Polyelectrolytes (PEs) are polymer molecules with ionizable functional groups that carry electrical charges in a polar solvent. Electrostatic interactions in solutions strongly modify the interactions of the PE molecules, which are screened by the dissolved ions.1 Therefore, conformation of PE molecules depends on the number of electrical charges and salt concentration in the solution. Weak PEs (where functional groups are weak acids or bases, e.g., carboxylic groups or amino functional groups) change the degree of ionization along with the pH of aqueous solutions. Both weak and strong PEs are sensitive to added salt. If the Debye screening length becomes comparable with the size of PE molecules, the PE coil shrinks in solutions at a high salt concentration. A further increase in salt concentration results in a change in the polymer coil conformation, which now approaches the conformation of a neutral coil in a good solvent. If water is a poor solvent for the neutral PE molecule, the coil shrinks to become a compact globule.2 This transition can be provoked by adding salt, so that the screening effect shifts the balance between electrostatic repulsion and hydrophobic attraction, that is, when the system undergoes phase separation at the solubility limit.
Conformational changes of biopolyelerctrolyte molecules, such as proteins and DNA, are involved in many natural phenomena in living systems. The response of PE molecules to changes in pH and ionic strength of aqueous solutions has been intensively explored for engineering man-made stimuli-responsive polymer systems. A range of properties of different materials were transformed and tuned based on pH- and salt-responsive behavior of PEs: wetting in mixed polymer brushes,3–6 adhesion and mechanical characteristics of thin films,7, 8 optical properties,9, 10 colloidal stability,11, 12 protein adsorption,13 transport of drugs and ions in PE gels,14, 15 brushes,16 and microfluidic channels,17 actuation and production of mechanical work,18 and templating single PE molecules for the fabrication of metallic nanoparticles of various sizes and shapes.19–22 In the examples listed above, the material properties were reversibly altered by changing the pH and salt concentrations in aqueous solutions. Even this brief listing unambiguously demonstrates how significant the PEs are in the sphere of materials and biomaterials science. Studies of the conformational transitions of PE molecules on single molecule level play important role for the understanding of PE behavior in the abovementioned materials and systems.
The range of important applications using PEs has drawn attention to the study of PEs' properties in changeable environments. The coil-to-globule transition phenomenon has been studied theoretically, using simulations and various experimental methods. The behavior of highly charged PE molecules in diluted solutions and at the surfaces is well understood and studied using methods like light-, X-ray- and neutron scattering (SANS). Very little information is available on transition of PE chains from extended coil to compact conformations. It was proposed that coil-to-globule transition of hydrophobic PEs takes place through a sequence of intermediate necklace-like states when the polymer chains were broken into charged smaller beads interconnected by polymeric strings.23, 24 As the charge density on the PE chain decreased, the number of beads decreased, whereas the diameter of the beads increased. This transition is rarely observed since the necklace-like structures are unstable. It was proposed that added salt could stabilize the necklace-like structures.25 These necklace-like intermediate states were observed using SANS experiments with poly(methacryloylethyltrimethylammonium methyl sulfate) by mixing aqueous solutions with acetone;26 with added CaCl2 salt to sodium polyacrylate aqueous solutions,27 and with added NaCl salt to sulfonated polystyrene in aqueous solutions.28, 29 Atomic force microscopy (AFM) experiments were used to monitor the shrinkage of poly(2-vinylpyridine) (P2VP)30 and poly(vinylamine)31 coils in aqueous solutions caused by increase in pH, and contraction of poly(methacryloyloxyethyl dimethylbenzylammonium chloride) by adding Na3PO4 salt to its aqueous solutions.32, 33 Necklace-like globules were reported in all these studies. However, in some AFM experiments, the necklace-like structures were visualized only if the polymer molecules were rapidly deposited on the mica substrates, as equilibration with the solution destroyed the necklace-like globules by adsorption forces.33 Although, details of the coil-to-globule transition mechanisms were reported in all these previous studies, a comparative analysis of dimensions of globules obtained in different coil-to-globule transition processes has not been conducted yet.
In this article, we compare structures of P2VP globules adsorbed on the mica surface from aqueous solution when the shrinking is brought about either by discharging the molecules at an elevated pH or by adding monovalent and polyvalent salts. We study the structure of the PE coils using in situ AFM experiments in aqueous solutions in a liquid cell. The effect of solid substrates on conformation is an area of major concern in the AFM experiments with single polymer molecules.34 There are two different possible cases: (1) polymer chains adsorb, and equilibrate on the surface. The sample represents an equilibrium conformation of the adsorbed polymer chain. (2) Polymer chains are irreversibly trapped by the substrate. In the latter case, the sample reflects the conformation, which appears as a result of the projection of 3D polymer coil on the substrate (3D-2D projected polymer coil). In both cases, the effect of the interaction between the polymer and the substrate may be very strong, thus resulting in the conformation of the adsorbed chain that is not reflecting the conformation in the solution. However, in some cases of several different hydrophobic PEs, it was a very good correlation between the necklace-structure of the polymer globules in solution and at surface,29, 33 although the interaction with the mica surface strongly affected the polymer chain conformation.33, 35 Strong hydrophobic forces in globules of hydrophobic PEs stabilized the globular conformation so that the coil-to-globule transition for adsorbed molecules was in accord with the transition in solutions.
In this article, we focus on the structure of the contracted polymer chains, which is considered as a final stage of the coil-to-globule transition. This conformation is not exhaustively studied because the phase separation and sedimentation of the polymer takes place in many cases. The AFM method studies single globules deposited on the substrate from very diluted solutions where adsorption of polymer globules takes place before their aggregation and precipitation.
Materials and Preparation of P2VP Solutions
Two stock P2VP, Mn 152,000, Mw/Mn 1.05 (Aldrich, MO) solutions (0.1 g/L) were prepared using Millipore water at pH 3.0 with HCl in the first solution and H3PO4 in the second solution. Then, the P2VP working solutions for the A series of experiments at different pH were prepared by diluting the stock solution with HCl solutions (in Millipore water) to adjust pH to a given value. The concentration of P2VP was adjusted to 5 × 10−4 g/L (or monomer units concentration [2VP] = 4.76 × 10−6 gequiv/L). The pH values of the working solutions were measured after the AFM experiments were completed (to avoid any contact between the working solution and the surface of the glass electrode). We excluded glass instruments and containers during preparation phase. All solutions were filtered using a Millex-LCR 0.45 μm (Millipore, MA). For the experiments in the B series, the samples of the first stock solution were diluted by NaCl solution at pH 3 (HCl). For the experiments in the C series, the samples of the second stock solution were diluted using Na3PO4 solution at pH3 (H3PO4). In both the B and C series, the concentrations of salts and acids were adjusted to prepare P2VP solutions of 500 mM ionic strength.
AFM images were recorded using a MultiMode Scanning Probe Microscope (Veeco Instruments, NY) equipped with a fluid cell and operated in tapping mode. To provide the appropriate conditions for the visualization of P2VP single molecules in liquid: (1) a high-grade substrate with atomically flat surface (V-1 grade muscovite mica 12–15 mm disks from Structure Probe, PA) was glued to the metal supporting disks with epoxy composition, (2) cleaved substrates were carefully checked for the surface quality and uniformity, (3) silicon nitride/silicon probes SNL (Veeco Instruments, NY) with a resonance frequency of ∼9 kHz in aqueous solutions were used, and (4) a thermal drift of scanner and mechanical drift induced by O-ring were well expressed (much stronger as compared with “in air” experiments). The drift was minimized by (1) incubation of the assembled cell with the mounted mica disk in the microscope for 0.5–2.5 h to equilibrate heat flows in the cell induced by the laser beam, and (2) precision placing of the O-ring to minimize lateral stresses. Polymer solutions were injected directly into the fluid cell and images were recorded in about 7 min post injection. The measurements were performed at amplitude set points ranging between 0.6 V and 2.2 V, tapping force of about 98% of the set points was used to minimize the effects of the tip on chain conformation.
Processing the Data
Self-developed software was used to process the images. Coordinates of the chains were recorded by dragging a cursor along the chain contour. The recorded coordinates were used to estimate the experimental values of rms end-to-end distance 〈r2〉1/2 and rms radii of gyration 〈s2〉1/2. Radii of gyration were calculated drawing the most probable path accounting for visible fragments. The values were averaged based on an analysis of 150–200 molecules.
RESULTS AND DISCUSSION
In Figure 1, we present the coil-to-globule transition of P2VP molecules adsorbed on the mica substrate from diluted aqueous solutions. The transition takes place due to the discharging of the P2VP coils if the pH changes from pH 3.0 to pH 4.8. In Figure 2, we show the globules formed in P2VP solutions at pH 3.0 with an added salt NaCl [Fig. 2(a)] and Na3PO4 [Fig. 2(b)]. Both the experiments with salt were conducted at the ionic strength of 500 mM. At this ionic strength, the Debye screening length approaches the dimension of a monomer unit (ca. 0.45 nm).
P2VP is a weak hydrophobic PE and water is a poor solvent for uncharged P2VP. The protonated polymer is soluble in water due to the release of counterions. The conformation of the P2VP coil is balanced by Coulomb repulsion and short range van der Waals attraction. Extended polymer coils undergo the coil-to-globule transition as the degree of protonation decreases or the solution's ionic strength increases. Although, the interaction between the P2VP coils and the oppositely charged mica surface affects the conformation of the adsorbed molecule, the coil-to-globule transition is well-pronounced for the adsorbed polymer.36 Specifically, in this study, P2VP chains were adsorbed at pH3, when mica is weakly charged, and at pH = 4.8, when P2VP is weakly charged. Thus, we expect a weak effect of the electrostatic polymer–substrate interactions on the conformation of the globules. It is likely that van der Waals interactions between P2VP and mica strongly contribute to the adsorption energy.
At pH 3.0, ionized P2VP approaches a flat conformation (2D coil) in less than 10 min [Fig. 1(a)] with a root mean square (rms) radius of gyration (〈s2〉1/2) = 23 ± 7 nm, and rms end-to-end distance (〈r2〉1/2) = 58 ± 25 nm. The conformation remains unchanged for hours. No diffusion, exchange, or desorption takes place. At pH 4.8 the compact globule appears as a spherical aggregate which combines several dense fragments. The details of the structure of the adsorbed 2D globules are well resolved and shown in magnified view in the AFM images (Fig. 3). Although, the globules are thicker than 2D coils, the fraction of segments in the loops exposed in Z-direction is small (ca. 15%). These loops are neglected in the estimations of rms radius of gyration and rms end-to-end distance using the 2D globule model (Table 1).
Table 1. Dimensions of P2VP (Mn 152,000 g/mol) 2D Coils Adsorbed from Aqueous Solutions at Different Salt Concentrations
The coil-to-globule transition is also well-pronounced in the presence of both monovalent and polyvalent salts (Fig. 2). In both cases, the shrunk coils appear as discoid structures, but they are less dense when compared with the discharged globules. The dimensions of the globules in the salted solutions are comparable with the size of P2VP coil in organic solvents (Table 1). These results are in agreement with previously reported conclusions that P2VP chains at high ionic strength possess a conformation with characteristics dimensions similar to an unperturbed polymer coil.2
The structures of the salted globule are well resolved in the AFM images [Fig. 3(c,d)]. In contrast with the discharged globules, the salted globules are swollen in aqueous solutions owing to the counterion condensation and formation of ion pairs along the polymer backbone. Thus, the salted solution dissolves the hydrophobic PE chain decorated with condensed counterions.
Very interesting results were observed from the comparison of globules obtained in the presence of monovalent and polyvalent counterions. The strong electrostatic interaction between multivalent ions and the PE backbone favors the ion condensation and chain collapse. Polyvalent ions make counterion condensation more efficient than condensation of monovalent ions. Multivalent ions play also role of the crosslinking agent by binding polymer chains together. It was found in simulation studies, that polyvalent ions are more efficiently affect transitions of PE chains from an extended to a more compact conformation: The transition takes place at lower salt concentrations for polyvalent ions and results in a more compact conformation of PE chains.37–40 However, in our experiments, the statistical analysis of the dimensions of the globules revealed no essential differences between NaCL and Na3PO4 solutions of the same 500 mM ionic strength. We did not observe any specific effects of multivalent counterions on the structure of the P2VP globules in these experiments. It seems that in the studied range of salt concentrations, we examined the PE chains in the conditions when the charges in the backbone are neutralized by condensed counterions and strong hydrophobic attractions screen a difference between contributions of monovalent and polyvalent counterions. We plan experiments for the study of salt concentration effects at their variable concentrations on conformations of P2VP chains in presence of polyvalent counterions to clarify the observed behavior of PEs in our future research project.
This study reports on the comparison of compact conformations of a weak hydrophobic PE chain in conditions of a low charge density or at high salt concentrations. We found a substantial difference in the dimensions of adsorbed globules of hydrophobic weak cationic PE prepared by using different mechanisms of coil-to-globule transitions. The abrupt coil-to-globule transition caused by pH changes and the accompanied discharge of polymer chains resulted in compact globules. If the pH corresponding to extended coil conformation remains unchanged, the coil shrinks due to the added salt. The size of the globule in the latter case corresponds to the unperturbed dimension of the polymer coil. There is no essential difference in the dimensions of the globules as obtained in the presence of monovalent and multivalent counterions for the studied ionic strength. It is likely that the reported differences between the discharged and salted globules can be observed only through the AFM experiments performed in situ in solutions, because deprotonated P2VP is not soluble in water at pH4.8. Also, in dry samples, the swollen globules shrink further and result in similar compact dry structures. Thus, the AFM experiments in a liquid cell provide a unique opportunity for the comparison of different types of polymer globules.