Article pubs.acs.org/Macromolecules
Interactions between Individual Charged Dendronized Polymers and Surfaces Lucie Grebikova,† Plinio Maroni,† Laura Muresan,† Baozhong Zhang,‡ A. Dieter Schlüter,‡ and Michal Borkovec*,† †
Department of Inorganic and Analytical Chemistry, University of Geneva, Sciences II, 30, Quai Ernest-Ansermet, 1205 Geneva, Switzerland ‡ Department of Materials, Institute of Polymers, Swiss Federal Institute of Technology, ETH Zurich, HCI J 541, 8093 Zurich, Switzerland ABSTRACT: Polymethacrylate-based amino-terminated dendronized polymers up to generation 5 were adsorbed on mica, hydrophobized mica, and gold and studied with the atomic force microscope (AFM). These studies were carried out in a mildly acidic electrolyte solution where these polymers are fully ionized. The polymers were imaged in intermitted contact mode in solution, and desorption forces of individual polymer chains were investigated by single molecule force spectroscopy. The adsorbed chains have a uniform envelope up to generation 4, but the generation 5 polymer has a pronounced pearl-necklace structure. Force spectroscopy clearly reflects this difference. While lower generation polymers show peeling events with a single force plateau or pulling event with a single spike, those of generation 5 are characterized by two different force plateaus as well as multiple pulling events. These features are probably related to the unwrapping of pearls in the polymer backbone. We suspect that the pearls result from the high hydrophobicity of the polymer backbone.
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devices30,31 or nanoparticles,32 supports for enzymes or nucleic acids,33−35 sensors for bacteria,36 and other biological uses.36,37 The atomic force microscope (AFM) represents a powerful tool to study dendrimers and DPs on surfaces. AFM imaging has shown that adsorbed dendrimers flatten substantially when adsorbed on solid substrates38−41 and that their swelling degree can be controlled by the nature of the substrate.42 Dendrimers adsorbed on oppositely charged substrates form loose monolayers with liquid-like structure.40,43 DPs are known to flatten in their adsorbed state, even though the flattening is less extensive than for the dendrimers.44 Covalent reactions could be induced by AFM nanomanipulation of individual DPs on surfaces.45,46 AFM studies also revealed that charged DPs can fold back on themselves to create duplex bundle structures.21,47 Mechanical properties of DPs were investigated with the AFM by means of single molecule force spectroscopy.25,48,49 This technique was pioneered by Gaub and co-workers50,51 and permits to study the extension of individual polymer chains upon applied force. One finds that the stiffness of DPs increases with increasing generation and that their mechanical properties may depend on the type of solvent or the salt level.25,48
INTRODUCTION Since their discovery in the 1980s, dendtritic architectures continue to capture the imagination of chemists.1−5 Globular dendrimers consist of several dendrons attached to a central core, and they represent the most widely investigated structures. While dendrimers of lower generation have relatively loose inner structure, higher generations are rather densely packed and uniform, even though this packing may be influenced by changing the solvent quality.3,6−8 Dendrimers have unusual properties;, for example, their specific viscosity goes through a maximum with increasing molecular mass,9 or their charge may build up in even−odd shell fashion.10 At higher concentrations, they assemble in liquid-crystalline structures.11,12 Dendrimers have been proposed for interesting applications, for example, as gene vectors,13,14 drug delivery systems,15−17 or catalysts.2,18,19 Dendronized polymers (DP) carry dendrons at each backbone repeat unit, and they represent the other important class of dendritic architectures.1,5 The properties of these polymers have been studied in lesser detail than dendrimers so far. Their radial density profile resembles the one from dendrimers, whereby crowding occurs at lower generations due to packing constraints.20 DPs assemble into a variety of structures including fibrils,21 vesicles,22 or liquid crystals.23 Potential applications of DPs are often exploiting their responsive behavior24−29 and include the fabrication of optical © 2013 American Chemical Society
Received: March 25, 2013 Revised: April 10, 2013 Published: April 18, 2013 3603
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After cleaning, the surfaces were immersed in DP solution and rinsed with electrolyte solution prior to imaging or force measurements. Mica and hydrophobized mica were incubated in the polymer solution for 40 s, while gold surfaces for 3 min. Whether the samples were kept wet or they were dried in between and rewetted had no effect on the AFM results. Imaging and Force Spectroscopy. AFM imaging was carried out in ac-mode with a Cypher (Asylum Research, Santa Barbara, CA). Imaging in solution was carried out with Biolever mini cantilevers (BLAC40TS, Olympus, Japan) with a nominal tip radius smaller than 9 nm and a resonance frequency of 25−36 kHz. Images were acquired with a scan rate of 4.88 Hz, free oscillation amplitude (FOA) of about 20 nm, and a set point corresponding to around 70% of the FOA. The root-mean-square (rms) roughness of the bare substrate was determined by imaging 1 μm2 of each samples, and they are reported in Table 1. To analyze lateral sizes and the volumes of the adsorbed molecules, a tip erosion algorithm was applied to the recorded images to correct for the tip convolution effect.58 This procedure requires the tip shape profile, which is obtained by imaging spherical particles in the electrolyte solution. For PG1, gold nanoparticles (Sigma-Aldrich) with diameter of about 5 nm were used, while for larger generations polystyrene particles (Nanosphere Size Standard, Thermo Scientific) with a diameter of around 30 nm. These particles were previously adsorbed on mica functionalized with polyethylenimine of a molar mass of 25 kg/mol (Polysciences, Eppenheim, Germany). The tip geometry was extracted by applying the erosion algorithm and by assuming that the particles are spherical with a diameter equal to their height, which can be accurately obtained by the AFM image.58 The volumes of the polymers were calculated with the Gwyddion software freely available at http://gwyddion.net/. Force spectroscopy measurements on individual DPs were carried out with the Cypher AFM. The Biolever mini cantilevers were silanized overnight in the gas phase in an evacuated container in the presence of (3-glycidoxypropyl)dimethylethoxysilane (Sigma-Aldrich, Switzerland). DPs were first imaged in the electrolyte solution based on the same protocol as described above. Because of tip broadening caused by the silanization process, the images obtained with functionalized tip had usually inferior lateral resolution than those acquired with a bare tip. To avoid thermal drifts during the force measurements, the DP were first continuously imaged with the AFM for about 1 h. When the image has stabilized, the piezo excitation of the cantilever was stopped and the tip was placed on a particular location on a selected molecule. Subsequently, a series of 300 approach-redraw force curves were recorded with a sampling rate of 10 kHz, whereby the deflection of the cantilever and the vertical piezo displacement were acquired as a function of time. To preserve the tip functionalization, a deflection set point toward the surface smaller than 30 nm was used. The cantilever spring constants were in the range of 0.04−0.09 N/m as measured through thermal fluctuations in air.59 The retraction velocity used was 200 nm/s. Retraction force curves were very similar when the velocity was varied in the range of 100−300 nm/ s. After these force measurements, the same region was imaged again. The cleanliness of the tip was checked by recording force curves in a part of the surface that was free of adsorbed molecules. A clean tip did not show any single molecule events. When such events were observed, the measurements were discarded. This procedure assures that the force measurements are truly performed at the single-molecule level and allows the detection of eventual lateral rearrangement after external mechanical excitation. High-Resolution Imaging. High-resolution scanning probe microscopy probes with tungsten spikes on the apex of a silicon tip (Hi’Res-W14/AIBS, μmasch, Tallinn, Estonia) were used for highresolution imaging in liquid. The declared spike radius was smaller than 1 nm. Images were acquired near the resonance frequency of the cantilevers in liquid at around 70 kHz and with a maximum scan rate of 0.5 μm/s and a maximum scan size of 250 μm. The FOA and the set point were adjusted to around 2 and 1.8 nm, respectively.
Force spectroscopy has not yet been used to study interactions of DPs with surfaces. Polymers weakly interacting with surfaces can be peeled from them, and this process leads to characteristic staircase-like force profiles.51−55 The peeling force represents a direct measure of the affinity between the polymer and the surface in the solvent in question. In this article, we report on investigations with AFM of the peeling of individual polymethacrylate-based DPs terminated with amine groups adsorbed to mica, hydrophobized mica, and gold from aqueous electrolyte solutions at pH 4. Under these conditions, all terminal amine groups of the DPs investigated are positively charged. Favorable conditions for peeling of DPs can be best realized on hydrophobized mica, and one finds that the peeling force strongly increases with increasing dendron generation. We also find that the DP of generation 5 peels from surfaces in a much more complex fashion than the lower generations, and we argue that this behavior is related to the pearl-necklace structure of this polymer.
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EXPERIMENTAL SECTION
Dendronized Polymers. The attach-to route was used to synthesize polymethacrylate-based DP of different generations (PGn, n = 1−5) terminated with amine groups as described elsewhere.56,57 The polydispersity of the side dendrons is less than 1% when expressed as the deviation from the theoretical number of the amine groups per repeat unit. The molar mass of the charged PG4 sample used in this study was determined by applying gel permeation chromatography (GPC) to its neutral direct precursor, a PG4 DP in which all terminal amine groups are tert-butyloxycarbonyl (Boc)-protected.56 This way the use of aqueous GPC could be avoided. Deprotection of Boc-protected DPs with trifluoroacetic acid proceeds quantitatively so that neither the molar mass nor the distribution is affected by this mode of operation. The GPC measurements were performed on a PL-GPC 220 instrument with a 2 × PL-Gel Mix-B LS column set (2 × 30 cm) equipped with refractive index (RI), viscometry, and light scattering, using DMF (LiBr, 1 g/L) as eluent at a temperature of 45 °C. Universal calibration was carried out using poly(methyl methacrylate) standards in the range of 2.7 × 103−1.5 × 106 g/mol (Polymer Laboratories Ltd., UK). Polymers were always dissolved in 10 mM KCl electrolyte solution of pH 4.0 unless noted otherwise. Some experiments were carried out in 1 and 100 mM KCl solutions. Eventual rinsing was done with the same electrolyte solution. Surfaces. Three types of surfaces were used, namely mica, hydrophobized mica, and gold. High grade mica was obtained from Plano (Wetzlar, Germany). Bare mica surfaces were obtained by cleaving prior to each experiment in air. A hydrophobized mica surface was obtained by vacuum silanization. More precisely, freshly cleaved mica was placed in an evacuated glass desiccator aside a 200 μL drop of dimethoxy(methyl)octylsilane (Sigma-Aldrich, Switzerland) for 30 min. Single crystal gold Au(111) surfaces grown on mica were obtained from Phasis (Geneva, Switzerland). The gold surfaces were rinsed with 2% Helmanex solution, washed with pure water, dried in a stream of nitrogen, and ozone cleaned with an UV-ozone cleaner (PSD Pro, Novascan, Ames, IA) in an oxygen-enriched atmosphere for 20 min. Contact angles of the different surfaces were measured with a homemade video-camera setup, and they are summarized in Table 1.
Table 1. Properties of the Surfaces Used surface
rms roughness (nm)
contact angle (deg)
bare mica hydrophobized mica gold
0.07 0.14 0.19
0 12 42 3604
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RESULTS AND DISCUSSION
Individual amino-functionalized DPs were investigated by AFM adsorbed on three different surfaces, namely bare mica, hydrophobized mica, and gold. They were investigated at pH 4 where the amino groups are fully ionized normally in 10 mM KCl electrolyte solution, unless indicated otherwise. In particular, images of the adsorbed molecules were acquired, and single molecule force experiments were carried out. Imaging. Different generations of DP adsorbed on mica were imaged with AFM in solution in the intermittent contact mode. Overview images of samples, where different generations were mixed, are shown in Figure 1. For generations up to PG4 one observes a uniform envelope of the adsorbed chains. However, PG5 features a characteristic pearl-necklace structure.
Figure 2. Adsorbed DPs on mica imaged by AFM in 10 mM KCl electrolyte of pH 4. Normal-resolution images (top row) and highresolution images (bottom row). PG4 (left column) and PG5 (right column).
probably somewhat underestimated due to their pearl-necklace structure. No dependence on the salt level of the heights and volumes of the single molecules could be established. Identical pearl structures for PG5 could be observed at different salt concentrations. The numbers reported in Table 2 compare relatively well with independent estimates. For the PG4 sample, the numberaverage molar mass Mn and PDI of 6.8 × 106 g/mol and 2.0 were determined by multiple-detection gel permeation chromatography, and they are in reasonable agreement with the present estimates. The somewhat smaller value of the molar mass obtained by AFM is probably due to errors in the deconvolution process. The average height and width of similar but neutral DP were studied with AFM in dry state and with cryo-field emission scanning electron microscopy.44 The presently reported heights compare very well with these values, while the observed widths are somewhat larger. The latter difference is probably due to the fact that the measurements are carried out on isolated molecules, while in the mentioned reference they were measured from the repeat distance between several aligned molecules. The average height of the PG5 molecule reported here is somewhat smaller than the one for PG4. This discrepancy has to do with the fact that the PG5 shows the pearl-necklace structure, while the unprotected polymer rather has a cylindrical form. The peak-to-valley height difference along the backbone is about 3 nm. We have also counted the number of pearls in all PG5 molecules imaged. By dividing the volume of each molecule by the corresponding number of pearls, we find an average volume of each pearl. Assuming a spherical shape, we find that the average diameter of a single pearl is about 11 nm. Given the known molar mass of a monomer and its density, we find that there are ∼71 monomers per pearl. The pearls appear larger in the normal-resolution AFM images due to convolution with the cantilever (see Figure 2, top). This analysis procedure of the normal resolution images does not provide any accurate information on the size distribution of the pearls. To obtain this information, we have analyzed the high-resolution images, where tip convolution effects are almost negligible (see Figure
Figure 1. Adsorbed dendronized polymers (DPs) of different generations on mica imaged by AFM in 10 mM KCl electrolyte of pH 4. (a) Structural formulas of the polymers and images of (b) a mixture PG1, PG2, and PG3 and (c) a mixture PG3, PG4, and PG5. The pearl structure of PG5 distinguishes it from PG4.
Figure 2 compares high-resolution images of adsorbed PG4 and PG5 chains obtained with very sharp AFM tips. These images clearly illustrate the different structures of the charged chains. Since very similar structures were also observed for chains adsorbed on hydrophobized mica and gold, we hypothesize that these structures exist in solution, too. One should note that uncharged PG5 does not show any pearnecklace structure.57 A large number of similar AFM images were analyzed quantitatively. The average heights and apparent lengths of the molecules were recorded. By applying a deconvolution algorithm, volumes of the individual molecules were estimated, too. Table 2 summarizes the resulting number-averaged molar mass Mn and of the polydispersity index (PDI) defined as Mw/ Mn, where Mw is the weight-averaged molar mass. The density of 1.3 g/mL was used for these molecules as reported earlier for neutral analogues.44 Dividing their volume by the apparent length furnishes the cross-sectional area. This area is converted to a width at the base of the molecule by means of a parabolic cross section, which turned out to represent a good approximation of the actual profile. The corresponding values are summarized in Table 2. Since we report the maximum heights for PG5, their width is 3605
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Table 2. Average Properties of the Dendronized Polymers (DP) Used no. of molecules analyzed PG1 PG2 PG3 PG4 PG5
99 125 188 104 73
mol mass (g/mol) (3.0 (8.0 (2.9 (5.8 (3.9
± ± ± ± ±
0.6) 0.9) 0.3) 0.5) 0.6)
× × × × ×
105 105 106 106 106
PDI 2.2 1.6 1.5 1.3 1.6
± ± ± ± ±
0.5 0.2 0.1 0.1 0.2
height (nm) 1.31 1.65 3.57 6.12 5.55
± ± ± ± ±
0.09 0.04 0.04 0.05 0.09
width (nm) 3.0 5.0 10.8 13.2 17.6
± ± ± ± ±
0.5 0.5 0.4 0.5 0.7
molecules. Any eventual lateral displacement of the molecule that may have occurred during the force scan can be detected. The individual force profiles vary substantially from one approach−retraction cycle to another. Rather frequently, no events are recorded at all during a cycle. For PG3, 96.3% of all cycles did show no events on average. In the remaining 3.7% of all cycles, single molecule events were observed. For PG4, events were observed more frequently, namely with a frequency of 23.7%. These events occur since the polymer is bridging the tip and the substrate, and when the tip is retracted, the polymer is progressively stretched. Two major types of events are observed, namely peeling and pulling. Peeling events are characterized by a constant plateau in the force curve, and they were observed in 3.0% of all retract curves for PG3 and in 20.9% for PG4. In this case, the molecule is more weakly attached to the substrate, and when the tip is retracted, the molecule is being peeled away. In the present case, the peeling force is about 25 pN. Note that only single plateaus are observed, and several plateaus were not detected. Several plateaus would be characteristic for peeling events involving loops within individual molecules or originating from several polymer chains. Sliding of the polymer on the surface might also lead to plateaus in the force profiles.64 Since the polymer images were mostly identical before and after the force experiments, we suspect that sliding of the polymer on the surface is unimportant. Pulling events were observed in 0.7% of all cases for PG3 and in 2.8% of all cases for PG4. They are associated with a characteristic spike and result when a molecule is strongly attached to tip and substrate. At larger extensions, the force increases strongly with the extension in a nonlinear fashion. The pulling force becomes at one point so large that the molecule detaches from the tip, and this event leads to the characteristic spike in the force curve. By imaging the polymer on the surface after the force experiments, one can ensure that the molecule has again detached from the tip. Detachment of an entire molecule from the surface was never observed, since single molecule events always occurred at a distance that was much smaller than the contour length of the molecule. Pulling of DP was investigated earlier and was shown to be sensitive to the salt level.25 This issue is complicated by the fact that DP may form duplex bundles,21,47 and their presence could affect the force extension relationships. We will address these aspects in a forthcoming article. Peeling from Different Surfaces. PG3 was used to investigate the differences in the peeling behavior involving various surfaces, namely bare mica, hydrophobized mica, and gold. Typical force curves are shown in Figure 4 (top). Histograms of the peeling forces for the three different surfaces shown in Figure 4 (left column) reveal that their distribution is approximately Gaussian. One also observes that the nature of the surface influences the magnitude of the peeling force substantially. The average peeling forces and the corresponding standard deviations are summarized in Figure 4a (right
2, bottom). By estimating the volumes of about 100 pearls in such images, we found that each pearl contains about 63 monomers on average, which is in good agreement with the value quoted above. This number suggests that the contour length of polymer backbone within one pearl is about 16 nm on average. We further find that the volume distribution of the pearls is relatively broad and can be characterized with PDI of about 1.2. Because of the limited set of high-resolution images available, these numbers only represent approximate estimates. The mechanism of pearl formation is probably related to the hydrophobic nature of the backbone, which is known to generate pearl-necklace structures in synthetic linear polyelectrolytes. With increasing hydrophobicity of the backbone, the polyelectrolyte undergoes a coil-to-globule transition whereby the intermediate structures have a pearl-necklace structure. For synthetic polyelectrolytes, this effect is well established by AFM imaging60,61 and with computer simulations.62,63 How this mechanism can be extended to dendronized polymers is not known yet in detail. Single Molecule Force Experiments. Typical experimental results with single DP molecules adsorbed on hydrophobized mica are shown in Figure 3. The polymers are
Figure 3. Single molecule force profiles on retraction for the molecule shown above before and after the experiment on hydrophobized mica. PG3 (left) and PG4 (right).
adsorbed on the surface and imaged in solution with the AFM with a silanized tip in intermittent contact mode. After the selection of a part of a suitable molecule (arrow), a series of about 300 approach−retract force profiles were recorded. After this experiment, the same region of the sample is imaged again. This procedure that was carried out within the same fluid cell assures that force profiles are recorded truly for individual 3606
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Generation Dependence of Peeling Forces. The generation of DP has also a substantial effect on the peeling forces. Figure 5a shows peeling events for different generations
Figure 4. Peeling of PG3 from different substrates in 10 mM KCl at pH 4 with typical force curves shown on the top. Left column shows histograms of the peeling forces for (a) bare mica, (b) hydrophobized mica, and (c) gold. Right column shows some characteristics of the peeling forces. (a) Average peeling force with error bars, (b) probability of events, and (c) relative probability of peeling and pulling.
Figure 5. Peeling of DP of various generations from hydrophobized mica in 10 mM KCl at pH 4 with typical force curves shown on the top. Left column shows histograms of the peeling forces for (a) PG3, (b) PG4, and (c) PG5. Right column shows some characteristics of the peeling forces. (a) Average peeling force with error bars, (b) probability of events, and (c) relative probability of peeling and pulling.
column). The peeling force increases in the sequence bare mica, hydrophobized mica, and gold. The contact angle and thus the hydrophobicity of the surface increases in the same sequence (see Table 1). The relative width of the distributions is about 25%. The present results are well comparable to peeling of other polymers, including poly(vinyl amine),51 poly(allyl amine),53 poly(acrylic acid),54 cellulose,55 or singlestranded DNA.65 These polymers show average peeling forces in the range of 50−100 pN and comparable widths of the corresponding distributions. The probability to observe an event shown in Figure 4b (right column) also increases in the same sequence. Both trends indicate that the forces responsible for the adsorption are principally of hydrophobic character. The relative probability to observe peeling versus pulling events as shown in Figure 4c (right column) does not indicate any clear trends, however. Hydrophobized mica shows the largest tendency for peeling. For bare mica, the probabilities to observe peeling or pulling are about comparable, while pulling is much more likely for gold. The polymer attaches to gold the strongest, which rationalizes its tendency to generate more frequent pulling events.
from hydrophobized mica, and one observes that the peeling force increases with increasing generation. Moreover, for PG5 one observes two rather distinct magnitudes of peeling forces. This situation is more clearly evident in the histograms of the force curves shown in Figure 5 (left column). PG3 and PG4 show the usual Gaussian distribution of peeling forces, while the distribution for PG5 shows two maxima. More detailed analysis of PG5 indicates that large peeling forces correspond to relatively short chains, while the smaller peeling forces include short as well as longer chains. We suspect that the smaller peeling forces originate from the unwrapping of the individual pearls, while the larger peeling forces correspond to the peeling of the polymer from the surface. Average peeling forces and the corresponding standard deviations are shown in Figure 5a (left column). One observes that the peeling force increases with increasing generation. This 3607
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trend can be understood since the polymers flatten in the adsorbed state, and this deformation leads to an increase in the number of contact points with increasing generation. This aspect can be also illustrated by considering the work of adhesion, which equal to the ratio between the peeling force and the width of the polymer. One obtains about 3.3 ± 0.6 mJ/ m2, and this value is independent of the generation. Interestingly, this value is about 1 order of magnitude lower reported for synthetic polyelectrolytes.52 This fact is also reflected in the adsorption energy per monomer, which was reported to be about 3−6 kT for synthetic polyelectrolytes, where kT refers the thermal energy. For DPs this number is substantially lower as it lies in the range of 0.7−2.9 kT, and it increases with the generation. This difference is probably related to the fact that only a part of the DP is exposed to the surface. The two values plotted for PG5 indicate the position of the two peaks in the histogram. This aspect is related to pearlnecklace structure of PG5 and will be discussed in the next section. Figure 5b (left column) shows the probability of all types of events versus the generation. While this probability roughly increases with increasing generation, PG3 shows the lowest probability among all. Figure 5c (left column) illustrates that peeling is much more likely than pulling for DP up to generation 4, while pulling becomes more frequent for PG5. Salt Dependence of Peeling Forces. The salt concentration was found to have a substantial effect on the observed peeling forces. Figure 6 summarizes these results for PG3. The histograms of the force plateaus are shown in the left column, and the average peeling force increases strongly with increasing salt concentration, as shown in Figure 6a (right column). The probability to observe an event also increases with increasing salt concentration (Figure 6b, right column). At low salt concentrations peeling is observed exclusively, while at high concentrations peeling and pulling events are roughly equally probable; see Figure 6c (right column). A similar increase of the peeling force with the salt concentration was also observed when bare mica was used as a substrate. The increase of the peeling force with increasing salt level may seem surprising at first. When only electrostatic forces would be present, one expects that the peeling force of a charged polyelectrolyte adsorbed at an oppositely charged substrate decreases with increasing salt level due to an increased screening of the electrostatic interactions by the salt ions. Such dependence was indeed observed for simple polyelectrolytes, such as poly(vinyl amine), poly(acrylic acid), or cellulose,51,53,55 and could be semiquantitatively explained with a simple Debye−Hückel model. However, a substantial nonelectrostatic contribution to the peeling force could be also identified, which may strongly depend on the substrate. In the present case, the nonelectrostatic contribution seems to dominate and even lead to the reversed trend. This trend is probably due to the hydrophobicity of the DP backbone. Since the hydrophilic charged amine groups are screened with increasing salt level, hydrophobic interactions between DP and the substrate are expected to become more important. However, it was also shown that shifts in ionization equilibrium upon adsorption of the polymer may also lead to a similar reversed salt dependence.53 Consequences of the Pearl-Necklace Structure of PG5. The highest generation polymer PG5 shows substantially different peeling behavior than all lower generations. We suspect that these differences are due to the pearl-necklace
Figure 6. Peeling of DP PG3 at various salt concentrations of KCl at pH 4 from hydrophobized mica. Typical force curves are shown on the top. Left column shows histograms of the peeling forces for (a) 1, (b) 10, and (c) 100 mM. Right column shows some characteristics of the peeling forces. (a) Average peeling forces with error bars, (b) probability of events, and (c) relative probability of peeling and pulling.
structure of PG5. Recall that the histogram of the peeling forces shows two maxima (see Figure 5). Another important difference between PG5 and the lower generation DPs is the presence of the complex multiple pulling events for PG5 shown in Figure 7. Such events are only observed for PG5 and not for lower generation polymers. Similar single molecule force events involving numerous spikes were reported for muscle and other proteins.50,66,67 These proteins show a very regular sequence of spikes, while the sequence of PG5 is random. Nevertheless, we suspect that the mechanisms are similar. While the spikes observed for proteins correspond to unwrapping of individual subdomains, those observed for PG5 probably correspond to unwrapping of individual pearls. The irregular sequence likely originates from the random coil structure of the individual pearls and random entanglement of the chain. Note that events involving multiple spikes have also been observed when several polymer molecules have been pulled at the same time.68 However, one can exclude this possibility since our method of imaging before and after the force experiments ensures that we deal with individual chains only. Figure 8 shows the distribution of the distances between individual spikes exceeding 50 pN. The distribution is relatively broad and has an average of about 16 nm and a standard deviation of 14 nm. This value is exactly the same as the estimated average contour length of the polymer within one 3608
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CONCLUSION Charged DP adsorbed on three different substrates were studied by AFM imaging and single molecule force spectroscopy. A pearl-necklace structure is observed for PG5, while the chains are uniform for the lower generations. This marked difference in conformations was also confirmed with force spectroscopy carried out on individual molecules. Lower generation polymers show peeling events with a single force plateau or pulling events with a single spike. On the other hand, PG5 shows two types of peeling events and frequent multiple pulling events. We suspect that these events are related to the unwrapping of the pearls from the PG5 backbone. The pearls observed in PG5 probably result from the large hydrophobicity of the polymer backbone. A similar mechanism has been demonstrated to lead to pearl-necklace structures for hydrophobic polyelectrolytes and was confirmed experimentally60,61 and theoretically.62,63
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Figure 7. Typical peeling force curves for PG5 together with the corresponding images of the molecules investigated. The arrow indicates the position of the force measurements. (left top to bottom) Peeling events illustrating the two different force plateaus, and multiple pulling event, and a single pulling event. (right) Combination of different pulling and peeling events.
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This research was supported by the National Research Program “Smart Materials” (NRP 62) and other programs of the Swiss National Science Foundation, University of Geneva, and ETH Zürich.
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REFERENCES
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Figure 8. Distribution of the distances between the spikes in the force curves for PG5. The inset shows a sample force curves with the distances indicated. The threshold to detect a spike was set at 50 pN.
pearl given above. The broadening of the distribution reflects most likely the polydispersity of the pearls. On the basis of the AFM images, we have concluded above that the pearls have a PDI of 1.2. The spike-to-spike distribution shown in Figure 8 suggests a PDI of 1.9. The fact that the distribution of spikes is wider than the distribution of the pearl volumes probably reflects the length distribution of segments attached to the substrate or our inability to resolve very small pearls with the AFM. Two different peeling forces reported in Figure 5 are also likely related to the pearl-necklace structure of PG5. Unwrapping of a single pearl may lead to a smaller peeling force, while desorption of an adsorbed polymer chain to a larger peeling force. 3609
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