NANO LETTERS
Simple Method to Isolate Single Polymer Chains for the Direct Measurement of the Desorption Force
2003 Vol. 3, No. 2 245-248
Shuxun Cui, Chuanjun Liu, and Xi Zhang* Key Lab for Supramolecular Structure and Materials, College of Chemistry, Jilin UniVersity, Changchun 130023, People’s Republic of China Received November 12, 2002; Revised Manuscript Received December 27, 2002
ABSTRACT We propose a simple method to isolate polymer chains individually at the quartz surface by utilizing the defects in the self-assembled monolayers of organosilane. This method allows us to measure the desorption force of a single polyelectrolyte chain from a substrate directly.
Single-molecule force spectroscopy (SMFS), based on atomic force microscopy (AFM), has become a versatile platform for studying intermolecular and intramolecular interactions with its extremely high force sensitivity.1 Using SMFS, a number of interesting topics such as protein unfolding,2 DNA unzipping,3 force-induced conformational transitions,4 hostguest interactions,5 single-polymer chain elasticity,6-8 and single-chain desorption from a substrate9 have been investigated. The preparation of a “single-molecule” sample is a key issue in SMFS. Using an extremely dilute solution of a target molecules for physical deposition on the substrate is the usual way to prepare a single-molecule sample. Another way is to mix the target molecule with thinner molecules in case chemical modification is needed. These methods sometimes work well for small molecules, but for polymers they often fail because of the entanglement of chains and surface concentration enrichment. Therefore, the “single polymer chain” experimental results first have to be filtered manually before statistical methods can be used to deduce the properties of single molecules. Recently, Weiss et al. have introduced a novel method to isolate small molecules individually by utilizing the defects in the self-assembled monolayers (SAM).10 We wonder whether this method can be used in the field of polymers, for example, for direct measurements of the desorption force between a single polymer chain and substrates. We modified a quartz slide with 3-aminopropyl-dimethylethoxysilane (APS, 97%, ABCR, Corp.). As usual, before silanization, the quartz slide was treated with a hot “piranha” solution (7:3 H2SO4/H2O2) to obtain a hydroxyl-modified surface and was then rinsed thoroughly with purified water (>18 MΩ). After drying, this slide was immersed in an * To whom correspondence should be addressed. E-mail:
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© 2003 American Chemical Society
APS-toluene solution (v/v ) 1:1000) for 12 h to obtain an organosilane SAM.11 This treatment will not generate a surface with absolute amino end groups and may leave some random defects where hydroxyl groups remain. Upon immersion in purified water, these defects made the surface heterogeneously charged (i.e., the amino group’s tailored area was positively charged whereas the polar hydroxyl groups’ tailored area was neutral). Therefore, the positively charged polyelectrolytes such as poly(diallyldimethylammonium chloride) (PDDA, 20% solution in water, Mw ) 4 × 105-5 × 105, Aldrich, Corp.) preferentially adsorb onto the hydroxyl groups’ tailored area12 because of the attractive chargedipole interaction. We immersed the heterogeneously modified quartz slide in a PDDA aqueous solution (v/v ) 1:100) for 5 min of physisorption and rinsed the slide twice by immersion in purified water (each time for 10 min). This quartz slide was dried by air flow and then used as a sample for a variety of characterization methods. The contact angle can be used as an indicator of the hydrophobicity of a surface. Compared with the hydroxylmodified quartz (∼7°), the heterogeneously modified quartz (∼67°) is relatively hydrophobic.13 The large discrepancy in the hydrophobicity of the samples before and after silanization suggests that most areas of the heterogeneously modified quartz are functionalized by APS. To study how much of the area of the substrate was modified by amino groups, X-ray photoelectron spectroscopy (XPS, VG ESCA LAB MKII, exciting source: Mg, KR ) 1253.6 eV) was used to characterize the surface of the substrate. There are two types of oxygen atoms on the surface of the quartz: type I is linked with one silicon atom whereas type II is linked with two silicon atoms. For the hydroxyl-modified quartz, the ratio of oxygen atoms between type I and type II is ∼1:1 whereas for the heterogeneously modified quartz the ratio
Figure 1. XPS spectra of oxygen atoms on the surface. The solid line and dashed line correspond to the samples before and after silane modification, respectively.
becomes 1:16.4. (See Figure 1.) This result indicates that most of the type I oxygen atoms have turned into type II after the silanization and that only ∼5.7% of the total number oxygen atoms on the surface come from hydroxyl groups. The average contact angle for this heterogeneously modified sample after PDDA adsorption shifted to ∼64°. The variation in the contact angle before and after PDDA adsorption is not significant (67 and 64°, respectively) and may be attributed to the small amount of adsorbed PDDA. A homebuilt SMFS with an AFM tip (Park, Sunnyvale, CA) was used1c to detect the existence of PDDA chain on the heterogeneously modified substrate. We used a standard sample to calibrate each tip, which has been described elsewhere.6 The spring constants of these cantilevers were in the range of 0.010-0.012 N/m. Prior to the force measurement, a drop of purified water, acting as a buffer, was injected between the substrate and the cantilever holder, whereupon both the substrate and the cantilever were immersed in water. By moving the piezo tube, we could bring the sample into contact with the AFM tip so that some polymer chains were adsorbed onto the tip because of nonspecific interactions, resulting in a number of “bridges”. As the distance between the tip and the substrate increased, the bridges were stretched, and the elastic force deflected the cantilever. A recorded deflection-piezo path curve was converted into a force-extension curve (force curve, in brief). The nonspecific interaction between the tip and the polymer chain can be up to a few nanonewtons in magnitude.1c,14 Therefore, using SMFS, we can measure the weak interaction force between a single polymer chain and the substrate. The stretching velocity used in this study was in the range of 460-4600 nm/s. A typical force curve obtained from the PDDA sample is shown in Figure 2 for the case of an individual PDDA chain being captured by the AFM tip. The sharp peak in the initial part of each force curve corresponds to the very strong nonspecific adhesion between the bare tip and the uncovered regions of the substrate.1a This peak is subsequently followed by a long plateau, and then the force drops to zero, indicating a complete detachment of the PDDA chain from the 246
Figure 2. Typical force curve obtained from the PDDA sample adsorbed on the heteromodified substrate. Inset: zoomed-in force curve.
Figure 3. Typical force curve showing two steps. Inset: scheme for the desorption of two strands of a single chain.
substrate. The long plateau suggests that the desorption process of the PDDA chain from the substrate is smooth and that it adopts a flat conformation at the interface.15,16 The desorption force remains ∼50 pN along the desorption process (seen more clearly from the inset). The rather low desorption force does not allow us to realize forward and backward stretching. However, we can verify whether the desorption/adsorption process is in equilibrium or not by change the stretching velocity. We find that the desorption force is independent of the stretching velocity from 460 to 4600 nm/s. This result suggests that the inherent desorption/ adsorption rate of PDDA at the interface is much faster than the rate we applied during experiments and that the desorption process is carried out in an equilibrium state.17,18 Thus, the adhesion force between PDDA and the substrate is equal to the desorption force obtained. The interaction between the PDDA chain and the hydroxyl groups’ covered area is a charge-dipole interaction or an electrostatic interaction in general. The length of the longest plateau obtained in our experiments is ∼500 nm, approxmately 1/3 of the contour length of the PDDA chain. In some cases, we obtained force curves with two sequential plateaus. (See Figure 3.) The two steps in the force curve have the same height of ∼50 pN, and the two steps may correspond to the desorption of two strands of a single chain. (See the inset of Figure 3.) The single PDDA chain was picked up by the AFM tip and was Nano Lett., Vol. 3, No. 2, 2003
Table 1. Analoguesa of the Groups Tailored on Heteromodified Quartz and Corresponding Behavior in Different Environments
Figure 4. (a) Ideal map of the heteromodified substrate. Black dots represent the area occupied by hydroxyl groups whereas the white background represents the area occupied by amino groups. (b) Ideal map of the heteromodified substrate after the adsorption of PDDA from solution.
separated into two strands. Considering the randomness of the position where the tip picks, for most cases, one strand is shorter than the other. The 2-fold height plateau corresponds to the simultaneous desorption of two strands whereas the 1-fold height plateau corresponds to the desorption of the longer strand following the complete detachment of the shorter one. During the force measurements, we did not observe a 3-fold height or even a higher plateau in the force curve, which should correspond to the behavior of multiple chains. This result confirms the extremely low polymer concentration on the heterogeneously modified substrate. In addition, SMFS measurements on this PDDA sample gave a very small probability (∼1/300) of catching PDDA chains from the surface, indicating that there were few PDDA chains adsorbed onto the substrate. As we can observe only single-chain behavior, we think that the polymer chain is isolated individually on the substrate by the unfavorable 3-aminopropyl “walls”. The organosilane used in this study contains only one ethoxy reactive group, which can avoid large defect domains during modification.11 For this reason, the ∼5.7% area of defects is randomly dispersed on the entire surface. AFM images (Nanoscope IIIa, tapping mode) of the heterogeneously modified quartz slide show no large domains, which supports the fact that the defects are dispersed well on the surface. Therefore, the PDDA chain is forced to adopt an extended flat conformation so that it can adsorb onto the dispersed defects. Our Monte Carlo simulation shows that when the area occupied by negative charge is ∼3% the polycation chain can be isolated individually. Figure 4a shows the ideal map of the heterogeneously modified substrate. The black dots (coverage 3%) represent the defects. Figure 4b illustrates the map of the same place after the adsorption of polycation. We have measured the proportion of the area tailored by hydroxyl groups by XPS to be ∼5.7%. Considering the fact that the amino groups’ tailored propyl brush would also cover some neighboring area that is tailored by hydroxyl groups, the area really “occupied” by hydroxyl groups, which is open for the polycation adsorption, should be less than 5.7%. This factor would reduce the final amount of adsorbed PDDA on the surface. From Table 1, we know that in purified water (pH 5.7) the heterogeneously modified substrate is covered by a Nano Lett., Vol. 3, No. 2, 2003
solution or buffer
pH value
ionized percentage of hydroxyl groups
pure water PDDA aqueous solution PDDA basic aqueous solution
5.7 4.7 13.0
∼1.5 × 10-4 ∼1.5 × 10-5 >99.9%
ionized percentage of amino groups >99.9% >99.9% ∼0.4%
a We selected orthosilicic acid (pK ) 9.5) and propylamine (pK ) a b 3.4) as analogues.
Figure 5. Typical force curve obtained from hydroxyl-modified quartz after the adsorption of PDDA.
predominant area of positively charged ammonium and a small area of neutral defects, which results in a trace amount of adsorbed PDDA. When the environment is alkalized (pH 13), the amino groups become neutral, containing, however, the negative end of the molecular dipole whereas hydroxyl groups become negatively charged. (See Table 1 for details.) Under this condition, the PDDA chain can adsorb onto both amino and hydroxyl groups’ tailored surfaces; namely, the entire surface of the substrate is open to PDDA adsorption. Actually, we observe only chaos force curves (see Figure 5) during SMFS measurements in this case, which is indicative of a large amount of PDDA on the surface. This finding shows that this single-molecule preparation method is sensitive to the pH. For the best performance, a neutral or acid environment is necessary during sample preparation and single-chain detection. As a comparison, we made a similar force measurement on the sample lacking the silanization process, namely, PDDA adsorbed onto a hydroxyl-modified quartz slide. Under this condition, we found that the probability to catch polymer chains was greater than 30%. The obtained complicated chaos force curves, which are attributed to the behavior of multiple PDDA chains such as the desorption of several chains from the substrate and the disentanglement and elastic elongation of chains, resemble the force curve shown in Figure 5. The difference between single-chain and multiple-chain stretching is very clear. Occasionally, we observed a short plateau at the end of the chaos force signals, which also had a height of ∼50 pN and is the same as the 247
Table 2. Contact Angle and Probability for Catching PDDA Chains in SMFS on Different Samples sample hydroxyl-modified quartz sample 1 with adsorption of PDDA sample 1 with silanization for 12 hours sample 3 with adsorption of PDDA sample 1 with silanization for 30 min sample 5 with adsorption of PDDA
sample contact probcode angle ability 1 2 3 4 5 6
∼7 ∼5 ∼67 ∼64 ∼55 ∼45
>30% ∼1/300 ∼1/80
result obtained from the heterogeneously modified substrate. This finding confirms our assumption that PDDA is adsorbed onto the hydroxyl-covered area of the substrate. The silanization process is a relatively long timescale process. This feature enables us to adjust the defect coverage simply by controlling the silanization time. The contact angle of the substrate with a shorter time (30 min) silanization is ∼55°. After PDDA adsorption, the value shifts to ∼45°. The data of the contact angle are summarized in Table 2. Compared with the result obtained from a long-time (12 h) silanized sample, the variation of the contact angle for the sample of short-time silanization is significant. This result indicates that a shorter time silanization would result in more PDDA adsorption because more of the hydroxyl groups’ tailored area is left. Actually, we found an enhanced probability (∼1/80) of catching polymer chains (force curves resemble that shown in Figure 2) on the sample of shorter time silanization. This also supports our assumption above. Moreover, it provides a way to control the amount of adsorbed polymer at the interface simply by controlling the time of chemical modification. In this paper, we present a method to prepare a singlemolecule sample. By silanization, the hydroxyl groups’ tailored surface can be covered by amino groups; however, the dispersed hydroxyl groups’ tailoring area remains because of the imperfection of the modification. These surface defects enable us to isolate polymer chains individually on the substrate. The SMFS measurements provide proof that PDDA chains are individually isolated by the unfavorable walls. Moreover, by controlling the time of silanization, we can adjust the amount of adsorbed polymer. There is a correlation between the force pattern and the adsorption conformation of the polymer. The long plateau in this case reflects a flat conformation of PDDA at the interface. This method would be significant for single-molecule chemistry and physics because of the high performance of single-
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molecule isolation and the ease of the preparing process as well as the potential for automatization of single-molecule detection. Acknowledgment. This study was supported by the Major State Basic Research Development Program (grant no. G2000078102), the Ministry of Science and Technology, and the Natural Science Foundation of China. We thank Professor Hermann E. Gaub for his kindly help in establishing the SMFS setup and Dr. Wenke Zhang, Mr. Chi Wang, and Mr. Bin Dong for their helpful suggestions. References (1) For the theory and instrumentation of SMFS, see (a) Hugel, T.; Seitz, M. Macromol. Rapid Commun. 2001, 22, 989. (b) Janshoff, A.; Neitzert, M.; Oberdo¨rf, Y.; Fuchs, H. Angew. Chem., Int. Ed. 2000, 39, 3212. (c) Oesterhelt, F.; Rief, M.; Gaub, H. New J. Phys. 1999, 1, 6.1. (2) (a) Rief, M.; Gautel, M.; Oesterhelt, F.; Fernandez, J.; Gaub, H. Science (Washington, D.C.) 1997, 276, 1109. (b) Li, H.; Linke, W.; Oberhauser, A.; Carrion-Vazquez, M.; Kerkvliet, J.; Lu, H.; Marszalek, P.; Fernandez, J. Nature (London) 2002, 418, 998. (3) Rief, M.; Clausen-Schaumann, H.; Gaub, H. Nat. Struct. Biol. 1999, 6, 346. (4) Marszalek, P.; Pang Y.; Li, H.; Yazal, J.; Oberhauser, A.; Fernandez, J. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 7894. (5) Scho¨nherr, H.; Beulen, M.; Bu¨gler, J.; Huskens, J.; van Veggel, F.; Reinhould, D.; Vancso, G. J. Am. Chem. Soc. 2000, 122, 4963. (6) Li, H.; Liu, B.; Zhang, X.; Gao, C.; Shen, J.; Zou, G. Langmuir 1999, 15, 2120. (7) Wang, C.; Shi, W.; Zhang, W.; Zhang, X.; Katsumoto, Y.; Ozaki, Y. Nano Lett. 2002, 2, 1169. (8) Bemis, J.; Akhremitchev, B.; Walker, G. Langmuir 1999, 15, 2799. (9) Hugel, T.; Grosholz, M.; Clausen-Schaumann, H.; Pfau, A.; Gaub, H.; Seitz, M. Macromolecules 2001, 34, 1039. (10) Donhauser, Z.; Mantooth, B.; Kelly, K.; Bumm, L.; Monnell, J.; Stapleton, J.; Price, D., Jr.; Rawlett, A.; Allara, D.; Tour, J.; Weiss, P. Science (Washington, D.C.) 2001, 292, 2303. (11) Haller, I. J. Am. Chem. Soc. 1978, 100, 8050. (12) The theoretical research on the adsorption of polyelectrolytes onto the heterogeneously charged surface has been demonstrated in the literature: Ellis, M.; Kong, C.; Muthukumar, M. J. Chem. Phys. 2000, 112, 8723. (13) The contact angle data consist of the results reported in Vezenov, D.; Noy, A.; Rozsnyai, L.; Lieber, C. J. Am. Chem. Soc. 1997, 119, 2006. (14) Li, H.; Zhang, W.; Xu, W.; Zhang, X. Macromolecules 2000, 33, 465. (15) Conti, M.; Bustanji, Y.; Falini, G.; Ferruti, P.; Stefoni, S.; Samorı`, B. ChemPhysChem 2001, 10, 610. (16) The relation between the adsorption conformation of a polymer chain and the pattern of the resulting force curve is described in Zhang, W.; Cui, S.; Fu, Y.; Zhang, X. J. Phys. Chem. B 2002, 106, 12705. (17) Evans, E. Annu. ReV. Biophys. Biomol. Struct. 2001, 30, 105. (18) Zapotoczny, S.; Auletta, T.; de Jong, M.; Scho¨nherr, H.; Huskens, J.; van Veggel, F.; Reinhoudt, D.; Vancso, G. Langmuir 2002, 18, 6988.
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Nano Lett., Vol. 3, No. 2, 2003
