NANO LETTERS
Imaging the Coil-to-Globule Conformational Transition of a Weak Polyelectrolyte by Tuning the Polyelectrolyte Charge Density
2004 Vol. 4, No. 1 149-152
Luke J. Kirwan, Georg Papastavrou, and Michal Borkovec* Department of Inorganic, Analytical and Applied Chemistry, UniVersity of GeneVa, 30, quai Ernest-Ansermet, GeneVa 1211, Switzerland
Sven H. Behrens Polymer Research Laboratory, BASF Aktiengesellschaft, Ludwigshafen, Germany Received October 20, 2003; Revised Manuscript Received November 5, 2003
ABSTRACT Conformational changes of poly(vinylamine) (PVA) upon adsorption onto mica were determined by AFM. The polyelectrolyte charge can be tuned through the solution pH. At pH 3, where the PVA is highly charged, an extended coil conformation is observed. When the pH is increased, the polyelectrolyte charge decreases and induces a segmental collapse through pearl-necklace structures. Above pH 9, where the PVA is only weakly charged, one can observe a globular conformation. By analyzing the volume of the imaged objects, it could be clearly demonstrated that single molecules have been observed. By examining PVA adsorbed in its most globular conformation, the molecular mass distribution has been determined. The PVA molecules could be also detected after being grafted to an epoxy terminated surface, which has been obtained through a silanization reaction. The observed polyelectrolyte collapse through a sequence of pearl-necklace structures is likely related to analogous conformations in solution, which are predicted on theoretical grounds.
Introduction. A highly charged flexible polyelectrolyte (i.e., charged polymer) dissolved in pure water has an extended coil conformation. At low charge density, on the other hand, a polyelectrolyte chain typically collapses into a compact globule. This coil-to-globule transition is driven by attractive hydrophobic interactions between the polymer segments that override the Coulombic repulsion forces at sufficiently small charge densities. One might suspect that this conformational transition should resemble the coil-to-globule transition of a neutral polymer chain. In this case the transition is sudden and can be induced by decreasing the solvent quality from a good solvent to a poor one.1 For a linear, flexible polyelectrolyte, however, the mechanism of this coil-to-globule transition might be quite different. It was argued that due to the longrange character of the Coulomb forces this transition should in fact resemble the Rayleigh instability of a charged droplet. The latter case is well understood. When the charge of a liquid droplet is being increased, the droplet will split up into several smaller droplets at a critical charge threshold. The higher the charge of the primary droplet, the larger is * Corresponding author. Tel: +41-22-702-6405, fax: +41-22-702-6069, email:
[email protected] 10.1021/nl034912l CCC: $27.50 Published on Web 11/26/2003
© 2004 American Chemical Society
the number of the secondary droplets formed. For the polyelectrolyte chain, one thus expects that the coil-to-globule transition will pass through a sequence of intermediate configurations, which consist of chains bearing individual smaller sub-globules along the chain - similar to a pearl necklace. The number of these globules will decrease with decreasing quality of the solvent, but also by decreasing the charge density of the chain or increasing the salt level (see Figure 1). The structure of intermediate configurations based on the analogy to the Rayleigh instability was suggested on theoretical grounds and later confirmed with computer simulations on the Debye-Hu¨ckel and primitive model levels.2-6 Direct experimental evidence for such pearl-necklace structures in the conformational transitions of polyelectrolyte chains has been lacking in the past. Exceptions include fluorescence images of large DNA molecules,7 in addition to indirect evidence for a cationic polyelectrolyte by smallangle X-ray scattering.8 In all cases, the pearl-necklace transition was found to result from a decrease of the solvent quality. Only recently have these structures been visualized for linear polyelectrolytes by imaging adsorbed poly(2-vinyl pyridine) and poly(methacryloyloxyethyl dimethyl benyl-
Figure 1. Schematic representation of the extended coil-to-globular conformational transition for linear flexible polyelectrolytes with the chemical structure of poly(vinylamine) (PVA).
ammonium chloride) (PMB) on mica with atomic force microscopy (AFM).9,10 These authors have used strong cationic polyelectrolytes, which were chosen to have a sufficiently bulky backbone to facilitate the AFM imaging. Since the charge on strong polyelectrolytes is fixed, the strength of the electrostatic repulsions was tuned by changing the salt level and thus by the extent of the screening of the Coulomb interactions. While variation of the polyelectrolyte charge would be the more direct way to induce this conformational transition, an experiment confirming this scenario is lacking so far. In this study, we present first experimental data of this kind by imaging poly(vinylamine) (PVA) adsorbed on mica by AFM as a function of solution pH. PVA is a weak cationic polyelectrolyte, and its charge density can be tuned through the pH from completely neutral (pH > 10) to fully ionized (pH < 3) as demonstrated by titration experiments.11 Imaging the Coil-to-Globule Transition. Poly(vinylamine) (PVA) of molecular mass 190 kDa was synthesized in the BASF (Ludwigshafen, Germany) and extensively dialyzed with deionized Milli-Q water. It was adsorbed onto freshly cleaved mica (Pelco Mica Sheets, Ted Pella, Inc., Cat. #53) from a 0.1 mg/L solution for 15 or 30 s, which pH was adjusted with HCl or KOH. After the adsorption, the mica was rinsed with Milli-Q water and dried under a stream of nitrogen. The samples were imaged in air with a Nanoscope IIIa AFM (Digital Instruments) with amplitude feedback operated with >80% of the free amplitude. The cantilevers used (Micro Cantilever, OMCL-AC160TS-WZ, Olympus) had a spring constant around 40 Nm-1, a resonance frequency of 300 kHz, and silicon tips with a tip radius below 10 nm as selected by imaging a Nioprobe standard (Aurora NanoDevices Inc.). The tips were functionalized with 3-aminopropyldimethyl-ethoxysilane by vapor deposition in a nitrogen atmosphere.12 Figure 2 shows representative AFM images (1 × 1 µm scans) visualizing the coil-to-globule transition of PVA as a function of pH. Selected zoomed sections of these scans are depicted in Figure 3. At pH 3.0 and 4.0, the PVA is fully ionized.11 The extended conformations of the chains can be noticed. A 150
Figure 2. AFM images showing the extended coil-to-globular conformational transition of poly(vinylamine) (PVA), achieved by adsorption onto mica from solutions of varying pH.
Figure 3. Expanded view (167 × 500 nm) of selected molecules represented in Figure 2 that highlight the structural transition as a function of solution pH. The pearl-necklace structures can be clearly seen at pH 4.0 and 4.9.
marked change in conformation is observed when going to pH 4.9 where a significant decrease in the degree of ionization of PVA sets in. This picture clearly confirms the existence of pearl-necklace structures during the conformational coil-to-globule transition of PVA. At pH 7.1 about half of the groups are ionized, and one observes more elongated, globular conformations. At pH 9.4 and 10 the Nano Lett., Vol. 4, No. 1, 2004
chain is only weakly charged, and collapsed globular structures can be seen. The images shown in Figures 2 and 3 taken at low pH reveal that even the fully ionized chain is not fully extended, but still features a pearl-necklace structure. The latter conclusion is supported by the fact that the apparent mean contour length was around 250 nm with a standard deviation (SD) of 120 nm, while the contour length of a fully stretched 190 kDa PVA chain is about 1 µm.13 We suspect that, taking into account the inherent noise level of the measurements performed in this study, a fully extended chain could remain invisible during a similar scan. In all images additional globular structures are apparent. A few small globules are present in samples adsorbed at low pH. For adsorption at higher pH, in addition to the frequent small globules, larger globular structures are present as well. Most probably these structures are artifacts attributable to sample drying and are examined in the following sections in detail. Determination of the Molecular Mass Distribution. The AFM scan in Figure 4 is a result of the adsorption of PVA onto mica from a pH 7.2 solution that was rinsed with deionized water, followed by rinsing with acetone to dehydrate the surface. The sample was then dried with nitrogen. These molecules depicted in the AFM image in Figure 4 feature a much more compact globular shape than the corresponding conformations obtained without acetone rinsing (see Figure 2, pH 7.1). Moreover, the conformations observed after an acetone treatment were independent of the solution pH from which the PVA is adsorbed. These observations indicate that acetone is a poor solvent for PVA, and induces the observed compact globular structures. This solvent further minimizes capillary drying effects, offering the possibility to easily distinguish individual PVA molecules. Therefore, these preparations permit the measurement of the height and width of each adsorbed molecule, allowing volumes to be estimated by applying a sphere-cap fit.14 From the measurement of 90 molecules, using a measured PVA density of 1.25 g cm-3, as determined from a dilution series with a Kratky-balance, the molecular mass distribution shown in Figure 4 can be obtained. From the latter, one can also estimate the number average molecular mass, Mn ) 254 kDa, and weight average molecular mass, Mw ) 421 kDa, and a polydispersity index Mw/Mn ) 1.66. The weight average molecular mass compares relatively well with the value of Mw ) 190 kDa determined with static light scattering and differential refractometry at pH 3. The remaining discrepancy probably originates from preferential adsorption of the polyelectrolytes of higher molecular mass. Most importantly, however, these results clearly demonstrate that single molecules are being imaged in contrast to polymer aggregates. This point also can be verified for extended coil conformations (i.e., adsorbed from pH 3 or pH 4 solution without acetone treatment). Seven molecules, which exhibited well resolved end points, were examined. The height and width of different cross-sectional areas along Nano Lett., Vol. 4, No. 1, 2004
Figure 4. Results of PVA adsorption on mica treated with acetone. Representative image (1 × 1 µm scan, top) and the corresponding molecular mass number distribution (bottom). From the histogram the number average molecular mass, Mn ) 254 kDa, and weight average molecular mass, Mw ) 421 kDa, can be estimated.
the molecules were measured, leading to an average width of 8.4 nm (SD 1.1 nm) and an average height of 0.29 nm (SD 0.07 nm). The length along the apparent backbone of these molecules was on average 250 nm (SD 120 nm). By approximating the cross-sectional areas of the adsorbed molecules by a circle segment one obtains the molecular volume, and then with the density of 1.25 gcm-3 one obtains a molecular mass of 301 kDa (SD 154 kDa). The latter value compares well with those determined for the globular structures in Figure 4. Therefore, this result shown again supports that single molecules are being imaged. Polyelectrolyte Grafting. In Figure 2 globular structures can be observed even for adsorption from low solution pH. These globular structures are most likely due to very weakly adsorbed molecules being forced into this conformation as a result of drying the sample prior to imaging. In addition, for adsorption from higher solution pH, some globular structures are very large, indicating possible aggregation of polymer molecules. At the higher solution pH, PVA is less charged and hence less strongly bound to the mica surface, 151
Figure 5. Adsorption of PVA onto (3-glycidoxypropyl)-trimethoxysilane modified mica surface (1 ×1 µm scans) from (a) pH 3.0 solution and (b) pH 6.8 solution.
and it is likely that the capillary action during drying causes the adsorbed polymer molecules to aggregate. To further investigate origin of these artifacts, the surface of the mica was modified with an epoxy silane in order to chemisorb the PVA to the surface.13 Mica was placed in a 5 wt % solution in 2-propanol of (3-glycidoxypropyl)-trimethoxysilane (ABCR GmbH & Co. KG) for 1 h, where the excess silane was removed by tempering the sample for 3 h at 80 °C.13 Imaging of such samples was carried out with the same silicon tips as before, albeit without the amino modification. The image for adsorption from pH 3.0 solution (Figure 5a) shows there is a significant increase in surface roughness as a result of the silanization procedure, precluding the detection of PVA molecules adsorbed in an extended conformation. A monofunctional ethoxy silane, (3-glycidoxypropyl)-dimethylethoxysilane (Fluorochem, UK) led to similar results. The observed globular structures are probably islands of the silanization product. For adsorption from pH 6.8 solution (Figure 5b), larger globular structures become visible, which correspond to the adsorbed PVA in the globular conformation. In particular, very large structures were not observed, indicating that this procedure inhibits the aggregation of the adsorbed polymer upon drying the sample. Conclusion. The collapse of poly(vinylamine) (PVA) was directly observed by AFM. Being a weak cationic polyelectrolyte, its charge density can be varied by adjusting the
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solution pH. The conformational transition was imaged in the adsorbed state with mica as a substrate. At low pH, where PVA is fully ionized, it adsorbs in an extended coil conformation, while at high pH the polyelectrolyte is completely deprotonated and has a globular conformation. The transitory conformations exhibited pearl-necklace structures, which do persist even for fully ionized molecules. This structural transition observed in the adsorbed state probably reflects a corresponding structural transition in solution, which was predicted on theoretical grounds.2-6 Acknowledgment. This work was supported by the BASF Aktiengesellschaft, Ludwigshafen, and the Swiss Commission for Technology and Innovation within the program TopNano21. References (1) Grosberg, A. Yu.; Khokhlov, A. R. Statistical Physics of Macromolecules; AIP Press: New York, 1994; Chapter 3. (2) Kantor, Y.; Kardar, M. Phys. ReV. E 1995, 51, 1299-1312. (3) Dobrynin, A. V.; Rubinstein, M.; Obukhov, S. P. Macromolecules 1996, 29, 2974-2979. (4) Micka, U.; Holm, C.; Kremer, K. Langmuir 1999, 15, 4033-4044. (5) Chodanowski, P.; Stoll, S. J. Chem. Phys. 1999, 111, 6069-6081. (6) Limbach, H. J.; Holm, C. J. Phys. Chem. B 2003, 107, 8041-8055. (7) Starodoubtsev, S. G.; Yoshikawa, K. J. Phys. Chem. 1996, 100, 19702-19705. Yoshikawa, K.; Yoshikawa, Y.; Koyama, Y.; Kanbe, T. J. Am. Chem. Soc. 1997, 119, 6473-6477. Ueda, M.; Yoshikawa, K. Phys. ReV. Lett. 1996, 77, 2133-2136. (8) Waigh, T. A.; Ober, R.; Williams, C. E. Macromolecules 2001, 34, 1973-1980. (9) Minko, S.; Kiriy, A.; Gorodyska, G.; Stamm, M. J. Am. Chem. Soc. 2002, 124, 3218-3219. (10) Kiriy, A.; Gorodyska, G.; Minko, S.; Jaeger, W., Sˇ teˇpa`nek, P.; Stamm, M. J. Am. Chem. Soc. 2002, 124, 13454-13462. (11) Katchalsky, A.; Mazur, J.; Spitnik, P. J. Polym. Sci. 1957, 23, 513532. (12) Riener, C. K.; Stroh, C. M.; Ebner, A.; Klampfl, C.; Gall, A. A.; Romanin, C.; Lyubchenko, Y. L.; Hinterdorfer, P.; Gruber, H. J. Anal. Chim. 2003, 479, 59-75. (13) Hugel, T.; Grosholz, M.; Clausen-Schaumann, H.; Pfau, A.; Gaub, H.; Seitz, M. Macromolecules 2001, 34, 1039-1047. (14) Pfau, A.; Schrepp, W.; Horn, D. Langmuir 1999, 15, 3219-3225.
NL034912L
Nano Lett., Vol. 4, No. 1, 2004
