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Coagulation and Stabilization of Colloidal Particles by Adsorbed DNA Block Copolymers: The Role of Polymer Conformation Harold W. Walker and Stanley B. Grant* Department of Civil and Environmental Engineering, University of California, Irvine, Irvine, California 92717 Received September 11, 1995. In Final Form: November 16, 1995X This study examines the relationship between polymer surface conformation and colloid stability using single-stranded DNA as a model polymer and a biochemical technique called hydroxyl radical footprinting (HRF). The DNA polymer (dT40B) has a diblock copolymer architecture with an uncharged and relatively hydrophobic block and an equally long negatively charged block. In a previous report, we showed that HRF could be used to probe the surface conformation of dT40B when the molecule is adsorbed to chargemodified latex particles at moderate salt concentrations (0.05 M NaCl). Here, we present additional HRF results for this model polymer and examine the relationship between the polymer’s surface conformation and its effect on latex particle coagulation rates. When the bare latex particles are positively charged, the DNA adsorbs in a flat conformation and particle stability depends on polymer surface coverage. In this case, the DNA influences particle stability by altering the net surface charge of the latex particles. When the bare particles are negatively charged and the salt concentration is high (1 M NaCl), the DNA diblock copolymer forms a polymer brush layer on the latex surface at high polymer surface density. The brush layer stabilizes the particles, presumably through steric interactions that develop on close particleparticle approach.
Introduction Polymer surface coatings play a key role in a broad spectrum of important engineering processes such as flocculation, stabilization, wetting, adhesion, coating, and lubrication.1-4 In many of these applications, block copolymers are used because these molecules can be “engineered” to provide specific solution or suspension properties. Block copolymers are characterized by an arrangement of polymer segments into regions (or blocks) with low solvent affinity (A) and blocks with high solvent affinity (B). Upon adsorption these molecules often adopt a three-dimensional conformation in the which the A block anchors the molecule to the surface and the B block extends into the liquid forming a “polymer brush”.5 While numerous experimental techniques are available for investigating the conformation of adsorbed polymers (e.g., NMR, SANS, IR spectroscopy, PCS),6 these methods generally do not provide direct information about the spatial arrangement of specific polymer segments at the solid-liquid interface. Therefore, little experimental data exist which directly relates the surface conformation of block copolymers to changes in macroscopic suspension properties such as colloid stability. We have been exploring the possibility of using singlestranded DNA as a model polymer for adsorption studies.7 These molecules represent an excellent model system for several reasons. Well-defined DNA molecules can be synthesized of exactly known length, with prespecified chemical and electrostatic architectures. Furthermore, the conformation of single-stranded DNA molecules at X
Abstract published in Advance ACS Abstracts, June 1, 1996.
(1) Bharat, B.; Isrealachvilli, J. N.; Landman, U. Nature 1995, 374, 607. (2) Napper, D. H. Polymeric Stabilization of Colloidal Dispersions; Academic Press: London, 1983. (3) Stumm, W.; Morgan, J. Aquatic Chemistry; John Wiley and Sons: New York, 1981. (4) Adachi, Y. Adv. Colloid Interface Sci. 1995, 56, 1. (5) Halperin, A.; Tirrel, M.; Lodge, T. P. Adv. Polym. Sci. 1992, 100, 31. (6) Cohen-Stuart, M. A.; Cosgrove, T.; Vincent, B. Adv. Colloid Interface Sci. 1986, 24, 143. (7) Walker, H. W.; Grant, S. B. Langmuir 1995, 11, 3772.
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the solid-liquid interface can be determined using a biochemical technique called hydroxyl radical footprinting (HRF). In a previous report, we used HRF to probe the conformation of a DNA diblock copolymer molecule (dT40B) adsorbed to the surface of positively and negatively charged polystyrene latex particles at moderate salt concentration (0.05 M NaCl).7 In this study, we present additional HRF results for this model DNA molecule at high salt concentration (1 M NaCl) and examine the relationship between the polymer’s surface conformation and its effect on particle coagulation rates. These results provide important new insight into the relationship between the structure of adsorbed polymer layers and the stability of aqueous colloidal dispersions. Materials and Methods Materials. The two model DNA molecules used in this study (see Figure 1) were synthesize using phosphoramidite technology by Oligo Etc. (Portland, OR). These molecules were received in lypholyzed form and, prior to use, the molecules were suspended in deionized water (Mill-Q water, 18 Ω cm, Millipore, Bedford, MA) and purified by column chromatography. Polystyrene latex particles, 480 nm amidine (lot no. 10-43-57) and 468 nm sulfate (lot no. 10-366-20), were synthesized by Interfacial Dynamics Corp. (Portland, OR). Under the neutral pH conditions used in the experiments described later, the amidine and sulfate particles have a positive (13.1 µC/cm2) and negative (4.6 µC/cm2) surface charge, respectively. All solutions were made with analytical grade reagents and deionized water. Electrophoretic Mobility. The electrophoretic mobility of the latex particles was determined using a Rank Brothers Mark II microelectrophoresis apparatus (Cambridge, England) fitted with a flat cell and a two electrode arrangement. Mobility measurements were carried out at 25 °C, at a pH of 6.8. Each reported mobility value represents the average of at least 20 measurements. Coagulation Kinetics. The coagulation kinetics of the latex particles was monitored using a photon correlation spectrometer (Malvern N4MD Particle Size Analyzer, Coulter Scientific Instruments, Amherst, MA). Measurements were conducted at a temperature of 22 °C and a scattering angle of 90°. The initial particle concentration for all stability experiments was 2.1 × 109 particles/mL. To carry out a stability experiment, 3 mL of an
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initially stable latex solution was added to a well-cleaned cuvette. DNA was then added (from 5 to 100 µL in volume) to the particle suspension and the electrolyte concentration adjusted by adding 1 mL of NaCl solution (either 0.2 M NaCl or 4 M NaCl, depending on the final NaCl concentration desired). The change in the average hydrodynamic radius of particle aggregates with time (dr/dt) was measured and related to the experimental rate constant for doublet formation (kexp) using the expression developed by Virden and Berg,8 namely
kexp )
1 dr Nr0R dt
(1)
where N is the initial particle concentration, r0 is the initial particle radius, and R is an optical factor estimated to be 0.5. Stability ratios were calculated by comparing the experimental coagulation rate constant to the rate constant for fast or Brownian coagulation (kb)
kb ) 4kT/3µ
(2)
where k is Boltzmann’s constant, T is the temperature, and µ is the solvent viscosity. The stability ratio, W, was calculated as the ratio of Brownian and experimentally observed rate constants: W ) kb/kexp. Hydroxyl Radical Footprinting. The details of the HRF method for determining the adsorbed conformation of singlestranded DNA molecules have been described previously.7 Briefly, DNA polymers are adsorbed to the surface of colloidal particles and then hydroxyl radicals are generated by the Fenton reaction.9 Because DNA strand scission by hydroxyl radicals is nearly diffusion-limited,10 segments of the DNA away from the surfacessay, in a polymer brushsare cut more frequently than segments directly adjacent to the surface. After the fragments are analyzed by polyacrylamide gel electrophoresis (PAGE), the cutting frequency of each DNA segment reveals the adsorbed molecule’s conformation. To carry out this procedure, radiolabeled DNA molecules are suspended in 10 µL of buffer (H2O, pH ∼ 7, at a desired [NaCl]) and latex particles are added to the suspension. The DNA is allowed to adsorb for 1 h, after which time unadsorbed DNA molecules are removed by centrifugation. DNA-coated particles are resuspended in DNA-free buffer, and hydroxyl radicals are generated by adding 3 µL of a solution containing FeIIEDTA, H2O2, and sodium ascorbate for final solution concentrations of 0.05 mM, 0.018%, and 5 mM, respectively. The cutting reaction is quenched after approximately 5 min and the DNA fragments are eluted from the particles and analyzed by PAGE (Model S2 sequencing gel apparatus, Biorad, Hercules, CA). Autoradiograms of the gels are analyzed by two-dimensional densitometry (Biorad Model GS-670 Imaging Densitometer, Biorad, Hercules CA). The relative degree of protection of an adsorbed nucleotide (or block of nucleotides) is determined by comparing the cutting frequency of DNA adsorbed to latex particles (“protection experiment”) to the cutting frequency of DNA in solution (“control experiment”). The relative cutting frequency, or RCF, is then calculated as RCF ) a/b, where a and b represent the band intensities of segments corresponding to the protection and control experiments, respectively.
Results Model DNA Molecules. The chemical and electrostatic characteristics of dT40A and dT40B are illustrated in Figure 1. Both molecules are 40 nucleotides in length and were synthesized with the base thymine at every nucleotide position. Under the neutral pH conditions used in our experiments the thymine base is uncharged, and therefore the electrostatic characteristics of the DNA molecules depend solely on the type and distribution of internucleotide linkages employed. dT40A was synthesized using only phosphodiester linkages, and so this (8) Virden, J. W.; Berg, J. C. J. Colloid Interface Sci. 1992, 149, 528. (9) Tullius, T. D. Nucleic Acids Mol. Biol. 1989, 3, 1. (10) Walling, C. Acc. Chem. Res. 1975, 8, 125.
Figure 1. Chemical structures and electrostatic architectures of the model DNA molecules used in this study.
Figure 2. Stability of negatively charged latex particles at 1 M NaCl, as a function of the dose of dT40A (4) and dT40B (O).
molecule is an acidic homopolymer characterized by one negative charge per nucleotide. The molecule dT40B contains ethylphosphonate linkages in which the negative charge on the phosphorus has been “turned off” and replaced with a hydrophobic ethyl group. Ethylphosphonate and phosphodiester linkages are arranged in dT40B so that the molecule has a diblock copolymer architecture, with an uncharged and relatively hydrophobic A block 19 nucleotides in length (MW ∼6300) and a negatively charged B block 21 nucleotides in length (MW ∼6900). Stability of Latex Microspheres. Initial experiments were conducted to investigate the effect of dT40A and dT40B on the stability of negatively and positively charged latex particles. The stability of the latex particles is presented in terms of the stability ratio, W. Physically, 1/W represents the fraction of particle-particle collisions which result in a sticking event. The effect of dT40A and dT40B on the stability of negatively charged latex at 1 M NaCl is shown in Figure 2. When dT40B is added to the suspension (circles in Figure 2), the latex particles are stabilized at polymer doses greater than 1 µg/mL, with stability ratios exceeding 100 at the highest polymer doses tested (>4 ng/cm2). In the presence of the homopolymer dT40A (triangles in Figure 2), on the other hand, the particles are unstable at all polymer doses tested. Because dT40A and dT40B differ solely in the number and spatial arrangement of linkage types (see Figure 1), the data in Figure 2 suggest that the electrostatic architecture of these molecules dramatically influences their ability to stabilize the negatively charged latex particles.
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Figure 3. (A) Stability of positively charged latex particles at 0.05 M NaCl (O) and 1 M NaCl (4), as a function of the dose of dT40B. (B) Effect of dT40B on the electrophoretic mobility of positively charged latex particles at 0.05 M NaCl.
The effect of dT40B dosage on the stability of positively charged latex particles is shown in Figure 3A. The particles are stable at 0.05 M NaCl (circles in Figure 3A) and low polymer dose (100 µg/mL). At 1.0 M NaCl (triangles in Figure 3A), the particles are unstable at all doses of dT40B tested. Figure 3B displays the electrophoretic mobility of positively charged latex particles in the presence of dT40B and 0.05 M NaCl. Increasing the dose of dT40B decreases the electrophoretic mobility of the particles and results in complete reversal in the sign of the mobility at sufficiently high polymer concentrations (>1 µg/mL). By comparing the data presented in parts A and B of Figure 3, we find that particle coagulation occurs at polymer doses for which the absolute mobility of the particles is close to zero. Hydroxyl Radical Footprinting. To determine how polymer conformation affects the coagulation kinetics of the latex particles, we probed the surface conformation of dT40B using HRF. The polyacrylamide gel in Figure 4 shows an HRF experiment conducted to investigate the conformation of dT40B adsorbed to negatively charged latex at 1 M NaCl and low polymer surface density (Γ ) 1 ng/cm2). Lane 1 of the gel shows purified dT40B. All of the DNA in this lane is contained in one distinct band at the top of the gel, indicating that prior to exposure to hydroxyl radicals dT40B was 40 nucleotides in length and highly monodisperse. Lane 2 shows the electrophoresis pattern for unadsorbed dT40B after exposure to hydroxyl radicals. In this case, cutting of the DNA results in a distribution of fragments ranging in size from mononucleotides at the bottom of the gel to full length dT40B molecules at the top. Lane 3 shows the hydroxyl radical
Figure 4. Hydroxyl radical footprinting experiment for dT40B adsorbed to negatively charged latex at 1 M NaCl and low polymer surface density (1 ng/cm2). Lanes of the autoradiogram represent purified dT40B (lane 1) and the hydroxyl radical cleavage of dT40B in solution (lane 2) and on the latex surface (lane 3).
cleavage pattern of dT40B, when the molecule is adsorbed to negatively charged latex particles at low surface coverage (Γ ) 1 ng/cm2). In this case, the entire molecule is protected from hydroxyl radical attack. The HRF gel presented above was quantified by twodimensional densitometry, and the resulting data are shown in terms of the relative cutting frequency, or RCF, in Figure 5. For a particular surface condition, the relative cutting frequency indicates how exposed a particular nucleotide (or block of nucleotides) is to hydroxyl radical attack. If the nucleotide is adsorbed close to the latex surface, then the RCF f 0; for nucleotides extending into solution, RCF f 1. For dT40B adsorbed to negatively charged latex at 1 M NaCl and low surface density, the average RCF values of the A and B blocks of the molecule are 0.05 and 0.06, respectively (Figure 5A). Thus, at low polymer surface density the entire dT40B molecule is adsorbed close to the latex surface where it is protected from hydroxyl radical attack. Additional experiments (gels not shown) were conducted for the same set of conditions described above (i.e., dT40B adsorbed to negatively charged latex at 1 M NaCl), except at a higher polymer surface density (Γ ) 35 ng/cm2). At high surface coverage the average RCF values on the A and B blocks of dT40B are 0.13 and 0.42, respectively (see Figure 5A). This differential cutting suggests that at least some of the
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Figure 5. (A) Average RCF values for the A and B blocks of dT40B when adsorbed to negatively charged latex at 1 M NaCl, for two different polymer surface densities (Γ ) 1 and 35 ng/ cm2). The surface density θ, expressed as a fraction of saturation coverage, is also shown. (B) The RCF of individual nucleotides in the B block of dT40B when adsorbed to negatively charged latex at 1 M NaCl and at two different surface coverages: 1 ng/cm2 (2) and 35 ng/cm2 (b).
dT40B molecules adopt a configuration in which the A block is in close contact with the latex surface and the B block is off the surface forming a polymer brush. The RCF values of individual nucleotides in the B block also appear to increase toward the 5′ end of the molecule, from RCF ∼ 0.2 near the junction of the A and B blocks to RCF ∼ 0.6 at the distal end of the molecule (circles in Figure 5B). By contrast, when dT40B is adsorbed at low surface coverage, the RCF values of individual nucleotides in the B block are roughly constant (triangles in Figure 5B). Thus, under the conditions studied in this set of HRF experiments, dT40B appears to undergo a transition from a relatively flat conformation at low surface coverage to a polymer brush conformation at high surface coverage. Discussion Relationship between Polymer Conformation and Colloid Stability. The HRF results presented above provide direct information about the conformation of dT40B on the surface of latex particles. Because strand scission of DNA by Fe[EDTA]2- is a result of hydroxyl radical attack on the sugar moiety in the DNA backbone,11,12 the HRF data can be used to elucidate the spatial arrangement of specific components of the DNA molecule (i.e., deoxyribose sugar, base, and internucleotide linkage) on the latex particles. In particular, the RCF values provide some measure of how close the sugar rings are to the latex surface. On the basis of the data presented in this paper and our previous study,7 we can develop the (11) Hertzberg, R. P.; Dervan, P. B. Biochemistry 1984, 23, 3934. (12) Prigodich, R. V.; Martin, C. T. Biochemistry 1990, 29, 8017.
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following model for how the conformation of dT40B surface coatings affects the stability of the latex particles. Stability of Negatively Charged Latex Particles. When dT40B adsorbs to negatively charged latex particles at 1 M NaCl, the conformation of the molecule depends dramatically on the polymer surface density. At low surface coverage the RCF values on both blocks of dT40B are low, suggesting that the molecule adsorbs flat and the sugar rings interact directly with the latex surface. When the surface coverage is increased, however, the amount of cutting on the B block of the molecule increases. At high coverage, the average amount of cutting on the B block is roughly 300% greater than the amount of cutting on the A block (see Figure 5A). As shown in Figure 6A, these data suggest that dT40B adopts a polymer brush configuration when adsorbed to negatively charged latex particles at high salt and high polymer surface density. One interesting characteristic of the data presented in Figure 5A is the apparent transition that dT40B undergoes from a flat conformation at low coverage to a polymer brush layer at high coverage. In our previous paper,7 we hypothesized that increasing the polymer surface density forces dT40B to adsorb to less electrostatically favorable adsorption sites on the charge-heterogeneous latex surface. This may account for the apparent transition to a polymer brush layer observed in the present study; in particular, at high coverage the B block of dT40B extends into solution to minimize direct interaction between negatively charged phosphodiester linkages and negatively charged patches on the latex surface. The influence of dT40B on the stability of the negatively charged latex particles at high salt (see Figure 2) can be explained as follows. At low doses of dT40B, the molecules adsorb flat, and the absence of strong steric and/or electrostatic interactions results in the rapid coagulation of the particles. As the polymer dose is increased, dT40B adopts a polymer brush configuration on the latex surface and the particles are stabilized, presumably as a result of steric interactions between polymer brush layers upon close particle-particle approach. The fact that the homopolymer dT40A has no effect on the stability of negatively charged latex particles at any polymer dose (see Figure 2) is further evidence that the stabilization of the particles at high doses of dT40B is directly related to the block copolymer architecture of this molecule. Stability of Positively Charged Latex Particles. Earlier we reported that dT40B molecules adsorbed to positively charged latex are highly protected from hydroxyl radical attack, suggesting that the sugar moieties in both blocks of the molecule interact directly with the latex surface (see Figure 6B).7 Because dT40B adsorbs close to the positively charged latex surface, this molecule influences colloid stability by altering the electrostatic character of the particle surface. At intermediate polymer doses, coagulation of the particles (see Figure 3) is due to charge neutralization brought about by ion pairing between the negatively charged linkages along the DNA backbone and amidine functional groups on the latex surface. The electrophoretic mobility data support this conclusion, showing that maximum coagulation occurs at polymer doses where the particles have low absolute mobility. At high polymer surface density, the net charge on the particles is reversed and the particles are electrostatically stabilized. The Effect of NaCl on Polymer Conformation. In our previous study,7 we found that dT40B did not form a polymer brush on negatively charged latex at moderate salt (0.05 M NaCl) and high polymer surface coverage. Instead, sugar rings in both the A and B blocks were equally susceptible to hydroxyl radical attack, suggesting
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Figure 6. Suspected conformation of dT40B on the surface of positively and negatively charged latex particles for each of the solution and surface conditions noted (left-hand column).
that adsorption occurred through base-surface interactions (see Figure 6C). For the set of experiments carried out at 0.05 M NaCl, the electrostatic repulsion between the B block of dT40B and the negatively charged latex surface is apparently accommodated by orienting the monomer units so that the sugar rings and phosphodiester linkages in the B block are off the surface. At this salt concentration, the intramonomer distance between phosphodiester linkage and thymine base is of the same order of magnitude as the characteristic Debye length (ca. 1 nm). Thus, by adsorbing in a “sugar up” orientation, the negative charges in the B block are apparently far enough off the surface so that the formation of a polymer brush is not favored in this case. However, when the NaCl concentration is raised to 1 M, both the Debye length and polymer solvency are decreased. The decrease in solvency and the fact that NaCl promotes hydrophobic interactions13,14 imply that hydrophobic moieties in dT40Bsnamely, the thymine bases and the ethylphosphonate linkagesswill have a greater tendency to partition to the latex surface. In our case, the enhanced hydrophobicity of the ethylphosphonate linkages apparently forces the sugar rings in the A block to adsorb close to the latex surface where they are protected from hydroxyl radical cleavage (i.e., the RCF values for the A block in Figure 5A are low). Perhaps this “sugar down” configuration of monomers in the A block, together with the well-documented rotational rigidity of singlestranded DNA,15-18 is the reason for the formation of the (13) Peng, W. Z.; Napper, D. H. Colloids Surf. 1995, 98, 93. (14) Einarson, M. B.; Berg, J. C. Langmuir 1992, 8, 2611. (15) Cantor, C. R.; Schimmel, P. R. Biophysical Chemistry: Part III, The Behavior of Biological Macromolecules; W. H. Freeman and Company: New York, 1980. (16) Wilson, D. H.; Price, H. L.; Henderson, J.; Hanlon, S.; Benight, A. S. Biopolymers 1990, 29, 357.
brush layer at high salt: if segments in the B block were to adsorb to the latex surface, they would be forced into a “sugar down” position which would be electrostatically unfavorable even at the high salt concentrations employed in this set of experiments. Alternatively, brush layer formation may be due to other (nonelectrostatic) polymer segment-segment, segment-surface, and/or segmentsolvent interactions that develop at high polymer surface density and high salt concentration. Effect of Secondary Structure. In the discussion above we have assumed that the secondary structure of dT40B in solution plays a minor role in determining this molecule’s conformation on the latex surface. At low to moderate salt concentrations (up to 0.1 M NaCl), electrostatic repulsion between adjacent nucleotides largely disrupts secondary structure in acidic single-stranded DNA molecules.16 At high salt concentrationsor for uncharged portions of dT40Bshydrogen bonding between adjacent bases could potentially occur, leading to “basestacking” interactions and a helical secondary structure.15 Rhodes and Klug19 found that when double-stranded DNA was adsorbed to calcium phosphate and then exposed to hydroxyl radical attack, the helicity of the molecule resulted in a modulated cutting pattern that was readily apparent when the DNA fragments were analyzed by PAGE. None of the HRF experiments we carried out with dT40B adsorbed to latex particles resulted in a modulated cutting pattern (see Figure 4 in this paper and Figure 5 in ref 7). This observation is consistent with the idea that the surface conformation of dT40B is determined primarily by the DNA-surface interactions and that any secondary (17) Schaper, A.; Urbanke, C.; Maass, G. J. Biomol. Struct. Dyn. 1991, 8, 1211. (18) Hagerman, P. J. Annu. Rev. Biophys. Chem. 1988, 17, 265. (19) Rhodes, D.; Klug, A. Nature 1981, 292, 378.
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structure exhibited by the molecule in solution is lost upon adsorption. Conclusions In this report, we employ DNA as a model polymer to investigate the role of polymer conformation in coagulating and stabilizing colloidal particles. Using a biochemical technique called hydroxyl radical footprinting we directly relate the surface conformation of DNA molecules to the stability of polystyrene latex particles. We find that when dT40B adsorbs to positively charged latex, it adopts a flat conformation, suggesting that the coagulation and stabilization of the particles at moderate salt concentrations (0.05 M NaCl) is due to electrostatic interactions. In this case, steric and bridging forces are of secondary importance. On the other hand, when dT40B adsorbs to negatively charged latex at high salt (1 M NaCl), the
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molecule adopts a polymer brush configuration at high surface coverage, and this brush layer stabilizes the particles through steric interactions between approaching polymer brush layers upon close particle-particle approach. Acknowledgment. We thank Professor Terese M. Olson for use of her photon correlation spectrometer and the department of Chemical and Biochemical Engineering at U.C.I. for use of their imaging densitometer. This work was funded by a grant from the Chemical and Thermal Systems Division of NSF under Contract Number CTS9208428. H.W.W. was also partially supported by a fellowship from the Irvine Ranch Water District and the School of Engineering, U.C.I. LA950757H
