Langmuir 1997, 13, 4795-4798
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High-Resolution Atomic Force Microscopy of Dextran Monolayer Hydration Richard A. Frazier,† Martyn C. Davies,*,† Gert Matthijs,‡ Clive J. Roberts,*,† Etienne Schacht,‡ Saul J. B. Tendler,*,† and Philip M. Williams† Laboratory of Biophysics and Surface Analysis, Department of Pharmaceutical Sciences, The University of Nottingham, University Park, Nottingham, NG7 2RD, U.K., and Biomaterial and Polymer Research Group, Department of Organic Chemistry, University of Gent, Krijgslaan 281, B-9000 Gent, Belgium Received March 17, 1997. In Final Form: July 10, 1997X Atomic force microscopy has been employed for the in situ investigation of the molecular hydration of thiolated dextran monolayers within a liquid environment. The studies have demonstrated the ability to measure corresponding changes in both monolayer morphology and elasticity due to the hydration state of dextran. Imaging in water allows the visualization of macromolecular swelling, matched by an increase in surface elasticity monitored via the analysis of force-distance curves. Subsequent imaging in a propanol environment leads to dehydration effects observed through a relaxation of swelling and a decrease in surface elasticity.
Introduction Dextran is a hydrophilic polysaccharide that has potential applications as a protein resistant surface coating,1-4 as a hydrogel matrix for site specific drug delivery,5-8 and as a monolayer coating for chromatography applications.9-11 Previously, we have explored the application of atomic force microscopy (AFM) to understand monolayer formation by imaging thiolated dextran monolayers chemisorbed to gold in air.12 We have elucidated the variation in size and coverage of individual molecules as a function of dextran molecular weight in the dry state. In order to achieve a better understanding of the interaction behavior of such a hydrophilic macromolecule monolayer with biomolecules within an aqueous environment, such as that presented in vitro and in vivo for biomedical applications, it is important to consider the hydration behavior. AFM may provide high-resolution * To whom correspondence should be addressed at The University of Nottingham. † The University of Nottingham. ‡ University of Gent, X Abstract published in Advance ACS Abstracts, August 15, 1997. (1) (a) O ¨ sterberg, E.; Bergstro¨m, K.; Holmberg, K.; Riggs, J. A.; Van Alstine, J. M.; Schuman, T. P.; Burns, N. L.; Harris, J. M. Colloids Surf. A: Physicochem. Eng. Aspects 1993, 77, 159. (b) O ¨ sterberg, E.; Bergstro¨m, K.; Holmberg, K.; Schuman, T. P.; Riggs, J. A.; Burns, N. L.; Van Alstine, J. M.; Harris, J. M. J. Biomed. Mater. Res. 1995, 29, 741-747. (2) Marchant, R. E.; Yuan, S.; Szakalas-Gratzl, G. J. Biomater. Sci., Polym. Ed. 1994, 6, 549. (3) Fournier, C.; Leonard, M.; Le Coq-Leonard, I.; Dellacherie, E. Langmuir 1995, 11, 2344. (4) Frazier, R. A.; Davies, M. C.; Matthijs, G.; Roberts, C. J.; Schacht, E.; Tasker, S.; Tendler, S. J. B. In Surface Modification of Polymeric Biomaterials; Ratner, B. D., Castner, D. G., Eds.; Plenum Press: New York, 1996; pp 117-128. (5) Langer, R. Acc. Chem. Res. 1993, 26, 537. (6) Kamath, K. R.; Park, K. Polym. Gels Networks 1995, 3, 243. (7) Kurisawa, M.; Terano, M.; Yui, N. Macromol. Rapid Commun. 1995, 16, 663. (8) (a) Brøndsted, H.; Hovgaard, L.; Simonsen, L. S.T.P. Pharma Sci. 1995, 5, 60. (b) Hovgaard, L.; Brøndsted, H. J. Controlled Release 1995, 36, 159. (9) Karim, M. R.; Janson, J.-C.; Takagi, T. Electrophoresis 1994, 15, 1531. (10) Petro, M.; Gemeiner, P.; Berek, D. J. Chromatogr., A 1994, 665, 37. (11) Oscarsson, S. J. Chromatogr., B 1995, 666, 21. (12) Frazier, R. A.; Matthijs, G.; Davies, M. C.; Roberts, C. J.; Schacht, E.; Tendler, S. J. B.; Williams, P. M. Submitted to Langmuir.
S0743-7463(97)00288-6 CCC: $14.00
images of surface topographies in three dimensions under liquid environments.13 Not only can AFM monitor surface topography, but it can also be utilized as a powerful tool for probing local surface properties such as adhesion14-17 and elasticity.18,19 Here, the hydration of individual dextran molecules has been explored by employing AFM within experiments to image a thiolated dextran monolayer under water and then propanol in situ. By imaging under water, it was intended that the effect of swelling could be detected from topographical changes in the AFM images. Imaging in propanol was undertaken with reference to previous work by Radmacher and co-workers,19 in which a repeatable reversal of gelatin film hydration was observed by replacing water with propanol. In parallel with the acquistion of topographical images, the surface elasticity of the thiolated dextran monolayers was monitored by the analysis force-distance curve measurements. Experimental Section Thiolated dextran (Mw ) 70 kDa, 4% -SH) was a 2-mercaptoethylcarbamoyl-dextran prepared via a 4-nitrophenylchloroformate activated dextran species as described previously.12,20 Samples were prepared by overnight incubation for at least 18 h of a 1 cm × 1 cm gold substrate, epitaxially grown on mica,21 in a 1 mg/mL aqueous solution of thiolated dextran. Immediately prior to study, the dextran monolayer coated gold substrate was removed from solution, rinsed with deioinized water, and dried by capillary action using filter paper. (13) (a) Duc, T. M. Surf. Rev. Lett. 1995, 2, 833. (b) Bottomley, L. A.; Coury, J. E.; First, P. N. Anal. Chem. 1996, 68, 185R. (14) Lee, G. U.; Kidwell, D. A.; Colton, R. J. Langmuir 1994, 10, 354. (15) Florin, E.-L.; Moy, V. T.; Gaub, H. E. Science 1994, 264, 415. (16) Noy, A.; Frisbie, C. D.; Rozsnyai, L. F.; Wrighton, M. S.; Lieber, C. M. J. Am. Chem. Soc. 1995, 117, 7943. (17) Allen, S.; Davies, J.; Dawkes, A. C.; Davies, M. C.; Edwards, L. C.; Parker, M.-C.; Roberts, C. J.; Sefton, J.; Tendler, S. J. B.; Williams, P. M. FEBS Lett. 1996, 390, 161. (18) Burnham, N. A.; Colton, R. J.; Pollock, H. M. Nanotechnology 1993, 4, 64. (19) Radmacher, M.; Fritz, M.; Hansma, P. K. Biophys. J. 1995, 69, 264. (20) Vandoorne, F.; Permentier, D.; Vercauteren, R.; Schacht, E. Macromol. Chem. Phys. 1985, 186, 2455-2460. (21) (a) DeRose, J. A.; Thundat, T.; Nagahara, L. A.; Lindsay, S. M. Surf. Sci. 1991, 256, 102. (b) DeRose, J. A.; Lampner, D. B.; Lindsay, S. M.; Tao, N. J. J. Vac. Sci. Technol., A 1993, 11, 776.
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Figure 1. A 1 µm2 gray scale AFM scan of an epitaxially grown gold film on mica. The surface topography consists of large atomically flat gold islands. AFM contact mode imaging and the acquisition of forcedistance curves was performed with a Topometrix TMX 2000 Explorer (Topometrix Corp., Saffron Walden, Essex, U.K.) using a silicon nitride probe mounted on a triangular cantilever.
Results and Discussion The epitaxially grown gold film substrate consisted of large atomically flat islands, typically extending over a few hundred nanometers as shown in Figure 1. Occasionally, as in this image, small globular contaminants
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were found at the adjoining edges of adjacent islands; however, the flat regions of the islands were always found to be clean and featureless. AFM images of the thiolated dextran layer were first taken in air before exposure to a liquid imaging environment. A typical image, which is consistent with AFM data described previously,12 is shown in Figure 2a and consists of a dense coverage of globular features forming a monolayer on the gold substrate. Contours in the image result from the influence of the underlying gold island topography. A force-distance curve recorded in air from this area, labeled “air” in Figure 3a, revealed a linear relationship between z-height (distance) and cantilever deflection (force) before and after a sharp probe-sample contact, which indicated a hard surface.18,19 After the presence of a complete and homogeneous dextran layer had been confirmed by achieving similar AFM images and force-distance curves from several areas of the same sample, it was then possible to commence imaging under liquid. Parts b-f of Figure 2 depict a series of AFM images of the dextran layer at sequential incubation time points in water. In Figure 3a, a parallel series of force-distance curves are shown, which were recorded alongside the acquistion of the images in Figure 2. The first AFM image, shown in Figure 2b, was recorded in water after a time period of 5 min exposure in water had elapsed. The topography of the dextran layer has changed from that typically observed in air, and a coverage of discrete, almost spherical features is now evident. After a time period of 8 min in water, a similar AFM scan was produced in Figure 2c. In this figure, however, deformation of the surface features in the probe’s scanning direction was evident, as well as a loss of discrete definition. This loss of feature definition continued as incubation time progressed through 35, 60, and 70 min as shown in parts d, e, and f of Figure
Figure 2. A series of 1 µm2 AFM images of a thiolated dextran layer on gold (a) in air and after (b) 5 min, (c) 8 min, (d) 35 min, (e) 60 min, and (f) 70 min incubation in water. As the hydration time increases, so the dextran features can be observed to swell and coalesce to form a continuous film.
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Figure 3. (a) Force-distance curves of probe z-height (distance) against cantilever deflection (force) taken in different imaging media during the hydration/dehydration of the thiolated dextran layer. (b) A schematic representation of the sample elasticity conditions influencing the approaching tip and the resultant force-distance curve.
2, respectively. Each image shows a further loss of feature definition until, after 1 h, evidence of any discrete dextran layer features was absent. The topography in parts e and f of Figure 2 was indicative of a flat and featureless dextran monolayer film, following the contoured outlines of the gold islands. The progression of image topography recorded during hydration of the thiolated dextran monolayer suggested that hydration caused a gradual swelling and coalesence of the discrete dextran molecules to form a continuous swelled film. A consequential increase in molecular volume led initially to improved image contrast prior to the onset of coalesence. Indeed, this was supported by inspection of force-distance curves recorded at time intervals of 1, 10, 20, 30, and 60 min during the hydration experiment. Figure 3a includes these curves as plots of probe z-height (distance) versus cantilever deflection (force). A gradual increase in the sample’s elastic modulus was noted as the incubation time in water increased. This was manifested as a loss of linearity in the contact region of the curves, which indicated deformation of the soft surface by the approaching probe’s tip, as shown schematically in Figure 3b. Unlike in air, the point of contact
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was ill-defined and did not result in a sharp gradient change. Instead, a gradual change in the curve’s gradient was observed, which occurred over a longer z-height scale as the hydration progressed. A similar series of events affecting topography and sample elasticity upon hydration in water has been previously reported for multilayer gelatin films on mica.19 It was summized that the increased water content in the swollen films led to a lowered elastic modulus, which in turn allowed a greater indentation of the sample by the imaging AFM probe and, thus, created a larger probesample contact area.22,23 Consequently, the changes in image topography were thought to arise merely due to a decrease in image resolution and not due to any intrinsic changes in the actual sample topography. A reversal of this phenomenon occurred by replacing water in the imaging chamber with propanol, upon which an increase in image resolution was observed. In a propanol environment, the gelatin layer was assumed to dehydrate and become stiffer, thereby reducing the probe-sample contact area. This work was of a multilayer gelatin film system,19 of thickness in the order of 50-60 nm measured from the indentation of the films by the AFM probe during forcedistance curve acquisition. However, for the hydration of dextran here, a tethered molecular monolayer was studied, which allowed the visualization, for the first time, of the effect of macromolecular hydration at such high resolution. To reverse the hydration process, water was replaced by propanol to cause dehydration.19 Figure 4 shows this in a series of images showing the changes in AFM image topography of the dextran layer upon incubation in propanol. After 5 min in propanol, Figure 4b shows a dramatic emergence of the resolution of discrete features as compared to the image taken after 70 min in water shown in Figure 4a. Discrete monolayer features are discernable in Figure 4 that were not detected in the previous image. Parts c and d of Figure 4 show the dextran layer after 10 and 20 min in propanol, respectively. In these AFM images, the resolution of discrete globular features has clearly improved. A dense and uniform coverage of discrete globular monolayer features was displayed in both images. The force curve recorded after 20 min in propanol, labeled “propanol” in Figure 3a, was almost identical to that recorded earlier in air. A linear deflection of the cantilever was measured upon a sharply defined probe-sample contact, indicating stiffness as a consequence of the significant dehydration of dextran. In summary, the use of a monolayer system in place of a multilayer film19 enabled the first application of AFM for the visualization of macromolecular hydration at high resolution in situ by both topography and force-distance measurement. In air the dextran monolayer consisted of a closely-packed coverage of globular surface units. Upon introduction of water into the imaging environment, the dextran monolayer was seen to swell, and its surface features coalesce to form a continuous film coverage. Force-distance measurements indicated a correspondingly gradual increase in sample elasticity with hydration. Replacement of water with propanol brought about a reversal in the hydration process, and the dextran layer was significantly dehydrated to revert to a stiff, globular morphology. The elasticity behavior observed within the measurement of force-distance data concurs with the previous literature reports of bulk gelatin film hydration.19 However, by employing a monolayer system, we were able (22) Weihs, T. P.; Nawaz, Z.; Jarvis, S. P.; Pethica, J. P. Appl. Phys. Lett. 1991, 59, 3536. (23) Shao, Z.; Yang, J. Q. Rev. Biophys. 1995, 28, 195.
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Figure 4. 1 µm2 AFM scans showing the dehydration of dextran following replacement of water with propanol. Images show the dextran layer after (a) 70 min incubation in water and (b) 5 min, (c) 10 min, and (d) 20 min incubation in propanol. The layer gradually dehydrates to re-form a globular morphology.
to overcome the adverse effects of bulk film hydration upon image resolution to interrogate the topographical changes associated with hydration at the molecular level.
Programme. R.A.F. thanks Courtaulds PLC for the funding of his PhD Research Studentship. S.J.B.T. is a Nuffield Foundation Science Research Fellow.
Acknowledgment. The authors acknowledge the funding of the European Community BRITE/Euram
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