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Molecular Resolution Imaging of Dextran Monolayers Immobilized on Silica by Atomic Force Microscopy S. Tasker, G. Matthijs,† M. C. Davies,* C. J. Roberts,* E. H. Schacht,† and S. J. B. Tendler* Laboratory of Biophysics and Surface Analysis, Department of Pharmaceutical Sciences, The University of Nottingham, University Park, Nottingham NG7 2RD, U.K., and Laboratory of Organic Chemistry, Department of Polymer Chemistry, University of Gent, Krijgslaan 281, B-9000 Gent, Belgium Received November 21, 1995. In Final Form: August 13, 1996X A range of dextrans (5, 10, and 70 kDa) were covalently bound to smooth silica slides and porous (500 Å) silica particles with an external diameter of 25 µm. AFM analysis of the slides in air has shown the presence of discrete, clustered, and in a few regions overlayered dextran molecules, which exhibit increasing diameter and decreasing packing densities with increasing molecular weight. Analysis of 10 and 70 kDa dextran bound to silica particles has revealed surface features consistent with those of the dextran molecules, superimposed upon the morphology of the underlying silica particle.
Introduction Dextran is a branched (1-6) linked R-D-glucan polysaccharide, one of a class of very flexible and extended polymers1 which have found uses as blood plasma extenders2-3 and gel media for bioseparation.5-7 Matrices based on cross-linked dextrans have for many years been used in aqueous size exclusion chromatography8-10 applications. Unfortunately, these soft gels cannot be used in high-performance liquid chromatography due to their low mechanical stability at high pressure. In contrast, silica is mechanically stable but contains acidic silanol groups that cause strong and often irreversible nonspecific adsorption of proteins in aqueous media.11-13 In recent years, coatings derived from polysaccharides have been recognized as efficient methods to prevent nonspecific adsorption of proteins at such surfaces.14-17 The coating leads to a reduction in interfacial energy and acts as a neutral, hydrated, steric barrier. In the field of chromatography, the coating of silica beads with polysaccharides * To whom correspondence should be addressed at The University of Nottingham. † University of Gent. X Abstract published in Advance ACS Abstracts, November 1, 1996. (1) Barham, P. J.; Arkinsw, E. D. T.; Nieduszynski, I. A. N. Polymer 1974, 15, 762. (2) Koester, K. H.; Schwarz, M.; Sele, V.; Sindrup, E. Lancet 1957, 262. (3) Shoemaker, W. C. Crit. Care Med. 1976, 4, 71. (4) Bergentz, S. E. World J. Surg. 1978, 2, 19. (5) Flodin, P.; Ingelman, B. Swedish Patent 169,293, 1959. (6) Flodin, P.; Porath, J. U.S. Patent 3,002,823, 1961. (7) Petro, M.; Gemeiner, P.; Berek, D. J. Chromatogr., A 1994, 665, 37. (8) Porath, J.; Flodin, P. Nature 1959, 183, 1657. (9) Determan, H. Gel Permeation Chromatography; Springer: Berlin, 1967. (10) Regnier, F. E.; Noel, R. J. Chromatogr. Sci. 1976, 14, 316. (11) Mizutani, T.; Mizutani, A. J. Chromatogr. Sci. 1976, 111, 216. (12) Unger, K. K. Porous Silica, Its Properties and Use as Support in Liquid Chromatography; Elsevier: Amsterdam, 1979. (13) Engelhardt, H.; Mathes, D. J. Chromatogr. 1977, 142, 31. (14) Mandenius, C. F.; Mosbach, K.; Welin, S.; Lundstro¨m, I. Anal. Biochem. 1986, 157, 283. (15) Elam, J. H.; Nygren, N.; Stenberg, M. J. Biomed. Mater. Res. 1984, 18, 953. (16) Malmsten, M.; Lindman, B.; Holmberg, K.; Brink, C. Langmuir 1991, 7, 2412. (17) Osterberg, E.; Bergstrom, K.; Holmberg, K.; Riggs, J. A.; Van Alstine, J. M.; Schuman, T. P.; Burns, N. L.; Harris, J. M. Colloids Surf., A 1993, 77, 159.
S0743-7463(95)01087-0 CCC: $12.00
has made it possible to combine the advantages of traditional soft gels with the mechanical properties of silica supports. The synthesis and evaluation of dextran bonded silicas has been reported previously by Matthijs et al.18 This work exploited the activation of dextran with amine reactive carbonates using p-nitrophenyl chloroformate which is coupled to an aminopropyl-modified silica. It has been demonstrated that the dextran layer formed an efficient neutralizing barrier between residual underlying propylamines and a range of proteins in solution, resulting in the elimination of non-size exclusion effects. However, little information is available on the nature and composition of the dextran layer on the silica surface. In this paper, we exploit the application of atomic force microscopy (AFM) for the analysis of the dextran coating at the silica interface. The invention of AFM19 in 1986 was a significant development in surface imaging technology, which has enabled the topographical analysis of nonconducting material surfaces under ambient conditions20 and liquids at the atomic and molecular level.21 The technique measures the short range interaction forces between an atomically sharp probe tip mounted upon a sprung cantilever and the surface of the material under investigation. Operating the apparatus in constant force mode and scanning the sample beneath the tip in a raster fashion allows a three-dimensional topographical map of the surface to be acquired. AFM has enabled a wider understanding of many interfacial phenomena. The AFM imaging of biological macromolecules adsorbed onto well defined substrates has provided valuable information about their conformational structure,22 molecular ordering in polymers has been observed,23 and the ability to image surfaces under liquids (18) Matthijis, G.; Schacht, E. J. Chromatogr., in press. (19) Binning, G.; Quate, C. F.; Gerber, C. Phys. Rev. Lett. 1986, 56, 930. (20) Drake, B.; Prater, C. B.; Weisenhorn, A. L.; Gould, S. A. C.; Albrecht, T. R.; Quate, C. F.; Cannell, D. S.; Hansma, H. G.; Hansma, P. K. Science 1988, 243, 332. (21) Weihs, T. P.; Nawaz, Z.; Jarvis, S. P.; Pethica, J. B. Appl. Phys. Lett. 1991, 59, 3536. (22) Cullen, D. C.; Lowe, C. R. J. Colloid Interface Sci. 1994, 166, 102. (23) Stocker, W.; Magonov, S. N.; Cantow, H. J.; Wittman, J. C.; Lotz, B. Macromolecules 1993, 26, 5915.
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has allowed the observation of dynamic events such as the degradation of blended polymer mixtures.24 Although several publications have used scanning probe microscopy (SPM) to investigate the fibrillar structure of polysaccharides such as cellulose25 and xanthan,26 no studies are known to have investigated polysaccharides covalently bound or adsorbed to a well defined surface using AFM. To date, the imaging of bonded polymersilica nanoparticulate systems has proven extremely difficult due to the size and roughness of the silica beads. Consequently, there is little understanding of how the polymer layer binds to the surface or how the immobilization process might affect the structure of a polysaccharide. Here, AFM has been used to image the surface topography of dextran coatings on two specific silica substrates: firstly, flat silicon wafers which provide a laterally smooth topography for AFM analysis and act as a model for the silica particles and, secondly, silica particles currently under development for chromatographic separations. The dextran layers were prepared from a series of dextrans of different defined molecular weights. In these studies, AFM analysis has attempted to probe the lateral distribution and thickness of the polysaccharide coating to molecular resolution.
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a
b
c
Experimental Section Materials. The spherical silica Nucleosil 25.500 (mean pore diameter, 50 nm; surface area, 35 m2/g; size, 25-40 µm) was purchased from Macherey-Nagel (Du¨ren, Germany). The glass slides were microscope coverslips of 16 mm diameter, manufactured by Chance Propper Ltd. (Smethwick, Warley, U.K.). The 5, 10, and 70 kDa dextrans were purchased from Pharmacia (Uppsala, Sweden) and used without further purification. (Dimethylamino)pyridine and (3-aminopropyl)triethoxysilane were obtained from Aldrich (Bornem, Belgium). p-Nitrophenyl chloroformate was from Merck (Darmstadt, Germany). Preparation of Dextran-Coated Silica Particles. The schematic for the preparation of the dextran-coated particles is shown in Figure 1. Phosphorus pentoxide-dried dextran (4 g) was dissolved in 200 mL of a dimethyl sulfoxide/pyridine mixture (1:1). (Dimethylamino)pyridine (0.2 g) and p-nitrophenyl chloroformate (2.5 g) were added at 0 °C. After 4 h, the activated dextran was precipitated in a mixture of dry ethanol/diethyl ether (4:1). The suspension was filtered and washed several times with an ethanol/diethyl ether mixture (4:1) until the wash solvent was free of p-nitrophenol, which was determined by its yellow color in alkaline solution. (Aminopropyl)silica was prepared as reported previously.18 The following reaction conditions were chosen in order to maximize the coverage of dextran for all cases. Only dry solvents were used, minimizing moisture exposure. (Aminopropyl)silica (5 g) was suspended in 60 mL of dimethyl sulfoxide/pyridine (1:1) and degassed using ultrasonic vibration. While the suspension was gently stirred, chloroformate-activated dextran (3.5 g), dissolved in 60 mL of dimethyl sulfoxide was added dropwise over a 1 h period. After 24 h of stirring, the silica beads were filtered and washed three times with 20 mL of dimethyl sulfoxide, ten times with 100 mL of water, and three times with 20 mL of ether, respectively. The dextran-coated silica beads were dried for 24 h at 65 °C prior to AFM analysis. Preparation of Dextran-Coated Silica Slides. The glass slides were previously cleaned in a boiling solution of 10% v/v nitric acid. After 1 h, the slides were washed with distilled water and dried at 110 °C. The immobilization of dextran on the silica slides was performed in an identical manner to that on silica particles except that the slides were mounted in an aluminium holder to avoid mechanical damage by the stirrer within the (24) Shakesheff, K. M.; Davies, M. C.; Tendler, S. J. B.; Shard, A. J.; Domb, A. Langmuir 1994, 10, 4417. (25) hanley, S. J.; Giasson, J.; Revol, J.-F.; Gray, D. G. Polymer 1992, 33, 4639. (26) Gunning, A. P.; McMaster, T. J.; Morris, V. J. Carbohydr. Polym. 1993, 21, 47.
Figure 1. Preparation of the immobilized dextran: amination of the silica substrate using (aminopropyl)triethoxysilane (a), modification of the dextran with 4-nitrophenyl chloroformate to produce the reactive carbonate (20% conversion) (b), and immobilization of the modified dextran on the aminated silica substrate (c). reaction vessel. The dextran-coated glass slides were allowed to dry in air for several days prior to analysis. Sample Analysis. The dextran-coated glass slides were cut into approximately 0.5 × 0.5 cm2 fragments for AFM analysis and mounted on 1 cm diameter AFM sample stubs using cyanoacrylate adhesive. The coated silica particles (25 µm diameter) were prepared for AFM analysis by embedding at 60 °C in thermoplastic adhesive in accordance with the procedure described by Shakesheff et al.,27 producing samples where the apex of the particles protruded above the surface level of the polymer film. AFM analysis was performed using a Polaron SP300 atomic force microscope (VG Microtech, Uckfield, U.K.), using prefabricated cantilevers (Park Scientific, CA) with theoretical spring constants of 0.01 and 0.03 nN/m. The AFM images were acquired in contact mode with nominal applied force of 0.2 nN at a scan frequency of 10 Hz. The images are presented as threedimensional gray scale representations. Four areas were selected at random for each of the samples analyzed by AFM; the images shown in the following section were chosen as the most representative in each case. Measurements to determine the average molecular sizes of the features were taken from all of the sampled areas.
Results and Discussion AFM Analysis of Dextran-Coated Silica Slides. A typical AFM image of the aminated silica slides is shown in Figure 2a, which displays a smooth topography broken up by a few areas of pitting. The flat interface highlights the suitability of this substrate for the visualization of immobilized dextran using AFM. The coupling of 5, 10, and 70 kDa dextran onto the silica slides resulted in a remarkably different topography, as observed in the AFM images in parts b, c, and d, respectively, of Figure 2. Ellipsoidal and spherical (27) Shakesheff, K. M.; Davies, M. C.; Jackson, D. E.; Roberts, C. J.; Tendler, S. J. B.; Brown, V. A.; Watson, R. C.; Barrett, D. A.; Shaw, P. N. Surf. Sci. Lett. 1994, 304, L393.
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Figure 2. AFM images (1 µm × 1 µm) of silica slides, following amination with H2NCH2CH2Si(OEt)3 (a), showing 5 kDa (b), 10 kDa (c), and 70 kDa (d) covalently bound dextran.
features were resolved in each case. These features were particularly stable under the imaging conditions employed, and we attribute them to individual dextran molecules. The images of 5 and 10 kDa dextran coupled to silica in parts b and c of Figure 2 show a high density of packing of the polymer molecules, and this suggests that complete monolayer coverage has been achieved. In the case of 70 kDa dextran shown in Figure 2d, a reduction in the packing density of the molecules can be observed and the distribution of features on the surface is more uneven compared to those of the lower molecular weight analogues. Areas of more densely distributed dextran molecules are apparent just to the right of center in Figure 2d (arrowed region), leading to features resembling clusters consisting of approximately 10-15 dextran molecules. The overall shape of the molecules deserves detailed consideration. The possible artifactual nature of the shape of the dextran molecules attributable to the AFM imaging process was investigated; however, repeated scanning of the sample using different scanning directions was found not to alter the acquired image. We were able to conclude that the observed images were a true reflection of the bound dextran layer. In order to understand the variation in molecular shapes observed on the surface, it must be realized that the degree of hydration of dextran plays a major role in defining its molecular conformation, which in turn affects its shape. Intramolecularly bound water molecules act as structural
bridging units between the hydroxyl groups in the molecule, and previous work28 has shown that fully hydrated dextran molecules adopt an almost spherical conformation, whereas ellipsoidally shaped molecules have been attributed to dehydrated dextran.29 Consequently the spherical and ellipsoidal shaped features observed in the images of immobilized dextran in Figure 2b-d could be taken to indicate dextran in varying hydration states which may arise due to adsorbed atmospheric water vapor (relative humidity ≈40%) present under these imaging conditions. Even so, given the long term exposure of the immobilized layer to atmospheric water vapor it would seem reasonable to assume that an equilibrium swelling of the dextran would be accomplished and that the degree of hydration in each case would be similar, giving rise to a regular distribution of molecular shapes and not the mix of spherical and ellipsoidal species observed in Figure 2b-d. An alternative explanation for the range of molecular shapes could be attributed to the random orientation of the polysaccharide molecules on the surface during the immobilization process. For example, endwise alignment of some of the molecules may cause them to appear spherical in shape whereas other orientations will result in ellipsoidally shaped molecules. This suggestion becomes more plausible when we consider that the dextran molecules were prepared under anhy(28) Ogston, A. G.; Woods, E. F. Trans. Faraday. Soc. 1954, 50, 635. (29) Ingelman, B.; Halling, M. S. Ark. Kemi 1949, 1, 61.
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drous conditions, should be largely dehydrated, and adopt ellipsoidal shapes. A final explanation for the range of molecular shapes could be found in the method by which the dextran was immobilized on the silica substrate. We have little information concerning the number of coupling reactions required to immobilize each dextran molecule although it seems likely that a variation in the number of covalent links between each molecule may lead to a distortion of its molecular structure once the solvent was removed. If we consider an ideally packed two dimensional arrangement of dextran molecules within the monolayer, then a pseudo-hexagonally close packed arrangement of molecules would be expected to enable the greatest density of bound molecules within a given area of the surface. This type of packing would minimize the proportion of unoccupied free space between the molecules and make most efficient use of the available binding sites. However the packing threshold of molecules in such a twodimensional array is determined by the molecular size of the species concerned; for example, larger molecules arranged within the same area of the sample would be expected to leave a greater proportion of the potential binding sites free. Consequently, limitations to this type of packing regime become apparent with the increasing molecular weight of the dextran. Close examination of the AFM images in Figure 2b-d reveals a small decrease in the packing efficiency on going from 5 to 10 kDa dextran; however, the immobilized 70 kDa dextran is significantly less densely packed in comparison to the previous two images, although this does not explain the presence of areas exhibiting higher and lower densities of dextran molecules. It is apparent that such a simple appraisal of the packing of dextran on the silica surface is inadequate, since it assigns equal probability to the reaction of each dextran molecule with the silica substrate and treats the problem purely in terms of the spatial limitations associated with arranging the molecules in a monolayer. In order to explain the nature of the monolayer formation in Figure 2b-d more effectively, the reactivity of the dextran with respect to the substrate must be considered. It must be pointed out that the polymeric structure of dextran in aqueous solution has been described as an extendable coil with a high degree of conformational freedom,30 and this mobility is derived from the relatively linear, unbranched nature of the dextran backbone. At higher molecular weights however the polymer chains become increasingly branched; this leads to reduced conformational mobility,31,32 and the polymer is less able to reorientate its structure. The flexibility of the polysaccharide may have twofold significance: firstly, reorientation of the dextran structure may enable more efficient derivatization, leading to a greater number of potential coupling sites being present on the molecule; secondly greater structural flexibility will allow the molecule to conformationally adapt in response to the interface, and this will enable more efficient binding, particularly at sterically hindered or poorly accessible sites of reaction. The average diameter of the dextran molecules was determined on the basis of n ) 20 measurements from each image. In the case of ellipsoidally shaped molecules a measurement of shortest axis was made in order to (30) Belder, A. N. In Industrial GumssPolysaccharides and their derivatives, 3rd ed.; Whistler, R. L., BeMiller, J. N., Eds.; Academic Press: New York, 1993. (31) Taylor, N. W.; Zobel, H. F.; Hillman, N. N.; Senti, F. R. J. Phys. Chem. 1959, 63, 599. (32) Taylor, N. W.; Clusby, J. E.; Senti, F. R. J. Phys. Chem. 1961, 65, 1810.
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Figure 3. Schematic representation of the AFM analysis of the dextran-silica system. Table 1. Average Values for the Diameters of the Dextran Microspheres Determined from the ATomic Force Micrographs of 5, 10, and 70 kDa Dextran Bound to Silica Slides Compared with the Molecular Diameter Derived from the Expression for the Theoretical Hydrodynamic Radius of Gyration (Rg)33 of the Respective Dextran in Solution molecular weight of dextran (kDa) 5 10 70
average diameter observed by AFM (nm) (n ) 20) 29 ( 6 43 ( 10 75 ( 12
diameter calculated from Rg (nm) 4.2 7.0 16.0
alleviate problems in determining the molecular dimensions which might arise due to the random orientation of the molecules on the surface. These data are presented in Table 1. These values are compared to the molecular diameters from the theoretical hydrodynamic radius of gyration calculated using the empirically derived expression33
Rg ) 0.66M0.43 where Rg is the radius of gyration (in Å) and M is molecular weight. The size of the molecules determined from the atomic force micrographs increases with increasing molecular weight, as expected. However, the average molecular diameters are approximately five times larger than the dimensions calculated from the theoretical hydrodynamic radii of the respective dextran molecules in aqueous solution. Several reasons could account for this discrepancy. Broadening of features during AFM analysis has been well documented34,35 and is attributed to the finite diameter of the probe tip. Interaction of the sample with areas of the tip other than the apex may lead to ‘tip selfimaging’ and prevent optimum profiling of the edges of molecular features. Therefore the image can to some extent be considered a convolution of the probe geometry and the ‘real’ surface topography. In addition, the high mechanical compliance of dextran molecules could lead to compression of the immobilized layer during the imaging process; this is illustrated schematically in Figure 3. A combination of these effects would reduce the apparent (33) Perez, E.; Proust, J. E. J. Phys. Lett. 1985, 46, 279. (34) Glaseby, T. O.; Batts, G. N.; Davies, M. C.; Jackson, D. E.; Nicholas, C. V.; Purbrick, M. D.; Roberts, C. J.; Tendler, S. J. B.; Williams, P. M. Surf. Sci. 1994, 318, L1219. (35) Williams, P. M.; Shakesheff, K. M.; Davies, M. C.; Jackson, D. E.; Roberts, C. J.; Tendler, S. J. B. Langmuir, submitted.
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Figure 4. AFM images of 10 kDa dextran on silica slides: 5 µm × 5 µm scan (a) and 2 µm × 2 µm scan (b) showing overlayer and cluster formation.
vertical height and exaggerate the lateral dimensions of the immobilized dextran. The AFM images presented in Figure 2b-d show covalently bound dextran molecules forming what appear to be monolayer coverages on the silica substrate. Continuous monolayer formation was observed for the majority of areas examined in each case, although a few areas were found to exhibit very poor coverages of dextran which could be attributed to mechanical damage during the preparation and handling of the samples prior to examination. More interestingly, a 5 µm × 5 µm scan of 10 kDa dextran is presented in Figure 4a, and a raised dextran-coated region 10-15 nm higher than the surrounding area is clearly apparent. A higher resolution image of the top right area of Figure 4a is presented in Figure 4b, and molecular features have again been clearly resolved in both the raised and lower areas which resemble the monolayer coverages found in Figure 2c. However, several densely clustered patches are present within the raised area which appear similar to those observed for the 70 kDa dextran sample discussed earlier. It is conceivable that the raised areas could be due to inhomogeneities in the silica substrate. The presence of terraced regions would be reflected in the overall topography of the dextran monolayer. However, thorough examination of the clean aminated substrate failed to confirm this, and inhomogeneities which could have caused the features observed in Figure 4a were not present in
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any of the regions examined. Surprisingly it seems likely that the raised areas in Figure 4 are due the formation of an adsorbed overlayer of dextran although it is not immediately clear how these regions arise and given their formation, why they were found in only a few parts of the sample surface. An examination of the published literature reveals that the aggregation of dextran in solution has been extensively studied36-38 and attributed to the formation of hydrogen-bonded networks which at high concentrations (ca. 40% w/v) have resulted in hydrogel formation. In light of these studies, it seems reasonable to suggest that the raised area could be a hydrogen-bonded layer of dextran. This may also explain the clustered regions which could have resulted from the formation of small aggregates of dextran in solution which have subsequently adsorbed. AFM Analysis of Embedded Dextran-Coated Silica Particles. Analysis of the uncoated 25 µm silica particles embedded in thermoplastic adhesive yielded images which are consistent with previous AFM studies of silica.27 Figure 5a represents a 1 µm × 1 µm scan of an uncoated silica particle from which it is apparent that the silica particle is a fused aggregate of silica nanoparticles with dimensions on the order of 50-100 nm. The pore structure of the silica particle can be seen in Figure 5a as deep channels in between the nanoparticulate species. AFM images of silica particles coated with 10 and 70 kDa dextran are presented in parts b and c, respectively, of Figure 5, and the change in the morphology of the coated compared to the uncoated particles is clearly demonstrated. The surface topography can be described as a combination of the gross morphology measured in the case of the uncoated silica particle and additional features attributable to the immobilized dextran layer superimposed over it. Despite the porous, heterogeneous nature of the substrate, individual dextran molecules have been successfully resolved for both of the 10 and 70 kDa dextran layers, and these are fairly evenly distributed across both the areas shown in Figure 5b and c. In agreement with the earlier observations for dextran immobilized on smooth silica slides, the increasing molecular weight of the polysaccharide has led to a reduction in the packing density and an increase in the size of the molecular features. Reassuringly, these results are in excellent agreement with the findings of Matthijis et al.,18 who showed lower coverages of the 70 kDa dextran bound to silica particles. This was inferred by the titration of the residual propylamine content using picric acid39 in dichloromethane. In Figure 5b the molecular features are so densely packed that distinguishing them is quite difficult, and it appears that the shape of the molecules has become distorted compared to the well defined spherical and ellipsoidal features observed earlier. Distortion of the 70 kDa dextran molecules shown in figure 5c is also apparent, although it is much less pronounced and some of their ellipsoidal shape has been retained. This supports the earlier evidence which suggested that, in order to maximize the density of molecules on the surface of the silica, dextran would have to alter its conformational structure in order to maintain an appropriate number of covalent links with the substrate. Consequently the distortion of the individual dextran molecules is much more pronounced when they are immobilized on a topographically hetero(36) Jeanes, A.; Schieltz, N. C.; Wilham, C. A. J. Biol. Chem. 1948, 176, 617. (37) Cadwallader, D. E.; Becker, C. H.; Winters, J. H.; Morans, D. J. Am. Pharm. Assoc. 1958, 47, 895. (38) Aizawa, M.; Suzuli, S.; Tatuo, K.; Nonyasu, N.; Yukata, I. Bull. Chem. Soc. Jpn. 1976, 49, 2061. (39) Alpert, A. J.; Reigner, F. E. J. Chromatogr. 1979, 185, 1895.
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agreement between the two systems; however, the small size of the molecules relative to the inhomogeneous nature of the substrate prevents the accurate measurement of the molecular dimensions. Conclusions
Figure 5. AFM images (1 µm × 1 µm) of silica particles, following amination with H2NCH2CH2Si(OEt)3 (a), showing 10 kDa (b) and 70 kDa (c) covalently bound dextran.
geneous silica particle, since the molecules are required to adopt more complex shapes to achieve the most energetically favorable configuration. In addition, the greater stability of the 70 kDa dextran suggests that the degree of molecular freedom is reduced for the higher molecular weight species. A qualitative comparison of the sizes of the molecules measured on the silica particles and those immobilized on the flat substrate shows good
Atomic force analysis of dextran-coated silica slides has clearly demonstrated the presence of a covalently bound dextran layer consisting of discrete spherical and ellipsoidal shaped polymer molecules, and these represent the first well resolved images of immobilized dextran monolayers. In common with many polysaccharides,40 the conformation of dextran is sensitive to the level of intramolecular hydrogen-bonded water, and this ultimately determines its supramolecular structure and accounts for the ellipsoidal shape of the dehydrated dextran molecules. Molecular resolution monolayer coverages of the dextran were observed, and this work has demonstrated an increasing molecular size and a diminishing surface density of molecules with increasing molecular weight, which is in agreement with other studies.18 The explanation of this phenomenon in terms of the ideal monomolecular packing of the dextran was shown to be inadequate, and this has led us to conclude that the supramolecular chemistry of dextran plays a significant role in determining the efficiency of the coupling reaction. The intramolecular freedom of the polymer chains affects the reactivity of the respective polysaccharide, both in terms of its degree of functionalization and its ability to adopt a suitable conformation to maximize the number of binding sites at the silica surface. Although the variety of information obtained from the embedded uncoated and coated silica particles was limited by the roughness of the underlying morphology, features consistent with those of the dextran have been observed on the particle surface. The sizes of these features compare favorably with those observed on the flat silica slides. The distortion of the shape in the case of the immobilized 10 kDa dextran but not the 70 kDa supports the theory that the higher molecular weight dextran with its highly branched and consequently more rigid structure may have lower intramolecular freedom. We have also suggested that the reduced flexibility of the dextran molecule at higher molecular weight could reduce the number of potential binding sites, and this theory is supported by the lower surface densities of 70 kDa observed in all cases. The AFM visualization of the monomolecular layers of dextran has provided a novel insight into the properties of the immobilized species, and this has direct implications for the wider understanding of their use in size exclusion chromatography systems. The future directions of this work will include the AFM analysis of the dextran monolayers under aqueous conditions, since this will provide a more accurate model of the surfaces in their chromatographic environment. Hydration of the polysaccharide layer has been shown to have a significant effect on the protein repellency of the silica surfaces. An interesting observation in the study conducted by Matthijis et al.18 was that silicas modified with high molecular weight dextran demonstrated the greatest protein resistance even though poorer coverages of dextran had been observed. This was explained by the tendency of the higher molecular weight dextran to swell to a greater degree in aqueous media than the low molecular weight analogues, leading to a more extended but less dense layer. Our intention therefore is to examine the interaction of (40) Nevell, T. P., Zeronian, S. H., Eds. Cellulose Chemistry and Its Application; Wiley: New York, 1985.
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proteins with the polysaccharide layers under aqueous conditions, and these findings will be reported in due course. Finally, this study has also been successful in demonstrating the flexibility of the embedding technique, which has provided a method of obtaining high-resolution images of a surface previously considered beyond the scope of scanning probe techniques. This is significant in that it presents the possibility of AFM becoming a more widely used technique for the investigation of chromatographically useful materials. On a wider perspective the experience gained in the analysis of these types of systems will be useful in governing future approaches in biosensor
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research. For example, this and other work in the literature further illustrate how AFM could be used in the design and characterization of specific molecular architectures at interfaces, particularly through the use of self-assembled monolayer technology. Acknowledgment. The authors would like to acknowledge the support of the BRITE/Euram programme, the EPSRC/DTI Nanotechnology LINK programme, and VG Microtech, and Professor P. Ferruti of the University of Brescia for kind discussions. LA951087I
