NANO LETTERS
Force Spectroscopy Study on Poly(acrylamide) Derivatives: Effects of Substitutes and Buffers on Single-Chain Elasticity
2002 Vol. 2, No. 10 1169-1172
Chi Wang, Weiqing Shi, Wenke Zhang, and Xi Zhang* Key Lab of Supramolecular Structures & Materials, College of Chemistry, Jilin UniVersity, Changchun 130023, P. R. China
Yukiteru Katsumoto and Yukihiro Ozaki Department of Chemistry, School of Science and Technology, Kwansei-Gakuin UniVersity, Sanda 669-1337, Japan Received July 10, 2002; Revised Manuscript Received August 22, 2002
ABSTRACT Single-chain elasticity of two important poly(acrylamide) derivatives, poly(N,N′-dimethylacrylamide) (PDMA) and poly(N,N′-diethylacrylamide) (PDEA), was investigated by an atomic force microscopy (AFM)-based single molecule force spectroscopy (SMFS). SMFS revealed that the single chain of PDEA was stiffer than that of PDMA due to different bulky substitutes. It was also found from the SMFS study that the elasticity of PDMA and PDEA was enhanced in urea buffer solutions, probably due to hydrogen-bond interactions between urea molecules and the side groups of poly(acrylamide) derivatives.
The deformation of a polymer chain is a classical problem in polymer science.1 However, in the past it was difficult to directly obtain information about nanomechanical properties of individual polymer chains with conventional experimental methods. With the recent advancement of piconewton instruments,2,3 an atomic force microscopy (AFM)-based technique single molecule force spectroscopy (SMFS) has been developed.4-6 Many elegant single-molecule experiments have been performed using this technique, providing information such as the elasticity of a single-polymer chain,7-10 force-induced conformational transition of an individual glucopyronose ring,4,6,11-14 single-molecule photomechanical cycle,15 details of helical structure splitting,16-19 unfolding of the immunoglobulin titin domain,5,20 host-guest interactions,21 and even the strength of single covalent bonds.22 N-Substituted water soluble poly(acrylamide) derivatives are of interest in fundamental research for protein modeling in bioscience, as well as in applied fields.23-25 In the present study, we investigated comparatively the single-chain elasticity of PDMA and PDEA with SMFS. Both polymers have exactly the same backbone structure but differ in the N-substituted groups, methyl and ethyl respectively, as shown * Corresponding author. E-mail:
[email protected]. 10.1021/nl0256917 CCC: $22.00 Published on Web 09/13/2002
© 2002 American Chemical Society
Scheme 1. The Chemical Structures of Poly(dimethylacrylamide) (PDMA) and Poly(diethylacrylamide) (PDEA)
in Scheme 1. Therefore, they offer a good example for a comparative study on the effects of different side groups on the elasticity of a single-polymer chain. Moreover, the SMFS experiments can be carried out under varied environmental conditions. Therefore, we also investigated the difference in the elastic behavior between PDMA and PDEA under different concentrations of urea buffer solutions. The home-built SMFS was set up in cooperation with Prof. Hermann Gaub in Muenchen; the experiment details have been described elsewhere.1,26 In brief, a polymer sample was first physisorbed onto a substrate, a glass slide in the present experiment. Then, an AFM cantilever was lowered to the substrate by the movement of a piezo, whereupon some molecules were absorbed onto the AFM tip, forming a polymer bridge in between. During the separation of the
Figure 1. Several typical force-extension curves of PDMA in an aqueous solution (pH ) 9), one of which is fitted by modified FJC (M-FJC) curve. The fit parameters are lk ) 1.3 nm, Ksegment ) 12000 pN/nm. The normalized force curves are superimposed and plotted in the inset.
cantilever and the substrate, the polymer chain then was stretched and the cantilever deflected; the deflection was detected by optical means. A deflection-extension curve was recorded and converted into a force-extension curve.1 The experiments were performed under different liquid buffer solutions, including an aqueous NaOH solution (pH ) 9) and aqueous urea solutions of 2 and 8 M. Silicon nitride cantilevers (PARK, Sunnyvale, CA) with spring constants of 12-60 pN/nm, as determined from thermal excitation, were used in the SMFS experiments.27 The concentrations of PDMA and PDEA for the sample preparation were approximately 5 × 10-4 mg/mL, and the solutions were stored at 0 °C before further use. Approximately 200 µL of the PDMA or PDEA aqueous solution was dropped onto a glass substrate and incubated, at room temperature, for approximately 25 to 30 min. The excess liquid was removed from the substrate, and the substrate with polymer was ready for SMFS measurement. Typical force-extension curves of PDMA in an aqueous solution (pH ) 9), obtained in different SMFS experiments with different cantilevers, are shown in Figure 1. The curves show similar characteristics; the force rises monotonically with extension and drops rapidly to zero until a rupture point reaching. Due to both the polydisperse nature of the polymer and the uncontrolled stretching point of an AFM cantilever, the observed contour lengths of PDMA vary, as shown in Figure 1. To compare the force-extension curves of the polymer for different contour lengths, the force curves were normalized under the same extension. The normalized force curves are superimposed in the inset of Figure 1. The normalization was carried out using a method reported in the literature.1,10 Here, the force was chosen to be 350 pN, and then the corresponding extensions was obtained from the force-extension curve and each curve was divided by its respective extension. From the superimposition of the normalized force curves, the elasticity of the polymer scales linearly with its extension. This strongly indicates that a single chain was stretched during the experiment. By keeping the stretching force lower than the rupture force, we repeatedly stretched and relaxed the same PDMA chain and recorded the deformation curves. Figure 2 shows a consecutive trace-retrace pair from the same of PDMA chain. No hysteresis between the stretching and relaxing 1170
Figure 2. Subsequent deformation curves of a same PDMA single chain.
traces was observed, suggesting that the single manipulation is done under equilibrium conditions. A modified freely jointed chain (M-FJC) model was used to describe the semiquantitative elasticity of single PDMA polymer chains.1,10 The modified FJC model, which is based on the extended Langevin function26,28 (see function below) treats a macromolecule as a chain of statistically independent segments. X(F) ) [coth((Flk)/(kBT)) - (kBT)/(Flk)](Lcontour + (nF)/Ksegment) Here, F is the external force upon an individual polymer chain, X represents the extension of polymer chain (end-toend distance), Lcontour is the length of the polymer chain under maximal extension, n is the number of segments being stretched, kB is the Boltzmann constant, and T is temperature. The deformation of segments is characterized by the segment elasticity, Ksegment. The length of the segment is Kuhn length, and the segments, which can be deformed under the stress, are freely joined together. The Kuhn length and the segment elasticity represent the elasticity of an individual polymer chain. The elasticity of a single chain is dominated by entropic and enthalpic contributions. In the low force region the elasticity of the polymer is mainly governed by entropy, including conformations of polymer chains, known as entropic elasticity. In the high force region the elasticity is predominantly controlled by enthalpy, including bond elongations and bond angle deformation, known as enthalpic elasticity. Although the contour lengths of the stretched PDMA chains are different, all force curves obtained in the SMFS experiments with different cantilevers can be well fitted to the extended FJC model. One of the experimental PDMA force curves and the fit curve are shown in Figure 1. The fit parameters for the modified FJC model for all of the PDMA force curves form a narrow distribution, lk ) 1.3 ( 0.1 nm and Ksegment ) 12000 ( 1000 pN/nm. Similarly, we explored single-chain stretching of PDEA in an aqueous solution (pH ) 9). Figure 3 shows a typical normalized force curve of PDEA and the corresponding model curve, which was also fitted to the modified FJC model. We obtained many force extension curves of PDEA, Nano Lett., Vol. 2, No. 10, 2002
Figure 3. Comparison of modified FJC fits and normalized force curves of PDMA and PDEA in aqueous solutions.
and they were all fitted well by the model. The fit parameters of elasticity were lk ) 1.6 ( 0.1 nm and Ksegment ) 17000 ( 1000 pN/nm for PDEA. For comparison of the single-chain elasticity, a normalized force-extension curve of PDMA in the aqueous solution and the corresponding model curve are also shown in Figure 3. We can clearly see that in the low force region the force curves of the two polymers superimpose, indicating that the entropic elasticity is similar. However, in the high force region, the elasticity of the two polymers, dominated by enthalpic contribution, differ; a single PDEA chain is stiffer than that of PDMA. It is probably because the two polymers exist in a similar conformational condition in the aqueous solutions, and thus the entropic elasticity of the two polymers is likely similar at low force. The elastic discrepancy in the high region may be attributed to the effects of the different side groups on the main polymer backbone. The PDEA side group is ethyl while that of PDMA is methyl. Thus it is well understandable that the side group effects of PDEA on the backbones are larger than those of PDMA. As a result, the single-polymer chain of PDEA is stiffer than that of PDMA. In addition, it is not only the bulkiness of the substituents but also the resulting different hydrophobicity of the polymers that determine their structure in water. Light scattering has confirmed that PDEA can form a microgel in aqueous solution due to the hydrophobicity of the substituents.29 However, tetrahydrofuran (THF) can decrease the hydrophobicity and lead to the breakage of the microgel. Therefore, the SMFS experiments were also carried out in THF, to avoid the influence of the microgel on single-chain stretching. When we compared the PDEA force curves obtained in aqueous solution and THF correspondingly, the enthalpic elasticity of a single-polymer chain was exactly the same, indicating that the hydrophobicity cannot affect the enthalpic elasticity in the high force region. Both the hydrogen bonds and the hydrophobicity of polyacrylamides in their solution might contribute to the entropic elasticity, but in this study for polyacrylamides with N-substituted groups there is no obvious discrepancy in their entropic elasticity in the low force region. In our previous study,10 it was shown that it is possible, by changing buffers, to study the interaction between small molecules and polymers using SMFS, since the experiment is carried out at a liquid-solid interface in a liquid cell. Here Nano Lett., Vol. 2, No. 10, 2002
Figure 4. Comparison of normalized force curves for aqueous solution and 2 M and 8 M urea buffer solutions: (a) PDMA, (b) PDEA.
we studied comparatively single-chain stretching of PDMA and PDEA in 2 and 8 M aqueous urea solutions. Figure 4a shows normalized force curves for PDMA in aqueous solution (pH ) 9) and 2 and 8 M urea buffer solutions. Of note in Figure 4a is that the elasticity of PDMA is enhanced in higher urea concentrations. The fit parameters of the modified FJC model for PDMA are the following; for the 2 M urea solution, lk ) 1.5 ( 0.1 nm, Ksegment ) 16000 ( 1000 pN/nm; for 8 M urea solution lk ) 2.8 ( 0.2 nm, Ksegment ) 28000 ( 1000 pN/nm. Comparing these fit parameters, we find that the stiffness of a PDMA polymer chain increased with urea concentration. This is attributed to the formation of hydrogen bonds between the PDMA and urea, by which urea molecules can enlarge the ethalpic elasticity of the polymer backbone by binding to the side groups directly. To understand the interaction between urea and PDMA, we measured infrared (IR) spectra of PDMA and the mixture of PDMA in the 2 and 8 M urea solutions. Figure 5 highlights the spectral changes in the carbonyl stretching vibration band region. Curve (a) is the IR spectrum of PDMA film, and a band at approximately 1639 cm-1 is attributed to the carbonyl stretching mode. Curve (b) is the IR spectrum of the mixture of PDMA in 8 M urea solution. Curve (c) is the difference FTIR spectrum calculated by subtracting the spectrum of 8 M urea buffer solutions from the spectrum of the mixture. In the IR spectrum of the mixture (b), there is a shoulder at approximately 1610 cm-1 region. While in the difference spectrum (c), the band at approximately 1619 cm-1 suggests the formation of hydrogen bond between a urea molecule and a carbonyl group of PDMA. In the case of the 2 M urea solution, FTIR spectra show similar results. The SMFS results obtained for PDEA were similar as for PDMA, as shown Figure 4b. The fit 1171
buffer solutions, as shown in Figure 6b, no discrepancy is seen. This indicates that when the concentration of urea solution is sufficiently high to become the dominating factor in determining the elasticity of a single-polymer chain, the discrepancy in the elasticity between PDMA and PDEA is shielded completely.
Figure 5. IR spectra in the 1800-1500 cm-1 region of PDMA: (a) PDMA film; (b) mixture spectrum of PDMA and 8 M urea solution; (c) mixture difference spectrum subtracted by 8 M urea solution.
Figure 6. Comparison of normalized force curves of PDMA and PDEA: (a) in 2 M urea solution; (b) in 8 M urea solution.
parameters for PDEA are lk ) 2.0 ( 0.1 nm, Ksegment ) 21000 ( 1000 pN/nm for the 2 M urea solution and lk ) 2.8 ( 0.2 nm, Ksegment ) 28000 ( 1000 pN/nm for the two corresponding concentrations of urea used. Thus, elasticity of PDMA and PDEA can be enhanced by the interaction between urea molecules and polymers, and the effect is more notable with increasing urea concentration. Of interest is that the effects of urea molecules on the elasticity of single PDMA and PDEA chains depend on the urea concentration. When comparing the elasticity between PDMA and PDEA in the 2 M urea buffer solution, the results in Figure 6a clearly show that the elasticity of PDEA is greater than that of PDMA as in the aqueous solution. The discrepancy in the high force region may be attributed as well to the different side groups. However, in the 8 M urea
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Acknowledgment. The study reported here was supported by the National Natural Science Foundation of China and Major State Basic Research Development Program (Grant G2000078102). References (1) Hugel, T.; Seitz, M. Macromol. Rapid Commun. 2001, 22, 1. (2) Binnig, G.; Quate, C. F.; Gerber, Ch. Phys. ReV. Lett. 1986, 56, 930. (3) Janshoff, A.; Neitzert, M.; Oberdo¨rfer, Y.; Fuchs, H. Angew. Chem., Int. Ed. 2000, 39, 3212. (4) Rief, M.; Oesterhelt, F.; Heymann, B.; Gaub, H. E. Science 1997, 275, 1295. (5) Rief, M.; Gautel, M.; Oesterhelt, F.; Fernandez, J. M.; Gaub, H. E. Science 1997, 276, 1109. (6) Marszalek, P. E.; Li, H. B.; Oberhauser, A. F.; Fernandez, J. M. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 4278. (7) Yamamoto, S.; Tsujii, Y.; Fukuda, T. Macromolecules 2000, 33, 5995. (8) Li, H. B.; Liu, B. B.; Zhang, X.; Gao, C. X.; Shen, J. C.; Zou, G. T. Langmuir 1999, 15, 2120. (9) Bemis, J. E.; Akhremitchev, B. B.; Walker, G. C. Langmuir 1999, 15, 2799. (10) Zhang, W. K.; Zou, S.; Wang, C.; Zhang, X. J. Phys. Chem. B 2000, 104, 10258. (11) Marszalek, P. E.; Oberhauser, A. F.; Pang, Y. P.; Fernandez, J. M. Nature (London) 1998, 396, 661. (12) Li, H. B.; Rief, M.; Oesterhelt, F.; Gaub, H. E.; Zhang, X.; Shen, J. C. Chem. Phys. Lett. 1999, 305, 197. (13) Marszalek, P. E.; Pang, Y. P.; Li, H. B.; Yazal, J. E.; Oberhauser, A. F.; Fernandez, J. M. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 7894. (14) Xu, Q. B.; Zhang, W. K.; Zhang, X. Macromolecules 2002, 35, 871. (15) Hugel, T.; Holland, B. N.; Cattani, A.; Moroder, L.; Seitz, M.; Gaub, H. E. Science 2002, 296, 1103. (16) Li, H. B.; Rief, M.; Oesterhelt, F.; Gaub, H. E. AdV. Mater. 1998, 10, 316. (17) Rief, M.; Clausen-Schaumann, H.; Gaub, H. E. Nature Struct. Biol. 1999, 6, 346. (18) Li, H. B.; Zhang, W. K.; Xu, W. Q.; Zhang, X. Macromolecules 2000, 33, 465. (19) Xu, Q. B.; Zou, S.; Zhang, W. K.; Zhang, X. Macromol. Rapid Commun. 2001, 22, 1163. (20) Marszalek, P. E.; Lu, H.; Li, H. B.; Carrion-Vazquez, M.; Oberhauser, A. F.; Schulten, K.; Fernandez, J. M. Nature 1999, 402, 100. (21) Scho¨nherr, H.; Beulen, M. J.; Bu¨gler, J.; Huskerns, J.; Van Veggel, F. C. J. M.; Reinhoudt, D. N.; Vancso, F. J. J. Am. Chem. Soc. 2000, 122, 4963. (22) Grandbois, M.; Beyer, M.; Rief, M.; Clausen-Schaumann, H.; Gaub, H. E. Science 1999, 283, 1727. (23) Mukae, K.; Bae, Y. H.; Okano, T.; Kim, S. W. Polym. J. 1990, 22, 250. (24) Bae, Y.; Okano, T.; Kim, S. W. Pharmacol. Res. 1991, 8, 624. (25) Katsumoto, Y.; Tanaka, T.; Sato, H.; Ozaki, Y. J. Phys. Chem. A 2002, 106, 3429. (26) Oesterhelt, F.; Rief, M.; Gaub, H. E. New J. Phys. 1999, 1, 6.1. (27) Butt, H.-J.; Jaschke, M. Nanotechnology 1995, 6, 1. (28) Smith, S. B.; Cui, Y.; Bustamante, C. Science 1996, 271, 795. (29) Itakura, M.; Inomata, K.; Nose, T. Polymer 2000, 41, 8681.
NL0256917
Nano Lett., Vol. 2, No. 10, 2002
