Quantitative analysis of atomic force microscopy topographs of biopolymer multilayers: Surface structure and polymer assembly modes
Introduction
The fabrication of thin polymer layers by the electrostatic assembly of oppositely charged polyelectrolytes has recently attracted much interest [1]. Similar to polyelectrolyte complexes [2], [3], the formation of these films is electrostatically driven, and favored by the release of counterions. The preparation is easy and versatile, and by alternating exposure to solutions of polycation and polyanion, multilayers can be prepared on surfaces varying in mechanical properties and topography. Polyelectrolytes, as well as proteins and nanoparticles can be incorporated, giving rise to a number of potential applications [4], [5], [6], [7], [8], [9], [10], [11], [12]. Potential applications for such multilayers include modification of surfaces to increase the biocompatibility, optical devices and separation membranes.
Structure and topography of material surfaces influence properties such as adhesion, biocompatibility, and optical properties. Quantitative characterization of the multilayers surfaces made from various polymer components is important both for practical purposes e.g. for evaluating the biocompatibility, and for the comparison of the influence of the polymer constituents on the multilayer features such as roughness and surface structure. One way to characterise a surface is the root-mean-square (rms) roughness, the variations in height of the surface around the mean height. However, the rms roughness only gives variations in height, and does not offer any information about the size and lateral separation of the various surface features. This information can be found by evaluating the autocorrelation function and the power spectral density of the surfaces. Such analysis has been carried out for thin films [13], [14], [15], [16], [17], material surfaces such as <100> silicon [18] and HgTe [19] as well as for polymer gel surfaces [20]. In this paper we explore the application of quantitative analysis of roughness and height autocorrelation function of polymer multilayer surfaces imaged by atomic force microscopy. Different polymer layer surfaces are obtained by alternating deposition of polycationic and polyanionic biopolymers (chitosan or poly(L-lysine) in combination with alginate), or by covalent linking of chitosan and modified scleroglucan in a layer-by-layer approach. The polymers investigated in this study are of interest from a biological/medical perspective. Alginate has previously been used to build multilayers in combination with poly(L-lysine) investigating their use for suppressing the interaction between surfaces and cells [21]. Alginate is also widely used for encapsulating cells [22], [23], [24], often in combination with either poly(L-lysine) or chitosan, while scleroglucan is a polymer that is showing immune-stimulating effects (for recent reviews, see e.g. [25], [26], [27]). Gels of scleroglucan derivatives, e.g. scleraldehyde, as well as the carboxylate derivative sclerox of scleroglucan, have previously been investigated with respect to gelation kinetics, swelling in various solvents and explored as a matrix for drug delivery [28], [29], [30]. In addition to extending the characterization of the porous structures of materials composed of these biopolymers from the bulk to the multilayer state, they additionally represent a series of biopolymers with persistence lengths from about 2 nm to 150 nm [31], [32], [33], [34], [35], [36].
The determination of the surface topography by atomic force microscopy (AFM) for further quantitative analysis is carried out for increasing number of layers of the investigated multilayers. The quantitative analysis by surface roughness and power spectral density of these multilayers aims at revealing information of the structure during build-up of the multilayers.
Section snippets
Polymer samples
Alginate from Laminara hyperborea stipe with a fraction of guluronic acid equal to 0.68 and an intrinsic viscosity [η] = 14.4 dl/g determined in 0.1 M NaCl [37] (Mw ~ 450 × 103 g/mol) (FMC Biopolymers, Drammen, Norway), was used without further purification. The alginate was dissolved in water (de-ionised water with resistivity 18.2 MΩ/cm, obtained using a Millipore system) overnight to a concentration of 1 mg/ml. Scleroglucan (Actigum CS11, Sanofi Bioindustries, France) was dissolved in water by
Chitosan–alginate multilayers
Atomic force microscopy of chitosan adsorbed on mica indicate that the first layer of adsorbed chitosan fully cover the surface (Fig. 2A). The surface roughness, calculated from the height information of the topograph is 0.1 ± 0.1 nm when the chitosan deposition was carried out at an ionic strength I = 5 mM. A similar result was obtained when the deposition of the first chitosan layer was carried out at an ionic strength of 150 mM (AFM topograph not shown, surface roughness equal to 0.1 ± 0.1 nm).
Discussion
The formation of multilayers of various kinds is receiving increased attention, and characterization of multilayer surfaces with AFM has revealed a variety of surface morphologies [55], [56], [57], [58], [59]. For a number of potential applications of multilayers it is important to also characterize the surface structure, as these are important for the overall properties. By studying the PSD of the surface, one obtains information on how the height fluctuations vary with length scale. If the
Conclusion
This study shows that the study of multilayer surfaces can be carried out by quantitative investigation of AFM topographs of the multilayer surfaces. By extracting not only the surface roughness, but also the correlation function and the power spectral density, information about the structural details of the surfaces can be obtained in a simple, automated fashion. In addition to the surface roughness, we suggest that further evaluation of the surface by analysis of the power spectral density is
Acknowledgements
This work is supported by The Norwegian Research Council (grants 134674/140 and 121894/420). Dr. Pawel Sikorski, Department of Physics, NTNU, is thanked for valuable discussions and help in implementing routines in IDL. We are grateful for the chitosan sample provided by Dr. Kjell Morten Vårum, Department of Biotechnology, NTNU.
References (67)
Curr. Opin. Colliod Interface Sci.
(2003)- et al.
Biosens.Bioelectron.
(1994) - et al.
Surf. Sci.
(1995) - et al.
Trends Biotechnol.
(1990) - et al.
Carbohydr. Polym.
(1995) - et al.
Polym. Gels Netw.
(1998) - et al.
Polym. Gels Netw.
(1998) - et al.
J. Control. Release
(1999) - et al.
Carbohydr. Polym.
(2001) - et al.
Carbohydr. Res.
(1991)