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Nanoscale Patterning of Adsorbed Amphiphile Films with an Atomic Force Microscope Probe Hideki Sakai,*,†,‡ Wakako Yokoyama,† James F. Rathman,§ and Masahiko Abe†,‡ Faculty of Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan, Institute of Colloid and Interface Science, Tokyo University of Science, 1-3 Kagurazaka, Shinjuku, Tokyo 162-8601, Japan, and Department of Chemical Engineering, The Ohio State University, 140th West 19th Avenue, Columbus, Ohio 43210 Received August 6, 2002. In Final Form: January 3, 2003 A contact mode scanning atomic force microscope (AFM) probe was found to allow the adsorbed film on mica of dialkyldimethylammonium bromides (DADBs) prepared from their vesicular suspensions to spread in a position-selective way. Such growth of an adsorbed film was shown to be peculiar to double-chain-type surfactants bearing a cationic moiety including DADB and dipalmitoylphosphatidylcholine, and neither cationic single-chain-type surfactants nor anionic double-chain-type amphiphiles exhibited such growth behavior. This type of film growth was suggested to arise from the breakdown of vesicles on the mica substrate caused by the scanning of the contact mode AFM probe because (1) the film growth depended on the magnitude of the force given by the probe and (2) it was observed with adsorbed films prepared from vesicular suspensions but not with those prepared by the Langmuir-Blodgett method. Moreover, this technique was shown to permit the nanoscale patterning of amphiphilic molecules including phospholipids.
1. Introduction Amphiphilic molecules (surfactants) having relatively high solubility in water form molecular assemblies such as micelles and vesicles and adsorbed films at solid/liquid interfaces. Since adsorption of amphiphilic molecules having useful functions provides the solid surface with new functions that it originally lacks, this phenomenon is diversely applied in stabilization of colloid particles, pneumomedicine, chemical separation, oil recovery, polymer chemistry, materials science, and so forth.1,2 In the case of water-soluble amphiphilic molecules with low water solubility such as double-tailed surfactants and phospholipids, the following two methods are generally used to prepare such adsorbed films: (1) transfer of a monomolecular film from an air/water interface using the Langmuir-Blodgett method3,4 and (2) adsorption from a vesicular suspension.5-7 Recently, the latter method has gathered significant attention because of its simplicity and is used to prepare adsorbed films with proteins such as hormone receptors.8 Adsorbed surfactant films have been studied by smallangle X-ray scattering,9,10 spectroscopic methods including * Corresponding author. Phone and Fax: +81-4-7122-1442. E-mail:
[email protected]. † Faculty of Science and Technology, Tokyo University of Science. ‡ Institute of Colloid and Interface Science, Tokyo University of Science. § Department of Chemical Engineering, The Ohio State University. (1) Fujii, M. J. Jpn. Oil Chem. Soc. 1996, 45, 181-188. (2) Sakai, K.; Matsuda, H.; Kawada, H.; Eguchi, K.; Nakagiri, T. Appl. Phys. Lett. 1988, 53, 1274. (3) Tamm, L. K.; McConnell, H. M. Biophys. J. 1985, 47, 105-113. (4) Merkel, T. M.; Sackmann, E. J. Phys. (Paris) 1989, 50, 15351555. (5) Bayer, T. M.; Bloom, M. Biophys. J. 1990, 50, 357-362. (6) Kalb, E.; Frey, S.; Tamm, L. K. Biochim. Biophys. Acta 1992, 1103, 307-316. (7) Mau, J.; Yang, J.; Huang, C.; Shao, Z. Biochemistry 1994, 33, 4439-4443. (8) Sui, S.-f.; Urumow, T.; Sackmann, E. Biochemistry 1988, 27, 74637469. (9) Clack, G. L.; Leppla, P. W. J. Appl. Phys. 1936, 58, 2199.
Fourier transform infrared spectroscopy11-13 and NMR,5,14 adsorbed amount measurement,15-17 and an atomic force microscope (AFM). The AFM has successfully been used for nanoscale observation of surfactant adsorbed films formed at solid/liquid interfaces18 since it has a high resolution even for organic substances and permits us to observe objects in solutions. In addition, the AFM has also been used to investigate dynamic changes in the state of proteins, liposomes, and polymers when they adsorb on the solid surface.19-24 Recent studies include measurements of surface forces and use of the probe as a manipulator,25-28 in addition to morphological observations. With use of the AFM probe, we can achieve nanoscale control of the state of adsorbed films on the solid surface and application for the preparation of biochips. In this study, we observed adsorbed films of double-tailed cationic (10) Mizushima, K.; Nakayama, T.; Azuma, M. Jpn. J. Appl. Phys. 1987, 26, 772. (11) Ellis, J. W.; Pauley, J. L. J. Colloid Interface Sci. 1964, 19, 755. (12) Kimura, F.; Umemura, J.; Takenaka, T. Langmuir 1986, 2, 96. (13) Radler, J.; Stery, H.; Sackmann, E. Langmuir 1995, 11, 45394548. (14) Quist, P. O.; Soderlind, E. J. Colloid Interface Sci. 1995, 172, 510. (15) Jackson, S.; Redoiras, M. D.; Lyle, I. G.; Jones, M. N. Faraday Discuss. Chem. Soc. 1986, 85, 291. (16) Esumi, K.; Yamada, T. Langmuir 1993, 9, 622. (17) Nollert, P.; Keifer, H.; Jahnig, F. Biophys. J. 1995, 69, 1447. (18) Sakai, H.; Nakamura, H.; Kozawa, K.; Abe, M. Langmuir 2001, 6, 1817-1820. (19) Drake, B.; Prater, C. B.; Weisenhorn, A. L.; Gauid, S. A. C.; Aldrecht, T. R.; Qest, C. F.; Chnnel, D. S.; Hansmma, H. G.; Hansmma, P. K. Science 1989, 243, 1586. (20) Egawa, H.; Furusawa, K. Langmuir 1999, 15, 1660-1666. (21) Stipp, S. L. S. Langmuir 1996, 12, 1884. (22) Thomson, N. H.; Collin, M. C.; Palin, K.; Parkins, D.; Roberts, C. J.; Tendler, S. J. B.; Williams, P. M. Langmuir 2000, 16, 4813-4818. (23) Doudevski, I.; Hayers, W. A.; Woodward, J. T.; Schwartz, D. K. Colloids Surf. 2000, 174, 233-243. (24) Kumar, S.; Hoh, J. H. Langmuir 2000, 16, 9936-9940. (25) Dufrene, Y. F.; Lee, G. U. Biochim. Biophys. Acta 2000, 1509, 14-41. (26) Biggs, S. Langmuir 1995, 11, 156-162. (27) Butt, H. J. Biophys. J. 1991, 60, 1438-1444. (28) Ducker, W. A.; Senden, T. J.; Pashley, R. M. Nature 1991, 353, 239, 241.
10.1021/la020699h CCC: $25.00 © 2003 American Chemical Society Published on Web 02/22/2003
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surfactants and phospholipids on mica substrates using the AFM. Furthermore, the nanopatterning of adsorbed films formed on the mica substrate using an AFM probe was demonstrated. 2. Experimental Section 2.1. Materials. Mica was used as the solid substrate. A clean surface of freshly cleaved mica was used for sample preparation. The surfactants used were dialkyldimethylammonium bromides, cationic double-chain-type surfactants (alkyl chain length: n ) 12 (DDAB), n ) 14 (DTDAB), n ) 16 (DHDAB), n ) 18 (DODAB)), and cetyltrimethylammonium bromide (CTAB, a cationic singlechain-type surfactant), all supplied by Tokyo Kasei Kogyo Co., Ltd. Dipalmitoylphosphatidylcholine (DPPC), an amphoteric phospholipid, and dipalmitoylphosphatidylglycerol (DPPG), an anionic phospholipid, were used as the lipids, both purchased from NOF Corp. The solvents used were distilled water for injection (Otsuka Pharmaceutics Co., Ltd.) and phosphate-buffered saline (PBS). 2.2. Preparation of Vesicle Dispersions. Dialkyldimethyammonium bromide vesicle dispersions were prepared by ultrasonic irradiation for 2 h (Branson Cleaning Equipment Co., model B-220, output 125 W, 40 kHz) of a surfactant/distilled water mixture at a temperature above the gel-to-liquid crystal transition temperature, Tc, for each surfactant (DDAB, 15 °C; DTDAB, 25 °C; DHDAB, 30 °C; DODAB, 45 °C). Phospholipid vesicles were prepared by the Bangham method using distilled water for DPPC and PBS for DPPG as solvents, and the vesicles prepared were sonicated for 2 h at a temperature above the Tc for each lipid (DPPC, 42 °C; DPPG, 42 °C). The particle size was determined by the dynamic light scattering method using a NICOMP 380ZLS (Particle Sizing Systems) to be about 100 nm for all vesicles. 2.3. Verification of Vesicle Formation. Vesicle formation was verified through transmission electron microscopic observations using the freeze fracture method. Solutions containing vesicles were frozen in a rapid freezing apparatus (model EM CPC, Leica), and their replicas were prepared using a freeze replica preparing apparatus (model FR-7000A, Hitachi Science System). The replicas thus prepared were observed with a transmission electron microscope (model JEM-1200EX, JEOL). 2.4. Preparation of Adsorbed Films. Adsorbed films were prepared by dipping a mica substrate for 10 or 60 min in each vesicle dispersion. The adsorbed films were washed in a large volume of water and dried in the air. Preparation of monolayers by the Langmuir-Blodgett method was also conducted. Lipids were dissolved in a chloroform solution at 1 mM. Monolayers were spread on deionized water at 30 °C. Monolayers were transferred at a surface pressure of 30 mN/m on a surface pressure meter (model HBM-A, Kyowa Interface Science Co., Ltd.). 2.5. ζ-Potential Measurement. The ζ-potential of vesicles was measured by the electrophoretic mobility method using a NICOMP 380ZLS (Particle Sizing Systems). 2.6. Contact Angle Measurement. Measurements of the static contact angle of water on mica (covered and uncovered with adsorbed films) were performed with a contact angle meter (Contact Angle Meter C-A, Kyowa Interface Co. Ltd.). Contact angle readings were made 10 times for each sample, and the average of the readings was taken as the contact angle. 2.7. AFM Observations. AFM observations were carried out at 20 °C with a SPI3800N/SPA300 (Seiko Instruments Inc.) using a 1000-µm-long Si3N4 or Au-coated Si3N4 cantilever having a spring constant of 0.08 N/m for the contact mode observation and a 200-µm-long Si cantilever having a spring constant of 16 N/m for the tapping mode observation.
3. Results and Discussion 3.1. Observations of Adsorbed Films in the Tapping Mode. Observations were made in the tapping mode in air on a mica substrate washed in a large volume of water and dried after being immersed into a 5 mM DODAB vesicle dispersion for 60 min. Figure 1 reveals that DODAB
Figure 1. A tapping mode AFM image of the DODAB adsorbed film on a mica substrate and a cross-sectional view of the film. The adsorbed film was prepared by being dipped in a DODAB vesicle dispersion for 60 min.
forms an islandlike adsorbed film. The area occupied by the adsorbed film increased with an increase in the concentration of DODAB and/or immersion time (data not shown). These results indicate that the vesicles consisting of DODAB molecules are disrupted to adsorb on the mica surface chemically through ion exchange with potassium ions of the mica.20 The thickness of the adsorbed film was about 2 nm on the basis of its cross-sectional view (Figure 1), and the contact angle of water on the film was about 75°, indicating that DODAB adsorbed film that was washed in water and then dried attaches to the substrate surface with its hydrophobic groups directed upward. It seems unlikely that the vesicle would still exist after drying, but perhaps drying leaves a concentrated gel phase where the vesicle was originally located. Spherical adsorbed objects that seem to be molecular assemblies still in vesicle form were also attached on the film surface as seen in Figure 1. 3.2. Growth of Adsorbed Film Caused by AFMProbe Scanning. Next, contact mode AFM observation (repulsive force region) was done for the DODAB adsorbed film prepared with the same procedure. Interestingly, scanning the probe in the contact mode was found to allow the area occupied by the DODAB adsorbed film to enlarge. Observations in the air of the substrate showed formation of an adsorbed film in the first scanning, a film similar to the one observed in the tapping mode (Figure 1), as in Figure 2a. Repeated scanning caused, however, an increase in the area occupied by adsorbed film as seen in Figure 2a-c. Zooming out the scanning area to 10 µm × 10 µm revealed uniform growth of the adsorbed film only in the 2 µm × 2 µm area in the central part that was scanned previously (Figure 2d). This is a very peculiar phenomenon in which the scanning AFM probe increases rather than decreases the area covered with an adsorbed film. The film thickness in the part that spread by the scanning was about 2 nm, which was the same as that of the original film, suggesting no change in the state of adsorbed alkyl chains before and after film growth.
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Figure 2. Contact mode AFM images of the DODAB adsorbed film on a mica substrate after being dipped in a DODAB vesicle dispersion for 60 min and growth of the adsorbed film by AFM scanning: (a) 1st scan (2 × 2 µm), (b) 5th scan (2 × 2 µm), (c) 10th scan (2 × 2 µm), and (d) scan area enlarged to 10 × 10 µm after the 15th scan. The spherical spots indicated by arrows disappear by the scanning.
3.3. Effect of Molecular Structure. Enlargement of the area occupied by the adsorbed film prepared from the vesicular suspensions using the same procedure as that for DODAB was observed only in the part scanned by the AFM probe for all of the compounds DDAB (n ) 12), DTDAB (n ) 14), and DHDAB (n ) 16) (Figure 3). This demonstrates that scanning the probe permitted the adsorbed films of dialkyldimethylammonium bromides (DADBs) to spread, irrespective of alkyl chain length. For all four DADBs including DODAB, islandlike domains expanded by the AFM scanning and finally merged together to form square and uniform adsorbed films. The surface of cleaved mica has potassium ions, and these alkali cations cause adsorption of inorganic and organic cations from solution through ion exchange.29-31 (29) Fujii, M.; Li, B.; Fukuda, K.; Kato, T.; Seimiya, T. Langmuir 2001, 17, 1138-1142. (30) Pashley, R. M.; McGuiggan, P. M.; Horn, R. G.; Ninham, B. W. J. Colloid Interface Sci. 1988, 126, 569. (31) Herder, C. E.; Ninham, B. W.; Christenson, H. K. J. Chem. Phys. 1989, 90, 5801.
This indicates an important role of the surface charge of vesicles when they are disrupted to form adsorbed films on the mica substrate. Vesicles were then prepared with cationic DHDAB, amphoteric DPPC, and anionic DDPG (see Chart 1), which have the same alkyl chain length but possibly exhibit different ζ-potentials, and the effect of the ζ-potential of the vesicles on the growth of the adsorbed film induced by scanning with the AFM tip was examined. Table 1 gives the ζ-potentials of the vesicles, the contact angles of water on the adsorbed films, and the presence or absence of film growth induced by the AFM tip. As expected, cationic DHDAB vesicles had a positive ζ-potential of +45 mV, whereas anionic DPPG vesicles exhibited a low negative potential of -5 mV. Thus, the cationic part of DHDAB is on the outer vesicle surface while the anionic part of DPPG lies in the interior of vesicles. The ζ-potential of amphoteric DPPC vesicles was +5 mV in distilled water at pH 5.9. The contact angles of water were around 70° on DHDAB and DPPC adsorbed films, showing a hydrophobic surface for the films, while
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Figure 3. Contact mode AFM images of dialkyldimethyammonium bromide adsorbed films that grew through probe scanning: (a) DDAB (20 × 20 µm), (b) DTDAB (10 × 10 µm), and (c) DHDAB (20 × 20 µm). These images were obtained after prescanning in areas of 10 × 10 µm (DDAB and DHDAB) or 2 × 2 µm (DTDAB).
Figure 4. Contact mode AFM images of DPPC adsorbed films that grew through probe scanning (2 × 2 µm) (left, 1st scan; center, 3rd scan; right, 12th scan). Chart 1. Molecular Structures of DHDAB, DPPC, and DPPG
Table 1. ζ-Potentials of Vesicles and Contact Angles of Water on the Adsorbed Films DHDAB DPPC DPPG
that on DPPG adsorbed film was too low to be measured, being indicative of a hydrophilic surface of the film. This would indicate that the positively charged quaternary ammonium ion at the terminal of the hydrophilic group for DHDAB and even DPPC adsorbs on mica through an ion-exchange reaction with K+ on the substrate surface. Figure 4 shows a growth phenomenon for DPPC adsorbed film observed when scanned in the contact mode with the AFM probe, which is similar to that obtained for DADB adsorbed film. The absence of spherical objects
ζ-potential (mV)
contact angle (deg)
+45 +5 -5
75 68 0
growth O O ×
(vesicles) in Figure 4, unlike DADB systems (Figure 1), may suggest that DPPC vesicles can be easily swept or broken down by the AFM tip, unlike DADB vesicles. In contrast, DPPG adsorbed film exhibited no growth phenomenon even after repeated probe scanning (the scanned central 2 µm × 2 µm area after being zoomed out to 10 µm × 10 µm is shown in Figure 5). These findings demonstrate that only adsorbed films that are constituted of molecules with a cationic group adsorbable to mica through ion exchange at the terminal of their hydrophilic group can spread on the substrate surface by AFM scanning. 3.4. Effect of the Force Given by the AFM Probe to the Substrate. The fact that the growth of adsorbed films produced by AFM probe scanning was observed in the contact mode but not in the tapping mode suggests a difference in the strength of the force from the probe between the two modes of scanning. Since the probe is in contact with the sample surface during measurement in the contact mode, it gives energy higher than that in the tapping mode to the sample. The effect on film growth of
Nanoscale Patterning of Amphiphile Films
Figure 5. A contact mode AFM image of the DPPG adsorbed film (10 × 10 µm).
Figure 6. A tapping mode AFM image of the DODAB adsorbed film observed by an oscillating cantilever with a large amplitude (5 × 5 µm). The film was prescanned in a 2 × 2 µm area in advance.
the magnitude of energy applied was then examined through controlling the amount of energy given to the probe by changing the amplitude of the cantilever in the tapping mode. Observations of DODAB adsorbed film while increasing the amplitude of the cantilever revealed that when the amplitude is increased, that is, the magnitude of energy given to the sample is increased, the film growth is observed by the tapping mode scanning as in the contact mode scanning (Figure 6). While spherical adsorbed objects that seem to be molecular assemblies keeping vesicle form were observed on DODAB adsorbed film for both tapping (Figure 1) and contact (Figure 2) modes, the absolute number of the objects after an AFM scanning was smaller in the contact mode compared to that in the tapping mode. This would be because the probe is scanned in such a way that it drags the sample surface in the contact mode, and the spherical objects are swept or broken. In addition, the growth process of the adsorbed film reveals the presence of spherical adsorbed objects that disappear or reduce in their size with adsorbed film growth (indicated by arrows
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Figure 7. A contact mode AFM image of the CTAB adsorbed film (2 × 2 µm).
in Figure 2). These results imply that the growth of adsorbed film during contact mode scanning is caused by breakdown of physically adsorbed vesicles due to the AFM cantilever scanning and ion exchange of the cationic amiphiphilic molecules with potassium ions on the mica substrate. Some vesicles physically (weakly) adsorbed on the monolayer surface may be swept and moved by the tip across the hydrophobic monolayer surface until they reach the bare mica surface and then broken down to form the monolayer film. Though the AFM scan was carried out in air, ion exchange may occur due to small amounts of water molecules hydrating the hydrophilic moieties of DADBs. 3.5. Importance of Vesicle Presence on the Film Growth. Contact mode observations were performed on adsorbed films of CTAB, a non-vesicle-forming singlechain-type cationic surfactant, and DHDAB monolayers transferred from the air/water interface to the mica substrate surface by the Langmuir-Blodgett method to examine the effect of the presence of vesicles on the growth of adsorbed film produced by AFM probe scanning. CTAB adsorbed film gave a uniform and smooth image (Figure 7), and the contact angle of water on this film was about 75°, showing the film surface to be hydrophobic. Similarly, the DHDAB monolayer prepared with the LB method exhibited a uniform and smooth image as shown in Figure 8 and the contact angle of water on the monolayer was about 75°, a sign indicating the hydrophobic nature of the monolayer surface. However, the AFM image of the films showed no change after repeated probe scanning in contact mode. This would support our view that the growth of adsorbed film is caused by vesicle breakdown due to contact mode AFM probe scanning. From the results mentioned so far, the increase in the area occupied by the adsorbed film induced by contact mode AFM scanning is brought about by the following mechanism. Initially, vesicles come into contact with mica and the outer cationic headgroups adsorb strongly to the substrate. This interaction disrupts the vesicle, causing it to break apart, leaving a bilayer adsorbed on the surface. Rinsing with water then washes off the outer layer, leaving a monolayer adsorbed to the surface. Vesicles can also adsorb in regions where the mica surface is already covered by a bilayer. It seems that vesicles that adsorb in this fashion maintain their structure and get “stuck” so that they are not washed away when the sample is rinsed with water. Finally, spread of the adsorbed film during contact
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Figure 8. A contact mode AFM image of a DHDAB monolayer transferred to the mica surface by the Langmuir-Blodgett method (2 × 2 µm).
Figure 9. Nanoscale patterning of the DHDAB adsorbed film by contact mode AFM scanning. The probe was scanned along an A-shaped path.
mode scanning occurs by breakdown of these stuck vesicles due to the AFM cantilever scanning and ion exchange of the cationic amiphiphilic molecules with potassium ions on the mica substrate. 3.6. Patterning of the Adsorbed Film by AFM Scanning. We attempted a nanoscale patterning of an adsorbed film using the adsorbed film growth phenomenon caused by AFM probe scanning and succeeded in drawing a figure of arbitrary shape with a line width of about 200 nm by scanning the probe on a programmed path. Figure 9 shows the figure obtained by scanning the probe along an A-shaped path on DHDAB adsorbed film. Scanning the probe at temperatures below the gel-to-liquid crystal transition temperature of amphiphiles is important to make lithographs with a higher resolution. The growth of the DDAB film seen in Figure 3 occurs in such a way that the film seems to be spread by probe scanning. This would result from a scanning temperature (20 °C) that is higher than the gel-to-liquid crystal transition temperature of DDAB (15 °C). In fact, DDAB is in the gel state at the scanning temperature and hence the fluidity of the DDAB film is higher than that in the liquid crystal state, thereby allowing the film to spread beyond the area scanned by the probe.
This technique should enable us to make nanoscale lithographs of amphiphiles and is expected to be applicable to preparation of biomaterials including biochips since a similar adsorbed film growth phenomenon is found with phospholipids of biological origin. 4. Conclusions Contact mode AFM probe scanning for DDAB adsorbed films prepared from vesicular suspensions was found to allow the area occupied by the adsorbed films to spread two-dimensionally. This phenomenon was verified to be caused by scanning-induced breakdown of vesicles that adsorbed on the mica surface and kept their form, and it was seen only with cationic double-chain-type amphiphiles whose cationic moieties are ion-exchangeable with the cations on the mica surface. The phenomenon also permitted us to make nanoscale lithographs with lipid molecules. Acknowledgment. This work was supported by a Grant-in-Aid for Scientific Research on Priority Areas (417) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of the Japanese Government. LA020699H
