with different head groups and phosphonic acid SAMs on Ti. The reactivity of trichlorosilane SAMs on Ti was significantly higher than that of any other SAM molecules. In another comparative study, the quality of monolayers formed from trichlorosilane molecules on Ti was better than that of phosphonic acid molecules.70 Though several studies in the past attributed the stability of silanes to Si-O-Si lateral bonds,1 Stevens showed that cross-polymerization cannot occur in highly organized and densely packed silane SAMs due to steric effects.71 Hence, the exact state of bonding responsible for the stability of silane SAMs on metal oxides is still debatable. On the basis of these arguments, in our study, the excellent stability of DDTS SAMs on Ti under physiological conditions is mainly attributed to stronger Si-O-Ti covalent bonds. The hydrogen bonds within the monolayer and the van der Waals interactions between the alkyl chains may also contribute to the stability of silanes. 3.4. Investigation of SAM Stability on Au in TBS and dd-H2O. High-resolution S 2p XPS spectra were obtained for the thiol/Au specimens that were immersed in TBS or dd-H2O for 1 and 7 days (SI Figure 2a,b). All the S 2p spectra showed the presence of doublet peaks at 161.9 ( 0.1 and 163.1 ( 0.1 eV with a ratio of the areas equal to 2. These doublet peaks confirm the presence of thiolate species which was undisturbed during immersion in TBS. No peaks for oxidized sulfur species (BE > 167 eV) were observed for up to 7 days. On the basis of these observations, the stability of phosphonic acid or phosphate SAMs on Ti (prepared by solution-phase deposition) is inferior to the stability of thiol SAMs on Au under physiological conditions. The stability of trichlorosilane SAMs on Ti is equivalent to that of thiols on Au for up to 7 days under the same conditions. If phosphonic acid or phosphate SAMs on Ti are to be useful for biomedical applications, there is a need to develop techniques that can impart stability to these SAMs on Ti under hydrolytic conditions. Annealing-based coating methods21,27 can be investigated to improve the stability of phosphonic acid or phosphate SAMs on Ti. On the basis of the previous studies, the stability of SAMs on Au is expected to be of no problem for up to three weeks under physiological conditions.10 The stability could be further improved by limiting the exposure of thiol SAMs to oxygen; however, the potential in vivo applications may be hindered because of the relatively large amount of oxygen in the human body. 3.5. Investigation of SAM Stability on Ti in Ambient Air. Table 1g shows the concentrations of C, O, Ti, and P atoms of Ti specimens exposed to ambient air for 1 and 14 days. No significant differences were observed in the atomic concentrations of any elements for up to 14 days of ambient air exposure. The P 2p:Ti 2p and C 1s:Ti 2p ratios were also calculated (Figure 5c). No significant difference was observed in the ratios after 1 day of exposure. On the basis of the P 2p:Ti 2p ratio, ∼20% of the SAM molecules were desorbed from the Ti surface after 14 days. Under ambient air exposure, the contact angle of OH-DDPA/Ti increased from 44° to 50° in 1 day and to 60° in 14 days. We would attribute this increase in contact angle (68) Cooper, E.; Leggett, G. J. Langmuir 1999, 15, 1024–1032. (69) Pawsey, S.; McCormick, M.; De Paul, S.; Graf, R.; Lee, Y. S.; Reven, L.; Spiess, H. W. J. Am. Chem. Soc. 2003, 124, 4174–4184. (70) Philippin, G.; Delhalle, J.; Mekhalif, Z Appl. Surf. Sci. 2003, 21212-213530-536. (71) Stevens, M. J. Langmuir 1999, 15, 2773–2778.

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Figure 5. (a) P 2p:Ti 2p peak area ratios and (b) C 1s:Ti 2p peak area ratios of OH-DDPA/Ti immersed in TBS at 37 °C and dd-H2O, (c) C 1s:Ti 2p and P 2p:Ti 2p peak area ratios of OH-DDPA/Ti exposed to ambient air, and (d) C 1s:Ti 2p and P 2p:Ti 2p peak area ratios of OH-DDPA/Ti exposed to UV irradiation.

values to the adsorption of impurities from the atmosphere when the specimens were exposed to ambient air.67 On the basis of these observations, the OH-DDPA SAM on Ti is stable for up to 14 days in ambient air without significant desorption. This indicates that the stability of the phosphonic acid SAM on Ti in ambient air is better than its stability under hydrolytic conditions. 3.6. Investigation of SAM Stability on Au in Ambient Air. XPS S 2p spectra of the thiol/Au specimens that were exposed to ambient air for 1 and 7 days were obtained (SI Figure 3a). We observed a nearly complete loss of peaks for bound thiolates (∼162 eV) in 1 day, and a new peak appeared at ∼168.5 eV. After 7 days, the peak at ∼162 eV was completely absent and the peak at ∼168.5 eV became stronger. Our study strongly suggests that most of the thiolates were oxidized in 1 day and all in 7 days. Subsequently, these oxidized sulfur species (sulfonates) were easily removed from Au specimens during cleaning. A significant increase in the ratio of O 1s to Au 4f was observed accompanied by a significant decrease in the C 1s:Au 4f and S 2p:Au 4f ratios after 1 and 7 days of ambient air exposure (SI Figure 4). In agreement with the results of other workers11–15 and on the basis of our observations, the stability of SAMs on Au is poor in ambient air conditions and the shelf life of SAM coated Au is less than 24 h. Hence, the ambient air stability of phosphonic acid SAM coated Ti is superior to that of thiol SAM coated Au. 3.7. Investigation of SAM Stability on Ti under UV Light. Figure 5d shows the changes in C 1s:Ti 2p and P 2p:Ti 2p ratios for Ti specimens that were exposed to UV radiation. The ratios significantly decreased after 1 and 12 h and suggest the removal of SAM molecules under UV exposure as a function of time (hours). The C 1s:Ti 2p ratio decreased from 6.8 ( 0.2 to 0.7 ( 0.2 in 12 h; however, the P 2p:Ti 2p ratio did not decrease at the same rate as the C 1s:Ti 2p ratio. On the basis of the P

2p:Ti 2p ratio, ∼25% and ∼58% of the SAM molecules were desorbed after 1 and 12 h, respectively. Table 1h shows a significant decrease in the concentration of carbon atoms accompanied by a significant increase in the concentrations of Ti and O atoms. No significant decrease was observed in the concentration of phosphorus atoms. These observations suggest that under UV exposure some alkyl chains in SAM molecules are photooxidized, leaving only the phosphonate groups on the Ti surface.37 Ti substrates can generate active oxygen species and hydroxyl radicals upon UV radiation exposure, and the radicals can decompose the organic molecules present on the substrates.72,73 Lee et al.74 investigated the photooxidation of trichlorosilane SAMs on TiO2 and SiO2. It was found that TiO2 decomposes the monolayers at a much faster rate than SiO2 with a gradual reduction in chain length. The siloxane groups remained on the TiO2 surface after the decomposition of the hydrocarbon chains. It was suggested that the reactive oxygen species generated from the Ti surface during UV irradiation exposure decomposes the alkyl chains.74 Ye et al.75,76 proposed that UV-induced oxidation of siloxane SAMs on silicon oxide surfaces occurs by the interaction of alkyl chains with atomic oxygen and/or hydroxyl radicals. We propose similar mechanisms for UV-light-induced oxidation on the OH-DDPA SAM coated Ti surface. Under direct UV light exposure, the degree of alkyl chain degradation is severe, and after 12 h, almost all the alkyl chains are (72) Ishibashi, K.-i.; Nosaka, Y.; Hashimoto, K.; Fujishima, A. J. Phys. Chem. B 1998, 102, 2117–2120. (73) Turchia, C. S.; Ollisa, D. F. J. Catal. 1990, 122, 178–192. (74) Lee, J. P.; Kim, H. K.; Park, C. R.; Park, G.; Kwak, H. T.; Koo, S. M.; Sung, M. M. J. Phys. Chem. B 2003, 107, 8997–9002. (75) Ye, T.; Wynn, D.; Dudek, R.; Borguet, E. Langmuir 2001, 17, 4497– 4500. (76) Ye, T.; McArthur, E. A.; Borguet, E. J. Phys. Chem. B 2005, 109, 9927– 9938.

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Figure 6. AFM contact mode images of SAM coated Ti specimens immersed in TBS at 37 °C for 1, 3, and 7 days.

decomposed, leaving only the phosphonate groups on the Ti surface. 3.8. Investigation of SAM Stability on Au under UV Light. The S 2p XPS spectrum of the thiol/Au specimens exposed to UV for 1 h showed peaks only for bound thiolates at 161.9 and 163.1 eV (SI Figure 3b). However, after 12 h, an additional peak for sulfonates (∼168.5 eV) was noticed with a concentration of ∼25% of the total S atoms. A significant decrease was observed in the C 1s:Au 4f ratio after 1 and 12 h. However, no significant decrease was observed in the S 2p:Au 4f ratio even after 12 h (SI Figure 4). This indicates the molecular decomposition of alkyl chains under UV exposure, followed by the oxidation of sulfur on the Au surface.17 This is also confirmed by the significant increase in the O 1s:Au 4f ratio only after 12 h and not in 1 h. In laboratories, metal substrates are exposed to UV light (for sterilization) typically for 12 h before biocompatibility experiments. On the basis of our observations, neither SAMs on Au nor those on Ti are stable for 12 h of UV light exposure under the conditions we used.

4. Conclusions In summary, the methyl- and hydroxyl-terminated phosphonic acid SAMs were coated on Ti from aqueous solution. The stability of dodecylphosphonic acid, dodecyl phosphate and dodecyl-

Figure 7. AFM roughness measurements of DDPA/Ti, DDPO4/Ti, DDTS/Ti, and OH-DDPA/Ti immersed in TBS at 37 °C.

trichlorosilane SAMs on Ti (coated using solution-phase deposition) was investigated in TBS at 37 °C. Under similar conditions, the stability of analogous thiol SAMs on Au was also investigated. On the basis of XPS, contact angle goniometry, and AFM, the

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stability of phosphonic acid or phosphate SAMs on Ti was inferior to the stability of thiol SAMs on Au. On Ti, extensive desorption of phosphonic acid or phosphate SAM molecules occurs within 1 day in TBS. Hence, aqueous-solution-based deposition may not be an appropriate method for producing stable phosphonic acid or phosphate SAMs on Ti for use in physiological conditions. The stability of the trichlorosilane SAM on Ti was on par with the stability of thiols on Au. Both the trichlorosilane SAM on Ti and the thiol SAM on Au were stable for up to 7 days in TBS. The phosphonic acid SAM on Ti was stable for up to 14 days in ambient air, while most of the thiol SAM molecules were lost from the Au surfaces after 1 day of exposure. Sterilization using UV radiation adversely affects the SAMs coated on Ti and Au specimens. After 12 h of UV exposure, the alkyl chains of the phosphonic acid SAM were decomposed, leaving primarily

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phosphonate groups on the Ti surface. Under similar conditions, the alkyl chains of the thiol SAM were decomposed on the Au surface followed by the oxidation of thiolates. Acknowledgment. We are grateful to Advanced Research Project No. 003659-0001-2006, VA Merit (M.D.F.), and the AHA Beginning Grant-in-Aid, Western Affiliates (D.P.), for financial support. Supporting Information Available: High-resolution XPS S 2p spectra of SAM coated Au (before and after rinsing) and SAM coated Au exposed to TBS, dd-H2O, ambient air, and UV irradiation and C 1s:Au 4f, O 1s:Au 4f, and S 2p:Au 4f ratios of SAM-coated Au exposed to various conditions at different time points. This material is available free of charge via the Internet at http://pubs.acs.org. LA8003646