Real-Time Dynamics of Single-DNA Molecules Undergoing Adsorption and Desorption at Liquid−Solid Interfaces

Real-Time Dynamics of Single-DNA MoleculesUndergoing Adsorption and Desorption atLiquid-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/desorptionbehavior of individual λ-DNA molecules at liquid-solidinterfaces were monitored by imaging within the evanes-cent field layer using total internal reflection fluorescencemicroscopy. At a fused-silica surface, molecular confor-mation and adsorption behavior were found to depend onboth pH and buffer composition. A histogram of individualλ-DNA adsorption durations measured by hydrodynami-cally flowing molecules along the interface exhibitedasymmetry nearly identical to that of the correspondingelution peaks found in capillary liquid chromatographyand capillary electrophoresis. The accessibility of thesurface to the molecules, which is proportional to thecapillary surface area-to-volume ratio, can be correlatedwith the capacity factor and the relative adsorption factor.At a C18 surface, the dynamics of individual DNA mol-ecules changed with the addition of organic solvent as wellas with pH. Hydrophobic interaction rather than electro-static interaction was the major driving force for adsorp-tion of individual DNA molecules.

The study of single-molecule adsorption, desorption, andmotion at a liquid-solid interface can provide insights intomolecular genetics,1-3 biosensor design,4-6 DNA biophysics,7-21

and basic separation theory.22-28 It is well known that bothelectrostatic and hydrophobic interactions govern protein adsorp-tion at liquid-solid interfaces.4,22 These fundamental interactionsare the foundation for many chromatographic protein separa-tions.29 Both rate theory and band broadening in chromatographyand electrophoresis have been explained by using statisticaltheory.30 Direct observation of individual molecular motion andinteractions at the liquid-solid interface would add valuable detailsregarding these and other related phenomena. How these sameinteractions affect DNA motion and adsorption is not as clear. Forinstance, on the basis of simple electrophoresis theory, it shouldnot be possible to separate DNA molecules greater than 10-20bp without the presence of a sieving medium. Surprisingly, sucha separation was clearly demonstrated.31 The separation mecha-nism was ascribed to electrostatic interactions between the DNAmolecule and the wall of the fused-silica capillary.

Imaging of individual DNA molecules labeled with fluorescentdyes has been possible for some time.32,33 However, not allmethods are applicable to real-time imaging of the random motionof individual DNA molecules in free solution. For example,

* Corresponding author: (tel) 515-294-8062; (fax) 515-294-0266; (e-mail)[email protected].

† Present address: Samsung Advanced Institute of Technology, P.O. Box1211, 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.

(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.

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scanning with a sharp optical probe provides high-resolutionimages but is not capable of capturing video rate motion.14 Recentadvances in intensified charge-coupled device (ICCD) cameradetection efficiency and speed have enabled temporal resolutionof 0.4 ms and spatial resolution of 0.3 µm.19,22 The motion of singlemolecules in free solution was captured in the form of movies.23,34

The total internal reflection (TIR) geometry14,35 provides high-contrast images of single molecules by virtually eliminating all ofthe 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-DNAmolecules labeled with YOYO-1. The motion and adsorption/desorption behaviors of these molecules at the fused-silica-waterinterface and the C18-water interface were analyzed as a functionof pH and buffer composition. We also assessed the driving forcefor adsorption of individual DNA molecules by the addition ofmethanol to modulate the strength of the hydrophobic interactionin aqueous solution.36 The results of the real-time imagingexperiments were compared to capillary electrophoresis (CE) andcapillary liquid chromatography (CLC) separations with referenceto band broadening and elution times.

EXPERIMENTAL SECTIONBuffer 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 (pH7-8), Tris/Tricine (pH 8-9), and sodium hydroxide/CHES (pH9-10). These regents were all purchased from Sigma ChemicalCo. (St. Louis, MO). Buffer solutions for measuring the dynamicsof adsorption/desorption were prepared at various pH values using1.0 M solutions of acetic acid, sodium acetate, and sodiumchloride. A.C.S. grade or higher glacial acetic acid, sodium acetate,and sodium chloride (all from Fisher Scientific, NJ) dissolved withultrapure 18-MΩ water. In each case, the final mass balance ofacetate 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 obtainedfrom Life Technologies (Grand Island, NY). All DNA samples were

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 pairsaccording to the manufacturer’s instructions. DNA samples wereprepared at a concentration of 200 pM. Samples were allowed toincubate for 5 min before further dilution and use. For the single-molecule imaging experiments, these DNA samples were furtherdiluted to 10-20 pM immediately prior to the start of theexperiment in the appropriate buffer. It was verified that theaddition of the Gly-Gly buffer did not noticeably change the pHof the final sample solution. For CE and CLC, DNA samples werediluted to 50 pM with 25 mM sodium acetate buffer immediatelyprior to injection into the capillary.

Preparation of C18 Monolayers at Gold Surface. Surfaceswere prepared on right-angle fused-silica prisms (Melles Griot,Irvine, CA; prism UVGSFS, A ) B ) C ) 25.4 mm) that wereprecleaned in methanol for ∼12 h. Following removal fromsolution, the surfaces were dried by using high-purity argon (AirProducts) and placed in a cryopumped E306A Edwards CoatingSystem. Next, the surfaces were primed with a ∼1.2-nm adhesivelayer 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-coatedsubstrates were either used immediately upon removal from theevaporator or stored under dry nitrogen. C18 monolayers weredeposited by their spontaneous adsorption from ethanolic solu-tions41,42 on the gold surface. Briefly, the gold-coated substrateswere 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 ofhigh-purity N2.

Evanescent Wave Excitation Geometry. The excitationgeometry was ostensibly similar to that previously reported(Figure 1).19,22 The sample chamber for single-molecule experi-ments was generated by sandwiching a 4-µL volume of samplesolution between a No. 1 (22-mm square) Corning glass coverslip and the hypotenuse face of a right-angle fused-silica prism ora gold-coated right-angle fused-silica prism with C18 monolayers.The thickness of the sample solution was ∼8 µm. A focused laserbeam was directed through the prism toward the prism-sampleinterface. The angle of incidence θi was greater than the criticalangle. In this system, θi was slightly greater than 66°.

Microscope and ICCD Camera. A Pentamax 512-EFT/1EIAintensified CCD camera was mounted on top of a Zeiss Axioskopupright microscope. The ADC rate of the camera was 5 MHz (12bits) with software controller gain set at 3 and hardware intensifiergain set at 10. The camera was operated in the externalsynchronous mode with the intensifier disabled open. The camerawas also in frame-transfer mode. The excitation source was aCoherent Innova-90 argon ion laser operated at 488 nm. Extrane-ous light (e.g., plasma lines) from the laser was eliminated with

(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: NewYork, 1967; pp 1-65.

(36) Tilton, R. D.; Robertson, C. R.; Gast, A. P. Langmuir 1991, 7, 2710-2718.

(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. ThinSolid 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.

Table 1. Preparation of Various 25 mM SodiumAcetate Buffers for Studying the Dynamics ofAdsorption/Desorption of DNA Molecules Starting from1.0 M Stock Solutions

pHacetic acid

(µL)sodium acetate

(mL)sodium chloride

(µL)water(mL)

4.0 2120 0.4 2120 95.44.5 1600 0.9 1600 95.95.0 900 1.6 900 96.65.5 377 2.1 377 97.16.0 133 2.4 133 97.46.5 43.7 2.5 43.7 97.57.0 14.0 2.5 14.0 97.57.5 4.4 2.5 4.4 97.58.0 1.4 2.5 1.4 97.5

1092 Analytical Chemistry, Vol. 73, No. 6, March 15, 2001

the aid of an equilateral prism and an optical pinhole at the exitof the laser system. The light was then focused with a 30-cm-focal length plano-convex lens such that the focal point was atthe fused-silica-water interface described above. The microscopeobjective used was a Zeiss 100× Plan-Neofluar (oil 1.3 NA). Theobjective was optically coupled to the cover slip with immersionoil (type FF, Cargille, Cedar Grove, NJ). Two 488-nm holographicnotch filters (Kaiser Optical, HNFP) with optical density of >6were used between the objective and the ICCD.

Single-Molecule Timing. Experimental timing was controlledwith a Stanford Research Systems model DG535 four-channeldigital delay/pulse generator. The ICCD camera was triggeredat time 0 ms with a 5-ms duration TTL pulse. Laser light waspassed through an Isomet model 1205C-2 acousto-optic modulatorthat was optimized for maximum output in the first orderaccording to the manufacturer’s instructions. The first-orderdispersion was used as the source for the experiments and thedigital delay generator was used to control the laser pulse durationand frequency. The camera integration time (software controlled)was estimated to be delayed ∼3 ms from the initial edge of thetrigger pulse. The laser pulse onset began at a time of +5 msrelative to the start of the trigger to the ICCD. Therefore, ∼2 msof dead time was present in each data frame.

CE and CLC. CE analyses of electroosmotic-driven flow wereperformed using a P/ACE MDQ capillary electrophoresis system(Beckman Coulter, Inc., Fullerton, CA) with laser-induced fluo-rescence detection (LIF). Various inner diameter (i.d.) fused-silicacapillaries (Polymicro Technologies, Inc., Phoenix, AZ) and etchedC18 capillaries with 50-µm i.d. (Unimicro Technologies, Inc., CA)were used. Running buffers were identical to the sodium acetatebuffers 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 theapplied electric fields were between +166.7 and +1,000 V/cm at25 °C. After each run, the capillary was rinsed in the following

sequence: water, 0.1 M NaOH, water, and finally running bufferfor 5 min at 20 psi.

In CLC analyses with pressure-driven flow, the DNA samplewas injected with low pressure for 3 s at the entrance end of thecapillary and eluted with low pressure at 25 °C without an appliedelectric field. The fluorescence signal was excited by means of alaser module (Beckman Coulter, Inc.) operating at 488 nm anddetected at an emission wavelength of 520 nm. P/ACE MDQsoftware (version 2.3) was used for system control, data collection,and data processing. All peaks were recorded by direct fluores-cence detection. However, unretained species in the CLC runningbuffer were recorded by indirect fluorescence detection43 by using1 µM fluorescein (Eastman Kodak Co., Rochester, NY).

RESULTS AND DISCUSSIONDNA at a Fused-Silica Surface. At basic pH, individual

λ-DNA molecules resembled random coils (Figure 2A). Themolecules appeared quite pliable as their structures fluctuatedrapidly from shape to shape (movie M1 in Supporting Informa-tion). They were also constantly moving between exposures. AtpH ∼5.5, λ-DNA molecules started to adsorb onto the fused-silicaprism surface. However, permanent immobilization on the surfacewas not observed and the structure of λ-DNA molecules was stillrandom coils. This contrasts a previous report that indicated thatλ-DNA has a compact supercoiled structure at pH 5.75.15 As thepH further decreased to 4.5, λ-DNA molecules were graduallydragged onto the fused-silica surface (Figure 2B), similar to theprocess of molecular combing.2,6,44 In Figure 2B, the moleculesappeared as short rods when freely moving in solution but as longstrands when adsorbed. λ-DNA has a 12-base unpaired region atboth ends of the molecule. This single-stranded portion appearedto 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.

Figure 1. Schematic diagram of the experimental setup at (A) fused-silica surface and (B) C18 surface for single-DNA molecules monitoringwithin the evanescent field layer.

Analytical Chemistry, Vol. 73, No. 6, March 15, 2001 1093

in the direction of fluid flow. Subsequently, the other end of themolecule 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 thefused-silica surface in a compact form (Figure 2C). Thesemolecules apparently took on the supercoiled structure. BelowpH 3.0, the DNA molecules were completely and permanentlyadsorbed 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 numbersof adsorbed λ-DNA molecules on the fused-silica surface increasedwith decreasing pH (Figure 3). This behavior can be attributedto a combination of hydrophobic and electrostatic interactions of

varying degrees.2,44 Near the pKa of fused silica, protonation ofthe silanoate groups occurs. The surface charge is neutralizedand the electrostatic repulsive force is decreased. Below the pKa,the molecules became completely adsorbed and likely precipitatedonto the fused-silica surface. The amount of negative charge onλ-DNA may also decrease slightly with decreasing pH. Decreasedcharge repulsion between the DNA and the surface, i.e., decreasedelectrostatic interaction, would facilitate but not directly causeadsorption. Since the bases in the unpaired region at both endsof the DNA are not hydrogen-bonded to their complement, theyare available for interaction in a way that the double-strandedportion is not. The unpaired region has a strong hydrophobic(nonpolar) character whereas the hydrophobic core of the pairedregion is, in effect, shielded by the charged groups from contact

Figure 2. Fluorescence image of λ-DNA molecules labeled withYOYO-1 at (A) 50 mM Tris/Tricine (pH 8.1), (B) 50 mM sodiumacetate/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 laserpower and 5-Hz exposure frequency. The concentrations of λ-DNAmolecule were 20 pM in (A) and (C) and 10 pM in (B).

Figure 3. Video images showing the shape of λ-DNA molecules ata 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 timewas 5 ms at 30 Hz.


with other molecules or with a surface. Our data show that it isthe ends of the λ-DNA molecule that are attached to the surfacefirst followed by adsorption of the middle section. This suggeststhat under these conditions hydrophobic (nonpolar) interactionsdominate adsorption compared to electrostatic (charge or polar)interactions to initiate adsorption. The overall shape of individualadsorbed λ-DNA molecules is determined by the direction of fluidmotion at the time of initial adsorption. The observed length ofindividual 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 variouslengths and were often cleaved with time (Figure 4). Thisfragmentation of DNA molecules was influenced by a combinationof pH, laser power, and velocity of fluid flow. The λ-DNA moleculestended to break more readily at high laser powers and in solutionsmoving at high velocities. After breaking, part of the DNA

molecule remained permanently adsorbed while the other portionwas swept away with the buffer solution as a random coil. Atcertain pH values (3.5-4.0), λ-DNA could be reproduciblystretched and adsorbed.

DNA at a C18 Surface. The pH effect on adsorption at theC18 surface was different from that at the fused-silica surface.Adsorption was observed at the entire range of pH 4-6. Althoughthe number of adsorbed molecules gradually increased withdecreasing pH, ∼6% of λ-DNA molecules remained not adsorbedon the C18 surface even at pH 4.0 (Table 2). Below pH 6.0,individual molecules tended to break in larger numbers comparedto those at a fused-silica surface. The molecules also showed arelatively high fraction of permanent adsorption at the C18 surface.These results again indicate that hydrophobic interaction iscontrolling the dynamics of adsorption and desorption of individualDNA molecules at the liquid-solid interface. Because the C18

surface was already covered with gold underneath, the electro-

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 fraction adsorbed fraction nonadsorbed

pH fused silica C18 fused silica C18 fused silica C18

8.2 18 ( 4 15 ( 3 0 ( 0 0.070 ( 0.033 1 ( 0 0.930 ( 0.0336.0 23 ( 6 27 ( 4 0.089 ( 0.035 0.720 ( 0.043 0.911 ( 0.035 0.280 ( 0.0435.5 17 ( 1 28 ( 4 0.186 ( 0.057 0.830 ( 0.056 0.814 ( 0.057 0.170 ( 0.0565.0 19 ( 4 60 ( 9 0.244 ( 0.067 0.925 ( 0.033 0.756 ( 0.067 0.073 ( 0.0334.5 60 ( 15 27 ( 8 0.960 ( 0.030 0.962 ( 0.019 0.040 ( 0.030 0.040 ( 0.0194.0 50 ( 3 21 ( 6 1 ( 0 0.934 ( 0.033 0 ( 0 0.066 ( 0.033

a Mean ( SD are for multiple experiments.


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 adsorbedλ-DNA molecules (Table 3). At 30% methanolic sodium acetatebuffer (pH 4.0), ∼76% of the DNA molecules were adsorbed onthe C18 surface, while all DNA molecules were adsorbed at thefused-silica surface. However, above 50% methanol, individualλ-DNA molecules precipitated on the C18 surface because of lowsolubility. This phenomenon can also be explained by hydrophobicinteraction between DNA molecules and C18 rather than electro-static interaction. The addition of methanol decreases adsorptionbecause hydrophobic interaction between the DNA molecules andthe solvent is enhanced.36 Silica is a classic normal-phase materialand is suitable for separating polar nonionic organic compounds.C18 is a classic reversed-phase (RP) material and is retentive fornonpolar solutes. As the methanol ratio in the buffer solutionincreased, the fraction of adsorbed λ-DNA molecule at the C18

surface decreased (Table 3). This result is in accordance with theretention times of analytes in chromatography because the C18

surface and the methanol-containing buffer can be considered asthe stationary phase and the mobile phase in RP-LC, respectively.

The adsorption of DNA molecules on the fused-silica surfacewas mediated by the addition of poly(vinylpyrrolidone) (PVP).DNA molecules in 50 mM sodium citrate buffer (pH 3.2) wereadsorbed and started to precipitate (Figure 5A). The adsorbedmolecule fraction was 1.0. However, in the same pH buffer with0.3% PVP, the polymer dynamically coated the surface anddecreased the adsorption of DNA molecules (Figure 5B). Theadsorbed molecule fraction was ∼0.89. Even at a low pH (3.2),the presence of 0.3% PVP effectively decreased the adsorption ofDNA molecules. This is in accordance with the fact the PVP is apolymer that has been shown to suppress electroosmotic flow(EOF) and to prevent the adsorption of DNA onto the inner wallof fused-silica capillaries in CE.45

Comparison with Chromatography. Since the ICCD cameracan be operated at 30 Hz, sequences of images can be recordedto follow the dynamics of adsorption/desorption of individual DNAmolecules at the solid-liquid interface. At pH 5.5, single λ-DNAmolecules on a fused-silica surface showed 33-267 ms (twoconsecutive frames to nine consecutive frames) adsorption (im-mobilization) duration and many molecules were adsorbed for 67ms (three consecutive frames) at the surface (Figure 6A). At thestart of each experiment, a drop of DNA solution was placed ona 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

microscope slide that was in the range of 20-200 nm/ms asdetermined by tracking the unadsorbed molecules. This degreeof flow should not affect the adsorption duration because the shearforce is not significant. The lack of correlation between bulkvelocity and adsorption duration is confirmed in Figure 6B.

Results of CLC of λ-DNA in fused-silica capillary displayed pH-dependent peak broadening which was consistent with adsorption.The asymmetry ratio (κ) increased with decreasing pH at boththe fused-silica capillary and the etched C18 capillary (Table 4).This asymmetry qualitatively follows the asymmetry in adsorptiontimes (Figure 6A) over the narrow pH range where the latter canbe measured with confidence. Below pH 6, the determination ofthe asymmetry ratio was not reliable for the etched C18 capillary.There, the reproducibility was poor and several peaks appearedbecause of memory effects due to strong adsorption of the DNAmolecules. We were able to clean the fused-silica capillary with0.1 M NaOH and water, but the etched C18 capillary was notproperly washed by using either water or methanol. These results(Table 4) are consistent with the single-molecule imaging datawhich show that the adsorption of DNA molecules at a C18 surfaceis stronger than that at a fused-silica surface in the range of pH4.5-6 (Table 2) and that adsorption to the capillary wall increasesas the pH decreases.

In a series of CLC runs at constant linear flow rate, λ-DNAshowed different peak shapes and retention times (Figure 7)depending on the inner diameter of the fused-silica capillary. The(45) Gao, Q.; Yeung, E. S. Anal. Chem. 1998, 70, 1382-1388.

Table 3. Effect of Methanol on Adsorption of λ-DNAMolecules at a C18 Surface and 25 mM Sodium AcetateBuffer (pH 4.0)

methanol(%)

molecule countper experiment

fractionadsorbed

fractionnonadsorbed

0 43 ( 4 0.923 ( 0.016 0.077 ( 0.01610 32 ( 4 0.921 ( 0.023 0.080 ( 0.02330 34 ( 3 0.760 ( 0.105 0.246 ( 0.10550a 51 ( 16 0.864 ( 0.080 0.136 ( 0.080

a At 50% methanol, precipitation rather than adsorption occurred.

Figure 5. Effect of PVP on adsorption of λ-DNA molecules on afused-silica surface: (A) 50 mM sodium citrate buffer (pH 3.2 with50 mM citric acid). (B) 50 mM sodium citrate buffer (pH 3.2 with 50mM citric acid) plus 0.3% PVP. Solid arrows show precipitation ofλ-DNA molecules. Dotted arrows show freely moving λ-DNA mol-ecules.


bulk velocities in CLC were deliberately set in the range of 7.4-22.7 cm/min to be comparable to those in the imaging experi-ments. Broader peaks and longer retention times indicate greateraccess to the column surface, which has identical adsorptionproperties in each case. The capillary surface area-to-volume ratio(2πrl/πr2l ) 2/r) is a quantitative measure of the accessibility ofthe capillary surface to the DNA molecules. The capacity factorof λ-DNA is found to be proportional to the capillary surface area-to-volume ratio (2/r) at pH 5.5 (Table 5). The regression curve isgiven 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 ofinteraction of the sample species with the packing material. Highercapacity factors are correlated with greater retention of solutes.46

In the same way, λ-DNA molecules here showed longer retentiontimes and broader peaks in relation to the capillary surface area-to-volume ratio. This indicates that the retention of DNA moleculesin CLC can be predicted from single-DNA molecule adsorption/desorption dynamics.

Comparison with CE. In the presence of EOF,47 the electro-phoretic velocity, Vep, in cm/min, is given by

where E is the applied electric field (V/cm) and µep is theelectrophoretic mobility given by

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

Figure 6. (A) Histogram of adsorption duration and (B) correlationof bulk flow velocity with adsorption duration of individual λ-DNAmolecules 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 froma Fused-Silica and an Etched C18 Capillary at VariouspHa

κb

pH 4.0 pH 4.5 pH 5.0 pH 5.5 pH 6.0 pH 8.2

fused silica 3 (1.0c) 2.7 2.4 1.8 1.7 1.3C18 1.8 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 thedetector); 0.5 psi of pressure-driven flow. Sample concentrations: 50pM λ-DNA and 100 nM coumarin 334; running buffer, 25 mM sodiumacetate. b Asymmetry ratio: ratio of the peak width at half-height (W1/2)before and after the peak maximum. c The symmetry ratio of coumarin334 eluted at pH 4.0; i.e., a perfectly symmetric peak was observed.

Figure 7. CLC chromatograms showing the effect of capillarysurface area-to-volume ratio on the elution of 50 pM λ-DNA at aconstant 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: 51µm i.d. and 0.8 psi, pressure-driven. Dotted line: 31 µm i.d. and 1.0psi, pressure-driven. All capillaries were fused silica with 30-cm totallength and 10-cm effective length. RFU, relative fluorescence units.

Table 5. Effect of Capillary Surface Area-to-VolumeRatio on the Capacity Factor of λ-DNA at a ConstantLinear Flow Rate in CLCa

31-µm i.d. 51-µm i.d. 74-µm i.d.

t0b (min) 0.09 0.10 0.10

tRc (min) 1.45 0.64 0.54

k′Rd 14.4 5.4 4.4Ve (cm/min) 6.92 15.58 18.452/r f (cm-1) 0.129 0.078 0.054applied pressure (psi) 1.0 0.8 0.5

a CLC conditions: 30-cm total length and 10-cm effective length;bare fused-silica capillary; running buffer, 25 mM sodium acetate (pH5.5). 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 thedetector (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.

Vep ) µepE (1)

µep ) q/3πηd (2)


introduce a new constant, the relative adsorption factor (K)

where Vapp is the observed velocity and Veof is the electroosmoticflow velocity. K therefore measures the decrease in migrationspeed due to adsorption.

The retention time and the peak shapes in CE also correlatedwith the adsorption dynamics of single λ-DNA molecules (Figure8). The spikes in the traces were from light scattering of DNAmolecules as they cross the laser beam. The DNA peaks becamemuch broader with decreasing pH. However, the asymmetry ratiowas much less than that in pressure-driven flow because of theEOF profile. The retention time also showed a large dependenceon the pH. In electroosmotic-driven flow, at a constant pH, therelative adsorption factor (K) was found to be also related to thecapillary surface area-to-volume ratio (Table 6). The regressioncurve is given by y ) 6.99x + 2.39 with a linear correlationcoefficient, R ) 0.9996, where y is the relative adsorption factorand x is the capillary surface area-to-volume ratio.

The linear velocities (0.8-1.4 cm/min) for the CE studies ofλ-DNA were smaller compared to those in the single-molecule

imaging experiments because of the relatively low applied electricfield (+166.7 V/cm, limited by Joule heating in the 51- and the74-µm-i.d. capillaries) and the opposing directions of electro-phoretic mobility and EOF. When +1 kV/cm was applied to a31-µm-i.d. capillary in the same buffer, the apparent velocity ofthe DNA molecules was 9.8 cm/min as predicted from Table 6.These results further confirm that the velocities of DNA moleculesin CE can be predicted by single-molecule imaging experimentsand that hydrophobic interaction as a function of pH is the primaryfactor controlling the migration velocities.

CONCLUSIONSDirect observation of the dynamics of adsorption/desorption

of individual DNA molecules was achieved by single-moleculeimaging at the solid-liquid interface. At basic and neutral pH,λ-DNA molecules exhibited rapid conformational fluctuations witha random coil structure. The molecules were gradually draggedand adsorbed onto the fused-silica surface with decreasing pH,until they finally precipitated and formed a compact supercoiledstructure. Adsorption was initiated at the unpaired hydrophobicends of the molecule. At pH 5.5, the kinetics of adsorption/desorption of individual DNA molecules was examined to elucidatethe balance between hydrophobic interaction and electrostaticrepulsion between the DNA molecules and the surface. At aconstant buffer condition, the retention time as well as peakasymmetry of DNA molecules was proportional to the capillarysurface area-to-volume ratio in both CE and CLC. Such behaviorreflects the relative accessibility of the surface to the DNAmolecule and can be predicted from the single-molecule imagingexperiments. At a C18 surface, the dynamics of adsorption/desorption of individual DNA molecules was altered with theaddition of methanol as well as with pH changes. Adsorption ofλ-DNA molecules at the C18 surface was much stronger than thatat the fused-silica surface. These results show clearly thathydrophobic interaction rather than electrostatic interaction is themajor driving force for DNA adsorption, even though bothinteractions have been implicated in adsorption.2,4,44

ACKNOWLEDGMENTWe thank Marc D. Porter and his group members, especially

Desiree Grubisha, for providing the C18 surfaces. The AmesLaboratory is operated for the U.S. Department of Energy by Iowa

(46) Weston, A.; Brown, P. R. HPLC and CE, Principles and Practice; AcademicPress: San Diego, CA, 1997; pp 8-9.

(47) Baker, D. R. Capillary Electrophoresis; John Wiley & Sons: New York, 1995;pp 19-52.

Figure 8. Electropherograms of 50 pM λ-DNA in 25 mM sodiumacetate buffer at pH (A) 6.5, (B) 6.0, (C) 5.5, and (D) 5.0. CEconditions: fused-silica capillary, 40 cm × 74 µm i.d. (30 cm to thedetector); applied voltage, + 10 kV; and RFU, relative fluorescenceunits.

K ) Vep + Veof - Vapp (3)

Table 6. Effect of Capillary Surface Area-to-VolumeRatio on the Migration of λ-DNA in CEa

31-µm i.d. 51-µm i.d. 74-µm i.d.

EOF b (min) 2.14 2.54 2.78MTc (min) 7.27 ( 0.06 10.14 ( 1.46 11.89 ( 2.76Vapp

d (cm/min) 1.377 0.986 0.841Veof

e (cm/min) 4.66 3.93 3.60K f 3.29 2.94 2.762/r g (cm-1) 0.129 0.078 0.054

a CE conditions: 30-cm total length and 10-cm effective length, barefused-silica capillary; applied electric field, +166.67 V/cm; runningbuffer, 25-mM sodium acetate (pH 5.5). b EOF, migration time of aneutral marker, 100 nM coumarin 334. c MT, migration time of λ-DNA.d Vapp, apparent velocity. e Veof, electroosmotic flow velocity. f K, relativeadsorption factor. g 2/r, capillary surface area-to-volume ratio.


State University under Contract No. W-7405-Eng-82. This workwas supported by the Director of Science, Office of Basic EnergySciences, Division of Chemical Sciences.

SUPPORTING INFORMATION AVAILABLETwo 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. Thismaterial is available free of charge via the Internet at http://pubs.acs.org.

Received for review November 20, 2000. AcceptedJanuary 22, 2001.

AC0013599


Documents

Real-Time Dynamics of Single-DNA Molecules Undergoing Adsorption and Desorption at Liquid−Solid Interfaces