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Langmuir 2008, 24, 9974-9978
Contact Angles of Oils on Solid Substrates in Aqueous Media: Correlation with AFM Data on Protein Adhesion Eun Chul Cho,*,† Do-Hoon Kim,‡ and Kilwon Cho§ Department of Biomedical Engineering, Washington UniVersity in St. Louis, 1 Brookings DriVe, St. Louis, Missouri 63130, Department of Chemical Engineering, Hanyang UniVersity, Seoul 133-791, Korea, and Department of Chemical Engineering, Pohang UniVersity of Science and Technology, Pohang 790-784, Korea ReceiVed May 9, 2008. ReVised Manuscript ReceiVed July 21, 2008 This study presents a method to measure the contact angles of oils on a substrate in water. Diiodomethane and perfluorodecalin were used as model oils. Self-assembled monolayers (SAMs) were prepared by adjusting the mole ratio of CH3- and OH-terminated alkanethiols. The contact angles of the two oils in water increased with increasing hydrophilicity of the SAMs, and the results are contrasted with the contact angles of oils on these surfaces in air. In addition, perfluorodecalin showed higher contact angles than diiodomethane on the same surface. On the poly(N-isopropylacrylamide) (PNiPAAM) monolayer surface, the contact angles of the two oils in water decreased sharply at the transition temperature of PNiPAAM (∼30 °C), but the surface retained fairly high hydrophilicity even after the transition. The above results are correlated with atomic force microscopy (AFM) measurements of the adhesion force between protein-immobilized AFM tips (human fibrinogen and bovine serum albumin) and these monolayers.
Introduction The adhesion/adsorption behavior between two bodies has been estimated by measuring water contact angles on substrates, where the cosine of the contact angle is directly related to the thermodynamic work of adhesion, according to the Young-Dupre equation.1 The contact angle of water on a flat, solid substrate shows good correlation with adhesion data in air or in an organic environment. It is also desirable to measure the contact angle of hydrophobic molecules adsorbed or adhered on a solid substrate in an aqueous system in order to estimate the interfacial behavior of these molecules more realistically. For example, the hydrophobic group of proteins is often crudely modeled as an organic molecule such as cyclohexane,2,3 hence the contact angle of hydrophobic molecules on a solid substrate in aqueous media will give important information about the adhesion/adsorption behavior of proteins. However, most adhesion data from atomic force microscope (AFM) measurements or adsorption data between hydrophobic (protein) molecules on solid substrates in aqueous media have been correlated by measuring the water contact angle in air or in an organic environment, not the contact angle of oil in aqueous media.3-5 Here we report a method to measure the contact angle of oil droplets in an aqueous system. A schematic of the experimental setup is shown in Figure 1. One of the main * Corresponding author. E-mail:
[email protected]. † Washington University in St. Louis. ‡ Hanyang University. § Pohang University of Science and Technology. (1) (a) Wu, S. Polymer Interface and Adhesion; Marcel Dekker: New York, 1982. (b) Israellachvili J. Intermolecular and Surface Forces; Academic Press: San Diego, CA, 1998. (2) (a) Creighton, T. E. Proteins: Structures and Molecular Properties, 2nd ed.; W. H. Freeman and Company: New York, 1993. (b) Norde, W. Colloids Surf., B 1994, 2, 517. (3) Sigal, G. B.; Mrksich, M.; Whitesides, G. M. J. Am. Chem. Soc. 1998, 120, 3464. (4) (a) Prime, K. L.; Whitesides, G. M. Science 1991, 252, 1164. (b) Elwing, H.; Welin, S.; Askendahl, A.; Lunstrom, I. J. Colloid Interface Sci. 1988, 123, 306. (5) Sethuraman, A.; Han, M.; Kane, R. S.; Belfort, G. Langmuir 2004, 20, 7779.
difficulties in measuring the contact angle of oil in an aqueous system is the fact that the densities of most oils are lower than that of water. Therefore, we chose two hydrophobic molecules having higher densities than water, diiodomethane (density ) 3.325) and perfluorodecalin (density ) 1.908). The contact angles of the two oils were measured on wettability-controlled selfassembled monolayers (SAM), and the results were compared with the contact angles of these oils in air. We also measured the contact angles on a thermally responsive monolayer, poly(N-isopropylacrylamide) (PNiPAAM), by varying the temperature of the water. These data are discussed in relation to AFM data measured between protein-immobilized tips and these surfaces.
Experimental Section Preparation of Monolayers on Gold. A gold-coated silicon wafer (SiO2 (300 nm)/Ti (10 nm)/Au (200 nm)) was cleaned sequentially in piranha solution (3:7 w/w H2O2/H2SO4), deionized water, and ethanol and then submerged in a 2 mM ethanolic solution of alkanethiolates (dodecanethiol (Aldrich) and 11-mercapto-1-undecanol (Aldrich)) under an excess flow of Ar for 18 h. The wettability of SAMs on the gold surface was controlled by adjusting the feed mole ratio of the two alkanethiolates (Table 1). After reaction, the SAMs on the gold surface were washed with excess ethanol and distilled water and then dried under vacuum. The thickness of the SAMs was measured by ellipsometry (M-2000V, J.A. Woolam Co., Inc.) and ranged from 1.5 to 1.6 nm, which is close to the previous results for SAMs of equivalent chain length.6 The arrangement of these SAMs, identified by reflection absorption FT-IR (Bruker IFS 66v FT-IR, Germany) spectroscopy, showed that all symmetric and asymmetric stretching CH2 peaks for these SAMs were below 2920 and 2850 cm-1, indicating that all of these SAMs were well-ordered structures (Supporting Information, Figure S1). The wettabilities of the SAMs were checked by measuring the contact angles of water in air. The preparation of the PNiPAAM (Mn ∼20 000 by GPC) monolayer on the gold-coated silicon wafer and its characterization were performed as described in the literature.7 Contact Angles of Oil Droplets on Monolayers. To measure the contact angles of oil drops on the monolayer surfaces in aqueous (6) Bain, C. D.; Troughton, E. B.; Tao, Y.-T.; Evall, J.; Whitesides, G. M.; Nuzzo, R. G. J. Am. Chem. Soc. 1989, 111, 321. (7) Cho, E. C.; Kim, Y. D.; Cho, K. Polymer 2005, 45, 3195.
10.1021/la801444q CCC: $40.75 2008 American Chemical Society Published on Web 08/20/2008
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Figure 1. Schematic for (a) the measurement of contact angles of the oils on a substrate and (b) the measurement of force-extension curves between protein-immobilized tips and a substrate in aqueous media. For the measurement of contact angles, a substrate was fixed in the homemade glass chamber, and the chamber was filled with water. After 1 h of rest, a drop of oil (∼5 µL) was placed on the substrate, and the contact angle of the drop was measured at room temperature or from 20 to 38 °C, which was controlled by a heating setup. Substrates were wettability-controlled self-assembled monolayers (SAMs) and the poly(N-isopropylacrylamide) (PNiPAAM) monolayer (thermally responsive monolayer) on gold surfaces. Force-extension curves between protein-immobilized tips and substrates were measured by using atomic force microscopy. A temperature controller was also used for the temperature-dependent force-extension curves between the tips and the PNiPAAM monolayer. Table 1. Contact Angles of Water and Oils (Diiodoemthane and Perfluorodecalin) Measured in Air on Self-Assembled Mixed Monolayers [OH]/([OH] + [CH3])(mol/mol)a contact angles (deg)
water diiodomethane perfluorodecalin
0
0.4
0.55
0.7
0.85
1
106 ( 1.5 64 ( 1.1 29 ( 1.2
85 ( 1.3 46 ( 0.7 14 ( 0.9
65 ( 2.5 35 ( 1.3 7 ( 0.5
49 ( 2.7 29 ( 2.1 5g
33 ( 1.3 22 ( 1.1 5g
17 ( 2.0 14 ( 0.5 5g
a [CH3] is dodecanethiol (HS(CH2)10CH3), and [OH] is 11-mercapto-1-undecanol (HS(CH2)11OH), which is used for the preparation of self-assembled monolayers.
Table 2. Contact Angles of Water Measured in Air on the Poly(N-isopropylacrylamide) (PNiPAAM)-Grafted Gold Surface PNIPAAM monolayer on gold surface 40 °C
room temperature gold (equilibrium)
equilibrium
advancing
receding
equilibrium
advancing
receding
90.0 ( 1.7°
60.1 ( 1.5°
63.2 ( 2.3°
28.7 ( 1.2°
73.0 ( 1.2°
79.6 ( 0.9°
32.4 ( 2.0°
media, we used two oils, diiodoemthane (Aldrich, density ) 3.325, surface energy (γsv) ) 50.8 mJ/m2 (γsvd ) 48.5, γsvp ) 2.3)) and perfluorodecalin (Aldrich, density ) 1.908, γsv ) 19.2 mJ/m2 (γsvd ) 15.4, γsvp ) 3.8)).8 As can be seen in Figure 1, we made a transparent glass chamber, attached a monolayer substrate to the bottom, and poured in deionized water. After 1 h of rest, a drop of oil (∼5 µL) was placed on the substrate, and the contact angle of the drop was measured at room temperature. We set up a temperature controller in the glass chamber to measure the contact angles on the PNIPAAM monolayers at various temperatures. The PNiPAAM monolayers were immersed in cold water for at least 1 day and then moved to the glass chamber and equilibrated in cold water. The PNiPAAM monolayer was equilibrated for 1 h at each temperature (over the range from 20 to 38 °C) before the contact angles were measured. Three different substrates were used to obtain average values and the standard deviation. Force-Extension Curve for the Interaction between Monolayers and Protein-Immobilized Tips. Force-extension curves between the monolayer and a protein-immobilized tip were measured by AFM (AutoProbe CR Research, Park Scientific Instruments). We used either bovine serum albumin (BSA, Mw ) 67 kDa, A8022 Sigma) or human fibrinogen (HF, Mw ) 340 kDa, F-4883 Sigma) for the immobilization of a protein on the AFM tips. A Si3N4 cantilever (Microlever A-type, Microscopes) was treated with oxygen plasma and then chemically modified with 10 mM γ-aminopropyltriethox-
ysilane toluene solution for 2 h at room temperature. This amineterminated AFM tip was further reacted with glutaraldehyde for 30 min, which was followed by reaction with the chosen protein (BSA or HF) in phosphate buffer saline (PBS, pH 7.4) for 40 min. Then the tips were washed with PBS and subsequently stored in PBS. We obtained force-extension curves between the proteinimmobilized tips and monolayer surfaces. The monolayer surface’s position was regulated by a piezoactuator, and as the surface approached the protein-immobilized AFM tip, an interaction was generated between the tip and the SAM surface, inducing a cantilever deflection. By multiplying the spring constant of the cantilever by the deflected distance (change in photodiode signals), the intermolecular force between the protein-immobilized AFM tip and the SAM surface could be calculated. The force could be detected in the same manner when the surface was retracted. A force-extension curve then could be constructed from these measurements. We used a spring constant of 0.05 N/m, supplied by the manufacturer. The applied set force was 1 nN for all measurements, and 1 µm/s was required to obtain the force-distance curve during the approach and retraction of the SAM surface from the protein-immobilized tip. In this study, we could obtain only qualitative information about the interactions between these surfaces because we could not obtain the exact spring constant,9 and the force-extension curves depend on
(8) Fowkes, F. M.; Riddle Jr, F. L.; Pastore, W. E.; Weber, A. A. Colloids Surf. 1990, 43, 367.
(9) Proksch, R.; Schaffer, T. E.; Cleveland, J. P.; Callahan, R. C.; Viani, M. B. Nanotechnology 2004, 15, 1344.
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Figure 2. Contact angles of the two oils (perfluorodecalin and diiodomethane) in water on (a) SAMs prepared with different [OH]/([OH] + [CH3]) and (b) PNiPAAM monolayers. We measured the contact angles on the PNiPAAM monolayer as a function of temperature.
the approaching/retracting speed,10 applied loading force, and other factors.11 In this experiment, all of the force-extension measurements were carried out in PBS solution (10 mM, pH 7.4). The force-extension curves between the protein-immobilized tips and the SAMs on the gold surfaces were measured at room temperature, and the temperature controller was used for the measurement of the force-extension curves on the PNiPAAM monolayers.
Results and Discussion Water Contact Angles on Monolayers in Air. Table 1 gives the equilibrium contact angles of water and the two oils on the SAMs (average values for five different SAM substrates for one composition). The contact angle of water for 100% CH3 (∼106°) is similar to reported values, and the contact angle of the SAM for 100% OH changed from 15 to 20 (average ∼17°), which is slightly higher than in other reports12 but close to the value of Evans et al. and Martins et al.13 The contact angles of diiodomethane and perfluorodecalin also show a decrease as the hydrophilicity of monolayer increases, but perfluorodecalin shows much lower values on these surfaces than diiodomethane. Table 2 shows water contact angles in air on the PNiPAM monolayer at two temperatures. The water contact angle on the PNiPAM surface was found to be 60° at room temperature, which is very different from the value of 90° observed for the bare gold surface. The difference between the water contact angles on the PNiPAM surface at room temperature and those at 40 °C indicate that the monolayer’s interfacial properties with water change in response to temperature. Although the receding water contact angle changes little, and both the equilibrium and the advancing water contact angles vary considerably from room temperature to 40 °C. This variation is in agreement with a previous report on the contact angle of the PNiPAM brush on a self-assembled monolayer-covered gold surface.14 Contact Angles of Oils on Monolayers in Water. Figure 2a shows the contact angles of diiodomethane and perfluorodecalin (10) Rief, M.; Gautel, M.; Oesterhelt, F.; Fernandez, J. M.; Gaub, H. E. Science 1997, 276, 1109. (11) Friedsam, C.; Wehle, A. K.; Kuhner, F.; Gaub, H. E. J. Phys.: Condens. Matter 2003, 15, S1709. (12) (a) Lestelius, M.; Liedberg, B.; Tengvall, P. Langmuir 1997, 13, 5900. (b) Engquist, I.; Lundstrom, I.; Liedberg, B J. Phys. Chem. 1995, 99, 12257. (c) Bain, C. D.; Evall, J.; Whitesides, G. M. J. Am. Chem. Soc. 1989, 111, 7155. (13) (a) Evans, S. D.; Sharma, R.; Ulman, A. Langmuir 1991, 7, 156. (b) Martins, M. C. L.; Ratner, B. D.; Barbosa, M. A. J. Biomed. Mater. Res. 2003, 67A, 158. (14) Balamurugan, S.; Mendez, S.; Balamurugan, S. S.; O’Brien, M. J., II; Lopez, G. P. Langmuir 2003, 19, 2545.
in water on SAMs. For diiodomethane, the contact angles increase as the hydrophilicity of the SAMs increases, from 43° for a SAM of 100% CH3 to 89° for a SAM of 100% OH. This tendency can also be found for perfluorodecalin, whose contact angles change from 54° (100% CH3) to 110° (100% OH) as the SAM becomes more hydrophilic. This result contrasts with the contact angles of diiodomethane and perfluorodecalin for the SAM of 100% OH measured in air (14 and e5°, respectively). The above results demonstrate that the oils on the SAMs behave differently in a water environment than they do in air. The contact angles of the two oils on the PNiPAM monolayers (Figure 2b) show a decrease as temperature increases (hydrophobicity increases). Because the contact angles for the SAM of 100% OH do not change for these temperatures within experimental error (data not shown), it is evident that these changes result from the change in the interfacial properties of the PNiPAM monolayer. The contact angles of the two oils drop sharply in the vicinity of the transition temperature of PNiPAAM, ∼30 °C. This value is similar to that of most PNiPAAM brushes, but the trend is different from that observed in air: they increase their water contact angles in air sharply around this temperature.15 Figure 2a shows that for all SAMs the contact angles of perfluorodecalin were larger than those of diidomethane by 10-20°. We cannot clearly explain the differences, but the higher density (3.325) and surface energy (50.8 mJ/m2) of diiodomethane than those of perfluorodecalin (1.908 and 19.2 mJ/m2) probably influence the contact angles in aqueous media. This trend can be also found on the PNiPAM surfaces (Figure 2b). Diiodomethane showed smaller contact angles than perfluorodecalin over the temperatures we measured. However, the deviations in the contact angles are smaller than those measured on the SAMs. In addition, the contact angles became similar as the temperature was lowered. The SAMs in this study are a well-ordered monolayer, and these monolayers change chemically only when the mole ratio of CH3- and OH-functionalized thiols changes. In contrast, PNiPAAM chains are known to change both chemically (15) Kikuchi, A.; Aoyagi, T.; Sakurai, Y.; Okano, T. Langmuir 1998, 14, 4657. (16) (a) Tiktopulo, E. I.; Bychkova, V. E.; Ricka, J.; Ptitsyn, O. B. Macromolecules 1994, 27, 2879. (b) Fujishige, S.; Kubota, K.; Ando, I. J. Phys. Chem. 1989, 93, 3311. (c) Zhu, P. W.; Napper, D. H. J. Phys. Chem. B 1997, 101, 3155. (17) (a) Petrash, S.; Sheller, N. B.; Dando, W.; Foster, M. Langmuir 1997, 13, 1881. (b) Nosonovsky, M. Langmuir 2007, 23, 3157. (c) Spori, D. M.; Drobek, T.; Zu¨rcher, S.; Ochsner, M.; Sprecher, C.; Mu¨hlebach, A.; Spencer, N. D. Langmuir 2008, 24, 5411.
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Langmuir, Vol. 24, No. 18, 2008 9977
Figure 3. Force-extension curves of proteins-immobilized tips (BSA and HF proteins) on the monolayer surfaces prepared with different [OH]/([OH] + [CH3]): (a) BSA-immobilized tips; (b) HF-immobilzed tips; numbers in panels a and b are the mole ratios of the SAMs ([OH] to [OH] + [CH3]). From top to bottom: [OH] 1 (100% OH), 0.85, 0.7, 0.55, 0.4, 0 (100% CH3). (c, d) Force-extension curves of protein-immobilized tips on PNiPAAM monolayers: (c) BSA-immobilized tips; (d) HF-immobilized tips. We measured the force-extension curves on the PNiPAAM monolayers as a function of temperature. All measurements were carried out in a phosphate buffer solution (10 mM, pH 7.4).
and physically with temperature.15,16 Therefore, it is thought that the contact angles of oils in aqueous media are influenced by both chemical and physical states, just as the contact angles in air are affected by both chemical and physical changes.17 Correlation of Contact Angles with AFM Data Measured in Water. Figure 3 shows the force-extension curves between protein-immobilized tips on the monolayer surfaces in aqueous media. As shown in Figure 3a,b, the two tips show a similar trend in the SAMs: as the hydrophobicity increases, the maximum pull-off force (during tip retraction) increases. However, there are dissimilarities in the force-extension curve profiles for the two tips as a result of the different structures of the proteins (Supporting Information).18 For the PNiPAAM monolayers (Figure 3c,d), we could detect pull-off (attractive) forces when the temperature was increased to 29 °C, and these forces increased as the temperature increased further. We also observed different force-extension curve profiles for the two tips on the PNiPAAM monolayers. The increase in the attractive force with the hydrophobicity of the SAMs in aqueous media is in line with previous results concerning protein adhesion/adsorption on organic surfaces.3,5 (18) (a) Brown, J. R.; ShockleyP. In Lipid-Protein Interactions; Jost, P. C., GrifffithO. H., Eds.; John Wiley & Sons: New York, 1982; Vol. 1, p 25. (b) Feng, L.; Andrade, J. D. In Proteins at Interfaces II; Horbett, T. A., Brash, J. L., Eds.; American Chemical Society: Washington, DC, 1995. (c) Weisel, J. W.; Stauffacher, C. V.; Bullitt, E.; Cohen, C. Science 1985, 230, 1388. (d) Privalov, P. L.; Medved, L. V. J. Mol. Biol. 1982, 159, 665.
This trend is also in agreement with our observations of the contact angles of the two oils on the SAMs in aqueous media. The decrease in the contact angles between the oils and the substrate indicates an increase in the thermodynamic work of adhesion between the two bodies.1 The consistency of the trends in adhesion with the contact angles also holds true for the PNiPAAM monolayer. The increase in the attractive force with increasing temperature is in accord with the trends in the contact angles of the two oils (Figure 2b). It is worth discussing the hydrophilicity of the PNiPAAM monolayer in an aqueous system by comparing the contact angles and the adhesion data with those on the SAMs. Below the transition temperature, we detected almost no or only weak adhesion during the retraction of the PNiPAAM monolayers from the protein-immobilized tips, which is almost the same as the SAM for 100% OH. The contact angles of the two oils were higher on the PNiPAAM monolayer than the SAM for 100% OH, which suggests that the PNiPAAM monolayer had similar or higher hydrophilicity than the SAM for 100% OH. Furthermore, the PNiPAAM monolayer still retained fairly high hydrophilicity after the phase transition, as evidenced by the observation that this surface at these temperatures had low attractive forces with protein-immobilized tips and had high contact angles with the two oils, and these are comparable with those for the 100% OH SAMs. The contact angles of diiodomethane and perfluorodecalin were 97 and 103°, respectively, at 38 °C on the PNiPAAM
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monolayer, and the 100% OH SAM had angles of 89 and 110°. Although it is probable that the contact angles could change for a long incubation (over 1 h) of the PNiPAAM monolayer at T > Ttransiton, our study revealed that most contact angles were stabilized within 1 h for these surfaces at each temperature (data not shown). On the basis of the current results, it is thought that the PNiPAAM chains retain a considerable number of water molecules around the chain by maintaining the hydrogen bond between the amide group of PNiPAAM and the water molecules, even after the phase transition.19 In conclusion, we propose that this method will be helpful in predicting the surface properties of substrates and in exploring the interfacial behavior between organic molecules or proteins and solid substrates in aqueous media. It is especially useful when explaining the unusual protein resistance of hydrophilic polymers such as PEO and PNiPAAM (below the transition temperature). These surfaces have usually been reported to have higher contact angles in the dried state than a 100% OH
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SAM,3,5,14,20 but they have different physicochemical properties when they are fully hydrated.21,22 Supporting Information Available: Characterization of the SAMs by reflection absorption FT-IR spectroscopy and an explanation for the force-extension curves in Figure 3. This material is available free of charge via the Internet at http://pubs.acs.org. LA801444Q (19) (a) Shibayama, M.; Mizutani, S.; Nomura, S. Macromolecules 1996, 29, 2019. (b) Bae, Y. H.; Feijen, J.; Kim, S. W. Macromolecules 1993, 26, 2496. (c) Cho, E. C.; Lee, J. Y.; Cho, K. Macromolecules 2003, 36, 9929. (20) (a) Liang, L.; Rieke, P. C.; Fryxell, G. E.; Liu, J.; Engehard, M. H.; Alford, K J. Phys. Chem. B 2000, 104, 11667. (b) Schmitt, F.-J.; Park, C.; Simon, J.; Ringsdorf, H.; Israelachvilli, J. Langmuir 1998, 14, 2838. (c) Ista, L. K.; Mendez, S.; Perez-Luna, V. H.; Lopez, G. P. Langmuir 2001, 17, 2552. (d) Kidoaki, S.; Ohya, S.; Nakayama, Y.; Matsuda, T. Langmuir 2001, 17, 2402. (21) (a) Wang, R. L. C.; Kreuzer, H. J.; Grunze, M. J. Phys. Chem. B 1997, 101, 9767. (b) Feldman, K.; Ha1hner, G.; Spencer, N. D.; Harder, P.; Grunze, M. J. Am. Chem. Soc. 1999, 121, 10134. (22) Mcpherson, T. B.; Lee, S. J.; Park, K. In Proteins at Interfaces II; Horbett, T. A., Brash, J. L., Eds.; American Chemical Society: Washington DC, 1995.
