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Phase-Separated Two-Component Self-Assembled Organosilane Monolayers and Their Use in Selective Adsorption of a Protein Jiyu Fang and Charles M. Knobler* Department of Chemistry and Biochemistry, University of California, Los Angeles, California 90095-1569 Received September 11, 1995. In Final Form: January 17, 1996X Stable mixed monolayers are prepared by combining Langmuir-Blodgett (LB) deposition with selfassembly. Islands of stable organosilane monolayers are formed on mica by the LB technique and the bare surface is then employed as a substrate for further selective self-assembly. When a surface covered by octadecyltrichlorosilane (OTS) monolayer islands is exposed to a solution of 1H,1H,2H,2H-perfluorodecyltrichlorosilane (FTS), self-assembled FTS monolayers form a continuous phase between the OTS islands. Similarly, mica surfaces covered by FTS islands can be used as substrates for self-assembly of OTS monolayers. Scanning force microscopy images of phase-separated OTS/FTS monolayers show how the composition of the monolayer and the size and surface density of the islands can be varied by controlling the conditions of deposition. Adsorption experiments with bovine serum albumin show that the protein is preferentially adsorbed on the CH3-terminated regions of the patterned monolayers.
Introduction Self-assembled monolayers (SAMs) of organosilanes are of interest for chemical modification of surfaces because of their high degree of organization and physical robustness.1 In these SAMs, trichlorosilane groups are bound covalently to substrate surfaces that have hydroxyl groups and cross-link to form a two-dimensional polymer network in which the alkyl chain axes are perpendicular to the substrate surface.2 The structural order and homogeneity of organosilane SAMs have been confirmed by infrared spectroscopy,3 low-angle X-ray reflectivity,4 ellipsometry,3,4 and atomic force microscopy (AFM).5 It is known that the nature of the terminal groups determines the surface physical, chemical, and biological properties of SAMs. Recently, several advances have been made in methods for distributing functional groups on monolayer surfaces. In one approach, ultraviolet irradiation through a mask is used to modify a SAM by photocleaving the terminal groups.6 Surfaces may also be patterned by using an AFM with a catalyst-coated tip to modify the terminal groups during scanning.7 Duschl and collaborators8 have used the Langmuir-Blodgett (LB) method to deposit phase-separated Langmuir films of a thiol and a lipid on gold surfaces. The lipid is then washed off, leaving a patterned SAM that can be used as a substrate for further self-assembly. Fujihara and Morita9 described experiments in which patterning was ac-
Figure 1. Formation of a phase-separated organosilane monolayer by combining LB and SA methods. The OTS and FTS substrates were prepared by the LB technique. Selfassembly of the FTS monolayer on the OTS substrate and selfassembly of the OTS monolayer on the FTS substrate yield surfaces patterned with CH3- and CF3-terminal groups.
X Abstract published in Advance ACS Abstracts, February 15, 1996.
(1) Ulman, A. An Introduction to Ultrathin Organic Films: From Langmuir-Blodgett to Self-Assembly; Academic Press: San Diego, CA, 1991. (2) Sagiv, J. J. Am. Chem. Soc. 1980, 102, 92. (3) Tillman, N.; Ulman, A.; Schildkraut, J. S.; Penner, T. L. J. Am. Chem. Soc. 1988, 110, 6136. (4) Wasserman, S. R.; Whitesides, G. M.; Tidswell, I. M.; Ocko, B. M.; Pershan, P. S.; Axe, J. D. J. Am. Chem. Soc. 1989, 111, 5852. (5) Schwartz, D. K.; Steinberg, S.; Israelachvili, J.; Zasadzinski, J. A. N. Phys. Rev. Lett. 1992, 69, 3354. Barrat, A.; Silberzan, P.; Bourdieu, L.; Chatenay, D. Europhys. Lett. 1992, 20, 633. (6) Dulcey, C. S.; Georger, J. H.; Krauthamer, V.; Stenger, D. A.; Fare, T. L. Calvert, J. M. Science 1991, 252, 551. (7) Mu¨ller, W. T.; Klein, D. L.; Lee, T.; Clarke, J.; McEuen, P. L.; Schultz, P. G. Science 1995, 268, 272. (8) Duschl, C.; Liley, M.; Vogel, H. Angew. Chem. Int. Ed. Engl. 1994, 33, 1274. Duschl, C.; Liley, M.; Corradin, G.; Vogel, H. Biophys. J. 1994, 67, 1229. (9) Fujihara, M.; Morita, Y. J. Vac. Sci. Tehnol. B 1994, 12, 1609.
Figure 2. Surface pressure-area isotherms for (a) an OTS monolayer at 18.6 °C and (b) an FTS monolayer at 28.4 °C. The subphase is water at pH 5.7.
complished by silanization of the bare areas of a surface covered by a microphotographically prepared pattern.
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Figure 3. Images of an OTS monolayer transferred onto acidtreated mica from the LE phase at a surface pressure of 5 mN m-1 and a transfer speed of 3 mm min-1. (a) A topographic image shows that the difference in height between the OTS islands and the bare mica is 2.8 ( 0. 2 nm. (b) Frictional force image, which was taken simultaneously with the topographic image, reveals higher frictional force (bright image) on the mica surface and lower frictional force (dark image) on the OTS islands.
Subsequent etching of the pattern allowed another silane to be deposited in its place. Here, we report a method to generate phase-separated organosilane monolayers with a well-defined distribution of two terminal groups. We have previously shown10 that stable organosilane monolayers on mica can be prepared by initially depositing them by the LB technique and that the size and number of islands of the silane can be controlled by varying the conditions of deposition. We describe here how these surfaces may be used as substrates for self-assembly from solution and how the combination of the two methods for preparation of monolayers makes it possible to vary the properties of the surface essentially continuously. Our work has been motivated in part by the desire to prepare surfaces on which proteins can be immobilized in (10) Fang, J.; Knobler, C. M. J. Phys. Chem., 1994, 99, 10425.
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Figure 4. Images of a phase-separated OTS/FTS monolayer. The OTS substrate system was prepared as described in Figure 3. (a) A topographic image shows that the difference in height between the CH3- and CF3-surfaces is 0.9 ( 0. 1 nm. (b) Frictional force image, which was taken simultaneously with the topographic image, reveals higher frictional force on the CF3-surface and lower frictional force on the CH3-surface.
isolated regions. We investigate this possibility by performing experiments in which we image phaseseparated monolayers onto which bovine serum albumin (BSA) has been adsorbed. The ease with which we can prepare surfaces with a range of island sizes, textures, and hydrophobicities allows us to examine factors that control the adsorption. Experimental Section Octadecyltrichlorosilane (OTS) (Aldrich, 95%), dodecyltrichlorosilane (DTS) (PCR, 97%), and 1H,1H,2H,2H-perfluorodecyltrichlorosilane (FTS) (PCR, 97%) were used as received. The solvents n-hexadecane, carbon tetrachloride, chloroform, benzene, and toluene (Fisher, g99%) were dried with Molecular Sieves prior to use. The OTS monolayers were prepared as previously described.10 A similar procedure was used for the DTS and the FTS. They were spread on Milli-Q water at pH 5.7 in a NIMA Type 611 trough from a benzene solution at a concentration of 0.2 mg mL-1. The temperature of the subphase was controlled at 28.4 °C by water flow from a thermostat that circulates through
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Figure 5. Topographic images of phase-separated OTS/FTS monolayers. Conditions of preparation of the OTS LB film: (a) in the LE phase at 10 mN m-1and a speed of 0.5 mm min-1; (b) in the LE phase at 2 mN m-1and 6 mm min-1; (c) in the LE-LC coexistence region at 15 mN m-1 and 1 mm min-1. The OTS islands are 0.9 ( 0.1 nm higher than the surrounding FTS monolayer.
Figure 6. Topographic images of phase-separated FTS/OTS monolayers. Conditions of preparation of the FTS LB film were as follows: (a) in the LE phase at a surface pressure of 0.5 mN m-1; (b) in the LE phase at 1 mN m-1; (c) in the LE-LC coexistence region at 3 mN m-1. In all cases the transfer speed was 1 mm min-1. The FTS islands are 0.9 ( 0.2 nm lower than the surrounding OTS monolayer. the base plate of the trough. After the benzene was allowed to evaporate for 5 min, the DTS and FTS monolayers were compressed at a speed of 1.5 Å2 min-1 and then transferred onto aqueous-HCl-treated mica after a preset pressure was reached. The samples were then heated in air in an oven at 100 °C for 30 min. OTS, DTS, and FTS monolayers prepared in this fashion were used as substrates for further selective self-assembly. Under a dry nitrogen atmosphere, the OTS-covered substrates were immersed in a 2.5 vol % solution of FTS in toluene for 60 min or for 30 min in a mixed solvent (in which the composition in vol % was as follows: DTS, 2; n-hexadecane, 80; carbon tetrachloride, 12; chloroform, 6). The FTS-covered substrates were immersed for 10 min in a mixed solvent (in which the composition in vol % was as follows: OTS, 2; n-hexadecane, 80; carbon tetrachloride, 12; chloroform, 6), and the DTS-covered substrates were immersed for 60 min in a 2.5 vol % solution of FTS in toluene. The samples were then withdrawn and rinsed with chloroform and toluene. Finally, they were heated again in an oven at 100 °C for 45 min. A diagram that describes the experimental procedures is shown in Figure 1. Contact angles were measured by the sessile drop method in the ambient environment. Advancing contact angles were obtained by increasing the volume of a water drop and recording the maximum value observed. Receding contact angles were obtained by decreasing the volume and recording the minimum value observed. The precision of the contact angle measurements is (2°. A scanning force microscope (Park Scientific Instruments Autoprobe) was used to characterize the phase-separated monolayers and observe the protein adsorption. In order to reduce protein deformation and tip damage, a small scan area was first examined and then the force was adjusted to the smallest possible value at which the protein could be imaged. All measurements
were carried out with a 100-µm scanner at room temperature in constant-force mode with a V-shaped silicon nitride cantilever with a normal spring constant of 0.05 N m-1. Two procedures were used to study the adsorption: (a) 50 µL of a 0.04 µg mL-1 solution of BSA (Sigma) was deposited on the surface with a syringe, the solution was allowed to evaporate in air, and the surface was rinsed several times with water; (b) the monolayer-covered substrate was immersed in a 0.05 µg mL-1 solution of BSA in a phosphate buffer (pH 7.0) for 1 h without stirring and was then gently rinsed with water. In both cases the rinsed samples were dried with nitrogen and stored in closed boxes in air before imaging. A buffer was not used in the samples evaporated to dryness because crystals of the salt obscured the images.
Results and Discussion Preparation and Nature of OTS/FTS PhaseSeparated Monolayers. Figure 2 shows a surface pressure-area isotherm for OTS spread on water (pH 5.7) at 18.6 °C. The isotherm exhibits a plateau at about 16 mN m-1 that we have attributed10 to coexistence between the liquid-expanded (LE) and a liquid-condensed (LC) phase. At this temperature there is no plateau in isotherms of FTS, but a coexistence region is evident at 28.4 °C, as seen in Figure 2. The plateaus diminish with time and the isotherms relax to the shape that has been observed11 on water acidified to pH 2, a result of the hydrolysis of the head group. (11) Ariga, K.; Okahata, Y. J. Am. Chem. Soc. 1989, 111, 5618. Barton, S. W.; Goudot, A.; Rondelez, F. Langmuir 1991, 7, 1029. Bourdieu, L.; Daillant, J.; Chatenay, D.; Braslau, A. Colson, D. Phys. Rev. Lett. 1994, 72, 1502.
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Table 1. Values of Advancing Contact Angle (θa) and Receding Contact Angle (θr) for Water Droplets on the OTS SAM, the FTS SAM, and OTS/FTS Monolayers at 24 °C area fraction of CH3-terminated surface 100 80 60 40 25 0
contact angle (deg) θa θr 110 110 108 106 104 103
99 92 91 89 86 92
Figure 3a is a topographic image of an OTS monolayer transferred onto mica from the LE phase at a surface pressure of 5 mN m-1 and a speed of 3 mm min-1. As can be seen, the monolayer breaks up into islands of nearly uniform size ( 1-2 µm in diameter) and the surface coverage is about 39%. We believe that the breakup of the LE phase into islands is caused by the dewetting of the water film that is transferred to the substrate. The islands are 2.8 ( 0.2 nm high, a value compatible with the length of a fully extended octadecane chain, indicating that the hydrocarbon chains are oriented perpendicular to the substrate surface. The exposed mica exhibits a frictional force about 9 times that of the OTS islands; see Figure 3b. A SAM of FTS can be formed from solution on the exposed mica. Figure 4 shows topographic and frictional force images for the resulting OTS/FTS monolayer. The height difference between the OTS and FTS regions is clearly visible in the topographic map, Figure 4a. The OTS islands are 0.9 ( 0.1 nm higher than the surrounding FTS monolayer, in agreement with the expected 0.88 nm difference in molecular length between all-trans alkyl chains. A frictional force image is shown in Figure 4b. The force on the CF3-terminated surface is three times that on the CH3-terminated surface, which is consistent with macroscopic measurements on OTS and FTS SAMs with a spherical glass slider in a pin-on-disk apparatus.12 The larger frictional force on the CH3-terminated surfaces has been attributed by Overney et al.13 to the difference in packing density between the CH3- and CF3-terminated regions. The less dense packing of the FTS allows the cantilever tip to penetrate more deeply and yields a larger frictional force. Defects in the FTS monolayer (dark spots) are visible in Figure 4a. The height profile shows that the defects are 1.8 ( 0.2 nm lower than the surrounding FTS monolayers, suggesting that they extend to the mica surface. The surface fraction of exposed mica is 0.05. The surface contrast is stable and reproducible even when scans are carried out with large forces (e.g., 70 nN); in contrast, the structure of fatty acid LB films can be imaged only with forces below 10 nN.14 The stability of the OTS islands in toluene is also evident from Figures 3 and 4, in which it can be seen that the size and number of the OTS islands are essentially unchanged after immersion in the FTS-toluene solution for 1 h and subsequent rinsing. This stability is not achieved without baking the initial LB film; OTS islands in an unbaked film do not survive the FTS self-assembly intact. The organization in phase-separated OTS/FTS monolayers can be controlled by changing the conditions under (12) DePalma, V.; Tillman, N. Langmuir 1989, 5, 868. (13) Overney, R. M.; Meyer, E.; Frommer, J.; Gu¨ntherodt, H. J.; Fujihira, M.; Takano, H.; Gotoh, Y. Langmuir 1994, 10, 1281. (14) Hansma, H. G.; Gould, S. A. C.; Hansma, P. K.; Gaub, H. E.; Longo, M. L.; Zasadzinski, J. A. N. Langmuir 1991, 7, 1051. Meyer, E.; Howald, L.; Overney, R. M.; Heinzelmann, H.; Frommer, J.; Gu¨ntherodt, H. J.; Wanger, T.; Schier, H.; Roth, S. Nature 1991, 349, 398.
Figure 7. Topographic images of BSA adsorbed on an OTS monolayer. The OTS islands were prepared from the LE phase at 8 mN m-1and 5 mm min-1. The images were taken after (a) one and (b) six scans.
which the LB film is prepared. For example, the size and number of the OTS islands can be changed by preparing the monolayer from the LE phase at different transfer pressures and speeds. In the OTS/FTS monolayer shown in Figure 5a, for which the OTS was deposited at 10 mN m-1 and a speed of 0.5 mm min-1, the OTS regions consist mainly of small islands (∼0.3 µm in diameter) and a few larger islands (1-1.5 µm in diameter) and the area fraction of CH3-terminated surface is about 0.40. Deposition at 2 mN m-1 and a speed of 6 mm min-1 leads to the monolayer shown in Figure 5b, in which the OTS islands are 2 µm in diameter and the surface fraction of the CH3-terminated surface is about 0.25. The shapes of the OTS islands can be controlled as well. When the OTS is deposited in the LE-LC coexistence region, it is possible to form OTS islands that are no longer compact, Figure 5c. The area fraction of CH3-terminated surface in the monolayer shown in the figure is 0.40. There is a coexistence region in the FTS isotherms, which means that the density of the FTS monolayer can also be controlled, allowing surfaces partially covered by islands of FTS to be used as substrates for selective
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Figure 8. Topographic images of BSA adsorbed on phase-separated OTS/FTS monolayers. Low-resolution images show that the patterned structure of the adsorbed BSA can be changed from small to large circular shapes by altering the conditions under which the OTS islands are prepared: (a) at 8 mN m-1 and 2 mm min-1; (b) at 8 mN m-1 and 5 mm min-1. (c) Higher-resolution image reveals that the adsorbed BSA is 25 ( 3 nm high and mainly aggregated.
self-assembly of OTS monolayers. Figure 6 shows topographic images for the resulting FTS/OTS monolayers. Height contrast between the OTS and FTS regions is readily seen in the topographic map. The FTS islands are more circular than the OTS islands shown in Figure 5. When the FTS monolayers are prepared from the LE phase, the size of the islands is about 0.4 µm at low compressions (Figure 6a) and 0.8 µm at higher compressions (Figure 6b). The area fractions of the CF3-terminated surface are 0.10 and 0.20, respectively. When the LB film is prepared in the LE-LC coexistence region, a mixture of small and large (6-8 µm in diameter) FTS islands that occupy about 40% of the surface can be formed, Figure 6c. Contact angles for water on SAMs of OTS and FTS and on the OTS/FTS monolayers have been determined at 24 °C; values for the advancing contact angle (θa) and receding contact angle (θr) are given in Table 1. The advancing contact angle is 103° on the FTS-covered surface and 110° on the OTS surface. DePalma and Tillman12 found contact angles of 105° and 111° for, respectively, FTS and OTS on silicon. The mixed monolayers have intermediate values and a somewhat larger hysteresis (17-18°), which may be attributed to their greater roughness and heterogeneity. The adhesive force between the Si3N4 tip and the CF3terminated surface is 27 ( 5 nN, less than that between the Si3N4 tip and CH3-terminated surface (36 ( 4 nN). The difference predominantly reflects the difference in hydrophilicity between the CH3- and CF3-terminated surfaces, which is seen in the water contact angles. Adsorption Studies. Most of the studies have been carried out by allowing drops of BSA solution to evaporate to dryness on the substrate. When the BSA is deposited on a surface that consists of OTS islands on mica, the protein is primarily adsorbed at the sides of the islands, Figure 7a. Repeated scans at applied forces of 2-5 nN leave the image unchanged, but when the force is increased to 10 nN, the BSA is displaced, as seen in Figure 7b in which the scan direction is from left to right. The BSA sheltered by the islands remains, while that at the leading edge is removed. In the case of the OTS/FTS surface, the protein adsorbs preferentially on the tops of the islands, parts a and b of Figure 8; even the smallest OTS islands, which are 0.2 µm in diameter, are covered by the BSA. The same preference for the tops of the islands is observed when the protein is adsorbed by immersion of the substrate in a buffered solution. BSA is a trimer that has the form of an equilateral triangle, 8 nm on a side; the height of the
Figure 9. Images of BSA adsorbed on a phase-separated DTS/ FTS monolayer. The DTS islands were prepared at 1.5 mN m-1 and 5 mm min-1. (a) A topographic image reveals that the adsorbed BSA is not uniformly distributed. (b) A frictional force image, which was taken simultaneously with the topographic image, shows that the BSA (bright spots) is adsorbed only on the DTS islands (dark).
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Figure 10. Topographic images of BSA adsorbed on phase-separated OTS/DTS monolayers. Low-resolution images show that the patterned structure of the adsorbed BSA can be changed from isolated circles to coalesced shapes by altering the conditions under which the OTS islands are prepared: (a) at 4 mN m-1 and 5 mm min-1; (b) at 12 mN m-1 and 5 mm min-1. (c) High-resolution image of BSA adsorbed BSA on a single OTS island.
molecule is 3 nm.15 The height of the dots in the images is 2.5 ( 0.3 nm, consistent with a single layer of the protein. While most of the dots represent aggregates of the protein, some of them are as small as 12 nm in diameter (Figure 8c) which, if one accounts for broadening by the AFM tip,16 correspond to isolated molecules. For the OTS/FTS surface, essentially none of the protein adheres to the sides of the OTS islands. In these monolayers the height of the CH2-terminated surface at the sides of the OTS islands is 0.9 nm (see Figure 4), only a third the size of the smallest dimension of BSA. In order to determine if the adsorption of the BSA onto the CH3-terminated islands is related to the difference in height, we studied the adsorption on two surfaces, one consisting of islands of DTS surrounded by FTS and the other in which islands of OTS are surrounded by DTS. Figure 9a shows a topographic image of the DTS/FTS surface after adsorption of BSA. The BSA is localized in islands. The slight difference in height between the FTS and DTS is not evident in the image, but when the system is examined in the frictional force mode, Figure 9b, it is clear that the protein (bright spots) binds preferentially to the CH3-terminated surface (dark islands). Adsorption on the OTS/DTS surface, on which there are differences in height but not in functionality, demonstrates that there is a preference for the island tops, as seen in Figure 10. Since the OTS islands in the OTS/DTS surface were prepared by the LB technique and the surrounding DTS was formed as a SAM, it seemed possible that the preference for the island tops resulted from differences in density or disorder between parts of the monolayers prepared by different methods. To explore this possibility, islands of OTS were deposited by the LB technique and the spaces between them were filled in with a SAM of OTS. The islands increase in size as the OTS is deposited from solution and defects remain where the islands have come into contact, Figure 11a; otherwise, the surface of the monolayer is smooth. As seen in Figure 11b, the BSA adsorbs at the defects, but there is no preference for the LB cores of the islands over the peripheral areas formed by self-assembly. Frommer et al.17 studied the adsorption of tobacco mosaic virus on mixed hydrocarbon-fluorocarbon LB films. They observed that the virus adsorbed preferentially at the hydrocarbon-fluorocarbon boundaries, points at which (15) He, X. M.; Carter, D. C. Nature 1992, 358, 209. (16) Allen, M. J.; Hud, N. V.; Balooch, M.; Tench, R. J.; Siekhaus, W. J.; Balhorn, R. Ultramicroscopy 1992, 42, 1095. (17) Frommer, J.; Lu¨thl, R.; Meyer, E.; Anselmetti, D.; Dreler, M.; Overney, R.; Gu¨ntherodt, H. J.; Fujihara, M. Nature 1993, 364, 198.
Figure 11. Topographic images of an OTS monolayer before (a) and after (b) BSA adsorption. The OTS monolayer was prepared by combining the LB and SA methods: First, the OTS islands were prepared as described in Figure 3a. The space between these islands was then filled in with a SAM of OTS.
the methylene groups were exposed, and they argued that the adsorption paralleled the surface energies, which from
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surface tension measurements18 are in the order CH2 > CH3 > CF3. The preference of BSA for the CH3-terminated regions over the CF3-terminated regions in the FTS/DTS monolayer is consistent with this order. In the OTS/FTS monolayer, in which methylene groups are exposed at the sides of the islands, the absence of significant adsorption at the boundaries might be attributed to the shortness of the exposed methylene surface. Adsorption at the sides of the islands is observed on the OTS/mica surfaces, for which the islands are twice as high. On the other hand, the preferences for the tops of the islands on the OTS/ DTS surface cannot be the result of differences in surface (18) Zisman, W. A. In Adhesion and Cohesion; Weiss, P., Ed.; Elsevier: New York, 1962; p 176.
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energy. We had surmised that it might result from the manner in which the droplets of the solution dried on the surface, but the fact that identical results were obtained in adsorption from solution shows that this is not the case. The protruding islands are likely to be more disordered than the rest of the monolayer and there may be a preference for the more disordered surface. Finally, we note that at higher concentrations, the protein adsorbs on all regions of the surfaces and the selectivity cannot be seen. Acknowledgment. This work was supported by the National Science Foundation. LA950751S
