Atomic Force Microscopy Study of E99P69E99 Triblock Copolymer

Atomic force microscopy (AFM) was employed to study the formation of Pluronic Polyol F127 oxyethylene99−oxypropylene69−oxyethylene99 (E99P69E99) ...
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Langmuir 2000, 16, 656-661

Atomic Force Microscopy Study of E99P69E99 Triblock Copolymer Chains on Silicon Surface Chunhung Wu,† Tianbo Liu,‡ Henry White,§ and Benjamin Chu*,‡,§ Department of Chemistry, Tamkang University, Tamsui, 25137, Taiwan, China, and Departments of Chemistry and Materials Science & Engineering, State University of New York at Stony Brook, Stony Brook, New York 11794-2275 Received June 8, 1999. In Final Form: September 7, 1999 Atomic force microscopy (AFM) was employed to study the formation of Pluronic Polyol F127 oxyethylene99-oxypropylene69-oxyethylene99 (E99P69E99) triblock copolymer micelles on surface-treated silicon substrates. The micellar size determined by dynamic light scattering and small-angle X-ray scattering techniques was 20-30 nm. The two-dimensional micelle size measured by ambient AFM on the modified silicon surfaces was somewhat distorted (20-50 nm in the xy dimension and ∼7 nm in the z dimension) under the environmental conditions present during scanning (i.e., copolymer chain collapsing in air and AFM tip distortion during scanning). Surface treatment was more important than solution concentration for micelle formation on the silicon surfaces. The results could be correlated with the effect of inner capillary surface coating on the resolution of double-stranded DNA capillary electrophoresis by using E99P69E99/1X TBE buffer as the separation medium.

Introduction Capillary electrophoresis (CE) has been demonstrated to be a superior method to conventional slab gel electrophoresis for the separation of biopolymers, such as proteins and DNA fragments.1 Several kinds of polymer solutions, such as linear polyacrylamide,2-6 poly-N,N-dimethylacrylamide,7 hydroxyalkylcellulose8,9 and polyoxyethylene (PEO),10-12 have been employed successfully as separation media. These polymer solutions improved the automation of CE when compared with permanent cross-linked gels, making it the best conventional analytical method for DNA analysis at this time. Recently, a novel separation medium for DNA analysis by using CE, a commercial triblock copolymer Pluronic F127 (E99P69E99, with E and P being oxyethylene and oxypropylene, respectively), has drawn considerable attention. Reported independently by our group13-16 and by * To whom all correspondence should be addressed. † Department of Chemistry, Tamkang University. ‡ Department of Chemistry, State University of New York at Stony Brook. § Department of Materials Science & Engineering, State University of New York at Stony Brook. (1) Adams, M. A.; Fields, C.; Venter, J. C. Automated DNA Sequencing and Analysis; Academic Press: London, 1994. (2) Heiger, D. N.; Cohen, A. S.; Karger, B. L. J. Chromatogr. 1990, 516, 33. (3) Guttman, A.; Cooke, N. Anal. Chem. 1991, 63, 2038. (4) Kleemib, M. H.; Gilges, M.; Schomburg, G. Electrophoresis 1993, 14, 515. (5) Carrilho, E.; Ruiz-Martinez, M. C.; Berka, J.; Smirnov, I.; Goetzinger, W.; Miller, A. W.; Brady, D.; Karger, B. L. Anal. Chem. 1996, 68, 3305. (6) Salas-Solano, O.; Carrilho, E.; Kotler, L.; Miller, A. W.; Goetzinger, W.; Sosic, Z.; Karger, B. L. Anal. Chem. 1998, 70, 3996. (7) Menchen, S.; Johnson, B.; Madabhushi, R.; Winnik, M. Progress in Biomedical Optics: Proceedings of Ultrasensitive Biochemical Diagnostics, 1996, San Jose, CA, SPIE-The International Society for Optical Engineering. (8) Grossman, P. D.; Soane, D. S. Biopolymers 1991, 31, 1221. (9) Baba, Y.; Ishimaru, N.; Samata, K.; Tsuhako, M. J. Chromatogr., A 1993, 653, 329. (10) Chang, H.-T.; Yeung, E. S. J. Chromatogr., B 1995, 669, 113. (11) Fung, E. N.; Yeung, E. S. Anal. Chem. 1995, 67, 1913. (12) Iki, N.; Yeung, E. S. J. Chromatogr., A 1996, 731, 273. (13) Wu, C.; Liu, T.; Chu, B.; Schneider, D. K.; Graziano, V. Macromolecules 1997, 30, 4574.

Rill et al.,17-19 E99P69E99 in 1X TBE buffer (Tris-Borate EDTA) was able to separate double-stranded DNA fragments and single-stranded oligonucleotide size markers. Being a typical amphiphilic block copolymer, E99P69E99 tends to self-associate into micellar structures in aqueous solution at room temperatures,13 with the hydrophobic P blocks forming compact micellar cores and the hydrophilic E blocks forming extended micellar shells. The spherical micelles will pack together at high polymer concentrations by the entanglements among the E chains and form a gel-like ordered fcc (face-centered cubic) structure.13 This immobile gel-like material has a sieving ability so that DNA fragments can be separated. The micellization and gelation behavior of F127 in 1X TBE buffer are pretty similar to those in aqueous solution. The major advantages of E99P69E99 for DNA separation include its unique viscosity-adjustable ability that enables it to be injected easily into capillary tubings and the selfcoating ability due to the electron-rich oxygen atoms along the polymer chains. For conventional separation media, as mentioned above, the polymer solution would usually have a very high viscosity at concentrations beyond the overlap concentration regime where the sieving ability begins to become acceptable. This inconvenience can be avoided by using an E99P69E99/1X TBE gel-like system. The micellization of E99P69E99 in aqueous solution (or in 1X TBE buffer) is strongly temperature dependent. At temperatures below 20 °C, the micelles are very difficult to form with a very high critical micelle concentration (cmc) value (over 6% (w/v)) and a very low association number (about only 2 in dilute solution), because the difference of solubility between E and P blocks is very small. Therefore, the gelation concentrations at low temperatures (e.g., 4 °C) are drastically higher than that (14) Wu, C.; Liu, T.; Chu, B. Electrophoresis 1998, 19, 231. (15) Wu, C.; Liu, T.; Chu, B. J. Noncryst. Solids 1998, 235-237, 605. (16) Liang, D.; Chu, B. Electrophoresis 1998, 19, 2447. (17) Rill, R. L.; Locke, B. R.; Liu, Y.; Van Winkle, D. H. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 1534. (18) Rill, R. L.; Liu, Y.; Van Winkle, D. H.; Locke, B. R. J. Chromatogr., A 1998, 817, 287. (19) Liu, Y.; Locke, B. R.; Van Winkle, D. H.; Rill, R. L. J. Chromatogr., A 1998, 817, 367.

10.1021/la990729p CCC: $19.00 © 2000 American Chemical Society Published on Web 10/28/1999

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at room temperature. This unique property suggests that over an appropriate polymer concentration range (from 15% (w/v)) to about 40% (w/v)), the E99P69E99/1X TBE system is a low-viscosity solution at low temperatures, but a gel-like immobile material at room temperature. The low-viscosity solution at low temperatures makes it easy to be injected into small quartz capillaries with inner diameters of only 50-100 µm. After the injection, the gellike materials that are capable of DNA separation can be formed by increasing the temperature gradually to about 25 °C. To suppress electroosmotic flow (EOF) and to prevent analyte adsorption, the inner wall coating of quartz capillaries with polymers is usually inevitable. The stability and the reproducibility of the coatings that usually involve multistep reactions are difficult to control. Several different physical adsorption coating methods, which could be performed before each run, or achieved by simply adding the coating material into the electrophoresis buffer, such as PEO,10-12 poly-N,N-dimethylacrylamide,7 poly(vinyl alcohol),20 and cellulose acetate,21 have been proposed. The first two can be served both as a separation medium and a coating material. From our earlier reports, 21.2% (w/v) E99P69E99 showed very good resolution in separating double-stranded DNA fragments and oligonucleotides. An inner-wall treatment of 1 N HCl was used to make the capillary inner wall acidic. The E99P69E99 polymer chains can be adsorbed at the acidic silicon surface so that EOF can be successfully suppressed. On the contrary, without the “acid wash”, E99P69E99 has a poorer separation resolution under similar experimental conditions. To elucidate the effects of surface treatment on the surface micelle formation of E99P69E99 and the subsequent effects on DNA separation, the micellar structures of E99P69E99 copolymer chains on modified silicon surfaces were investigated by using atomic force microscopy (AFM). AFM provides the possibility to take a direct look at the morphology of block copolymers on different surfaces and can provide a better understanding on what has occurred on coated quartz capillaries. Experimental Section Sample Preparation. Commercial Pluronic Polyol F127 was obtained as a gift from BASF Corp., New Jersey, and was used without further purification. F127 powder was prepared in 1X TBE buffer (89 mM tris(hydroxymethyl)aminomethane, 89 mM boric acid, and 2 mM EDTA in deionized water) to the desired polymer concentrations (all buffer reagents bought from Sigma Chemical Company, St. Louis, MO). The mixture was then stirred at around 0 °C for about 1 h. The ΦX174 DNA-HaeIII digest was purchased from New England Biolabs, Inc. (Beverly, MA). The dsDNA sample was diluted to 10 µg/mL by using the 1X TE (Tris-EDTA) buffer. Capillary Electrophoresis (CE). Fused-silica capillaries (Polymicro Technologies, Phoenix, AZ), 12-cm long, 98 µm i.d., and 364 µm o.d., were used for CE. To make comparisons, the capillaries were used under two different conditions: one without any further treatment and the other flushed with 1 mL of 1 N HCl over a period of about 10 min. In both cases, a detection window of 2 mm was opened at 10 cm from the cathodic end by stripping the polyimide coating. Both cathode and anode reservoirs (2.6 mL volume) were filled with a running buffer of 1X TBE and 3 µg/mL ethidium bromide (Sigma). The E99P69E99 copolymer solution was filled into the capillary tubing at 4 °C. The detailed descriptions on the experimental setup and procedure have been reported elsewhere.22 (20) Gilges, M.; Kleemiss, M. H.; Schomburg, G. Anal. Chem. 1994, 66, 2038. (21) Busch, M. H. A.; Kraak, J. C.; Poppe, H. J. Chromatogr., A 1995, 695, 287. (22) Wu, C.; Quesada, M. A.; Schneider, D. K.; Farinato, R.; Studier, F. W.; Chu, B. Electrophoresis 1996, 17, 1103.

Langmuir, Vol. 16, No. 2, 2000 657 Atomic Force Microscopy. Sample Preparation. Hydrophobic, hydrophilic (HCl treatment), and untreated silicon substrates were prepared and once dipped in and out of solution to transfer the solution morphology to a suitable substrate for AFM studies. The silicon (111) substrates used in this experiment were cut from P-type boron-doped wafers (polished on one side to a mirror finish) purchased from Semiconductor Processing Company. All AFM studies were performed on the polished side. The hydrophobic surface treatment involved washing the silicon substrate in a solution containing 4:1:1 H2O/H2O2/NH4OH at 80 °C for 5 min, rinsing in deionized water, washing at room temperature in a solution containing 3:1 H2O/HF, and finally rinsing again in deionized water to wash away residuals. The hydrophilic surface treatment (modified Shiraki technique23,24) involved preparing a hydrophobic surface and then washing this surface in a solution containing 5:1:1 H2O/H2O2/HCl at 80 °C for 5 min, rinsing in deionized water, and repeating the procedure again starting from the dilute HF treatment until a surface with a contact angle with water of zero was achieved. The gel-like E99P69E99 samples on a silicon surface were prepared by dipping the silicon chip into E99P69E99 solution at low temperatures (e.g., around 0 °C). A thin film of polymer solution will be attached onto the surface. A low temperature was preferred for this experiment because E99P69E99 solution had very low viscosity under that condition, as we reported before.13,14 Then the silicon chip was raised to room temperature. This procedure was to simulate the conditions when we prepared E99P69E99 separation mediums for capillary electrophoresis. The polymers on surfaces were dried at room temperature for AFM measurements. Atomic Force Microscopy. Polished single crystal silicon (111) wafers were employed as the substrate for AFM measurements. Following surface treatment and E99P69E99 deposition, the surfaces were allowed to dry and were scanned in air using the Digital Nanoscope III atomic force microscope in the contact mode. A Si3N4 tip (radius approximately 10 nm) with a force of approximately 18 nN was used for all measurements. All AFM images were flattened, and micelle height and width dimensions (x, y, and z) were measured by random cross section analysis in the x-y plane of the surface. All AFM images were illustrated using three-dimensional surface plots to best reveal the dimensions (x, y, and z) of the micelles. The micelle dimensions measured by AFM were distorted by the scanning environment (tip in contact with the copolymer surface and air environment causing collapse of the copolymer chains); however, the corrected micelle dimensions were comparable to those measured in solution by small angle X-ray scattering (SAXS) and dynamic light scattering (DLS). The nature of the surfaces after treatment can be calibrated by measuring the contact angles of water on different surfaces. The contact angle θ is defined as25

cos θ ) (γsv - γsl)/γ1v

(1)

where γsv, γsl, and γ1v represent the interfacial surface tension of solid-vapor, solid-liquid, and liquid-vapor interfaces, respectively. A surface is considered as wetting to a liquid with contact angle θ approaching to zero, while with θ > 90° the surface is dewetting with that liquid. A Kernco Instruments Co. contact angle device (model 2MG) was used for determining the contact angles of water on the silicon surfaces with different treatments. The sample thickness on silicon surface (film) was estimated by ellipsometry. A Rudolph Research Inc. Auto El null point ellipsometer was used to measure the analyzer and polarizer angles of the silicon substrate at 632.8 nm. The complex refractive indices for the substrate were then determined. After film formation, the samples were again analyzed and the film thickness was determined using a refractive index of 1.6 for the film and the previously measured complex indices for the substrate. (23) Liu, Y.; Zhao, W.; Zheng, X.; King, A.; Singh, A.; Rafailovich, M. H.; Sokolov, J.; Dai, K. H.; Kramer, E. J.; Schwarz, S. A.; Gebizlioglu, O.; Sinha, S. K. Macromolecules 1994, 27, 4000. (24) Recipe for cleaning silicon wafers developed by Julia M. Philips, AT&T Bell Laboratories, Murray Hill, NJ. (25) Adam, N. K. The Physics and Chemistry of Surfaces; Oxford University Press: Oxford, 1941.

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Figure 1. AFM measurement of 1 wt % E99P69E99 (above its cmc at room temperature) on untreated silicon surface with contact angle of 20° for H2O. No ordered micellar structure can be observed.

Results and Discussions Effect of Surface Treatment on Silicon (1% (w/v) E99P69E99 in Water). Atomic force microscopy (AFM) measurements were performed at first on the untreated silicon surface with 1% (w/v) E99P69E99 aqueous solution. The triblock copolymer E99P69E99 has a critical micelle concentration (cmc) of 6.1 mg/mL in aqueous solution at 25 °C, and the cmc value shows a very strong temperature dependence.13 The 1% (w/v) concentration of E99P69E99 was a little higher than the cmc, and the micelles should have a small association number (about 2) in solution at room temperature. The untreated silicon surface had a contact angle of 20° for H2O, indicating a hydrophilic surface mainly due to the native oxide surface. In Figure 1, AFM measurement showed that there was no obvious micellar structure on the silicon surface. On the contrary, some large, irregular domains and small dewetting valleys were observed, suggesting that due to the inhomogeneous nature of the untreated surface, E99P69E99 block copolymer chains could not be attached to the silicon surface uniformly. Therefore, no ordered structure of micelles could form on the surface. Figure 2a shows the AFM result of 1% (w/v) E99P69E99 aqueous solution on the hydrophobic surface treated with HF washing. The effect of a hydrophobic surface to the formation of block copolymer micelles is similar to that of adding another immiscible hydrophobic solvent into the aqueous copolymer solution, e.g., xylene, which will induce micelle formation at lower copolymer concentrations. In Figure 2a, some micellar structures can be observed. It is reasonable to assume that the micelles are formed on the surface by attaching hydrophobic P blocks to the surface with the hydrophilic E blocks extending into the solution. Figure 2b is a magnified view of Figure 2a. The micelles on the surface were comparatively uniform in size, averaging 20-50 nm in diameter (x-y dimension), but the height (z dimension) of the micelles was only 2-5 nm, suggesting that the micelles had been collapsed onto the silicon surface. The size of micelles on the surface was comparable with that in dilute E99P69E99 solutions from DLS measurements and that at higher polymer concentrations from SAXS measurements. In both cases, a size range of 20-30 nm for the spherical copolymer micelles was obtained.13 More uniform (x, y, and z dimensions) micelles can be formed on a hydrophilic surface following the Shiraki treatment, as shown in Figure 3. Shiraki-treated surfaces

Figure 2. (a) AFM measurement of 1% (w/v) E99P69E99 aqueous solution on hydrophobic silicon surface (with HF treatment). Some micelles can be found on the surface. (b) A magnified view of Figure 2a that gives a clearer picture.

Figure 3. AFM measurement of 1% (w/v) E99P69E99 aqueous solution on hydrophilic silicon surface (with Shiraki treatment).

have a contact angle of 0° for H2O, indicating the hydrophilic nature of the surface. In this case, E99P69E99 micelles should use the hydrophilic E blocks to attach onto the silicon surface. Figure 3 shows that the micelles on the surface are uniform in size and more closely packed than those in Figure 2a. Ellipsometer measurements averaged over the surface of the sample showed a solid polymer layer of about 70 Å (7 nm). Considering that the size of E99P69E99 micelles in aqueous media was around 20-30 nm, the difference of micellar size on the hydrophilic surface can be attributed to two major factors. First, the dehydration effect on the surface makes the micellar chains (especially micellar shells) more compact, while in

Triblock Copolymer Morphology

Figure 4. (a) AFM measurement of 1% (w/v) E99P69E99 aqueous solution on hydrophilic silicon surface (with Shiraki treatment), different magnitude to Figure 3. (b) AFM measurement of 1% (w/v) E99P69E99 aqueous solution on hydrophilic silicon surface (with Shiraki and HCl treatments). Similar micelles were observed in the plot, with some of them seemed to be “taller” on the surface than those in Figure 4a.

solution the micellar chains are more extended. Second, AFM imaging of micelles on the surface usually creates a distortion on the micellar morphology which will make spherical micelles having more extended dimensions along the surface but lower height over the surface. Effect of HCl Acid Treatment. AFM measurements were performed on the silicon surfaces with the Shiraki treatment as well as with HCl treatment. After the routine Shiraki treatment, the silicon substrate was dipped in and removed from a 1 N HCl solution and then was taken out. The purpose of the HCl wash was to clean and to create an acidic surface in order to increase the affinity of the polymer (E block) for the surface. HCl washing has become an important step in DNA capillary electrophoresis when using the E99P69E99 block copolymer as the separation medium.14 The effect of HCl washing after the Shiraki treatment on the E99P69E99 micelles on silicon surfaces is shown in Figure 4. In Figure 4a, normal (seen previously in Figure 2b, approximately 20-50 nm in width (x-y dimension) and 2-4 nm in height (z dimension) micelles can be observed. In Figure 4b (a scan from the same surface in a different area at a slightly larger scan size), it is noted that after the HCl treatment, some larger micellar structures (z-dimension) appeared with the height up to 7 nm. Complete desorption of E99P69E99 polymer chains

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from the hydrophilic silica surface at pH ) 10.4-10.8 has been reported in the literature, which was mainly due to the repulsion between the negative silanol groups and the electron-rich oxygen atoms in the polymer backbone.26 Therefore, it is important to keep the inner surface in an acidic environment so that the E99P69E99 polymer chains could have the desirable coating effect. AFM Study of E99P69E99 Aqueous Solution at Higher Polymer Concentrations. The phase diagram of E99P69E99/water had a very strong temperature dependence, especially when gelation occurred. This special property made it easy to inject the separation material into capillaries for DNA capillary electrophoresis. The gelation concentration for E99P69E99 in aqueous solution at room temperature is about 18% (w/v).14 While at low temperatures (around 0-4 °C), even a 35% (w/v) E99P69E99/ water system is still in the solution state and can be easily injected into thin capillaries (100 µm i.d.); at room temperatures it can form an immobile gel-like structure which has sieving ability. In Figure 5a, the AFM study of 10.6% (w/v) E99P69E99/ water (below gelation concentration at any temperature) is presented. Figure 5b is a magnified view of Figure 5a. It is noted that regular micelles (as described previously at lower concentrations) are formed at a size of about 50 nm along the silicon surface. In comparison with AFM measurements at lower copolymer concentrations (see Figures 2b, 3, and 4a), the number of micelles obviously increased. Figure 6 shows the AFM measurement of 21.2% (w/v) E99P69E99/water system on a modified silicon surface with Shiraki and HCl treatment. This was the best E99P69E99 concentration for separating double-stranded DNA fragments by capillary electrophoresis. As this concentration was already higher than the gelation concentration of the E99P69E99/water system at room temperature, the sample was prepared by dipping the silicon substrate into a 21.2% (w/v) E99P69E99/water micellar solution at 0 °C. Then the substrate was allowed to reach room temperature before AFM measurements. The AFM result shows that the micelles were more closely packed on the surface when compared with those in Figure 5a, with sizes in the range of 30-50 nm along the silicon surface (x-y dimensions). The micellar size of E99P69E99 micelles on the silicon surface showed essentially no copolymer concentration dependence, in agreement with earlier scattering results that the micellar size remained about the same at polymer concentrations above the cmc. Effects of Surface Treatment on DNA Capillary Electrophoresis. Figure 7 shows a comparison of electropherograms obtained at two different capillary conditions but using the same double-stranded DNA sample (ΦX174 DNA-HaeIII digest) and the same E99P69E99 concentration (21.2% (w/v) in 1XTBE buffer). Figure 7a shows the result using untreated capillary, while Figure 7b corresponds to the experiment with a HCl-treated capillary. The DNA separation resolution by using fused silica capillary column without acid wash treatment was not as good as those with the acid wash. As mentioned in the previous section for the discussion of the effect of HCl treatment on hydrophilic surface, we understand that after acid wash the triblock copolymer micelles could adsorb on the inner wall of the fused silica column more efficiently and therefore greatly suppress the occurrence of EOF. The existence of EOF could totally destroy the electrophoretic effect in many cases. In the current case, it did (26) Malmsten, M.; Linse, P.; Cosgrove, T. Macromolecules 1992, 25, 2474.

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Figure 6. AFM measurement of 21.2% (w/v) E99P69E99/water system (above gelation concentration) on silicon surface with Shiraki and HCl treatment. The dimension of the micelles on surface after correction was comparable with those from DLS and SAXS measurements.

Figure 5. (a) AFM measurement of 10.6% (w/v) E99P69E99 aqueous solution (much higher than its cmc but below the gelation concentration at room temperature) on hydrophilic silicon surface (with Shiraki and HCl treatments). (b) A magnified view of Figure 5a that gives a clearer picture.

not have a very strong negative effect, which could be attributed mainly to the surfactant behavior of E99P69E99 block copolymer. However, for the small DNA fragments, such as 72 and 118 base-pair fragments, it was hard to separate them by using untreated capillaries (Figure 7a) while they could be separated easily by using the HCltreated capillaries (Figure 7b). Moreover, the presence of EOF seemed to reverse the electrophoretic mobility of small DNA fragments (e.g., the 72 and 118 base-pair fragments in Figure 7a). However, the misplaced fragments returned to the normal sequence after HCl treatment. Conclusions Atomic force microscopy (AFM) measurements were employed to provide a direct observation of the E99P69E99 micellar morphologies on silicon surfaces with different surface treatments. E99P69E99 block copolymer chains form core-shell micelles in an aqueous solution. In the presence of an untreated silicon surface, micelles could not be

Figure 7. Electropherograms of φX174 DNA-Hae III digest in capillary electrophoresis at 25 °C by using 21.2% (w/v) F127 in 1X TBE buffer as a separation medium: (a) without any innerwall washing; (b) with HCl washing. Peak identifications from right to left in base pairs are 1353, 1078, 872, 603, 310, 281, 271, 234, 194, 118, and 72. Electrophoresis conditions are described in the text.

formed effectively on the surface. Normal collapsed micelles (∼20 to 50 nm in width (x-y dimension) and 7 nm in height (z dimension)) formed on both hydrophobic and hydrophilic surfaces. Micelles in solution are spherical, while micelles on surfaces that were imaged in air were

Triblock Copolymer Morphology

distorted by the AFM tip and appeared collapsed. The total volume of the micelles after correction for the difference in water content was comparable with those obtained from light scattering and small-angle X-ray scattering results. The surface treatments and the corresponding differences in the micellar formation on the silicon surface with different treatments had noticeable effects on the resolution of double-stranded DNA capillary electrophoresis by using the E99P69E99 gel-like material as the separation medium.

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Acknowledgment. B.C. gratefully acknowledges the support of this work by the National Human Genome Research Institute (2R01HG0138604) and the National Science Foundation (MRSEC on Engineered Polymer Interfaces DMR 9632525), as well as useful discussions with Miriam Rafailovich, Director of the MRSEC on Engineered Polymer Interfaces at Stony Brook. C.W. thanks the support of National Science Council of Taiwan. LA990729P