Anal. Chem. 2001, 73, 1091-1099
Real-Time Dynamics of Single-DNA Molecules Undergoing Adsorption and Desorption at Liquid-Solid Interfaces Seong Ho Kang,† Michael R. Shortreed,‡ and Edward S. Yeung*
Ames LaboratorysUSDOE and Department of Chemistry, Iowa State University, Ames, Iowa 50011
The conformational dynamics and adsorption/desorption behavior of individual λ-DNA molecules at liquid-solid interfaces were monitored by imaging within the evanescent field layer using total internal reflection fluorescence microscopy. At a fused-silica surface, molecular conformation and adsorption behavior were found to depend on both pH and buffer composition. A histogram of individual λ-DNA adsorption durations measured by hydrodynamically flowing molecules along the interface exhibited asymmetry nearly identical to that of the corresponding elution peaks found in capillary liquid chromatography and capillary electrophoresis. The accessibility of the surface to the molecules, which is proportional to the capillary surface area-to-volume ratio, can be correlated with the capacity factor and the relative adsorption factor. At a C18 surface, the dynamics of individual DNA molecules changed with the addition of organic solvent as well as with pH. Hydrophobic interaction rather than electrostatic interaction was the major driving force for adsorption of individual DNA molecules.
The study of single-molecule adsorption, desorption, and motion at a liquid-solid interface can provide insights into molecular genetics,1-3 biosensor design,4-6 DNA biophysics,7-21 * Corresponding author: (tel) 515-294-8062; (fax) 515-294-0266; (e-mail)
[email protected]. † Present address: Samsung Advanced Institute of Technology, P.O. Box 1211, Suwon 440-600, Korea. ‡ Present address: Department of Chemistry, University of Wisconsin, Madison, WI 53706. (1) Herrick, J.; Michalet, X.; Conti, C.; Schurra, C.; Bensimon, A. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 222-227. (2) Herrick, J.; Bensimon, A. Chromosome Res. 1999, 7, 409-423. (3) Lyubchenko, Y. L.; Shlyakhtenko, L. S. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 496-501. (4) Chan, V.; McKenzie, S. E.; Surrey, S.; Fortina, P.; Graves, D. J. J. Colloid Interface Sci. 1998, 203, 197-207. (5) Chan, V.; Graves, D. J.; Fortina, P.; McKenzie, S. E. Langmuir 1997, 13, 320-329. (6) Jordan, C. E.; Frutos, A. G.; Thiel, A. J.; Corn, R. M. Anal. Chem. 1997, 69, 4939-4947. (7) Bensimon, A.; Simon, A.; Chiffaudel, A.; Croquette, V.; Heslot, F.; Bensimon, D. Science 1994, 265, 2096-2098. (8) Bensimon, D.; Simon, A. J.; Croquette, V.; Bensimon, A. Phys. Rev. Lett. 1995, 74, 4754-4757. (9) Xue, Q.; Yeung, E. S. Nature 1995, 373, 681-683. (10) Houseal, T. W.; Bustamante, C.; Stump, R. F.; Maestre, M. F. Biophys. J. 1989, 56, 507-516. 10.1021/ac0013599 CCC: $20.00 Published on Web 02/08/2001
© 2001 American Chemical Society
and basic separation theory.22-28 It is well known that both electrostatic and hydrophobic interactions govern protein adsorption at liquid-solid interfaces.4,22 These fundamental interactions are the foundation for many chromatographic protein separations.29 Both rate theory and band broadening in chromatography and electrophoresis have been explained by using statistical theory.30 Direct observation of individual molecular motion and interactions at the liquid-solid interface would add valuable details regarding these and other related phenomena. How these same interactions affect DNA motion and adsorption is not as clear. For instance, on the basis of simple electrophoresis theory, it should not be possible to separate DNA molecules greater than 10-20 bp without the presence of a sieving medium. Surprisingly, such a separation was clearly demonstrated.31 The separation mechanism was ascribed to electrostatic interactions between the DNA molecule and the wall of the fused-silica capillary. Imaging of individual DNA molecules labeled with fluorescent dyes has been possible for some time.32,33 However, not all methods are applicable to real-time imaging of the random motion of individual DNA molecules in free solution. For example, (11) Auzanneau, I.; Barreau, C.; Salome, L. C. R. Acad. Sci., Ser. III 1993, 316, 459-462. (12) Strick, T. R.; Allemand, J.-F.; Bensimon, D.; Croquette, V. Biophys. J. 1998, 74, 2016-2028. (13) Fan, F.-R. F.; Bard, A. J. Science 1995, 267, 871-874. (14) Funatsu, T.; Harada, Y.; Tokunaga, M.; Saito, K.; Yanagida, T. Nature 1995, 374, 555-559. (15) Chiu, D. T.; Zare, R. N. J. Am. Chem. Soc. 1996, 118, 6512-6513. (16) Nie, S.; Chiu, D. T.; Zare, R. N. Science 1994, 266, 1018-1021. (17) Yokota, H.; Saito, K.; Yanagida, T. Phys. Rev. Lett. 1998, 80, 4606-4609. (18) Enderlein, J. Biophys. J. 2000, 78, 2151-2158. (19) Xu, X.; Yeung, E. S. Science 1997, 276, 1106-1109. (20) Dickson, R. M.; Norris, D. J.; Tzeng, Y.-L.; Moerner, W. E. Science 1996, 274, 966-969. (21) Ma, Y.; Shortreed, M. R.; Yeung, E. S. Anal. Chem. 2000, 72, 4640-4645. (22) Xu, X.-H.; Yeung, E. S. Science 1998, 281, 1650-1653. (23) Shortreed, M. R.; Li, H.; Huang, W.-H.; Yeung, E. S. Anal. Chem. 2000, 72, 2879-2885. (24) Smith, S. B.; Aldridge, P. K.; Callis, J. B. Science 1989, 243, 203-206. (25) Ueda, M. J. Biochem. Biophys. Methods 1999, 41, 153-165. (26) Zullli, S. L.; Kovaleski, J. M.; Zhu, X. R.; Harris, J. M.; Wirth, M. J. Anal. Chem. 1994, 66, 1708-1712. (27) Swinton, D. J.; Wirth, M. J. Anal. Chem. 2000, 72, 3725-3730. (28) Wirth, M. J.; Swinton, D. J. Anal. Chem. 1998, 70, 5264-5271. (29) Poole, C. F.; Poole, S. K. Chromatography Today; Elsevier: New York, 1991, (30) Tallarek, U.; Rapp, E.; Scheenen, T.; Bayer, E.; Van As, H. Anal. Chem. 2000, 72, 2292-2301. (31) Iki, N.; Kim, Y.; Yeung, E. S. Anal. Chem. 1996, 68, 4321-4325. (32) Morikawa, K.; Yanagida, M. J. Biochem. 1981, 89, 693-696. (33) Matusumoto, S.; Morikawa, K.; Yanagida, M. J. Mol. Biol. 1981, 152, 501516.
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Table 1. Preparation of Various 25 mM Sodium Acetate Buffers for Studying the Dynamics of Adsorption/Desorption of DNA Molecules Starting from 1.0 M Stock Solutions pH
acetic acid (µL)
sodium acetate (mL)
sodium chloride (µL)
water (mL)
4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0
2120 1600 900 377 133 43.7 14.0 4.4 1.4
0.4 0.9 1.6 2.1 2.4 2.5 2.5 2.5 2.5
2120 1600 900 377 133 43.7 14.0 4.4 1.4
95.4 95.9 96.6 97.1 97.4 97.5 97.5 97.5 97.5
scanning with a sharp optical probe provides high-resolution images but is not capable of capturing video rate motion.14 Recent advances in intensified charge-coupled device (ICCD) camera detection efficiency and speed have enabled temporal resolution of 0.4 ms and spatial resolution of 0.3 µm.19,22 The motion of single molecules in free solution was captured in the form of movies.23,34 The total internal reflection (TIR) geometry14,35 provides highcontrast images of single molecules by virtually eliminating all of the background. Excitation is confined to the evanescent field layer (EFL), where the motions of individual molecules are recorded. In this study, we monitored the dynamics of single-DNA molecules labeled with YOYO-1. The motion and adsorption/ desorption behaviors of these molecules at the fused-silica-water interface and the C18-water interface were analyzed as a function of pH and buffer composition. We also assessed the driving force for adsorption of individual DNA molecules by the addition of methanol to modulate the strength of the hydrophobic interaction in aqueous solution.36 The results of the real-time imaging experiments were compared to capillary electrophoresis (CE) and capillary liquid chromatography (CLC) separations with reference to band broadening and elution times. EXPERIMENTAL SECTION Buffer Solutions. The various pH buffer systems used were as follows: dihydrogen phosphate/phosphoric acid (pH 2-3), sodium citrate/citric acid (pH 3-4), sodium acetate/acetic acid (pH 4-6), MES/sodium chloride (pH 6-7), Tris/Tris-HCl (pH 7-8), Tris/Tricine (pH 8-9), and sodium hydroxide/CHES (pH 9-10). These regents were all purchased from Sigma Chemical Co. (St. Louis, MO). Buffer solutions for measuring the dynamics of adsorption/desorption were prepared at various pH values using 1.0 M solutions of acetic acid, sodium acetate, and sodium chloride. A.C.S. grade or higher glacial acetic acid, sodium acetate, and sodium chloride (all from Fisher Scientific, NJ) dissolved with ultrapure 18-MΩ water. In each case, the final mass balance of acetate was 25 mM as was the nominal ionic strength (Table 1). All solutions were filtered through a 0.2-µm filter prior to use. DNA Sample Preparation. λ-DNA (48 502 bp) was obtained from Life Technologies (Grand Island, NY). All DNA samples were (34) Ma, Y.; Shortreed, M. R.; Li, H.; Huang, W.; Yeung, E. S. Electrophoresis, in press. (35) Harrick, N. J. Internal Reflection Spectroscopy; John Wiley & Sons: New York, 1967; pp 1-65. (36) Tilton, R. D.; Robertson, C. R.; Gast, A. P. Langmuir 1991, 7, 2710-2718.
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prepared in 10 mM Gly-Gly (Sigma Chemical Co.) buffer, pH 8.2. DNA samples were labeled with YOYO-1 (Molecular Probes, Eugene, OR) at a ratio of one dye molecule per five base pairs according to the manufacturer’s instructions. DNA samples were prepared at a concentration of 200 pM. Samples were allowed to incubate for 5 min before further dilution and use. For the singlemolecule imaging experiments, these DNA samples were further diluted to 10-20 pM immediately prior to the start of the experiment in the appropriate buffer. It was verified that the addition of the Gly-Gly buffer did not noticeably change the pH of the final sample solution. For CE and CLC, DNA samples were diluted to 50 pM with 25 mM sodium acetate buffer immediately prior to injection into the capillary. Preparation of C18 Monolayers at Gold Surface. Surfaces were prepared on right-angle fused-silica prisms (Melles Griot, Irvine, CA; prism UVGSFS, A ) B ) C ) 25.4 mm) that were precleaned in methanol for ∼12 h. Following removal from solution, the surfaces were dried by using high-purity argon (Air Products) and placed in a cryopumped E306A Edwards Coating System. Next, the surfaces were primed with a ∼1.2-nm adhesive layer of chromium37-40 at 0.1 nm/s followed by the deposition of ∼30 nm of gold (99.99% purity) at 0.1-0.2 nm/s. The gold-coated substrates were either used immediately upon removal from the evaporator or stored under dry nitrogen. C18 monolayers were deposited by their spontaneous adsorption from ethanolic solutions41,42 on the gold surface. Briefly, the gold-coated substrates were immersed in ∼1 mM solution of sodium n-octadecanethiolate (Aldrich) under ambient conditions for at least 12 h. Afterward, the samples were rinsed with ethanol and dried in a stream of high-purity N2. Evanescent Wave Excitation Geometry. The excitation geometry was ostensibly similar to that previously reported (Figure 1).19,22 The sample chamber for single-molecule experiments was generated by sandwiching a 4-µL volume of sample solution between a No. 1 (22-mm square) Corning glass cover slip and the hypotenuse face of a right-angle fused-silica prism or a gold-coated right-angle fused-silica prism with C18 monolayers. The thickness of the sample solution was ∼8 µm. A focused laser beam was directed through the prism toward the prism-sample interface. The angle of incidence θi was greater than the critical angle. In this system, θi was slightly greater than 66°. Microscope and ICCD Camera. A Pentamax 512-EFT/1EIA intensified CCD camera was mounted on top of a Zeiss Axioskop upright microscope. The ADC rate of the camera was 5 MHz (12 bits) with software controller gain set at 3 and hardware intensifier gain set at 10. The camera was operated in the external synchronous mode with the intensifier disabled open. The camera was also in frame-transfer mode. The excitation source was a Coherent Innova-90 argon ion laser operated at 488 nm. Extraneous light (e.g., plasma lines) from the laser was eliminated with (37) Hampy, R. E.; Yost, F. G.; Ganyard, F. P. J. Vac. Sci. Technol. 1979, 16, 25-30. (38) Audino, R.; Destefanis, G.; Gorgellino, F.; Pollino, E.; Tamagno, S. Thin Solid Films 1976, 36, 343-347. (39) Terry, L. E.; Wilson, R. W. Proc. IEEF 1969, 57, 1580-1586. (40) Widrig, C. A.; Chung, C.; Porter, M. D. J. Electroanal. Chem. 1991, 310, 335-359. (41) Nuzzo, R. G.; Allara, O. L. J. Am. Chem. Soc. 1983, 105, 4481-4483. (42) Walczk, M. M.; Alves, C. A.; Lamp, B. D.; Porter, M. D. J. Electroanal. Chem. 1995, 396, 103-114.
Figure 1. Schematic diagram of the experimental setup at (A) fused-silica surface and (B) C18 surface for single-DNA molecules monitoring within the evanescent field layer.
the aid of an equilateral prism and an optical pinhole at the exit of the laser system. The light was then focused with a 30-cmfocal length plano-convex lens such that the focal point was at the fused-silica-water interface described above. The microscope objective used was a Zeiss 100× Plan-Neofluar (oil 1.3 NA). The objective was optically coupled to the cover slip with immersion oil (type FF, Cargille, Cedar Grove, NJ). Two 488-nm holographic notch filters (Kaiser Optical, HNFP) with optical density of >6 were used between the objective and the ICCD. Single-Molecule Timing. Experimental timing was controlled with a Stanford Research Systems model DG535 four-channel digital delay/pulse generator. The ICCD camera was triggered at time 0 ms with a 5-ms duration TTL pulse. Laser light was passed through an Isomet model 1205C-2 acousto-optic modulator that was optimized for maximum output in the first order according to the manufacturer’s instructions. The first-order dispersion was used as the source for the experiments and the digital delay generator was used to control the laser pulse duration and frequency. The camera integration time (software controlled) was estimated to be delayed ∼3 ms from the initial edge of the trigger pulse. The laser pulse onset began at a time of +5 ms relative to the start of the trigger to the ICCD. Therefore, ∼2 ms of dead time was present in each data frame. CE and CLC. CE analyses of electroosmotic-driven flow were performed using a P/ACE MDQ capillary electrophoresis system (Beckman Coulter, Inc., Fullerton, CA) with laser-induced fluorescence detection (LIF). Various inner diameter (i.d.) fused-silica capillaries (Polymicro Technologies, Inc., Phoenix, AZ) and etched C18 capillaries with 50-µm i.d. (Unimicro Technologies, Inc., CA) were used. Running buffers were identical to the sodium acetate buffers used for the single-DNA molecule imaging experiments. The λ-DNA sample was introduced with low pressure (0.5 psi ) 3.4 × 103 Pa) for 3 s at the anodic end of the capillary, and the applied electric fields were between +166.7 and +1,000 V/cm at 25 °C. After each run, the capillary was rinsed in the following
sequence: water, 0.1 M NaOH, water, and finally running buffer for 5 min at 20 psi. In CLC analyses with pressure-driven flow, the DNA sample was injected with low pressure for 3 s at the entrance end of the capillary and eluted with low pressure at 25 °C without an applied electric field. The fluorescence signal was excited by means of a laser module (Beckman Coulter, Inc.) operating at 488 nm and detected at an emission wavelength of 520 nm. P/ACE MDQ software (version 2.3) was used for system control, data collection, and data processing. All peaks were recorded by direct fluorescence detection. However, unretained species in the CLC running buffer were recorded by indirect fluorescence detection43 by using 1 µM fluorescein (Eastman Kodak Co., Rochester, NY). RESULTS AND DISCUSSION DNA at a Fused-Silica Surface. At basic pH, individual λ-DNA molecules resembled random coils (Figure 2A). The molecules appeared quite pliable as their structures fluctuated rapidly from shape to shape (movie M1 in Supporting Information). They were also constantly moving between exposures. At pH ∼5.5, λ-DNA molecules started to adsorb onto the fused-silica prism surface. However, permanent immobilization on the surface was not observed and the structure of λ-DNA molecules was still random coils. This contrasts a previous report that indicated that λ-DNA has a compact supercoiled structure at pH 5.75.15 As the pH further decreased to 4.5, λ-DNA molecules were gradually dragged onto the fused-silica surface (Figure 2B), similar to the process of molecular combing.2,6,44 In Figure 2B, the molecules appeared as short rods when freely moving in solution but as long strands when adsorbed. λ-DNA has a 12-base unpaired region at both ends of the molecule. This single-stranded portion appeared to attach first while the remainder of the molecule was extended (43) Xue, Q.; Yeung, E. S. J. Chromatogr., A 1994, 661, 287-295. (44) Allemand, J.-F.; Bensimon, D.; Jullien, L.; Bensimon, A.; Croquette, V. Biophys. J. 1997, 73, 2064-2070.
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Figure 2. Fluorescence image of λ-DNA molecules labeled with YOYO-1 at (A) 50 mM Tris/Tricine (pH 8.1), (B) 50 mM sodium acetate/acetic acid (pH 4.5), and (C) 50 mM sodium citrate/citric acid (pH 3.0) buffer solutions. Images were acquired with 2.2-mW laser power and 5-Hz exposure frequency. The concentrations of λ-DNA molecule were 20 pM in (A) and (C) and 10 pM in (B).
in the direction of fluid flow. Subsequently, the other end of the molecule became adsorbed (movie M2 in Supporting Information). This adsorption process occurred in the pH range of 4.5 ( 1.0. At pH 3.0, the DNA molecules were strongly adsorbed on the fused-silica surface in a compact form (Figure 2C). These molecules apparently took on the supercoiled structure. Below pH 3.0, the DNA molecules were completely and permanently adsorbed in the compact form at the fused-silica surface. In another set of experiments with a constant acetic acid/ acetate mass balance and constant ionic strength, the numbers of adsorbed λ-DNA molecules on the fused-silica surface increased with decreasing pH (Figure 3). This behavior can be attributed to a combination of hydrophobic and electrostatic interactions of 1094 Analytical Chemistry, Vol. 73, No. 6, March 15, 2001
Figure 3. Video images showing the shape of λ-DNA molecules at a fused-silica-water interface in 25 mM sodium acetate buffers: (A) pH 6.0, (B) pH 5.0, (C) pH 4.5, and (D) pH 4.0. ICCD exposure time was 5 ms at 30 Hz.
varying degrees.2,44 Near the pKa of fused silica, protonation of the silanoate groups occurs. The surface charge is neutralized and the electrostatic repulsive force is decreased. Below the pKa, the molecules became completely adsorbed and likely precipitated onto the fused-silica surface. The amount of negative charge on λ-DNA may also decrease slightly with decreasing pH. Decreased charge repulsion between the DNA and the surface, i.e., decreased electrostatic interaction, would facilitate but not directly cause adsorption. Since the bases in the unpaired region at both ends of the DNA are not hydrogen-bonded to their complement, they are available for interaction in a way that the double-stranded portion is not. The unpaired region has a strong hydrophobic (nonpolar) character whereas the hydrophobic core of the paired region is, in effect, shielded by the charged groups from contact
Figure 4. Time progress of cleavage of a λ-DNA molecule at a fused-silica surface in 50 mM sodium acetate buffer (pH 5.5 with acetic acid): (A) 1.4, (B) 2.4, (C) 2.6, and (D) 2.8 s. Exposure time was 10 ms at 5 Hz. The arrows show the fragmentation of one λ-DNA molecule. Table 2. Degree of Adsorption of λ-DNA Molecules at Different Surfaces and pHa molecule count per expt
a
fraction adsorbed
fraction nonadsorbed
pH
fused silica
C18
fused silica
C18
fused silica
C18
8.2 6.0 5.5 5.0 4.5 4.0
18 ( 4 23 ( 6 17 ( 1 19 ( 4 60 ( 15 50 ( 3
15 ( 3 27 ( 4 28 ( 4 60 ( 9 27 ( 8 21 ( 6
0(0 0.089 ( 0.035 0.186 ( 0.057 0.244 ( 0.067 0.960 ( 0.030 1(0
0.070 ( 0.033 0.720 ( 0.043 0.830 ( 0.056 0.925 ( 0.033 0.962 ( 0.019 0.934 ( 0.033
1(0 0.911 ( 0.035 0.814 ( 0.057 0.756 ( 0.067 0.040 ( 0.030 0(0
0.930 ( 0.033 0.280 ( 0.043 0.170 ( 0.056 0.073 ( 0.033 0.040 ( 0.019 0.066 ( 0.033
Mean ( SD are for multiple experiments.
with other molecules or with a surface. Our data show that it is the ends of the λ-DNA molecule that are attached to the surface first followed by adsorption of the middle section. This suggests that under these conditions hydrophobic (nonpolar) interactions dominate adsorption compared to electrostatic (charge or polar) interactions to initiate adsorption. The overall shape of individual adsorbed λ-DNA molecules is determined by the direction of fluid motion at the time of initial adsorption. The observed length of individual completely stretched λ-DNA molecules was 16.3 µm, or 98.9% compared to the theoretical value of 16.5 µm (48,502 bp, 1 kb ) 0.34 µm). The adsorbed DNA molecules were stretched to various lengths and were often cleaved with time (Figure 4). This fragmentation of DNA molecules was influenced by a combination of pH, laser power, and velocity of fluid flow. The λ-DNA molecules tended to break more readily at high laser powers and in solutions moving at high velocities. After breaking, part of the DNA
molecule remained permanently adsorbed while the other portion was swept away with the buffer solution as a random coil. At certain pH values (3.5-4.0), λ-DNA could be reproducibly stretched and adsorbed. DNA at a C18 Surface. The pH effect on adsorption at the C18 surface was different from that at the fused-silica surface. Adsorption was observed at the entire range of pH 4-6. Although the number of adsorbed molecules gradually increased with decreasing pH, ∼6% of λ-DNA molecules remained not adsorbed on the C18 surface even at pH 4.0 (Table 2). Below pH 6.0, individual molecules tended to break in larger numbers compared to those at a fused-silica surface. The molecules also showed a relatively high fraction of permanent adsorption at the C18 surface. These results again indicate that hydrophobic interaction is controlling the dynamics of adsorption and desorption of individual DNA molecules at the liquid-solid interface. Because the C18 surface was already covered with gold underneath, the electroAnalytical Chemistry, Vol. 73, No. 6, March 15, 2001
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Table 3. Effect of Methanol on Adsorption of λ-DNA Molecules at a C18 Surface and 25 mM Sodium Acetate Buffer (pH 4.0) methanol (%)
molecule count per experiment
fraction adsorbed
fraction nonadsorbed
0 10 30 50a
43 ( 4 32 ( 4 34 ( 3 51 ( 16
0.923 ( 0.016 0.921 ( 0.023 0.760 ( 0.105 0.864 ( 0.080
0.077 ( 0.016 0.080 ( 0.023 0.246 ( 0.105 0.136 ( 0.080
a
At 50% methanol, precipitation rather than adsorption occurred.
static repulsion between λ-DNA molecules and silanoate ions (SiO-) at the fused-silica surface does not exist at the C18 surface. The addition of methanol decreased the fraction of adsorbed λ-DNA molecules (Table 3). At 30% methanolic sodium acetate buffer (pH 4.0), ∼76% of the DNA molecules were adsorbed on the C18 surface, while all DNA molecules were adsorbed at the fused-silica surface. However, above 50% methanol, individual λ-DNA molecules precipitated on the C18 surface because of low solubility. This phenomenon can also be explained by hydrophobic interaction between DNA molecules and C18 rather than electrostatic interaction. The addition of methanol decreases adsorption because hydrophobic interaction between the DNA molecules and the solvent is enhanced.36 Silica is a classic normal-phase material and is suitable for separating polar nonionic organic compounds. C18 is a classic reversed-phase (RP) material and is retentive for nonpolar solutes. As the methanol ratio in the buffer solution increased, the fraction of adsorbed λ-DNA molecule at the C18 surface decreased (Table 3). This result is in accordance with the retention times of analytes in chromatography because the C18 surface and the methanol-containing buffer can be considered as the stationary phase and the mobile phase in RP-LC, respectively. The adsorption of DNA molecules on the fused-silica surface was mediated by the addition of poly(vinylpyrrolidone) (PVP). DNA molecules in 50 mM sodium citrate buffer (pH 3.2) were adsorbed and started to precipitate (Figure 5A). The adsorbed molecule fraction was 1.0. However, in the same pH buffer with 0.3% PVP, the polymer dynamically coated the surface and decreased the adsorption of DNA molecules (Figure 5B). The adsorbed molecule fraction was ∼0.89. Even at a low pH (3.2), the presence of 0.3% PVP effectively decreased the adsorption of DNA molecules. This is in accordance with the fact the PVP is a polymer that has been shown to suppress electroosmotic flow (EOF) and to prevent the adsorption of DNA onto the inner wall of fused-silica capillaries in CE.45 Comparison with Chromatography. Since the ICCD camera can be operated at 30 Hz, sequences of images can be recorded to follow the dynamics of adsorption/desorption of individual DNA molecules at the solid-liquid interface. At pH 5.5, single λ-DNA molecules on a fused-silica surface showed 33-267 ms (two consecutive frames to nine consecutive frames) adsorption (immobilization) duration and many molecules were adsorbed for 67 ms (three consecutive frames) at the surface (Figure 6A). At the start of each experiment, a drop of DNA solution was placed on a microscope slide. A cover slip was then put on top of the solution. The weight of the cover slip created a bulk flow across the (45) Gao, Q.; Yeung, E. S. Anal. Chem. 1998, 70, 1382-1388.
1096 Analytical Chemistry, Vol. 73, No. 6, March 15, 2001
Figure 5. Effect of PVP on adsorption of λ-DNA molecules on a fused-silica surface: (A) 50 mM sodium citrate buffer (pH 3.2 with 50 mM citric acid). (B) 50 mM sodium citrate buffer (pH 3.2 with 50 mM citric acid) plus 0.3% PVP. Solid arrows show precipitation of λ-DNA molecules. Dotted arrows show freely moving λ-DNA molecules.
microscope slide that was in the range of 20-200 nm/ms as determined by tracking the unadsorbed molecules. This degree of flow should not affect the adsorption duration because the shear force is not significant. The lack of correlation between bulk velocity and adsorption duration is confirmed in Figure 6B. Results of CLC of λ-DNA in fused-silica capillary displayed pHdependent peak broadening which was consistent with adsorption. The asymmetry ratio (κ) increased with decreasing pH at both the fused-silica capillary and the etched C18 capillary (Table 4). This asymmetry qualitatively follows the asymmetry in adsorption times (Figure 6A) over the narrow pH range where the latter can be measured with confidence. Below pH 6, the determination of the asymmetry ratio was not reliable for the etched C18 capillary. There, the reproducibility was poor and several peaks appeared because of memory effects due to strong adsorption of the DNA molecules. We were able to clean the fused-silica capillary with 0.1 M NaOH and water, but the etched C18 capillary was not properly washed by using either water or methanol. These results (Table 4) are consistent with the single-molecule imaging data which show that the adsorption of DNA molecules at a C18 surface is stronger than that at a fused-silica surface in the range of pH 4.5-6 (Table 2) and that adsorption to the capillary wall increases as the pH decreases. In a series of CLC runs at constant linear flow rate, λ-DNA showed different peak shapes and retention times (Figure 7) depending on the inner diameter of the fused-silica capillary. The
Figure 7. CLC chromatograms showing the effect of capillary surface area-to-volume ratio on the elution of 50 pM λ-DNA at a constant linear flow rate in 25 mM sodium acetate buffer (pH 5.5). Solid line: 74-µm i.d. and 0.5 psi, pressure-driven. Dashed line: 51 µm i.d. and 0.8 psi, pressure-driven. Dotted line: 31 µm i.d. and 1.0 psi, pressure-driven. All capillaries were fused silica with 30-cm total length and 10-cm effective length. RFU, relative fluorescence units. Table 5. Effect of Capillary Surface Area-to-Volume Ratio on the Capacity Factor of λ-DNA at a Constant Linear Flow Rate in CLCa
b
Figure 6. (A) Histogram of adsorption duration and (B) correlation of bulk flow velocity with adsorption duration of individual λ-DNA molecules at a fused-silica surface in 25 mM sodium acetate buffer (pH 5.5). In (B), each set of symbols represents a different experiment. Table 4. Asymmetry Ratio of λ-DNA Peak Eluted from a Fused-Silica and an Etched C18 Capillary at Various pHa
t0 (min) tRc (min) k′Rd Ve (cm/min) 2/r f (cm-1) applied pressure (psi)
31-µm i.d.
51-µm i.d.
74-µm i.d.
0.09 1.45 14.4 6.92 0.129 1.0
0.10 0.64 5.4 15.58 0.078 0.8
0.10 0.54 4.4 18.45 0.054 0.5
a CLC conditions: 30-cm total length and 10-cm effective length; bare fused-silica capillary; running buffer, 25 mM sodium acetate (pH 5.5). b t0, the time taken for unretained species to reach the detector (void time). c tR, the time taken for a specific solute to reach the detector (retention time). d k′R, the capacity factor ) (tR - t0)/t0. e V, linear velocity ) effective length of capillary/tR. f 2/r, surface area/ volume ) 2πrl/πr2l ) 2/r.
κb
fused silica C18
pH 4.0
pH 4.5
pH 5.0
pH 5.5
pH 6.0
pH 8.2
3 (1.0c)
2.7
2.4
1.8
1.7 1.8
1.3 1.5
a CLC conditions: hydrodynamic injection for 3 s at 0.5 psi () 3.4 × 103 Pa); bare fused-silica capillary 30 cm × 50-µm i.d. (20 cm to the detector); 0.5 psi of pressure-driven flow. Sample concentrations: 50 pM λ-DNA and 100 nM coumarin 334; running buffer, 25 mM sodium acetate. b Asymmetry ratio: ratio of the peak width at half-height (W1/2) before and after the peak maximum. c The symmetry ratio of coumarin 334 eluted at pH 4.0; i.e., a perfectly symmetric peak was observed.
bulk velocities in CLC were deliberately set in the range of 7.422.7 cm/min to be comparable to those in the imaging experiments. Broader peaks and longer retention times indicate greater access to the column surface, which has identical adsorption properties in each case. The capillary surface area-to-volume ratio (2πrl/πr2l ) 2/r) is a quantitative measure of the accessibility of the capillary surface to the DNA molecules. The capacity factor of λ-DNA is found to be proportional to the capillary surface areato-volume ratio (2/r) at pH 5.5 (Table 5). The regression curve is given by y ) 140.4x - 4.2 with a linear correlation coefficient, R ) 0.9726, where y is the capacity factor and x is the capillary
surface area-to-volume ratio. In liquid chromatography separations, the capacity factor, k′R, is a direct measure of the strength of interaction of the sample species with the packing material. Higher capacity factors are correlated with greater retention of solutes.46 In the same way, λ-DNA molecules here showed longer retention times and broader peaks in relation to the capillary surface areato-volume ratio. This indicates that the retention of DNA molecules in CLC can be predicted from single-DNA molecule adsorption/ desorption dynamics. Comparison with CE. In the presence of EOF,47 the electrophoretic velocity, Vep, in cm/min, is given by
Vep ) µepE
(1)
where E is the applied electric field (V/cm) and µep is the electrophoretic mobility given by
µep ) q/3πηd
(2)
where q is the charge of the ionized solute, η is the buffer viscosity, and d is the solute diameter. If there is adsorption, we can Analytical Chemistry, Vol. 73, No. 6, March 15, 2001
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Table 6. Effect of Capillary Surface Area-to-Volume Ratio on the Migration of λ-DNA in CEa
EOF b (min) MTc (min) Vappd (cm/min) Veof e (cm/min) Kf 2/r g (cm-1)
31-µm i.d.
51-µm i.d.
74-µm i.d.
2.14 7.27 ( 0.06 1.377 4.66 3.29 0.129
2.54 10.14 ( 1.46 0.986 3.93 2.94 0.078
2.78 11.89 ( 2.76 0.841 3.60 2.76 0.054
a CE conditions: 30-cm total length and 10-cm effective length, bare fused-silica capillary; applied electric field, +166.67 V/cm; running buffer, 25-mM sodium acetate (pH 5.5). b EOF, migration time of a neutral marker, 100 nM coumarin 334. c MT, migration time of λ-DNA. d V , apparent velocity. e V , electroosmotic flow velocity. f K, relative app eof adsorption factor. g 2/r, capillary surface area-to-volume ratio.
imaging experiments because of the relatively low applied electric field (+166.7 V/cm, limited by Joule heating in the 51- and the 74-µm-i.d. capillaries) and the opposing directions of electrophoretic mobility and EOF. When +1 kV/cm was applied to a 31-µm-i.d. capillary in the same buffer, the apparent velocity of the DNA molecules was 9.8 cm/min as predicted from Table 6. These results further confirm that the velocities of DNA molecules in CE can be predicted by single-molecule imaging experiments and that hydrophobic interaction as a function of pH is the primary factor controlling the migration velocities. Figure 8. Electropherograms of 50 pM λ-DNA in 25 mM sodium acetate buffer at pH (A) 6.5, (B) 6.0, (C) 5.5, and (D) 5.0. CE conditions: fused-silica capillary, 40 cm × 74 µm i.d. (30 cm to the detector); applied voltage, + 10 kV; and RFU, relative fluorescence units.
introduce a new constant, the relative adsorption factor (K)
K ) Vep + Veof - Vapp
(3)
where Vapp is the observed velocity and Veof is the electroosmotic flow velocity. K therefore measures the decrease in migration speed due to adsorption. The retention time and the peak shapes in CE also correlated with the adsorption dynamics of single λ-DNA molecules (Figure 8). The spikes in the traces were from light scattering of DNA molecules as they cross the laser beam. The DNA peaks became much broader with decreasing pH. However, the asymmetry ratio was much less than that in pressure-driven flow because of the EOF profile. The retention time also showed a large dependence on the pH. In electroosmotic-driven flow, at a constant pH, the relative adsorption factor (K) was found to be also related to the capillary surface area-to-volume ratio (Table 6). The regression curve is given by y ) 6.99x + 2.39 with a linear correlation coefficient, R ) 0.9996, where y is the relative adsorption factor and x is the capillary surface area-to-volume ratio. The linear velocities (0.8-1.4 cm/min) for the CE studies of λ-DNA were smaller compared to those in the single-molecule (46) Weston, A.; Brown, P. R. HPLC and CE, Principles and Practice; Academic Press: San Diego, CA, 1997; pp 8-9. (47) Baker, D. R. Capillary Electrophoresis; John Wiley & Sons: New York, 1995; pp 19-52.
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CONCLUSIONS Direct observation of the dynamics of adsorption/desorption of individual DNA molecules was achieved by single-molecule imaging at the solid-liquid interface. At basic and neutral pH, λ-DNA molecules exhibited rapid conformational fluctuations with a random coil structure. The molecules were gradually dragged and adsorbed onto the fused-silica surface with decreasing pH, until they finally precipitated and formed a compact supercoiled structure. Adsorption was initiated at the unpaired hydrophobic ends of the molecule. At pH 5.5, the kinetics of adsorption/ desorption of individual DNA molecules was examined to elucidate the balance between hydrophobic interaction and electrostatic repulsion between the DNA molecules and the surface. At a constant buffer condition, the retention time as well as peak asymmetry of DNA molecules was proportional to the capillary surface area-to-volume ratio in both CE and CLC. Such behavior reflects the relative accessibility of the surface to the DNA molecule and can be predicted from the single-molecule imaging experiments. At a C18 surface, the dynamics of adsorption/ desorption of individual DNA molecules was altered with the addition of methanol as well as with pH changes. Adsorption of λ-DNA molecules at the C18 surface was much stronger than that at the fused-silica surface. These results show clearly that hydrophobic interaction rather than electrostatic interaction is the major driving force for DNA adsorption, even though both interactions have been implicated in adsorption.2,4,44 ACKNOWLEDGMENT We thank Marc D. Porter and his group members, especially Desiree Grubisha, for providing the C18 surfaces. The Ames Laboratory is operated for the U.S. Department of Energy by Iowa
State University under Contract No. W-7405-Eng-82. This work was supported by the Director of Science, Office of Basic Energy Sciences, Division of Chemical Sciences. SUPPORTING INFORMATION AVAILABLE Two AVI movie files: movie M1, radom coil motion of λ-DNA molecules in Figure 2A; movie M2, sequential adsorption of the
two ends of λ-DNA molesules with stretching in Figure 2B. This material is available free of charge via the Internet at http:// pubs.acs.org. Received for review November 20, 2000. Accepted January 22, 2001. AC0013599
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