Single Molecular Pair Interactions between Hydrophobically Modified

Jun 4, 2009 - ... and Medical Technology, Department of Physics, The Norwegian ... association between these hydrophobes in HMHEC and amylose ...
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Single Molecular Pair Interactions between Hydrophobically Modified Hydroxyethyl Cellulose and Amylose Determined by Dynamic Force Spectroscopy Makoto Takemasa, Marit Sletmoen, and Bjørn T. Stokke* Biophysics and Medical Technology, Department of Physics, The Norwegian University of Science and Technology, NTNU, NO-7491 Trondheim, Norway Received March 18, 2009. Revised Manuscript Received April 30, 2009 Interactions among HMHEC (hydrophobically modified hydroxyethyl cellulose) and between HMHEC and amylose were investigated by means of dynamic force spectroscopy of single molecular pairs. The technique was realized using a scanning probe based platform, and the molecular pair interactions were investigated in aqueous solutions over a range of force loading rates. Both hydrophobic interactions among hydrophobe C16 alkyl side chains in HMHEC and association between these hydrophobes in HMHEC and amylose showed a stretching type peak. The distribution analysis of rupture force based on Bell-Evans’s model revealed that the peaks had a most probable rupture force ranging from 27 pN at a force loading rate rf = 0.43 nN/s to 125 pN at rf = 170 nN/s for HMHEC-HMHEC, and from 13 pN at rf = 0.20 nN/s to 34 pN at rf = 33.7 nN/s for HMHEC-amylose interactions. The distance of the energy barrier relative to the minimum, xβ, and the apparent lifetime in the absence of external force, τ, were found to depend on the force loading rate, and the average values are estimated to be 0.99 nm and 0.89s for HMHEC-HMHEC and 0.31 nm and 0.075s for HMHEC-amylose interactions. The obtained data for these pairwise molecular interactions are underpinning the associative behavior of the macroscopic properties of aqueous solutions of these polysaccharides.

Introduction Gels have been extensively studied using a variety of experimental techniques1-3 and theoretical approaches.4-9 However, physically cross-linked gels are less well understood than the chemically cross-linked gels. One reason for this is the additional complexity introduced by the continuous rearrangement of the network structure caused by a finite lifetime of the cross-links. According to the theoretical approaches to describe gels,4,6,10 there are several important factors dominating the properties of gels. These factors include the number of cross-links, the polymer segment length between cross-links, the strength of the crosslinks, as well as the lifetime of the cross-link. For chemically crosslinked gels, the former two are the key factors, which can be controlled by choosing monodisperse samples with different molar masses, end-selective reactive polymers, and cross-linkers with well-defined functionalities. For the latter two, the strength of the covalent cross-links is sufficient to maintain a lifetime that is considered infinitely large. However, for physically cross-linked gels, the strength and the lifetime of the cross-links have finite values, and the methods to characterize them are limited. Due to these fundamental differences between covalent and physical gels, *Corresponding author. Telephone: +47-73593434. Fax: +47-73597710. E-mail: [email protected]. (1) Nijenhuis, K. T. Thermoreversible Networks: Viscoelastic Properties and Structure of Gels; Springer: Berlin, 1997; Vol. 130. (2) Clark, A. H.; Ross-Murphy, S. B. Adv. Polym. Sci. 1987, 83, 57–192. (3) Guenet, J. M. Thermoreversible Gelation of Polymers and Biopolymers; Academic Press: London, 1992. (4) Tanaka, F.; Koga, T. Bull. Chem. Soc. Jpn. 2001, 74(2), 201–215. (5) Tanaka, F. Macromolecules 2004, 37(2), 605–613. (6) Tanaka, F. Polym. J. 2002, 34(7), 479–509. (7) Tanaka, F. Macromolecules 2003, 36(14), 5392–5405. (8) Tanaka, F. Colloids Surf., B 2004, 38(3-4), 111–114. (9) Tanaka, F. Macromol. Symp. 2004, 207, 125–130. (10) Tanaka, F. Phys. A 1998, 257(1-4), 245–255.

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the progress in the understanding of physical gels has been limited from the experimental point of view. For physically cross-linked gels, the probability of the crosslink formation and the lifetime of the cross-link dominate the overall properties of the system, that is, whether it becomes liquid, gel, or intermediate between them. In this paper, we describe studies of the properties for interactions between polymers reported to form physical gel networks. The aim is to obtain relevant data characterizing the physical interactions supporting the gel formation, such as the relaxation time of the molecular interconnections or their ability for reconstruction of their network structure. Atomic force microscopy is a powerful technique to determine the surface topography as well as to determine forces between pairs of macromolecules. The force acting between the molecules is determined by allowing the molecules to approach each other, and then separating them, while continuously recording the force acting between the molecules. By forcing the molecules to separate at different speeds, additional information could be obtained, including the lifetime of association, probability of the interaction, and energy landscapes.11-13 Single molecule interactions have been determined for a variety of molecular pairs, such as protein-protein (receptor-ligand) interactions,14-16 protein-polysaccharide interactions,17 lipid-lipid interactions,18 conformational changes of polymers, such as unwinding of the (11) Evans, E. Faraday Discuss. 1998, 1–16. (12) Evans, E. B. Biophys. Chem. 1999, 82(2-3), 83–97. (13) Evans, E. Annu. Rev. Biophys. Biomol. Struct. 2001, 30, 105–128. (14) Florin, E. L.; Moy, V. T.; Gaub, H. E. Science 1994, 264(5157), 415–417. (15) Moy, V. T.; Florin, E. L.; Gaub, H. E. Science 1994, 266(5183), 257–259. (16) Merkel, R.; Nassoy, P.; Leung, A.; Ritchie, K.; Evans, E. Nature (London) 1999, 379(6714), 50–53. (17) Sletmoen, M.; Skjak-Braek, G.; Stokke, B. T. Biomacromolecules 2004, 5 (4), 1288–1295. (18) Wieland, J. A.; Gewirth, A. A.; Leckband, D. E. J. Phys. Chem. B 2005, 109 (12), 5985–5993.

Published on Web 06/04/2009

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double helix (helix-coil transition of DNA19,20 and polysaccharides21-23), unfolding of proteins,24-26 and boat-chair transitions of monosaccharides.27,28 This approach has given new insights into the mechanisms of molecular interactions on the single molecular pair level. Determination of the unbinding force as a function of force loading rate (dynamic force spectroscopy, DFS) supports elucidation of characteristic parameters of the molecular interaction, such as lifetime of the association among molecules.11-13,17,29 In this study, we apply DFS for the characterization of the cross-link formation between molecules forming physical gels, focusing on the interactions among hydrophobically modified hydroxyethyl cellulose (HMHEC) molecules, and between HMHEC and amylose. HMHEC is an amphiphilic polymer and one of the water-soluble cellulose derivatives. By introducing hydroxyethyl groups to cellulose, yielding hydroxyethyl cellulose (HEC), it becomes water-soluble. Grafting longer alkyl chains to HEC, as shown in Figure 1, enhances the hydrophobic characteristics of HEC. The alkyl side chains are referred to as hydrophobes because they dominate the hydrophicity of the polymer. The viscosity of solutions of associative polymers, including HMHEC, is much higher than expected for a nonassociating polymer at the same Mw and chain flexibility. In the case of HMHEC, the driving force of the association is hydrophobic interactions among the hydrophobes. By changing the solvent quality, or by adding molecules that interact with the hydrophobes competitively, such as R-cyclodextrin, the hydrophobic association among hydrophobes could be influenced, and a decrease of the apparent molar mass, and the viscosity of the system, can result.30 The hydrophobes of HMHEC make complexes with a variety of molecules, including surfactants, which are widely used in industries to control the macroscopic properties, such as viscosity. For instance, the hydrophobes of HMHEC and surfactants make a hybrid micelle, and the stability of the micelles dominates the overall properties of the system even at low polymer concentrations due to interconnecting different micelles.31,32 Hence, the mixed micelle acts as a physical cross-link, and any other polymer which can act as a cross-linker among hydrophobes, such as amylose, can also dominate the overall behavior of the system. Amylose, R-(1,4)-linked D-glucose, is a water-soluble polysaccharide. It is reported that amylose also makes a complex with a (19) Rief, M.; Clausen-Schaumann, H.; Gaub, H. E. Nat. Struct. Biol. 1999, 6 (4), 346–349. (20) Pope, L. H.; Davies, M. C.; Laughton, C. A.; Roberts, C. J.; Tendler, S. J. B.; Williams, P. M. Eur. Biophys. J. Biophys. Lett. 2001, 30(1), 53–62. (21) Li, H.; Rief, M.; Oesterhelt, F.; Gaub, H. E. Appl. Phys. A: Mater. Sci. Process. 1999, 68(4), 407–410. (22) Xu, Q. B.; Zou, S.; Zhang, W. K.; Zhang, X. Macromol. Rapid Commun. 2001, 22(14), 1163–1167. (23) Rief, M.; Oesterhelt, F.; Heymann, B.; Gaub, H. E. Science 1997, 275 (5304), 1295–1297. (24) Fisher, T. E.; Marszalek, P. E.; Oberhauser, A. F.; Carrion-Vazquez, M.; Fernandez, J. M. J. Physiol. (London) 1999, 520(1), 5–14. (25) Carrion-Vazquez, M.; Oberhauser, A. F.; Fowler, S. B.; Marszalek, P. E.; Broedel, S. E.; Clarke, J.; Fernandez, J. M. Proc. Natl. Acad. Sci. U.S.A. 1999, 96 (7), 3694–3699. (26) Rief, M.; Gautel, M.; Oesterhelt, F.; Fernandez, J. M.; Gaub, H. E. Science 1997, 276(5315), 1109–1112. (27) Marszalek, P. E.; Pang, Y. P.; Li, H. B.; El Yazal, J.; Oberhauser, A. F.; Fernandez, J. M. Proc. Natl. Acad. Sci. U.S.A. 1999, 96(14), 7894–7898. (28) Li, H. B.; Rief, M.; Oesterhelt, F.; Gaub, H. E.; Zhang, X.; Shen, J. C. Chem. Phys. Lett. 1999, 305(3-4), 197–201. (29) Koch, S. J.; Wang, M. D. Phys. Rev. Lett. 2003, 91(2), 028103. (30) Karlson, L.; Thuresson, K.; Lindman, B. Carbohydr. Polym. 2002, 50(3), 219–226. (31) Piculell, I.; Thuresson, K.; Lindman, B. Polym. Adv. Technol. 2001, 12(1-2), 44–69. (32) Egermayer, M.; Norrman, J.; Piculell, L. Langmuir 2003, 19(24), 10036– 10043.

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variety of molecules, such as iodide,33-35 and alkyl chains.36 These molecules are positioned inside a helical conformation called V-amylose. Upon complexation of the alkyl side chain of HMHEC with amylose, the overall properties of the system change significantly. For example, the steady shear viscosity of a HMHEC aqueous solution increases drastically by addition of amylose, and this is explained by cross-linking due to the complexation of hydrophobes of HMHEC and amylose,37 which is also supported by competition studies of surfactants or cyclodextrin.32,36,38 Details of the mechanism of the interaction at the molecular level, and the relation between the molecular characteristics and macroscopic properties, are not well-known. In this study, we performed single molecular pair interaction studies by dynamic force spectroscopy to focus on the molecular characteristics, such as the strength and lifetime of the molecular interaction.

Materials and Methods Polymers. The HMHEC sample (Natrosol Plus grade 331, Aqualon, Hercules) molecular weight was 2.5  105.32 The sample was supplied from Wilbur-Ellis, Japan. C16 alkyl chains were linked to 1.7 mol % of the anhydroglucose units in the cellulose backbone, which corresponded to a degree of hydroxyethyl group substitution equal to 3.3.39 Amylose is a linear polymer consisting of R-(1,4) linked D-glucose, but amylose extracted from natural sources, such as starch, includes a small fraction of branches.40 In this study, enzymatically synthesized amylose,41,42 having a strictly linear polymer chain, was used. The sample used had Mw = 8.2  105, and Mw/Mn =1.08 as determined by size exclusion chromatography coupled to a multiangle laser light scattering (SECMALLS) detector. The contour length of HMHEC was estimated to be 340 nm based on the molecular weight and the substitution ratio of hydrophobe and hydroxyethyl groups, and roughly 2000 nm for amylose (1640-2160 nm, depending on the mass per unit length ML).43-45 Methods. An atomic force microscope (AFM), Multimode AFM system (NanoscopeIIIa, Digital Instruments, Santa Barbara, CA), equipped with a Picoforce module was used. All the force measurements were performed using a piezoelectric scanner, type-PF, and liquid cell (model MTFML, Veeco). The AFM cantilevers used in this study were of the type Si3N4 contact mode tip Nanoprobe NP-S from Veeco. The softest of the four cantilever in each chip, with a length of 196 μm, width of 23 μm, and a nominal spring constant of 0.06 N/m, was used. The actual spring constant of the cantilever after immobilization of polysaccharides on the tip was estimated in water by noise analysis of the thermally driven fluctuation. All the measurements were performed at room temperature and in Milli-Q (MQ)-water. (33) Various, A. J. Am. Chem. Soc. 1943, 65(9), 1707–1710. (34) Handa, T.; Yajima, H. Biopolymers 1981, 20(10), 2051–2072. (35) Yu, X. C.; Houtman, C.; Atalla, R. H. Carbohydr. Res. 1996, 292, 129–141. (36) Egermayer, M.; Karlberg, M.; Piculell, L. Langmuir 2004, 20(6), 2208– 2214. (37) Chronakis, I. S.; Egermayer, M.; Piculell, L. Macromolecules 2002, 35(10), 4113–4122. (38) Karlberg, M.; Piculell, L.; Ragout, S. Langmuir 2006, 22(5), 2241–2248. (39) Nilsson, S.; Thuresson, K.; Hansson, P.; Piculell, L.; Lindman, B. Abstr. Pap. Am. Chem. Soc. 1998, 216, 025-CELL. (40) Hizukuri, S.; Takeda, Y.; Yasuda, M.; Suzuki, A. Carbohydr. Res. 1981, 94 (2), 205–213. (41) Whelan, W. J.; Bailey, J. M. Biochem. J. 1954, 58(4), 560–569. (42) Husemann, E.; Pfannemuller, B. Makromol. Chem. 1963, 69, 74–96. (43) Nakanishi, Y.; Norisuye, T.; Teramoto, A.; Kitamura, S. Macromolecules 1993, 26(16), 4220–4225. (44) Goebel, K. D.; Brant, D. A. Macromolecules 1970, 3(5), 634–&. (45) Roger, P.; Axelos, M. A. V.; Colonna, P. Macromolecules 2000, 33(7), 2446–2455.

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Figure 1. Schematic illustration of (a) the structure used for reducing-end-selective immobilization of polysaccharides to mica substrates or AFM tips. In panel a, the polysaccharide is depicted as amylose, but the immobilization strategy is the same for HMHEC, whose structure is shown in (b). The distance between the surface of the mica substrate (or AFM tip) and the second sugar residue from the reducing end is about 4 nm in the extended form. The sugar residue at the reducing end of the polysaccharides, both (backbone of) HMHEC and amylose, is kept open after immobilization reaction as illustrated in this figure (see text). It has been reported that this thermal noise analysis for the determination of the absolute value of the spring constant might cause 30-40% error for long cantilevers such as the ones used in this study (196 μm in the nominal value), due to the finite size and uncorrected position effects of the laser spot.46 Due to the error in the determination of the spring constant, combining the data sets obtained with different cantilevers for statistical analysis potentially increases the experimental error.47 In this study, only one cantilever was therefore used during data collection for statistical analysis for each sample, in order to minimize the error in the estimation of the rupture force and force loading rate. It was confirmed that different cantilevers gave essentially the same results but sometimes gave rise to a shift in force probably due to the error of the estimation of the spring constant. Force-distance curves were collected with the following conditions: tip-substrate contact time of 500 ms; scanning range of the distance, 1000 and 3000 nm for HMHEC-HMHEC and HMHEC-amylose interactions, respectively; and the pushing force against substrate after contact was fixed at 600pN, which was controlled by a feedback mechanism implemented in the Picoforce controller. AFM topographs were collected by tapping mode in air as previously described48 using a cantilever (PPP-NCH-W, Nanosensors) with nominal resonance frequency in the range 204-497 kHz.

Covalent Immobilization Procedure of Molecules on AFM Tip and Mica. Functionalization of the mica substrate and AFM tip were performed based on a previously proven method for polysaccharides.17 Briefly, freshly cleaved mica and AFM tips were cleaned for 30 min by immersion in 1:1 v/v concentrated (37%) HCl/ MeOH and rinsed in MQ-water. Then, the surface of the mica and the tip were silanized by soaking in a freshly prepared 1% (v/v) solution of trimethoxysilylpropyldiethylenetriamine (Aldrich) in 1 mM acetic acid for 20 min at room temperature. After rinsing with MQ-water, the aminesilanized layer on mica was transformed to an aldehyde layer by incubating with glutaraldehyde (diluted from 50% solution of glutaraldehyde from Sigma-Aldrich) of 12.5% (v/v) in MQ-water for 14 h and then rinsed in MQ-water. Amine-functionalization of polysaccharides was performed by a reducing-end-selective method.49 A total of 0.5 mL of (46) Proksch, R.; Schaffer, T. E.; Cleveland, J. P.; Callahan, R. C.; Viani, M. B. Nanotechnology 2004, 15(9), 1344–1350. (47) Ray, C.; Brown, J. R.; Kirkpatrick, A.; Akhremitchev, B. B. J. Am. Chem. Soc. 2008, 130(30), 10008–10018. (48) Stokke, B. T.; Falch, B. H.; Dentini, M. Biopolymers 2001, 58(6), 535–547. (49) Gray, G. R. Methods Enzymol. 1978, 50, 155–60.

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5 M Na BH3CN (5.0 M solution of sodium cyanoborohydride in aqueous ∼1 M sodium hydroxide, Sigma-Aldrich), 1 μL of 60 wt % aqueous solution of 1,6-hexanediamine (Acros organics), 1.5 mL of acetate buffer (pH = 5.5), 0.5 mL of 0.1% (w/w) solution of polysaccharide, and 1.5 mL of MQ-water were mixed and incubated for 48 h. Polysaccharides were covalently anchored to the glutaraldehyde on AFM tips and glutaraldehyde on mica substrate by leaving the tips or surfaces in contact with the above-mentioned amine-functionalized polysaccharides in solution for 17 h at room temperature. The AFM tips or mica surfaces were then transferred to MQ-water and stored in this solution until use in order to avoid drying of the surfaces. The final concentrations of polysaccharides used for immobilization were 2 μg/mL for the AFM tips and 5 μg/mL for the mica. Figure 1 depicts the structure of the polysaccharide immobilized on the mica or AFM tip. The total length of the linker connecting the mica substrate (or AFM tip) to the reducing end of the polysaccharide, which is the sum of the lengths of trimethoxysilylpropyl-diethylenetriamine, glutaraldehyde, and hexanediamine, is about 4 nm in the extended form. This is much shorter than the lengths of the polysaccharides, which are about 300 and 2000 nm for HMHEC and amylose, respectively, and it is also much shorter than that the tip-substrate separation distances where most of the unbinding events were observed in the forcedistance curves.

Results and Discussion Homogeneity of the Sample Surface. The surface topographs of the mica substrate (Supporting Information Figure 1) were determined by AFM in tapping mode following each reaction step for the immobilization of the polysaccharide. The surface roughness increased after each reaction step, but the surface was judged homogeneous at the micrometer scale. This is in accordance with previously reported data were the same conditions were used.17,50 After the immobilization of the polysaccharides, a larger height difference was seen, but this could be caused by the immobilization of the molecule itself. It was confirmed that different locations on mica had comparable surface roughness and gave rise to comparable height profiles of AFM topographs, reflecting a comparable density of immobilized molecules. (50) Sletmoen, M.; Skjak-Braek, G.; Stokke, B. T. Carbohydr. Res. 2005, 340 (18), 2782–2795.

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Classification and Stability of the Unbinding Events of the Molecules. Three essentially different types of events have previously been observed in force-distance curves: adhesion, stretching, and peeling.51,52 Pairwise interactions between polymers similar to the entanglement concept in more concentrated polymer solutions may represent a further interaction mechanism. However, preliminary studies of nonassociative high molecular weight polysaccharides using DFS (data not shown) and employing similar grafting densities have revealed that technique does not yield frequent anchoring events and unbinding signatures. Interactions by possible entanglements appear therefore not likely in the present case. In this study, the peeling type of unbinding event, which is characteristic for very fast dissociation and reassociation equilibrium processes, was not observed. This means that the time scale of the unbinding events observed in this study, that is, adhesion and stretching peaks, is slower than the time scale of the pulling speed of the molecules produced by the retraction of the cantilever. We define adhesion and stretching peaks as follows (Figure 2): an “adhesion peak” is defined as a deflection peak in the forcedistance curve having a slope that is constant and identical to that of the contact region with the mica substrate. Additionally, an adhesion peak should have a slope that does not change until the rupture distance, lr, at which a sudden decrease of the force is observed (Figure 2, inset). By “stretching peak”, we refer to a peak for an unbinding event with an increasingly larger force increasing non-monotonically with increasing separation just prior to unbinding (Figure 2). The slope for the stretching peaks in the force-distance curves increases with increasing extension ratio of the molecules. This characteristic behavior is in accordance with models of polymer elasticity which includes changes in entropy occurring upon stretching, and the observed peaks in the force-distance curves can be fitted by models53 such as the freely jointed54,55 or wormlike chain model.56,57 However, quantitative fitting of the appropriate force-elongation polymer models to such experimental data has not yet proven to be a viable route toward estimation of persistence lengths of the polymers being studied.17 Figure 2 shows the rupture forces, fr, of adhesion and stretching peaks plotted against the number index of the force-distance curves collected for HMHEC-amylose interactions. Although some fluctuations are observed, the overall behavior of both adhesion and stretching unbinding peaks, such as the probability for their occurrence, their mean value of fr, and the distribution of fr, remained at a constant level during the 2000 force curves, especially for the stretching peaks, which is the focus in the detailed analysis below. This means that these unbinding events are not caused by breaking of covalent bonds of the anchoring nor the glycosidic linkage in the polysaccharides (Figure 1), but by the dissociation of the physical interactions between the molecules immobilized on the mica substrate and the AFM tip. Force-Extension Profiles. Figure 3 shows typical force curves obtained (a) when allowing HMHEC molecules immobilized on the tip to interact with HMHEC immobilized on mica substrates and (b) when allowing HMHEC immobilized on mica to interact with amylose molecules immobilized on the tip. (51) Hugel, T.; Seitz, M. Macromol. Rapid Commun. 2001, 22(13), 989–1016. (52) Friedsam, C.; Seitz, M.; Gaub, H. E. J. Phys.: Condens. Matter 2004, 16 (26), S2369–S2382. (53) Wei, H.; van de Ven, T. G. M. Appl. Spectrosc. Rev. 2008, 43(2), 111–133. (54) Kuhn, W. Kolloid Z. 1934, 68, 2–11. (55) Guth, E.; Mark, H. Monatsh. Chem. 1934, 65, 93–121. (56) Kratky, O.; Porod, G. Recl. Trav. Chim. Pays-Bas 1949, 68, 1106–1122. (57) Fixmann, M.; Kovac, J. J. Chem. Phys. 1973, 58, 1564–1568.

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Figure 2. Stability of unbinding events, rupture forces, fr, of adhesion, and stretching types of peaks plotted as a function of index of force curve. The probability and the pattern of events, including the distribution of each type of peak, remained similar in all the force curves observed. This means that the molecules immobilized on the AFM tip and on mica substrate are tough and remain intact over 1000 force curve measurements. The definition of the rupture force of adhesion and stretching peaks in the force-distance curves are illustrated in the inset figure and further explained in the text.

The lateral position of the tip over the mica surface was not changed during the collection of the data for each sample. Most of the retrace curves contained peaks as shown in Figure 3a. The well isolated force jumps observed in the curves indicate that the chosen immobilization procedure gives rise to a density of molecules on the tip that is sufficiently small to allow a focus on pairwise interactions. The curves also indicate that the probability of interaction is high under the conditions used in this study. In the retrace force-distance profiles a-1 to a-5, adhesion peaks are observed, in addition to numerous, not well resolved peaks in the substrate-tip separation range from 0 to about 600 nm. No peaks were observed for substrate-tip separations larger than 700 nm. Nearly identical stretching profiles were occasionally observed, as illustrated by the series of force curves from Figure 3a-6 to a-10. The force curves from a-1 to a-10 were obtained at the same position on mica. Therefore, the reason why the anchoring probability changed over time is thought to be caused by the fluctuation of the direction of molecules on tip and on substrate. Although the contact position of the tip on mica might change during the experiments due to drift of the piezo-scanner, it is reasonable to assume that it remained unchanged while collecting the curves shown in Figure 3a-6 to a-10, since these curves where collected within a short time interval. The HMHEC-amylose (Figure 3b) and HMHEC-HMHEC (Figure 3a) interactions share the following similarities: (1) overlapped peaks were more often observed close to the substrate and (2) stretching peaks having fr ∼100 pN were observed far from the surface. On the other hand, the following main differences among these molecular pairs were observed: the unbinding event of the stretching peak occurred for a tip-surface separation up to 2000 nm for HMHEC-amylose interactions, whereas it was only up to 600 nm for the HMHEC-HMEC pairs. Second, for HMHEC-amylose systems, the total areas of overlapped peaks that appeared close to the surface were, in most cases, much larger than those for HMHEC-HMHEC (Figure 3). It is interesting to note that the latter five force curves for the HMHEC-amylose pairs, indices b-6 to b-10, Figure 3, show two stretching type peaks at almost the same distance (1500 and 1750 nm of tip-surface separation). This reproducibility in the position of the unbinding events in the force curve suggests that these two stretching events are due to the interaction among the same molecules (and same hydrophobes). DOI: 10.1021/la9009515

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Figure 3. Typical force-distance curves obtained (a) between HMHEC molecules and (b) between HMHEC and amylose. The horizontal and vertical scales are identical in panels (a) and (b). The blue curve is the approach curve, and the red curve is the retrace curve. The curves from b-6 to b-10 are obtained in a series with only a short waiting time between the collection of subsequent curves, showing two stretching peaks at almost the same distances from the substrate surface (see text).

There was a tendency to observe larger adhesion peaks in curves having larger R work, as illustrated in Figure 3. Work is here defined as w = F dz, where z is the distance from the mica surface and F is the force in the z direction, and is calculated based on the total area between the baseline (approach curve) and the retrace curve. Larger work reflects higher probability of anchoring of molecules. This correlation tendency suggests that this adhesion is caused not by unspecific interactions between the naked mica substrate and the AFM tip but by the molecular interactions among HMHEC. Collection of Peaks for Statistical Analysis. The force curves a-8 and a-9 in Figure 3a show an unbinding event well separated from the other peaks with a fr value around 100 pN. These peaks represent examples of peaks included in the detailed analysis of single molecular interactions. The other peaks visible in these force curves were not included in the analysis in order to avoid observations due to two or more molecules. Overlapped peaks were observed close to the surface, approximately from 0 to 200 nm (Figure 3a-6 to a-10), and were probably the results of overlapping of stretching peaks. Due to the exclusion of the overlapped peaks, the actual probability of unbinding events is probably higher than the apparent probability given in figures. Relation between Rupture Force and Rupture Distance. A clear tendency for an increase in rupture force fr with increasing distance lr was observed for the adhesion type unbinding forces, whereas the magnitude and distribution of fr of the stretching peaks was found to be independent of lr (Figure 4). All the adhesion peaks were observed close to the substrate (lr < 50 nm), and they were all characterized by a linear relation between force and distance at the rupture point. The slope of the spring deflection due to adhesion forces between the tip and the surface almost agreed with the line with a slope of k. This indicates that the HMHEC functionalized mica surface has a higher stiffness than the AFM tip and immobilization of HMHEC on mica did not have an important impact on the mechanical properties of the mica surface. Distribution of Rupture Forces. The distribution of unbinding forces obtained when analyzing the stretching peaks (Figure 5) displays one maximum. For the adhesion peaks, the distribution 10178 DOI: 10.1021/la9009515

Figure 4. Relation between force and distance at rupture point observed (a) among HMHEC molecules and (b) HMHEC (on surface) and amylose (on tip). The number shown in the legend reflects the total number of observations for each type of peak. The dashed line represents a line having a slope equal to the spring constant of 0.041 nN/nm for HMHEC-HMHEC and 0.028 nN/nm for HMHEC-amylose.

of fr of for HMHEC-amylose interactions is narrower than that for HMHEC-HMHEC interactions. The most probable value of fr for the stretching unbinding events obtained by Gaussian fitting are 64 and 26 pN for HMHEC-HMHEC and HMHEC-amylose interactions, respectively. Assuming that stronger adhesion reflects a higher probability of anchoring of molecules, the average number of molecules contributing to the adhesion could be given by the ratio of the mean rupture force of adhesion and stretching, Æ f *æadhesion/ Æ f *æstretching, and is around 3 and 10 for HMHEC-HMHEC and HMHEC-amylose interactions, respectively. Langmuir 2009, 25(17), 10174–10182

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Figure 5. Histogram of rupture force for (a) HMHEC-HMHEC and (b) HMHEC-amylose interactions. The open bars represent the stretching peak, and the closed bars the adhesion. The conditions used to make this histogram are as follows: For HMHECHMHEC interactions, the rupture forces ranging from 0 to 1500 pN were divided into 150 sections, and the number of each type of event was calculated for each section. For HMHECamylose interactions, rupture forces ranging up to 3000 pN were included in the analysis, and 300 subsections were created and further analyzed. The insets represent the enlarged view.

The unbinding forces of the stretching peaks are in most cases lower than 150 pN. According to the literature,58 the force required to rupture covalent bonds ranges from 2.0 ( 0.3 nN for Si-C (from force experiments) and 4.1 nN for C-C (calculation) to 4.3 nN for C-O bond (calculation). Although the effect of the hydrophobic interaction among hydrophobes in HMHEC on fr is not clear, the magnitude observed here can be compared to that reported by Ray et al., who used dynamic force spectroscopy to determine fr between C14 alkane chains from 40 to 140 pN depending on force loading rate.47 This fr value is much smaller than the adhesion force found in this study but comparable to the unbinding forces in the stretching peaks. Distribution of Rupture Distances. The distribution of lr for HMHEC-HMHEC interactions was very broad, and no clear peak appeared (Figure 6a). Nevertheless, the distribution of fr with lr showed a declining incidence of unbinding events with increasing lr. This broad distribution supports the interpretation that this molecular interaction can occur almost everywhere in the molecule and there is no specific region with high probability of the interaction, as would be the case for, that is, end-selective interaction. In the case of HMHEC-HMHEC interactions, the maximum lr for the stretching peak is around 600 nm, which is almost twice of the length of the individual HMHEC chain. In this study, immobilization of polysaccharides, both HMHEC and amylose, was, both on the substrate and on the tip, done by reducing endselective reaction procedure. Since an interaction can occur everywhere along the molecules, the maximum length of the rupture event is expected to be the sum of the contour length of (58) Grandbois, M.; Beyer, M.; Rief, M.; Clausen-Schaumann, H.; Gaub, H. E. Science 1999, 283(5408), 1727–1730.

Langmuir 2009, 25(17), 10174–10182

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Figure 6. Histogram of rupture distance, lr, for (a) HMHECHMHEC and (b) HMHEC-amylose interactions. Open bars represent the number of stretching peaks, and filled ones adhesion. The inset is an enlarged view of each of the histograms. The arrows in inset (b) represent periodic peaks that appear in the histogram and are given as a guide for eye.

the molecules attached to the substrate and the tip. Twice the contour length of HMHEC is 680 nm, and the sum of the contour length of HMHEC and amylose molecules is 2340 nm. These estimates are in good accordance with the maximum lr shown in the histograms (Figure 5). In the case of HMHEC-amylose interactions, most of the lr values ranged from 500 to 2500 nm. Note, however, that the peaks observed in the separation range from 0 to 500 nm contained a large fraction of overlapped peaks, making the estimation of fr and the location of separated peaks impossible in this distance range. This explains why almost no data of stretching peaks are plotted in the distance range lower than 500 nm. As for HMHEC-amylose interactions, the unbinding events showed a tendency of appearing at certain tip-surface separation distances, with a constant separation distance (= 250 nm) (Figure 6b). This observation is not common in dynamic force spectroscopy, as far as we know. From the AFM topographs of hydrated (data not shown) and dried samples (Supporting Information Figure 1), no orientation of the polymers or structure of the order of 250 nm was identified. Although we do not at present have a clear explanation for this phenomenon, we suggest some possible explanations below. A bloblike necklace structure, which is proposed for some polyelectrolytes,59 might be formed in amylose due to the intramolecular forces. The separation distance between the peaks, ∼250 nm, is comparable to the contour length of HMHEC (equal to 340 nm when estimated based on the weight average molecular weight). This suggests that the peaks in the histogram might correspond to unbinding of the amylose molecule attached to the tip from successive HMHEC molecules that are simultaneously interacting with the same amylose. Dynamic Force Spectroscopy. Basis of Analysis. The rupture forces, fr, and the other characteristic properties of molecular interactions studied at the single molecular level depend on the force loading rate, rf = dF/dt, which describes (59) Jeon, J.; Dobrynin, A. V. Macromolecules 2007, 40, 7695–7706.

DOI: 10.1021/la9009515

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Figure 7. Rupture force of stretching peaks plotted against loading force rate observed for HMHEC-HMHEC interactions. A total of 2163 peaks were collected and analyzed. Open circles represent the most probable rupture force f * calculated based on eq 1 with fitted parameters.

the actual increase in force exerted on the molecules as they are being forced apart. The retraction speed of the AFM cantilever, dz/dt, where z is the distance between the mica substrate and the AFM cantilever, was fixed for each force-distance curve, but the actual dF/dt value varies for each stretching peak due to the springlike action of the polymers. Therefore, a more detailed analysis was performed for different data sets obtained at a fixed dF/dt. According to Bell, and Evans and Ritchie,52,60,61 based on Kramers’ rate theory, the intermolecular potential is affected by the external pulling force applied to the molecules and results in the decrease of the activation energy and a subsequent increase of the dissociation rate. Under the constant force loading rate rf, the probability density P( fr) to observe a bond rupture as a function of pulling force f is given by62 8 9 "    #