DNA Compaction at Hydrophobic Surfaces Induced by a Cationic

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DNA Compaction at Hydrophobic Surfaces Induced by a Cationic Amphiphile† Marite´ Ca´rdenas,* Alan Braem, Tommy Nylander, and Bjo¨rn Lindman Physical Chemistry 1, Center for Chemistry and Chemical Engineering, University of Lund, P.O. Box 124, SE-221 00 Lund, Sweden Received October 24, 2002. In Final Form: February 3, 2003 Cetyltrimethylammonium bromide (CTAB) induces partially irreversible compaction of DNA-adsorbed layers on hydrophobic silica. Additionally, there is a synergistic increase in the adsorbed amount when both CTAB and DNA are present as compared to the surface excess concentration of either of the individual components. In this study of the DNA adsorption and DNA-CTAB coadsorption by ellipsometry, emphasis has been placed on the DNA molecular weight as well as its conformation (single and double stranded). The DNA molecular weight and conformation have a large effect on the surfactant-free DNA adsorption behavior but not on the mixed DNA-CTAB adsorption behavior. Comparison between interfacial and bulk complexation has been made where possible. The DNA-CTAB complexes at the interface are neutral despite the large excess of DNA in the bulk. The final structure of the adsorbed layer was found to be dependent on the history of complex formation and DNA size.

Introduction Complexes between DNA and cationic surfactants or polymers have attracted a large amount of interest lately due to the possible use of these systems for gene transfection. In these so-called lipoplexes, DNA molecules (or any drug molecule) are protected from environmental factors and are therefore more efficiently delivered to the target cells.1 Although viral vectors have higher transfection efficiency, lipoplexes are less prone to provoke inmunological responses and do not represent any infection threat to patients.1,2 The factors that control DNA compaction upon interaction with cationic surfactants and polymers as well as multivalent ions have been extensively studied lately.3,4 The nature of both the DNA5 and the surfactant6-8 has been found to influence the phase separation limits as well as the structure of the DNAcationic surfactant complexes. The proposed mechanism for the in vivo uptake of these lipoplexes involves transport into cells by endocytosis or fusion and transfer into the cytoplasm in the form of endosomes.9 Furthermore, lipoplexes must be designed to circulate in the blood stream for hours in order to deliver DNA (or the drug) systematically.2 Here, lipoplexes will encounter different types of interfaces such as walls of protein vessels, blood cells, proteins, and other “colloidal” particles in the blood, which are likely to interact and * To whom correspondence may be addressed. E-mail: Marite. [email protected]. † Part of the Langmuir special issue dedicated to neutron reflectometry. (1) Somia, N.; Verma, I. M. Nature 1997, 389, 239. (2) Lasic, D. D.; Templeton, N. S. Mol. Biotechnol. 1999, 11, 175. (3) Santerre J. P.; Hayakawa, K.; Kwak, J. C. T. Biophys. Chem. 1983, 17, 175. (4) Lindman, B.; Mel’nikov, S.; Mel’nikova, Y.; Nylander, T.; Eskilsson, K.; Miguel, M.; Dias, R.; Leal, C. Prog. Colloid Polym. Sci. 2002, 120, 52. (5) Shirahama, K.; Takashima, K.; Takisawa, N. Bull. Chem. Soc. Jpn. 1987, 60, 43. (6) Gorelov, A. V.; McLoughlin, D. M.; Jacquier, J. C.; Dawson, K. A. Nuovo Cimento 1998, 20, 2553. (7) Dias, R.; Mel’nikov, S.; Lindman, B., Miguel, M. Langmuir 2000, 16, 9577. (8) Koltover, I.; Salditt, T.; Ra¨dler, J. O.; Safinya, C. R. Science 1998, 281, 78. (9) Lasic, D. D.; Templeton, N. S. Adv. Drug Delivery Rev. 1996, 20, 221.

therefore influence the uptake behavior of the lipoplexes. Hence, the interfacial behavior of lipoplexes will partly determine how efficiently DNA-surfactant complexes are delivered to the target cells. Despite this, the interfacial behavior of DNA-surfactant complexes has received relatively little attention. In this paper, cetyltrimethylammonium bromide (CTAB) is used because its behavior in the bulk and at interfaces has been well characterized.10 Furthermore, earlier studies have shown that CTAB is an effective compacting agent for DNA.6,7 Previously we have shown that ellipsometry is an efficient technique to study the interfacial behavior of DNA and DNA-surfactant complexes.11 This behavior could be correlated to the complex formation in bulk solution. On the basis of the results of Eskilsson et al.,11 the present study was undertaken with the following main objectives: (1) verify the relationship between the interfacial behavior and complex formation/compaction in bulk solution, using well-defined, monodisperse samples of different molecular weight; (2) determine the effect of double-stranded (ds) DNA denaturation into singlestranded (ss) DNA on (a) the interaction with hydrophobic surfaces and (b) the interaction with cationic surfactants as reflected in the interfacial properties of the formed complexes. For these purposes different well-defined DNA samples have been used, which include (1) long ds-DNA (2000 base pairs), (2) long ss-DNA (heat-denatured 2000 bases), (3) short ds-DNA (146 base pairs), and (4) short ss-DNA (100 bases). Experimental Section Materials. Salmon sperm DNA was purchased from Gibco, BRL, and used as received. As stated by the manufacturer, this DNA is double stranded and 2000 ( 500 base pairs (bp) in length, as determined by 1% TAE Agarose gel analysis, and free from Dnase and Rnase. Short mononuclesomal DNA, 146 bp as determined by 7.5% Acrylamide gel analysis, was kindly provided by D. McLoughlin and was dialyzed against 10 mM NaBr solution prior to use. The 100 bases (b) ss-DNA (MWG-Biotech AG) was used as received. All DNA and CTAB solutions were prepared in solutions containing 10 mM NaBr solution. The DNA (10) Runimg, D.; Holland, P. M. Cationic Surfactants. Physical Chemistry; Mercel Dekker: New York and Basel, 1991; p 527. (11) Eskilsson, K.; Leal, C.; Lindman, B.; Miguel, M.; Nylander, T. Langmuir 2001, 17, 1666.

10.1021/la026747f CCC: $25.00 © 2003 American Chemical Society Published on Web 03/15/2003

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concentration was measured spectrophotometrically by its absorbance at 260 nm, using an extinction coefficient of 33 µg/mL for ss-DNA and 50 µg/mL ds-DNA.12 The ratio of absorbance at 260 and 280 nm was about 1.8-1.9 and the absorbance at 320 nm was negligible, indicating the absence of protein contamination.12 Salmon sperm DNA was thermally denatured to produce ss-DNA by heating at 85 °C for 10 min and then cooling rapidly by injecting the sample into a beaker that is cooled by immersion into a mixture of cold ice and ethanol.13 The conformation of DNA in aqueous solution, that is whether the chains are single or double stranded, was verified by circular dichroism (CD) measurements.14 Cetyltrimethylammonium bromide (Merck pa quality), sodium dodecyl sulfate (Sigma), and sodium bromide (Aldrich, extra pure quality) were used as received. Water purified by a Milli-Q system (Millipore Corporation, Bedford, MA) was used in all measurements. The silica surfaces were kindly provided by Stefan Klintstro¨m. Surface Preparation. The silicon wafers (p-type, borondoped, resistivity 1-20 Ω‚cm) were thermally oxidized in an oxygen atmosphere at 920 °C for ∼1 h, followed by annealing and cooling in argon flow. This procedure yields a SiO2 layer of ∼300 Å thickness. The oxidized wafers were cut into slides with a width of 12.5 mm and cleaned in a mixture of first NH4OHH2O2, followed by HCl-H2O2 as described earlier.15 To prepare the hydrophobized silica surfaces, the wafers were dried under vacuum (0.001 mbar) and treated in a plasma cleaner (Harrick Scientific Corporation, model PDC-3XG) in low-pressure air (0.001 mbar) for 5 min. Then they were placed in a reactor that contained 2 mL of dimethyloctylchlorosilane and were subjected to vacuum for 24 h at room temperature. The hydrophobized silica surfaces were then sonicated first in ethanol and then in tetrahydrofuran. The surfaces were then stored in ethanol. Before use, the surfaces were dried under vacuum (0.001 mbar). Ellipsometry. An automated Rudolph Research thin-film null ellipsometer, type 43603-200E was used as described by Tiberg et al.15 to measure the adsorbed amount and thickness of adsorbed layers in situ. The optical properties of hydrophobized silica surfaces, i.e., the refractive index of the silicon and the oxide layer as well as the oxide layer thickness, were characterized at the beginning of each experiment by measuring the ellipsometric angles, Ψ and ∆, in two different ambient media, air and 10 mM NaBr solution, as described earlier.16 To avoid any air film sticking to the hydrophobic surface, ethanol was pumped through the ellipsometric cuvette before the aqueous solution was added. Once the optical properties were measured, the DNA and/or surfactant sample was injected by aliquots of a concentrated stock, typically ranging from 10 to 100 µL, to 5 mL of NaBr solution. All measurements were performed with a light-source wavelength of 4015 Å and with an angle of incidence of ∼68°. A 5 mL thermostated cuvette was used at 25.1 ( 0.1 °C and agitated with a magnetic stirrer at about 300 rpm. The recorded ellipsometric angles were evaluated using a four-layer optical model, assuming isotropic media and planar interfaces. The mean refractive index, nf, and the ellipsometric thickness, df, of the adsorbed layer were calculated numerically as described elsewhere.17,18 The adsorbed amount, Γ, was calculated from nf and df using the formula

Γ)

(nf - n0) df dn/dc

(1)

where n0 is the refractive index of the bulk solution and dn/dc is the refractive index increment as a function of bulk concentration. For DNA solutions,19 dn/dc ) 0.134 g/cm3, and for CTAB (12) Cantor, C. R.; Schimmel, P. R. Biophysical Chemistry; W.H. Freeman and Co.. San Francisco, CA, 1980; p 1371. (13) Krasna, A.-I. Biopolymers 1970, 9, 1029. (14) Gray, D. M.; Ratliff, R. L.; Vaughan, M. R. Methods Enzymol. 1992, 211, 389. (15) Tiberg, F.; Landgren, M. Langmuir 1993, 9, 927. (16) Landgren, M.; Jo¨nsson, B. J. Phys. Chem. 1993, 97, 1656. (17) Jenkins, T. E. J. Phys.: Appl. Phys. 1999, 32, R45. (18) Azzam, R. M. A.; Bashara, N. M. Ellipsometry and polarized light; North-Holland Publishing Co.: Amsterdam, 1977; p 529. (19) Bhattacharya, S.; Mandal, S. S. Biochim. Biophys. Acta 1997, 1323, 29.

solutions, dn/dc ) 0.15 g/cm3. In the data presented below for the mixed systems, surface excess concentrations are calculated using the dn/dc of DNA. The highest relative error in adsorbed amount due to this simplification was ∼10% (calculated by assuming the layer was purely CTAB). For typical experimental conditions, the errors in Ψ and ∆ are normally distributed with standard deviations of 0.001° and 0.002°, respectively. The relative errors in refractive index and thickness are rather high for small surface excess concentrations (Γ < 0.5 mg/m2) decreasing rapidly to values of approximately 5-10% for Γ > 1 mg/m2. However, the relative error in surface excess concentration is much smaller: 15% at ∼0.1 mg/m2 and less than 1% for Γ ) 2 mg/m2.20

Results and Discussion Surfactant-Free DNA. All the different types of DNA (long and short, single and double stranded) adsorbed to the hydrophobic silica surface. Although DNA is a highly charged polymer, with a linear charge density corresponding to one charge per 1.7 Å in the case of B-DNA,21 it also contains nitrogenous bases that are hydrophobic in nature. Interactions between the hydrophobic surface and the hydrophobic parts of the DNA molecule are therefore a plausible driving force for adsorption to the hydrophobic surface. The adsorption isotherms, expressed as adsorbed amount versus mass concentration of DNA, of the different DNA samples in 10 mM NaBr solution on hydrophobic surfaces are shown in Figure 1A. After each adsorption experiment, the DNA solution was replaced with 10 mM NaBr solution. No detectable desorption occurred in any case. For both the long and short ds-DNA samples, the adsorption increases progressively with concentration within the range studied (0.01-0.1 mg/mL). Both long and short ss-DNA adsorb to a larger extent than any dsDNA. To explain this observation, the differences between double and single stranded DNA molecules are considered. These are, ss-DNA has greater flexibility (lower persistence length22), lower linear charge density, and greater hydrophobicity as compared to ds-DNA. It can be argued that the greater hydrophobicity is the main factor that causes the ss-DNA to adsorb to a greater extent than dsDNA: 1. Flexibility. In general, higher flexibility implies larger entropy loss upon adsorption due to the loss of translation and rotation degrees of freedom. This would imply a lower adsorbed amount of the ss-DNA. 2. Charge Density. Although ss-DNA has a lower linear charge density as compared to ds-DNA, the final state of the ss-DNA absorbed layers has at least nine times smaller volume per base (or nine times larger number of charges per unit volume) than what was observed for ds-DNA (Table 1). The local electrostatic repulsion between polyelectrolyte segments adsorbed at the hydrophobic interface invokes condensation of counterions. This gives an entropic penalty due to the loss of translation freedom for the polymer’s counterions. This penalty will be larger for adsorbed layers with a larger number of charges per volume unit. If this would be the factor that controls the adsorption, ss-DNA would be expected to adsorb to a lesser extent than ds-DNA. On the basis of these arguments, we conclude that the greater hydrophobicity of the ss-DNA plays a determining role in the adsorption process. That is, the larger segmental adsorption energy overcomes the entropy loss due to both flexibility and counterion condensation. (20) Tiberg, F. J. Chem. Soc., Faraday Trans. 1996, 92, 531. (21) Gelbart, W. M.; Bruinsma, R. F.; Pincus, P. A.; Parsegian, V. A. Phys. Today 2000, 53, 38. (22) Tinland, B.; Pluen, A.; Sturm, J.; Weill, G. Macromolecules 1997, 30, 5763.

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Figure 1. (A) Adsorption isotherm of different DNA samples at the hydrophobic silica-water interface in 10 mM NaBr solution. Plus signs and open circles correspond respectively to 2000 bp ds-DNA and 146 bp ds-DNA. Closed triangles and circles correspond respectively to 2000 b ss-DNA and 100 b ss-DNA. Lines are drawn as guides for the eye. (B) Adsorbed amount as a function of time for different DNA samples in 10 mM NaBr solution where the bulk DNA concentration is fixed at 0.02 mg/mL. The symbols are the same as those used in part A. (C) Adsorbed amount as a function of time at hydrophobic surfaces for long ds-DNA. A 0.03 mg/mL portion of DNA was injected at 0 min, followed by the addition of 1 × 10-6 M SDS at 80 min. Rinsing with 10 mM NaBr solution was performed at 125 min.

The higher affinity of ss-DNA as compared to that of ds-DNA is also confirmed by the initial slope of the

Ca´ rdenas et al.

adsorption isotherm, when plotted as a function of polymer molar concentration (data not shown). The affinity of DNA to the surface also increases with the size of the molecule for both single- and double-stranded DNA. This effect of molecular weight on the surface behavior is the expected one for polymers adsorbing from a good solvent.23 The ellipsometric thicknesses of surfactant-free DNA layers at a DNA bulk concentration of 0.02 mg/mL are given in Table 1. At this bulk concentration, long ds-DNA forms a 312 ( 14 Å thick layer. Thinner adsorbed layers are found for long ss-DNA (96 ( 5 Å), short ds-DNA (105 ( 13 Å), and short ss-DNA (20 ( 3 Å) at the same bulk concentration. If the concentration is raised to 0.1 mg/mL DNA, no significant change in layer thickness is observed for either long single- or double-stranded DNA. However, for short ds-DNA the layer thickness reduces from 200 at the lowest concentration used to 30 Å at the highest. Thus, short chains seem to adsorb in a more parallel orientation relative to the surface as the bulk DNA concentration is raised. Short ss-DNA, on the other hand, forms a ∼20 Å thick layer onto the surface regardless of the bulk concentration. The diameter for B-DNA has been found to be around 20 Å.24 If one layer of strongly associated water is taken into account, the diameter of a DNA helix is expected to increase to 30 Å.24 Therefore, short DNA indeed adsorbs flat along its entire length instead of having loops and tails as in the case of long DNA. Interestingly, the length of both short ds and short ss DNA is approximately equal to the corresponding persistence length of ss22 and ds25 DNA at the ionic strength used in this work. In other words, the polymer segments will preserve the same direction along the whole DNA chain. Loop and tail formation is therefore less likely, and DNA can be expected to preferentially adapt a flatter conformation on the surface. The calculated volume per base of adsorbed material is also presented in Table 1. For ds-DNA, the volume occupied by one base is nearly 10 times larger than the volume per base of ss-DNA for large molecular weights and almost 36 times larger in the case of small molecular weights. Therefore, the layer density (volume per base-1) increases with decreasing DNA size and is significantly higher for single-stranded chains. The results presented here correspond qualitatively to DNA adsorption isotherms that were previously reported by Gani et al.26 On activated carbon, a hydrophobic surface, the surface excess concentration was found to increase with nucleotide concentration until adsorption saturation was reached at a bulk concentration of 0.014 mg/mL. Thus, it was proposed that DNA molecules would adsorb forming a monolayer, and then further accumulation would occur leading to molecular aggregation or interfacial coagulation at the solid surface. Additionally, both heat- and acidtreated ss-DNA were found to adsorb to a larger extent than ds-DNA. The differences in surface excess concentration were explained by the different conformations that ds and ss chains would adopt: ds-DNA lies flat on the surface and is oriented in random fashion, leaving vacant surface area in contact with the solvent. On the other hand, ss-DNA could pack more efficiently due to the more flexible nature of single-stranded chains. These observa(23) Fleer, G. J.; Cohen Stuart, M. A.; Scheutjens, J. M. H. M.; Cosgrove, T.; Vincent, V. Polymers at Interfaces, 1st ed.; Chapman & Hall: London, 1993. (24) Stryer, L. Biochemistry, 4th ed.; W.H. Freeman and Co.: New York, 1995. (25) Odijk, T.; Tijdschr, N. Natuurk. 1998, A50, 53. (26) Gani, S. A.; Chattoraj, D. K.; Mukherjee, D. C. Langmuir 1999, 15, 7130.

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Table 1. Equilibrium Surface Excess Concentration, Volume per Base, Layer Thickness, and Area per DNA Molecule at Hydrophobic Silica Surfacesa DNAb

DNA-CTAB complexesc rinsingd

surface excess concn (mg/m2) vol per base (nm3) layer thickness (Å) area per DNA molecule (nm2) surface excess concn (mg/m2) layer thickness (Å) surface excess concn (mg/m2) vol per base (nm3) layer thickness (Å) area per DNA molecule (nm2)

long ds-DNA

short ds-DNA

0.20 84.2 312 ∼11000 2.04 33-48 ∼1.00 ∼4.9 ∼90 ∼2200

0.22 25.6 105 ∼716 1.97 32-41 ∼1.60 ∼1.1 ∼32 ∼98

long ss-DNA 0.54 9.6 96 ∼2000 2.04 42 ∼1.24 ∼4.4 ∼100 ∼870

short ss-DNA 0.96 0.7 20 ∼56 1.97 32 ∼1.19 ∼0.7 ∼15 ∼45

a As a reference, a 13 Å thick CTAB monolayer (0.72 mg/m2) forms at 1 × 10-6 M CTAB. b Bulk concentration 0.02 mg/mL DNA. c Bulk concentration 0.02 mg/mL DNA and 1 × 10-6 M CTAB. d Rinsing with 10 mM NaBr solution.

tions and their interpretation are in agreement with the current paper. However, one important difference is that the extent of adsorption was found to be 1 order of magnitude higher in our case than in the work reported by Gani et al., perhaps due to the different surfaces used. Figure 1B shows the kinetics of DNA adsorption (the adsorbed amount as a function of time) for all DNA samples at a DNA bulk concentration of 0.02 mg/mL in 10 mM NaBr solution. The largest initial adsorption rate is found for short ss-DNA followed by long ss-DNA. In the case of ds-DNA, the initial adsorption rates (Figure 1B), as well as steady-state surface excess concentration (Table 1), of long and short ds-DNA are found to be roughly the same. To check if the differences in initial rate can be explained by differences in mass transfer rates, the apparent diffusion coefficients were computed from the initial adsorption rates for both long and short ds-DNA, using an unstirred layer thickness of 47 µm, as described by Corsel et al.27 The calculated diffusion coefficient of 2000 bp ds-DNA (∼5 × 10-8 cm2/s) was found to be similar to the self-diffusion coefficient for 2311 bp ds-DNA in bulk reported in the literature (4.56 × 10-8 cm2/s),28 indicating the initial adsorption process is probably transport limited. For 146 bp ds-DNA, the calculated diffusion coefficient (∼5 × 10-8 cm2/s) was found to be less than the selfdiffusion coefficient reported for 367 bp ds-DNA (15.8 × 10-8 cm2/s).28 Thus, the transport of DNA molecules from the bulk to the interface is not the rate-limiting step of the adsorption process at least in the case of short dsDNA; a slower process at the interface controls the kinetics of adsorption. Furthermore, the slow kinetics of adsorption could also be determined by an increasing electrostatic repulsion created as the DNA layer is built up. When a certain concentration is reached, this repulsive electrostatic contribution to the energy of adsorption could begin to dominate the attractive hydrophobic contribution and hence limit further DNA adsorption to the surface. To test whether the adsorption of DNA results in a negatively charged surface, we attempted to adsorb the anionic surfactant sodium dodecyl sulfate (SDS) to the preadsorbed DNA layers. Figure 1C gives the adsorbed amount as a function of time for the sequential adsorption of long ds-DNA and SDS to the hydrophobic silica. A 0.03 mg/mL portion of DNA was first injected at 0 min followed by the addition of 1 × 10-6 M SDS at 80 min. As can be observed, SDS does not adsorb to the preadsorbed layer. In other words, the DNA layer imparts a negative charge to the surface that leads to electrostatic repulsions between the negatively charged SDS molecules and the surface. (27) Corsel, J. W.; Willems, G. M.; Kop, J. M. M.; Cuypers, P. A.; Hermens, W. T. J. Colloid Interface Sci. 1986, 111, 544. (28) Sorlie, S. S.; Pecora, R. Macromolecules 1990, 23, 487.

Figure 2. Adsorption isotherm for different DNA-CTAB complexes at the hydrophobic silica-water interface. At [CTAB] ) 0, the equilibrium value for the surface excess concentration at 0.02 mg/mL DNA is given Table 1. For short ss-DNA at 3 × 10-5 M CTAB, the surface excess concentration increases to ∼8 mg/m2. The adsorption isotherm corresponding to CTAB alone (crosses) is also included. Plus signs and open circles correspond respectively to ds-DNA and 146 bp ds-DNA, closed triangles and circles correspond respectively to 2000 b ss-DNA and 100 b ss-DNA. Lines are drawn as guides for the eye.

Finally, at 125 min rinsing with 10 mM NaBr solution was performed with no detectable desorption. DNA-Surfactant Complexes. Figure 2 shows the coadsorption isotherm for DNA and CTAB mixtures as a function of surfactant concentration. These isotherms were recorded as follows. At zero time, 0.02 mg/mL DNA solution was injected. Once steady state was reached, aliquots of CTAB were added to reach the desired concentration. The adsorbed amount was allowed to reach steady state between each new addition. Figure 2 also includes the adsorption isotherm of CTAB at hydrophobized silica for comparison. Although both CTAB and surfactant-free DNA readily adsorb to hydrophobic surfaces, a large synergistic increase in adsorbed amount is observed when both CTAB and DNA are present. This increase in surface excess concentration is observed regardless of the type of DNA. A plateau of ∼3 mg/m2 is reached at very low surfactant concentration (1 × 10-5 M CTAB), also regardless of the type of DNA. Furthermore, the same adsorption plateau was obtained independent of the method of reaching the desired CTAB concentration. That is, there was no difference between experiments where CTAB was added in one step to the DNA solution and experiments where

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small aliquots were added progressively to reach the same final bulk concentration. CTAB readily adsorbs to the bare hydrophobic surface at any of the surfactant concentrations used in the coadsorption experiments. However, due to electrostatic interactions, CTAB is expected to preferentially interact with DNA rather than the surface. Thus, most surfactant molecules should be complexed to DNA instead of directly adsorbed to the surface. Supporting this idea is the observation that the adsorbed amount and layer thickness is the same for the case in which CTAB is adsorbed prior to the addition of DNA (data presented in the section to follow) as it is for the case in which the surfactant is added to a preadsorbed DNA layer. Therefore, in the following discussion we find it justified to treat the mixed adsorbed layers as layers of DNA-CTAB complexes, rather than layers built up by two components adsorbing independently of each other from the DNA-CTAB mixture. One final noteworthy observation about the coadsorption isotherm is that the short ss-DNA-CTAB mixtures (closed circles) deviate from those recorded for the other types of DNA-CTAB mixtures, which all show a plateau in the adsorbed amount as a function of surfactant concentration. Further addition of CTAB to short ss-DNA just at the point of macroscopic phase separation leads to a sharp increase in both the surface excess concentration (from ∼3 to ∼8 mg/m2) and layer thickness (∼30 to ∼60 Å). At this molar ratio, the solution became turbid. However, in contrast to the other samples, the turbidity was sufficiently low to allow further ellipsometric measurements. The large increase in both adsorbed amount and layer thickness is indicative of surface precipitation or multilayer formation. The adsorbed amount (open circles) and layer thickness (crosses) are plotted as a function of time in Figure 3A for long ds-DNA and CTAB complexes in 10 mM NaBr solution. At time zero, 0.02 mg/mL DNA is added. At 80 min, once steady state is reached, 1 × 10-6 M CTAB is injected. Finally, rinsing with 10 mM NaBr solution is performed at 150 min. This figure clearly shows that the increase in adsorbed amount produced by addition of cationic surfactant is accompanied by a simultaneous compaction of the adsorbed layer from a thickness of >200 to ∼40 Å. The steady-state values for surface excess concentration and layer thickness in each step related to Figure 3A are summarized in Table 1. The data for all the different DNA samples are also included in this table. A similar behavior was obtained for all the types of DNA studied, indicating that the structure of the mixed adsorbed layer is independent of DNA type and length. A study performed in our group29 permits us to compare the present results with the bulk precipitation behavior of the DNA-CTAB system. The structure of the electroneutral precipitate formed by both ds-DNA and denatured ss-DNA (due to the absence of salt) with CTAB was studied by means of small-angle X-ray diffraction. Both these types of DNA were found to assemble into a hexagonal array, although the ss-DNA-CTAB structure is more disorganized. Therefore, the structure of DNA-CTAB complexes is found to be largely independent of the DNA conformation both in the bulk and on hydrophobic surfaces. It is also useful to compare the preprecipitation compaction data in the bulk with the compaction behavior at the interface. In bulk, CTAB induces compaction of large DNA molecules.30,31 Mel’nikov et al.31 observed that giant, individual DNA molecules undergo a discrete transition (29) Dias, R. S.; Lindman, B.; Miguel, M. G. Prog. Colloid Polym. Sci. 2001, 118, 163.

Ca´ rdenas et al.

Figure 3. (A) Adsorbed amount (open circles) and layer thickness (crosses) as a function of time for DNA-CTAB complexes at hydrophobized silica surface. A 0.02 mg/mL portion of 2000 bp ds-DNA was added at 0 min, followed by 1 × 10-6 M CTAB at 80 min. Finally, the surface was rinsed with 10 mM NaBr solution starting at 150 min. Arrows refer to changes in bulk concentration. (B) At 0 min, 0.02 mg/mL long ds-DNA was added. CTAB (1 × 10-6 M) was added at 60 min followed by the addition of 1 × 10-6 M SDS at 175 min. The concentration of SDS was raised to 2 × 10-6 M at 232 min. Rinsing with 10 mM NaBr solution was performed at 254 min. (C) At 0 min, 0.02 mg/mL long ds-DNA was added. CTAB (1 × 10-6 M) was added at 60 min followed by rinsing with 10 mM NaBr solution at 110 min. Arrows refer to changes in bulk concentration.

from coil to globule state, starting at 9.4 × 10-6 M CTAB for 6 × 10-7 M DNA in nucleotide. They observed only globules at the point of precipitation, which occurred at

DNA Compaction at Hydrophobic Surfaces

a CTAB concentration of 2 × 10-5 M. Although the DNACTAB molar charge ratio (FDNA/CTAB) is different in the present work, the coil-globule transition should have similar properties to the above work. That is, the transition should be sharp and occur far below the critical micelle concentration (cmc) of the surfactant. In the present work, compaction of the preadsorbed layer and its saturation occurs within a very narrow surfactant concentration range (1 × 10-6 to 1 × 10-5 M CTAB). Furthermore, these concentrations are far below both the cmc and the concentration corresponding to an equimolar DNA-CTAB charge ratio. Although the two experiments cannot be compared directly, it is noteworthy that in the experiments of Mel’nikov et al.,31 globule formation did not occur until there was a great excess of CTAB. Thus, the surface appears to facilitate rather than inhibit DNA compaction. To determine the mixed DNA-CTAB layer charge, we attempted to adsorb SDS to the preadsorbed mixed layers. Figure 3B shows the effect of SDS addition to a hydrophobized silica surface covered by DNA-CTAB complexes in terms of adsorbed amount (open circles) and layer thickness (crosses). As in Figure 3A, 0.02 mg/mL long ds-DNA was injected at time zero, followed by surfactant compaction with 1 × 10-6 M CTAB at 60 min. Then, 1 × 10-6 M SDS was added at 175 min. The concentration of SDS was doubled at 232 min, and finally rinsing with 10 mM NaBr solution was performed at 254 min. In this case, SDS addition to the bulk solution leads to the deposition of SDS on the layer of DNA-CTAB complexes. The adsorbed layer becomes ∼12 Å thicker, corresponding to the formation of a SDS monolayer. This is a clear indication that the DNA-CTAB complexes do not carry a net negative charge, but rather the adsorbed layers are neutral in nature. This observation is especially striking given that the DNA-CTAB complexes in bulk solution are far below the point of charge neutrality (FDNA/CTAB ) 62). Two possible explanations are proposed: (1) the surface induces changes in the composition of the complexes once they adsorb, or (2) there is a coexistence of neutral and charged complexes in the bulk, where neutral complexes are preferentially adsorbed. The first explanation is supported by results from studies of other polymersurfactant systems32,33 that show that the complexation at surfaces occurs over a different concentration range than complexation in the bulk. Therefore, at a given concentration, the surface complexes are expected to have a different composition than the bulk complexes. This is logical, given that the concentration of adsorbing species will be greatly enhanced at the interface compared to the bulk. On the other hand, fluorescence microscopy studies on the compaction of giant DNA by CTAB support the “uneven distribution” hypothesis (explanation 2). Mel’nikov et al.31 showed that both random coils and globules coexist at surfactant concentrations below the point of phase separation. Another study that supports the uneven distribution of surfactant was performed by Dias et al.7 They studied the phase diagram for the DNA-CTABH2O system, showing that precipitation occurs at very low surfactant concentrations as compared to DNA concentration. This was also interpreted as a coexistence of two different types of complexes: (1) the DNA molecules (30) Ghirlando, R.; Wachtel, E. J.; Arad, T.; Minsky, A. Biochemistry 1992, 31, 7110. (31) Mel’nikov, S. M.; Sergeyev, V. G.; Yoshikawa, K. J. Am. Chem. Soc. 1995, 117, 2401. (32) Braem, A. D.; Prieve, D. C.; Tilton, R. D. Langmuir 2001, 17, 883. (33) Dedinaite, A.; Claesson, P. M. Langmuir 2000, 16, 1951.

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that are already saturated with surfactant and (2) DNA molecules that are not binding with surfactant to an appreciable extent. A definite proof of either possibility is beyond the scope of the current work. Effect of Rinsing with Salt. To study the response of the DNA-CTAB mixed layer upon removal of both DNA and CTAB from the bulk, the DNA-CTAB mixed solution was replaced with 10 mM NaBr solution once steady state was reached. As shown in Figure 3B, replacing the DNACTAB solution with salt solution leads to a reduction of the surface excess concentration to ∼1 mg/m2 as well as expansion of the layer to a thickness to ∼90 Å. This thickness is less than the thickness of a DNA layer that has not been exposed to cationic surfactant. Thus, DNA compaction with CTAB is not a fully reversible process. The area per DNA molecule (Table 1) diminishes by a factor of 5 for long ds-DNA after compaction with surfactant and subsequent rinsing. Similar behavior was obtained for all the studied types of DNA (Table 1). We contend that the decrease in adsorbed amount and increase in layer thickness upon rinsing is mainly due to surfactant desorption. To corroborate this idea, 1 × 10-6 M SDS was added to the cuvette long after the DNACTAB mixture had been replaced with 10 mM NaBr solution (Figure 3C). No change in adsorbed amount or thickness was observed, indicating that the layer is now negatively charged as a consequence of the rinsing with salt. This indicates that rinsing removes the cationic surfactant molecules from the adsorbed layer, leaving only DNA molecules in a conformation different to the one adopted when DNA adsorbs from a surfactant-free solution. Moreover, rinsing results in the removal of half of the adsorbed amount. As already shown, the mixed layer is neutral, and thus half of its mass corresponds to surfactant molecules (since a DNA base and a CTA+ ion have approximately the same molecular weight). To summarize, the addition of CTAB leads to a significant change in the structure of an adsorbed DNA layer, causing it to change into a more compact and dense layer structure. Once flushing with salt displaced CTAB molecules, the DNA chains tend to relax back to their original conformation, but the system is kinetically trapped in a denser nonequilibrium conformation. That is, the existence of an energy barrier prevents the DNA chains from decompacting or desorbing from the surface. Supporting this idea, Braem et al.32 found a similar behavior for a neutral polymer and negatively charged surfactant system at water-silica interfaces in which the surfactant (SDS) could tune the conformation of the adsorbed polymer layer (Pluronic F108) as measured by reflectometry. In this case, the structure of the surfactant-free polymer layer could be transformed into a different conformation by the addition of surfactant as revealed by changes in surface excess concentration. Moreover, processing with surfactant induced the simultaneous compaction of the adsorbed polymer layer as determined by atomic force microscopy and streaming current techniques.34 Effect of the Surfactant and DNA Order of Addition on the Mixed Layer Structure. As is common for other polymer-surfactant systems,32 the data above suggest that the layer formation is kinetically controlled. To further investigate possible nonequilibrium effects, the effect of order of addition of CTAB and DNA on the coadsorption behavior was studied. In this series of experiments, 0.02 mg/mL 146 bp or 2 kbp ds-DNA and 1 × 10-6 M CTAB have been used. The three cases studied were (34) Braem, A. D.; Biggs, S.; Prieve, D. C.; Tilton, R. D. Langmuir, in press.

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Table 2. Effect on the DNA-CTAB Mixed Layer Thickness upon Rinsing with 10 mM NaBr Solutiona long ds-DNA short ds-DNA

a

order of addition

effect of rinsing on layer thickness

simultaneous addition DNA adsorbed first CTAB adsorbed first simultaneous addition DNA adsorbed first CTAB adsorbed first

layer relaxation (from ∼45 to ∼90 Å) layer relaxation (from ∼45 to ∼90 Å) layer relaxation (from ∼45 to ∼90 Å) layer compaction (from ∼125 to ∼90 Å) no change (∼45 Å) no change (∼45 Å)

In all cases, a similar reduction in adsorbed amount was observed.

Figure 4. Layer thickness of DNA-CTAB complexes as a function of time. A 0.02 mg/mL portion of 146 bp ds-DNA and 1 × 10-6 M CTAB in 10 mM NaBr solution was used. DNACTAB complexes were simultaneously added at 0 min (triangles), CTAB was added at 0 min to a surface previously covered with 146 bp DNA (circles), and DNA was added at 0 min to a surface previously covered with CTAB (crosses).

(1) DNA adsorption followed by CTAB addition, (2) CTAB adsorption followed by DNA addition, and (3) simultaneous addition of a premixed DNA-CTAB solution. The adsorbed amount was nearly the same in all experiments, reaching a plateau at ∼2 mg/m2, regardless of both the order of addition and the DNA molecular weight. For ds-DNA, the layer thickness was not dependent on the history of complex formation, being roughly the same (∼40 Å) for all cases. However, the layer thickness did change depending on the history of formation for short ds-DNA. Figure 4 shows the layer thickness as a function of time for short ds-DNA and CTAB adsorbed simultaneously (open triangles) and in steps by adding either the DNA first (open circles) or the CTAB first (crosses). The layer thickness for the DNA-CTAB complexes is roughly the same (∼38 Å) when the surface has been previously covered either by DNA or by CTAB but three times larger in the case when both DNA and CTAB are added at the same time. In other words, a less dense layer is formed by simultaneous addition of DNA and CTAB than in the case of sequential experiments. In the former case, the complexes are formed before exposing them to the interface. Thus, complexes will adsorb directly, rather than being formed at the interface. In the sequential experiments, however, the complexes are formed at the interface, where attractive interactions with the surface may limit the mobility of the DNA molecules. The lower layer thicknesses for the cases with sequential adsorption clearly

demonstrate the importance of kinetic trapping in determining the ultimate layer structure. We also wanted to investigate the effect of the order of addition on the layer structure upon rinsing. To do this, rinsing with 10 mM NaBr solution was performed after every adsorption experiment (sequential or simultaneous) described above. As expected, the extent of desorption (the decrease in the adsorbed amount) was roughly the same in all cases, regardless of the size of ds-DNA. However, the effect in layer thickness was dependent on the DNA molecular weight as given in Table 2. For long ds-DNA, relaxation of the adsorbed layer to larger thickness was observed in all cases. However, for short dsDNA there was a pronounced order of addition effect seen in layer thickness. The thickness of the adsorbed layer did not change after rinsing when DNA and CTAB were added in steps. However, when both short ds-DNA and CTAB are added simultaneously to the cuvette, the decrease in adsorbed amount due to rinsing with salt solution was also accompanied by a reduction of the adsorbed layer thickness (Table 2). For the cases where the complexes are formed at the interface, short ds-DNA molecules are kinetically trapped on the surface and therefore their mobility is reduced even upon the displacement of the surfactant molecules. However, when the complexes are the adsorbing species, the desorption of surfactant leads to the accommodation of the DNA layer into a conformation (∼90 Å) similar to that of the DNA layers that have not been compacted with CTAB (∼105 Å). Conclusions The structure of the adsorbed layer of DNA on hydrophobized silica surface depends on the polyelectrolyte conformation (whether DNA is single or double stranded) and molecular weight. Addition of CTAB to a preadsorbed layer induces both a large increase in adsorbed amount as well as significant layer compaction. In contrast to surfactant-free DNA layers, the structure of the mixed adsorbed layer does not depend on the DNA conformation or molecular weight. The adsorbed layer is neutral despite the large excess of negative charges in the bulk. Rinsing with salt solution induces desorption of the cationic surfactant, leading to a denser DNA layer structure at the interface than those formed by surfactant-free DNA. The final structure of the adsorbed layer depends on the history of formation of the complexes and the length of the DNA. Acknowledgment. We thank Daragh McLoughlin for kindly providing the 146 bp ds-DNA sample. We also thank Stefan Klintstro¨m for kindly supplying the silica plates. The Swedish Foundation for Strategic Research Program Colloid and Interface Technology financially supported this work. LA026747F