Langmuir 1993,9, 1115-1120
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A Scanning Tunneling Microscopy and Transmission Electron Microscopy Study of Poly(ethy1ene oxide) Films G. J. Leggett, M. J. Wilkins, M. C. Davies, D. E. Jackson, C. J. Roberts, and S. J. B. Tendler The VC SPM Laboratory for Biological Applications, Department of Pharmaceutical Sciences, University of Nottingham, University Park, Nottingham NC7 2RD, United Kingdom Received September 28,1992. In Final Form: February 2,1993 We present images of poly(ethy1eneoxide) films cast onto freshly cleaved mica and prepared for analysis by coating with a platinum-carbon film. A number of structural forme were observed, including globular, fibrous, and branched types. The basic unit from which all of these structures were formed was a rope of diameter 20 nm. The branched features were observed to be formed from folds of this rope. Particularly striking images were obtained from boundaries between domains, showing a sharp separation between regions of the sample in which the polymer is found in the fibrous and the branched forms. Examination of the same samples by transmission electron microscopy (TEM)revealed identical structures. However, comparison of the scanning tunneling microscopy (STM) and TEM images demonstrates that the TEM images exhibit lower vertical contrast and fail to provide some of the structural detail which is apparent in the STM images.
Introduction Poly(ethy1ene oxide), PEO, is a polymer of considerable biomedical significance. Of particular importance are ita exhibition of minimal protein adsorption1and ita suggested passive nonthrombogenicity.2 As a consequence of these characteristics, PEO is finding a wide range of biomedical applications,- in many of which it is surface-mediated biointeractions which are of crucial importance. Recent years have seen the application of surface-analytical techniques, including X-ray photoelectron spectroscopy (XPS), Fourier transform infrared spectroscopy (FTIR), and secondaryion mass spectrometry (SIMS), to the study of PEO in a number of systems;- the application of STM to studies of PEO structure therefore seems appropriate and, potentially, very valuable. Besides this basic motivation, however, there are a number of other reasons which have prompted the present STM study. The casting of films from aqueous solution is a common method for sample preparation in STM, not only for polymeric samples but also for a wide range of materials including biomolecules. There are a number of phenomena associated with film formation from solution which are of significance, including, for example, network formation, which has been observed during the casting of films of materials as diverse as polystyrene,6 xanthum gum: and DNA* under a range of conditions. We are interested in the interactions of PEO with proteins, and, in particular we are interested in co-crystallization of the polymers with proteins. As an essential precursor to studies of polymer/ protein co-crystallization, it is necessary to obtain an
understandingof the kinds of structure which are exhibited by the polymer following film casting from aqueous solution (aqueousconditions are preferable because of the need to handle proteins in an aqueous buffer solution). The present study attempta to characterize these structural forms for PEO cast onto mica surfaces. We are also interested in exploring methods by which the twin problems of poor sample conductivity and tipinduced sample movement may be overcome for polymeric samples. There is good evidence that the application of a conducting coating can provide a highly effective means by which macromoleculesmay be immobilized, facilitating the acquistion of high-resolution STM images@-1° with very good reproducibility. Gold coating has been employed in STM studies of polyethylene single crystals,ll and platinum-carbon (Pt/C) coating has been employed to facilitate the imaging of polyethylene (PE) crystallized from solution.12 In the present study, we utilize Pt/C coating to prepare samples of PEO (molecular weight 9OOOOO) cast from aqueous solution onto mica. An essential component of our work is the validation of data by comparison with other appropriate techniques, including transmission electron microscopy (TEM), and in this paper we present a combined STM-TEM study of platinum-carbon coated samples of PEO.
Experimental Section PEO, molecular weight 900 OOO, was obtained from Sigma (Poole, Dorset, U.K.). Solutions were prepared with a concentration of 0.025% w/v in filtered deionized water. The polymer was used as received. Immediately following dissolution of the polymer, samples were prepared by depositing 2 p L drops onto wafers of freshly cleaved mica (Agar, Stansted, Essex, U.K.). The samples were dried rapidly under vacuum and coated with a layer of platinum-carbon by electron beam evaporation in a
(1) Gregonis, D. E.; Buerger, D.E.; van Wagenen, R. A,; Hunter, S. K.; Andrade, J. D. Tram. SOC.Biomater. 1984, 7,766. (2) Merril, E. W.; Salzman, E. W. Am. SOC.Art. Int. Org. 1983, 60. (3) Tingey, K. G.; Andrade, J. D.; Zdrahala, R. J.; Chittur, K. K.; Gendreau, R. M. In Surface Characterisation of Eiomateriafs;Ratner, B. D.,Ed.;Elaevier: Amsterdam, 1988, pp 255-270. (4) Grainger, D. W.; Okano, T.; Kim, S. W.; Castner, D. G.; Ratner, B. D.; Briggs, D.; Sung, Y. K. J. Biomed. Mater. Res. 1990, 24, 547. (9) Zasadzinski, J. A. N.; Schneir, J.; Gurley, J.; Lings, V.; Hansma, P. K. Science 1988,239, 1013. (5) Brindley, A.; Davies, M. C.; Lynn, R. A. P.; Davis, S. S.; Hearn, J.; Watta, J. F. Polymer 1992,33, 1112. (10) Amrein, M.;Stasiak, A.; Gross, H.;Stoll, E.;Travaglini, G. Science 1988,240, 514. (6)Stange, T. G.; Mathew, R.; Evans, D. F.; Hendrickson, W. A. (11) Piner,R.;Reifenberger,R.;Martin,D.C.;Thomas,E.L.;Apkarian, Langmuir 1992,8, 920. R. P. J. Pofym.Sci. C: Polym. Lett. 1990, 28,399. (7) Wilkins,M.J.;Davies,M.C.;Jackson,D.E.;Mitchell,J.B.;Roberts, C. J.; Stokke, B.; Tendler, S. J. B. Ultramicroscopy, in press. (12) Reneker, D. H.; Schneir, J.; Howell, B.; Harary, H. Pofym. Commun. 1990, 31, 167. (8) Williams,P. M. University of Nottingham, unpublisheddata, 1992.
0743-7463/93/2409-1115$04.00/0Q 1993 American Chemical Society
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nm
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Figure 1. STM image of globular form of PEO. Scan size: 945 X 945 nm. Balzers 360M freeze-etching unit. The grain size obtained for coatings prepared in this way was typically of the order of 4-5 nm. STM analysis was performed on a VG STM 2000 system (VG Microtech, Uckfield, U.K.) using a cut platinum/iridium tip in the constant current mode. Typical experimental conditions comprised a tunneling current of 50 pA and a sample bias voltage of +1.3 V. Following STM analysis, the same samples were prepared for TEM analysis. The samples were coated with a carbon layer by resistive evaporation from a carbon rod, in order to provide them with sufficient mechanical strength to facilitate transfer of the intact replica to a copper grid by flotation on a water droplet. Copper grids of 200 mesh (Agar) were used. TEM was then performed on a Philips EM 300 electron microscope with an acceleratingvoltage of 80 kV and a nominal electron magnification of between 49 818X and 80 224X.
Results We observed several different types of polymer structure, which we have grouped into three categories: globular, fibrous, and branched. In addition, we have observed quite distinct boundaries between domains of the polymer exhibiting the branched and fibrous types of structure. We discuss each form of the polymer separately in sections (a-d) below. (a) Globular Structures. The first form of the polymer consisted of “globular” features ranging in size and shape from small approximately circular features, with diameters of the order of 50-100 nm, to larger, more irregular shapes, with largest dimensions of the order of 400 nm. The image in Figure 1exhibits a representative collection of clumps of varying shapes and sizes. Some substructure is evident but is generally rather indistinct. (b) Fibrous Structures. The fibers which formed the second structural type were large (typically some 20 nm in diameter)and were closely packed to form a dense array. The magnitude of the fibers clearly suggested that they were formed from a number of PEO molecules bound together. In some regions, the fibers possessed a degree of ordering, for example, in the image in Figure 2a they form an interwoven pattern. Smaller scale scans (for example, Figure 2b) allowed a more accurate estimation of the diameter of the fibers but did not reveal any more detailed structural information. It is possible that finer structural detail was obscured by particles of the coating. In other regions, the fibers were lesswell ordered, although similar dimensions were observed (Figure 2c). Figure 3 shows a TEM image of a fibrous region of the sample. The fiber diameter was again in the region of
21.08
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29.97
nm Figure 2. STM images of fibrous form of PEO. Scan size: (a, top) 945 X 945 nm; (b, middle) 307 X 307 nm; (c, bottom) 945 X 945 nm.
20-25 nm, giving good agreement with the value obtained from the STM images. The arrangement of the fibers at the surface was also very similar to that observed in the STM images, although the contrast was not quite so great and no three-dimensional information was directly available. (c) Branched Structures. This third category is the most broadly defined. It covers a range of structures, which vary in detail from denselybranched structures to dendritic forms with high fractal dimensions. Figure 4 shows the densely branched form. The branches have a somewhat uneven topography, suggesting a complex substructure. Several branches seem to be formed from loops; others
STM and TEM Study of PEO Films
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Figure 3. TEM image of fibrous form of PEO. Scan size: 882 X
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nm Figure 5. Large scale STM images of the dendritic form of PEO. Scan size: (a, top) 2906 2882 nm.
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Figure 4. STM image densely branched form of PEO. Scan size: 945 X 945 nm.
seem to be formed from thick entangled deposits of the polymer. Figure 5 shows two images of the dendritic variant of the branched form. An arrangement of long backbone dendrites is observed, each of which exhibits many side branches at right angles to its main axis. These side branches have, in turn, many smaller side branches at right angles to their central axes. The dendrites often make abrupt turns resulting in a number of geometrical forms. Variations in contrast were also evident within the dendrites and were made clearer by examining images obtained with increased resolution. In Figure 6a, darker regions may be seen in the center of the dendrites, bordered by narrower features of high contrast. These high contrast margins give the impression of a number of loops. In segments where the dendrite diameter is small, the sides of the loops are very close together, and there is little change in contrast as one moves acrossthe dendrite. Where
X
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the dendrite broadens, however, the loop becomes clearly visible (see Figure 6b). The fiber diameter was typically 20-30 nm, clearly similar to the value obtained for the fibers of fibrous form of the polymer. In some regions, a feature of high contrast is observed which suggests the presence of a clump or similar dense collection of material. Such features are often accompanied by a particularly closely packed and less well ordered group of dendrites, radiating out from them. One example is highlighted in Figure 5a. A possible explanation for this observation is that these features represent points at which a number of polymer chains become tangled. The untangled portions of the fibers radiate outward from the point of high contrast. The entangling of the fibers would account for the unusually high density of dendrites around such points. TEM images were also obtained of the branched form of, the polymer. Figure 7 shows two examples. The structures are very similar to those observed in the STM images. However, the substructual forms,although clearly evident in the STM images, are not seen in the TEM images. (d) Boundaries between Domains of the Branched and Fibrous Forms of the Polymer. STM images were obtained of boundaries between domains of the fibrous and branched forms of the polymer. These boundaries were strikingly sharp, with a very clear separation between the two forms of the polymer (see Figure 8). In the lower
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Figure 7. TEM image of PEO branched structures. Scan size: 882
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nm Figure 6. (a, top) Higher resolution STM images of a region of the image in Figure 5b. The loops which form the fingers of the dendrites are more clearly evident. The inset shows a cross section along the dashed line between the two arrows. Scan size: 661 X 661 nm. (b, bottom) Detailed image showing the looping of polymer fibers to form a dendrite. Scan size: 248 X 248 nm.
portion of the image the polymer exists in a fibrous form and the image is very similar to those in Figures 2 and 3, whereas the upper portion of the image matches closely the structures in Figures 4 and 8. TEM images again showed the same clear separation between domains (see Figure 9). The structure of the polymer film was found to depend upon position in relation to the center of the drop originally placed upon the substrate. The fibrous form of the polymer was observed nearer to the center of the drop than was the branched form. Densely branched forms of the polymer were observed near to regions where the fibrous form was observed (see the images of domain boundaries in Figures 8 and 9), whereas the dendritic variant was observed much further from the center of the drop. (e) Examinationof Uncoated Material. Finally, we examined films prepared from the same PEO solution by drying onto freshly-cleaved highly oriented pyrolitic graphite (HOPG). We were unable to obtain a steady tunneling current, indicative of the presence of a polymer film which was sufficientlythick for tip-sample conduction to be restricted. Thus the application of a conducting overlayer has, in this case, facilitated the imaging by STM of materials which would otherwise be inaccessible to the technique.
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nm Figure 8. STM image of a boundary between domains occupied by the branched and fibrous forms of the polymer. Scan size: 945 X 945 nm.
Discussion A number of studies of the molecular structure of crystallinePEO have been reported in the literature. These studies have utilized X-ray diffraction and IR spectroscopy and have shown that the PEO molecule has a helical structure in the crystalline state.13J4 The fiber identity period was found to be of length 1.93 nm and to contain seven repeat units and two turns. Upon crystallization, the polymer forms lamellar single crysta1s.l4-l6 Recently, (13) Tadokoro, H.; Chatani, Y.; Yoshihara,T.; Tahara, S.; Murahashi, S . Makromol. Chem. 1964, 73, 109. (14) Arlie, J. P.; Spegt, P.; Skoulios, A. Mukromol. Chem. 1967,104, 212. (15) Kovacs, A. J.; Straupe, C.; Gonthier, A. J. Polym. Sci., Polym. Symp. 1977,59,31. (16) Barnes, W . J.; Price, F. P. Polymer 1964, 5, 283.
STM and TEM Study of PEO Films
Figure 9. TEM image of a boundary between domains occupied by the branched and fibrous forms of the polymer. Scan size: 882 X 1096 nm.
atomic force microscopy (AFM) has been used to study these lamellar structures.l7 The images obtained by using the AFM were found to be in good agreement with images of PEO lamellae obtained by electron microscopy.16 The AFM studies revealed17 that slow disintegration of the lamellae occurred in air, to yield “treelike” patterns. The authors postulated that chain refolding was occurring during the disintegration process. However, despite a resemblance between the disintegrated PEO lamellae and some of our images (particularly the densely branched structures of “form C”), there is a considerable difference in scale, and, thus, no obvious common explanation for the branched features we have observed. A study of negatively stained PEO by electron microscopy suggested that very large helical structures could be formed, with a diameter greater than 1pm.18 In a different study, Yang et al.19 observed complex features which appeared to be formed from thick strands of the polymer. In particular, they observed in one image a feature of diameter 23 nm, which twisted back upon itself at one point to form a second segment of diameter 33 nm. They suggestedthat the basic structural unit of this feature was a polymer strand some 4.3 nm in diameter (composed of 32 PEO chains) coiled to form a helix of diameter 23 nm. Such thick strands are clearly of similar dimensions to those which we have observed, although we were not able to observe a helical structure. Of all the reported forms of PEO, these are the closest in dimensions both to the fibrous structures we have described under (b) above and to the fibers which fold to form the loops of the dendritic structures. However, Yang et al. did not report the ~~
~
(17) Snetivy, D.; Vancso, G. J. Polymer 1992, 33, 432. (18) Deryagin, B. V.; Lyashev, K. F.; Novik, 0. G. Dokl. Akad. Nauk SSSR 1975,218,599. (19) Yang, R.; Yang, X. R.; Evans, D. F.; Hendrickson, W. A.; Baker, J. J . Phys. Chem. 1990,94,6123.
Langmuir, VoZ. 9, No. 4, 1993 1119 observation of any of the extensive structures we have reported above. In separate studies of PEO-salt complexes,20 they have presented images of layers composed of many closely packed parallel chains which bear some similarity to our images of form B of the polymer, but the diameter of the chains in their images was only 2.5 nm, an order of magnitude smaller than that which we have observed. Thus none of the reported studies of PEO provide us with a suitable explanation for the variety and the physical characteristics of the different forms of PEO we have observed using STM. Reneker et al. have reported images of PE films on mica.12 Their samples were prepared by entrapping a solution of PE in hot xylene between mica sheets which were then rapidly cooled to room temperature, resulting in the precipitation and crystallization of PE onto the mica sheets. Using a similar approach to ours, they shadowed the samples with Pt/C and imaged them using STM and TEM. They observed a wide variety of structural forms, includingdendritic features which bore a superficial resemblence to those presented in Figure 5 above (likened by the authors to growths of moss). These features were attributed to the rapid cooling step involved in the preparation of samples. However, careful analysis of their results suggests that the detailed morphology of the PE dendritic forms is substantially different from that observed in our PEO images. In particular, the PE dendritic structures were formed from elongated patches some 20 nm in width and 100nm in length. Conversely, the fingers of the dendrites in our images (Figures 5 and 6) are clearly shown to be formed from continuous, folded ropes of polymer material. Furthermore, Reneker et al. note that, on examination of high-resolution images, “most of the surface [was found to be] covered with elongated patches of material”.12 In the case of PEO, however, despite our achieving resolution at least as good as that reported by Reneker et al., we obtained no evidence for any polymerrelated morphology in regions of the surface other than those occupied by the dendrites. Indeed, the most likely interpretation of our images (due to its very low surface corrugation) is that the underlying surface on which the dendritic structures rest is simply the mica substrate. The parameter most likely to exert control over the film structure would be the concentration of the polymer at the surface during film formation. The clearest indication for this is the observed variation in PEO film morphology across the region covered by the original drop of solution. Dendritic forms of the polymer were observed near the edge of the drop; the more densely branched forms were observed near the center of the drop. Dendritic crystal growth is often associated with crystallization from supersaturated solution, and if supersaturation occurred near the edge of the drying drop (either during drying or prior to solvent evaporation, depending upon whether or not solvent evaporation was a necessary prerequisite for crystallization), then dendritic crystal structures might be expected to result. A further possibility, particularly in relation to the fibrous domains of the polymer, is that fibrillar crystals of PEO form during the rapid drying process under vacuum. Fibrillar crystals exhibit diameters of the order of 5-20 nm,21consistent with the values of ca. 20 nm which we have recorded. However, this discussion must, to some extent, remain speculative in the absence of further relevant data. In summary, what we can conclude with (20) Yang, R.; Yang, X. R.; Evans, D. F.; Hendrickson, W. A.; Baker, J. J . Phys. Chem. 1991,95,3765. (21) Pennings, A. J. J . Polym. Sei., Polym. Symp. 1977, 59, 55.
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more certainty is that PEO dries onto mica surfaces from aqueous solutions to form f i structures of some complexity, the basic unit of which is arope or fiber of diameter ca. 20 nm comprisinga large number of polymer molecules twisted together (after Yang et al.19). These ropes are observed as interwoven fibers or as dendritic or branched forms, depending upon the position within the deposited drop at which an image is recorded. Nearest the edge of the drop, a highly ordered and geometrically precise arrangement of dendrites,formed from folds of the polymer fibers, is observed. A densely branched structure is observed closer to the center of the drop, but this also is possibly formed from loops or folds of the PEO rope. Domains of the polymer with this kind of structure are observed close to the edges of domains in which the polymer adopts the fibrousstructure, and the two types of structure are separated by sharp boundaries. Finally, in relation to our initiallystated objectives,it is clear that film formation by solvent evaporation, while not reproducing the network structures observed for other macromolecules, does lead to considerablelateral inhomogeneityin the film structure. There is scope for the further investigation of the formation of these structural features. In particular, it would be valuable to investigate the effect of polymer molecular weight and casting solution concentration on film structure. Recent studies have clearly shown the role of these variables in controlling polystyrene film structure during spin-casting.6 In relation to improving the lateral homogeneity of the film, the investigation of spraydeposition of the polymer solution seems pertinent. In studies of samples of xanthan gum, spraying has proved a valuable means of reducing network formation; for PEO samples,it is possible that the deposition of alarge number
of small droplets, rather than a single large drop, will effectivelydecrease the lateral variation in f i i structure accrw a substrate of dimensions of the order of several mm2. We are presently pursuing such studies for PEO.
Conclusions STM images have been recorded of PEO films coated with a platinum-carbon layer. The images obtained were essentiallysimilar to images obtained of the same samples using transmission electron microscopy, although the vertical contrast was superior in the STM images. PEO was observed to adopt a variety of structural forms, includingglobular,fibrous,and branched forms. The basic unit from which these structural features are formed is a rope of diameter ca. 20 nm comprising a large number of polymer molecules twisted together. Complex dendritic structures are observed in regions close to the edge of the deposited drop of polymer solution. The fiigers of these dendrites are formed from loops or folds in the polymer ropes. Densely branched structures were also observed to be formed from loops of the polymer rope. The polymer morphology is thought to be controlled by the drying process, with the most structurally distinct form (the dendritic form) being observed nearest the edge of the drop, and the least structurally distinct form (the globular form) being observed near to the center of the drop.
Acknowledgment. We acknowledge the support of the SERC/DTI Link Protein Engineering programme, Glaxo Group Research, Ltd.,and VG Microtech. M.J.W. acknowledges the support of the MAFF/DTI Link Hydration (HYDRA) programme.
