The Effect of Divalent Cations - American Chemical Society

Sep 6, 2013 - School of Physics and Astronomy, University of Edinburgh, Edinburgh EH9 3JZ, Midlothian, Scotland, United Kingdom. ∥. Materials and ...
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Interactions of Hyaluronan Layers with Similarly Charged Surfaces: The Effect of Divalent Cations Lei Jiang,*,† Simon Titmuss,§ and Jacob Klein*,‡,∥ †

State Key Laboratory of Heavy Oil Processing, Center for Bioengineering and Biotechnology, China University of Petroleum, 66 Changjiang West Road, Qingdao, Shandong 266580, P. R. China ‡ Physical and Theoretical Chemistry Laboratory, University of Oxford, Oxford OX1 3QZ, United Kingdom § School of Physics and Astronomy, University of Edinburgh, Edinburgh EH9 3JZ, Midlothian, Scotland, United Kingdom ∥ Materials and Interfaces Department, Weizmann Institute of Science, Rehovot 76100, Israel S Supporting Information *

ABSTRACT: We used colloidal probe atomic force microscopy to measure the normal forces between the surface of a silica colloidal particle and a sparse layer of hyaluronan (hyaluronic acid, HA, MW ≈ 106 Da) covalently attached to a planar silica surface, both across pure water and following the addition of 1 mM MgCl2. It was found that in the absence of salt the HA layer repelled the colloidal silica surface during both approach and retraction. The addition of the MgCl2, however, changes the net force between the negatively charged HA layer and the opposing negatively charged silica surface from repulsion to adhesion. This interaction reversal is attributed to the bridging effect of the added Mg2+ ions. Our results provide first direct force data to support earlier simulation and predictions that such divalent cations could bridge between negative charges on opposing surfaces, leading to an overall reversal of force from repulsion to attraction.

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attraction mediated by divalent ions.4,15 Interestingly, it has been realized that the divalent counterions’ role could be more than just enhancing the adsorption of charged polyelectrolyte at similarly charged surfaces. From stiff polyelectrolytes with large persistence lengths such as DNA to flexible polymers with low persistence lengths such as sodium polystyrenesulfonate (PSS), experimental data have emerged that, under certain conditions, divalent ions can reverse the original repulsive force to give an attraction between similarly charged polymer surfaces.4,15 In the present study, the normal forces acting between a surfaceattached layer of the semiflexible anionic biopolymer hyaluronan (also known as hyaluronic acid or HA) and an opposing negatively charged solid surface are considered. In particular, the effects of added divalent ions (Mg2+) on bridging and in modulating the overall interfacial forces are probed. This study, for the first time to our knowledge, directly measures and compares the forces with and without the added divalent counterions between a negatively charged HA-coated surface with an opposing (bare) negatively charged surface, complementing earlier simulation and scanning microscopy data.4,12 Since HA layers are ubiquitous in biological systems, as are divalent ions, and since most biological surfaces (e.g., cartilage

INTRODUCTION The effect of salt on aqueous polyelectrolytes in solution has been studied for decades.1 Most surfaces and macromolecules are charged in an aqueous biological environment, and divalent ions (Ca2+, Mg2+, etc.) play important roles in mediating interactions between biomacromolecules in cellular environments.2 In industrial practice, salts have frequently played an important role in controlling the viscosity and the on-surface functionalities of polyelectrolytes such as carbohydrate and cellulose.3 Previous studies have shown that divalent counterions can lead to a surface charge reversal of a negatively charged solid surface,4,5 while for bulk macromolecular solution, it has been demonstrated that they can induce aggregation or precipitation of charged surfactants and proteins by screening the electrostatic repulsion.6 Although divalent ions have commonly been added to solutions to help the polyelectrolyte adsorb to solid surfaces,7,8 for example in the application of lime to soil clay9 and EDTA on glassware cleaning,10 most studies on their effect of modulating the forces and interactions between charged polymers and surfaces are indirect, relying on theoretical simulations or observations made with scanning microscopy.4,8,11,12 There were only a few direct force measurements in which only the counterion valency is varied.13,14 Recently more in-depth studies have started to focus on a more simplified model system of polyelectrolytes at solid surfaces, seeking experimental support for the predicted © 2013 American Chemical Society

Received: May 22, 2013 Revised: August 28, 2013 Published: September 6, 2013 12194

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surfaces or surfaces of the extracellular matrix16) are negatively charged, these results have clear bearing also on interactions in biological environments.

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cantilever results in a thermal noise spectrum which is used at the beginning of each experiment to calibrate the spring constant of the cantilever. The magnitude of force exerted on the cantilever can be determined by multiplying the deflection of the cantilever by the spring constant (Hooke’s law, usually between 60 and 120 mN/m). Attaching a silica bead of radius R = 7.5 μm to the cantilever tip provided a well-defined interaction geometry, permitting comparison with theory through the Derjaguin approximation and accurate calculation of interaction forces.23 Figure 2 is the schematic MFP measurement setup of normal forces between a silica colloidal probe and a planar silica surface. Each MFP measurement was repeated at least three times.

EXPERIMENTAL SECTION

Materials. All water was of high purity obtained by treating tap water with an activated charcoal filter followed by a Millipore water purification system, comprising of a RiOs and a Milli-Q Gradient A10 stage. The resistivity of water used was 18.2 MΩ·cm, conductivity was ≤0.055 μS/cm, and the concentration of the total organic compounds (TOCs) was ≤4 ppb at 25 °C. Hyaluronan (HA), of molecular weight 1 × 106 Da, was purchased from Genzyme Corporation (Cambridge, MA), and all other chemicals, including magnesium chloride (≥98%), toluene (≥99.0%), 4-(2 hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), N-(3-(dimethylamino)propyl)-3-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), and 3-aminopropyltrimethoxysilane (APTMS) were purchased from Sigma-Aldrich Ltd. (UK). Silica beads of uniform diameter 15 μm were purchased from Polysciences and glued onto the tip of Si3N4 AFM cantilever using UV curable glue (Norland 81, Norland Company, Chambersburg, PA) under an optical microscope. Colloid Probe Cleaning. Before each experiment, the cantilever with attached sphere was soaked in ethanol overnight as a preliminary cleaning step. Immediately before the start of each experiment, the probe was treated by 40 min oxygen−plasma using a Biorad PT7125 plasma etcher (1 mbar, 50 W net power). Oxygen−plasma treatment is believed to be an effective way to remove possible contamination from the surface of the sphere and to increase the number of reactive hydroxyl groups at the glass surface.17,18 Grafting HA to a Silica Surface. 300 μm thick silicon wafers, with a surface layer of SiO2 (silica), were cleaned in piranha solution (H2O2:H2SO4 = 3:7) and rinsed thoroughly with water. To amine functionalize the surface of the wafer, it was soaked in a 9:1 v/v mixture of 80 °C toluene/3-aminopropyltrimethoxysilane (APTMS) for 4 h, rinsed 4 times in dry toluene, and sonicated for 30 s to break any silane network at the silica surface. To couple HA to the aminefunctionalized substrate, 1 mg/mL HA in 10 mM 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) buffer was mixed with 0.2 M N-(3-(dimethylamino)propyl)-3-ethylcarbodiimide hydrochloride (EDC) and 0.05 M N-hydroxysuccinimide (NHS) 5 min before adding the silicon wafer and incubating for 12 h. Afterward, the wafer was rinsed thoroughly and kept in water for 5 days, exchanging the water daily. This is to ensure that all the noncovalently bound HA were removed from the surface and all the free APTMS had been fully hydrolyzed before the force measurement.19,20 A schematic of the HA layer structure on silica is shown in Figure 1.

Figure 2. Schematic of the MFP measurement of normal forces between a silica probe and a planar silica surface. Fine separation control in MFP is achieved by expanding and contracting a piezoelectric crystal by application of a voltage. Unlike the surface force balance (SFB)24 which uses an interferometric measurement of the absolute separation between atomically smooth mica surfaces, MFP employs a linear variable differential transducer (LVDT) system to provide an independent and very linear measurement of displacement. Unlike traditional AFM, in MFP the laser beam strikes the cantilever at an angle of 11° to the normal, eliminating the effect of interference with laser light reflected from the substrate surface. Ellipsometry. A Beaglehole picometer ellipsometer (New Zealand) was used to measure the thickness of APTMS layer 1.5−2 nm, consistent with the 2.5 ± 1.2 nm thickness in the previous literature.20 Assuming that each bond contributes 0.1 nm to the chain length, this 1.5−2 nm APTMS film suggests that it was composed of 3−4 layers, amine groups from the top layer exposed outward to bind HA. After HA grafting, the total layer thickness measured by ellipsometry ∼5 nm in air and ∼100 nm in water. For HA of 106 Da molecular weight and contour length ∼2 μm, the 100 nm layer thickness measured suggests that the polymers form loops, trains, and tails on the solid surface, as discussed later. This 100 nm layer thickness in water is in agreement with previous measurements using similar grafting methods.25,26 If taking APTMS as ∼2 nm, the 5 nm total thickness corresponds to ∼3 nm highly hydrated HA layer such that it may not provide a direct measure of surface excess of “dry” HA on the surface. However, the amount of HA can be estimated with a simplified model, as proposed in the Discussion. Atomic Force Microscopy (AFM). The setup and principle of AFM have been reported previously.22 Image was obtained in air with a Multimode 8 (Nanoscope Inc.) microscope using silicon cantilever in tapping mode. The AFM images (data not shown) suggest that the APTMS layer is relatively smooth with a roughness ∼0.5 nm. To image the HA conformation on solid surface, the sample was prepared by dipping bare mica surface in 10−3 mg/mL HA and 1 mM MgCl2 solution for 10 min before drying with N2 jet flow.

Figure 1. Illustration of how HA is covalently grafted to the silica surface after the silanization of silica surface and 5 days hydrolysis in water. Molecular Force Probe (MFP). The MFP-1D, as optimized for force measurement, was purchased from Asylum Research Company (Santa Barbara, CA). The basic force measurement principle is similar to that used in atomic force microscopy (AFM).21,22 A silicon nitride cantilever is mounted rigidly, and a laser beam is focused on the back of the cantilever and reflected onto a quadrant photodiode via a mirror. Deflection of the cantilever spring results in a displacement of the light spot vertically across the face of the photodiode, leading to a change in the voltage across the diode. Brownian displacement of the

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RESULTS Control Force Measurements across Water and MgCl2 Solution. The normal forces between bare silica surfaces, 12195

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across water and across 1 mM MgCl2 solution, were first measured and are shown in Figure 3a. The open triangle

= 54 mV, corresponding to an effective surface charge density σ of 0.0005 C m−2 (∼1e/321 nm2) via the Grahame equation.35 This surface potential value is in line with the expectation of ψ0 > 40 mV for a silica surface in 1.1 × 10−5 M 1:1 electrolyte solution.36,37 At D < 5 nm marked by the dark arrow is in Figure 3a, there is an additional repulsion over and above that predicted from DLVO theory (solid line). This additional repulsion has been observed for silica and other surfaces interacting across aqueous solution and it has been suggested that a layer of polysilic acid “hairs” is present at the surface, forming a layer of silica gel.38 As an additional control, a force profile was measured between the bare silica surfaces across 1 mM MgCl2 solution prior to grafting HA polymer to the substrate surface, indicated as the open (compression) and closed (decompression) square symbols in Figure 3a. The dashed line in Figure 3a represents the DLVO fit (κ−1 = 3.19 nm, ψ0 = 20 mV), consistent with extensive surface force balance (SFB) measurements (e.g., a value of κ−1 = 3.4 nm for a 1.15 mM 1:2 electrolyte solution39), suggesting the system is largely free from contamination by other electrolytes. Compared to the no-added salt case, Mg2+ reduces the surface charge density and significantly decreases the Debye screening length as expected. The possible influence of APTMS on normal forces was also considered. The roughness of APTMS layer is ∼0.5 nm from AFM characterization (data not shown). After 5 days water hydrolysis, it is expected that most silanized layer have been hydrolyzed.19,20 Because of its low molecular weight and hydrophobic carbon backchain, APTMS is likely to behave rigidly in aqueous solution with thickness almost unchanged. Therefore, the 100 nm thick HA layer will probably dominate the surface interaction and ensure the 2 nm APTMS layer at the bottom will not get near to the opposite probe even under compression. Despite the hydrolysis of APTMS, the free amine groups may still be protonated and positively charged in the experimental solution of pH 6.5. In order to study their possible effect on surface interaction between HA and the opposing surface, the forces between the amine functionalized silica surface and the bare silica probe across 1 mM MgCl2 and 1 mM NaCl were measured as shown in Figure 3b. In these control experiments the double-layer repulsion was screened due to the addition of salt and on separation there were an adhesion of ∼−0.02 mN/ m at D < 10 nm between the surfaces. This adhesion may attribute to the attraction between negatively charged probe and weakly positively charged amine groups or the attractive hydrodynamic force (∼−0.015 mN/m for D = 10 nm) as calculated in the Discussion. These data from sample without HA enable us to compare to further experiments with HA grafted surfaces. Force Measurements between Grafted HA and Colloid Probe across Water. HA is then chemically grafted to the planar silica surface, as described in the Experimental Section. It is worth to note that before grafting HA an earlier attempt to physically adsorb HA to negatively charged surfaces in both water and MgCl2 has been made. The results (data not shown) suggested that the Mg2+-assisted adsorption was too weak so that the polymers were squeezed out of gap when the opposite surface came close into contact. In order to obtain a stabilized layer of HA on surface, we chemically grafted HA in this experiment to study the adsorption of HA to silica mediated by divalent ion bridging.

Figure 3. (a) F/R vs distance D profile measured between a bare silica surface and a silica colloidal probe across water (triangle) and 1 mM MgCl2 solution (square), respectively. The open symbols indicate the approach profile with the closed ones for separation. The lines are DLVO theory fit to the data for 1:1 solution (solid line) and 1:2 solution (dashed line). (b) F/R vs distance D profile measured between the amine functionalized surface and a silica colloidal probe across 1 mM MgCl2 (square) and 1 mM NaCl (circle) solution, respectively. The open symbols indicate the approach profile with the closed ones for separation.

symbols were measured on compression (in-profile) and closed symbols were measured on decompression (out-profile), both in water. The continuous solid line represents the prediction of the Derjaguin−Landau−Vervey−Overbeek model,27,28 F(D)/R = (64πkB2T2e−2εrε0/κ) tanh2(eψ0/4kBT)e−κD − AH/6D2, where kB is Boltzmann’s constant, T the absolute temperature, εr and ε0 the relative permittivity (≈81 for pure water) and permittivity of free space, respectively, κ−1 the Debye length, ψ0 the constant surface potential, e the electronic charge magnitude, and AH the nonretarded Hamaker constant for silica across water. With Hamaker constant A = 0.8 × 10−20 J for silica,29,30 we used the approximate equation with constant potential boundary, instead of full numerical solution,31 for the sake of general estimation of Debye length and surface potential. The slope of the salt-free water profile gives a Debye length κ−1 = 91.6 nm, corresponding to a concentration of 1:1 electrolyte of 1.1 × 10−5 M. This value is within the range of effective electrolyte concentrations previously measured in pure water ((0.5−1.5) × 10−5 M), arising from CO2 dissolved from air and possibly ions leached from glassware, and indicates that the solution is free of contamination.32−34 Ducker and Horn have previously measured the surface potential of a hydrophilic silica surface in 1:1 aqueous solution to be 23, 28, 32, and 40 mV with decreasing electrolyte concentration 10−1, 10−2, 10−3, and 10−4 M. The DLVO fit here provides an effective surface potential ψ0 12196

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of water. As presented in the Discussion, this leads to a consistent picture in terms of surface interactions. Force Measurements between Grafted HA and Colloid Probe across MgCl2 Solution. 1 mM Mg2+ ions were then added, and three sets of force profiles are shown as red lines in Figure 4a. The addition of 1 mM Mg2+ ions has significantly screened the double-layer repulsion. For example, in water the normal forces onset at D ∼ 200 nm (where F/R ≥ 0.01 mN/m), and when separation D is reduced to ∼50 nm, the force increases to F/R ∼ 0.13 mN/m; while in 1 mM MgCl2 the repulsion did not commence (where F/R ≥ 0.01 mN/m) until D ∼ 50 nm. The measured force from ∼50 nm is mainly from steric repulsion between HA layer and the opposing colloidal probe surface. Therefore, it is taken that the thickness of grafted HA layer across 1 mM Mg2+ solution is ∼50 nm. In comparison with the ∼100 nm ellipsometric thickness without added salt, this represents a 50% reduction in the measured thickness of the polyelectrolyte layer. This is most simply understood in terms of the osmotic compression of the HA layer by the added 1 mM Mg2+, as discussed in the next section.29 This is also in agreement with previous reports on the effect of added electrolyte on the conformation of adsorbed polyelectrolyte.25,41,42 For further comparison, 1 mM NaCl were added to water and the force profile was illustrated in Figure 4b. The 1 mM monovalent counterions would not only compress the HA layer thickness but also partly screen the DLVO repulsion, although its screening effect is not as strong as divalent ions. The repulsion commenced at about 100 nm and out-profile is different from the in-profile with a possible hysteresis. We believe this difference was mainly attributed to the hydrodynamic force, as detailed in the Discussion. Figure 5 compares the forces measured on compression in Mg2+ solution with and without the HA grafted to the surface.

Figure 4a shows the measured interaction between silica surfacesone bare and the other grafted with HAboth

Figure 4. (a) F/R vs distance D profile between colloidal probe and HA grafted silica surface across water (dark lines) and 1 mM MgCl2 (red lines). The arrow represents the in and out profiles. (b) F/R vs distance D profile between colloidal probe and HA grafted surface across 1 mM NaCl solution with open symbols for in-profile and closed ones for out-profiles.

across water (dark lines from two repeat experiments) and across 1 mM MgCl2 solution (red lines from three repeat experiments). In water, a monotonic repulsion commences at ∼200 nm, which can be ascribed to a long-range double-layer repulsion between the negatively charged bare silica surfaces and the negatively charged HA layer. As the separation is reduced to below 100 nm, which equals the thickness of HA layer measured by ellipsometry, one might expect an additional steric interaction due to compression of the surface attached HA layer. However, the compression profiles measured in water with and without HA layers grafted show no significantly differences. That is, the water profile with HA (open and closed triangles in Figure 3a) is very similar to the profile without HA as thicker solid lines in Figure 4a. More measurements have been repeated at each condition to minimize the experimental errorsonly two sets of in-water profiles are displayed in Figure 4a for a clear comparison, and the full data profiles are provided in the Supporting Information Figure S1. In previous surface force studies with surface-attached polymers the extra steric repulsion was normally taken as the main evidence of polymers’ presence at surfaces, adsorbed or grafted.33,40 As we demonstrate below, the lack of such extra repulsion should not be taken to imply that there is no HA at the surface. HA has been grafted to silica using a wellestablished method with an ellipsometric thickness ∼100 nm, similar to the previously reported value.25 We attribute the absence of a measured steric interaction to a rather low surface excess of highly hydrated HA on the surface. Concomitant with the low volume fraction of HA at the surface is a high volume fraction of water: HA is strongly associated with water, and the ∼3 nm HA thickness measured in air contains a large amount

Figure 5. Comparison of F/R vs D compression profiles between colloidal probe and silica surface across 1 mM MgCl2, with and without HA grafted. Red lines indicated repeated measurements with HA grafted while the square symbols represent the force without HA.

In 1 mM Mg2+, when the two surfaces are approaching, the presence of the grafted HA results in the repulsion commencing at a larger separation. For example without HA, the repulsion between two negatively charged silica surfaces reaches F/R ∼ 0.01 mN/m at D ∼ 15 nm while in the graftedHA scenario this force is reached at D ∼ 50 nm. On separation of the surfaces in 1 mM MgCl2, the effect of HA is even more striking. Without HA there is a short-ranged repulsion (closed square symbols in Figure 3a), while with HA the net force has changed to attraction, as in Figure 4a. Taking the MgCl2 profiles out of Figure 4a, Figure 6 shows that on separation of the compressed surfaces, over a range of 12197

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positively charged in solution, the small adhesion between amine groups and silica probe in Figure 3b suggests that these charges might be too weak to draw the partly ionized HA47,48 flat on surface. HA has been reported to be highly hygroscopicit has the capacity to hold water up to 1000 times more than its own volume.49 Even if dried and stored over months, HA is still able to hold some bound water. This is probably due to its potential for high ionizationsimilar effects have been observed in other hygroscopic polymers such as the dipolar poly(2(methacryloyloxy)ethylphosphorylcholine) (pMPC) brushes.50 Considering the bound-water effects and the 3 nm in-air and 100 nm in-water ellipsometric thickness of hydrated HA layer, we propose the structure of HA shown in Figure 7a where each

Figure 6. F/R vs D profile between colloid probe and HA-grafted silica in 1 mM MgCl2, which was selected from Figure 4a for a focus on separation profile. The upper inset is a schematic illustration of polymer compression, pulling, and stretching, which is suggested to occur during the force measurement between the probe and grafted HA across 1 mM MgCl2. The arrow represents the in- and out-profiles.

250 nm, a long-ranged attraction was observed, as several continual attractive wells. This attraction is somewhat similar to some previous surface force measurements, in which polymer adsorption and bridging forces were thought to be important, although most bridging effects were observed between oppositely charged or neutral polymers and surfaces.43,44 The attractive force F/R is at the level of −0.1 to −0.2 mN/m over a range of 200 nm, much larger than any possible attractive hydrodynamic effect (∼−15 μN/m, calculation in the Discussion).45,46 It is also larger and longer-ranged than the attraction between amine-functionalized surface and silica probe as in Figure 3b. It confirms the presence of grafted HA and the attraction difference is likely to be from the surface interactions between HA and silica probe. At the same time the form of the attraction suggests that it is mainly due to stretching of the grafted polymer chains that undergo Mg2+mediated adsorption to the opposing bare colloidal probe surface, as illustrated in Figure 6, upper inset. On compression of the HA between the surfaces, divalent counterions bridge the HA segments to opposing silica surfaces, and on separation the HA is progressively pulled off, resulting in the characteristic adhesion profiles.

Figure 7. (a) Schematic drawing of the HA polymer conformation on silica surface. (b) A simple type of bridging mechanism involves units of a negatively charged polymer loop, on the one side covalently grafted on surface A and on the other side adsorbing to the opposing surface B mediated by the divalent counterions.

polymer could be grafted to the surface in more than one location (“trains”) with “loops” in between the trains and free ends (“tails”). The radius of gyration Rg of HA with molecular weight MHA = 106 is of the order of 200 ± 50 nm,51,52 where each molecule covers an area A ≈ (2Rg)2 and the molecules are close-packed on the surface. The ellipsometrically measured thickness LHA = 100 nm, less than Rg, is the “averaged” height of HA layer, which is used also for force measurement and calculation. In Supporting Information Figure S2, we show an AFM image of HA adsorbed to the negatively charged mica with the help of Mg 2+ counterions, where the polymers were “combed”53 to stretch in a preferential orientation.54 Further height analysis of HA images has been conducted which clearly shows some height fluctuation along the polymer chain. Although the AFM was from an adsorbed HA chain in air, instead of grafted one in water, the up-and-down feature along the chain has suggested that HA may adopt a “loop” and “train” conformation at solid surfaces. Some morphology of HA polymer is similar to the “blobs-on-a-string” structure which had been often observed55 and numerically studied56 in viscoelastic polymer fluid. As HA is a polymer of high viscoelasticity,25 this conformation may be formed on the adsorbed HA during the drying step by N2 jet flow before AFM imaging, while in water such structure may not occur or interfere with the overall surface interactions. Forces between HA and Colloid Probe. For a polymer conformation as in Figure 7a, taking Rg ≈ 200−250 nm, a

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DISCUSSION Conformation of the Grafted HA Layer. The main qualitative finding of this study is the effect of the divalent cations in changing the repulsion between the HA layers and the opposing similarly charged silica surface to an attraction mediated by divalent ion bridging. First, we try to identify the conformation of the grafted HA on the surfaces and estimate the surface excess of HA. For HA immersed in water the layer thickness LHA measured by ellipsometry is ∼100 nm. As in Figure 3a between the HA-coated surface and the silica across water, the repulsion is F/R ≈ 0.13 mN/m for D ≈ 50 nm, of which the repulsion is mainly attributed to counterion osmotic pressure Πc/i. As derived in the calculation in the Supporting Information, the effective surface excess of HA is 0.032 mg/m2. Taking the density of HA as 1−1.5 g/cm3, the corresponding “dry thickness” of HA on silica surface would be 0.02−0.032 nm, much smaller than the measured ∼3 nm thickness by ellipsometry in air. Therefore, we conclude that HA remains highly hydrated in air and the ellipsometrically measured air thickness is indeed for a layer with a significant amount of water bound, of which the volume fraction of HA is ϕ ∼ 0.01. Although the free amine groups underneath HA may be 12198

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a constant approaching velocity ν of 1 μm/s, as used in this experiment, the hydrodynamic repulsion on approach (and attraction on retraction) is ∼3 μN/m. This ignores any perturbing effect of the HA layers on the flow of liquid as any such effect would decrease the effective D and increase FH.58 We use a value of 50 nm because this is the point, in the 1 mM salt, at which the HA layers just touch the opposing surface, and so we would expect the bridging to start. This hydrodynamic force may also account for the hysteresis or force difference between in- and out-profiles in 1 mM NaCl solution in Figure 4b. For example at D = 10 nm, the repulsion between HA and silica probe is ∼0.06 mN/m on compression and ∼0.025 mN/m on separation. The 0.035 mN/m difference between the two profiles is mainly attributed to the hydrodynamic force, which is 0.015 mN/m on compression and −0.015 mN/m on retraction based on the calculation above. Bridging Force. An attractive bridging force is an additional force that could contribute to the interaction free energy between grafted HA and the opposing surfaces. It may be estimated as in Figure 7b. We assume that the initial bridging attraction is dominated by a single large loop, for each polymer, of height L, with a monomer size l, and a fraction f of the monomers negatively charged. Each charged monomer is able to attach to the opposing negatively charged silica surface via an Mg2+ ion, with an adhesive energy ε that is essentially the free energy associated with counterion release, such that ε ≈ 1 kBT at most. Then a decrease in D by an increment l will enable 2f charged monomers (one on each side of the loop) to attach to the surface, with an energy gain 2fε. In this experiment, the bridging starts at surface separation LHA ≈ 50 nm in the 1 mM salt and at a separation D < L, the total bridging energy per unit area Ebridge(D) ≈ [(L − D)/l](2fε/s2). Applying the Derjaguin approximation the force due to bridging is Fbridge(D)/R = 2πEbridge(D). Substituting f = 0.2, LHA = 50 nm, D = 40 nm (compression of the layer by 10 nm), l = 1 nm (size of an HA disaccharide unit), ε = 1 kBT, and spacing between each polymer s = 2Rg = 400 nm, the bridging force is Fbridge(D=40 nm)/R ≈ 0.6 μN/m. This value is smaller than the magnitude of hydrodynamic force FH/R at D ≈ 50−40 nm. Even taking into account the rather rough nature of the calculation and our assumptions, we see that the hydrodynamic repulsion is of order of, or larger than, the bridging attraction in the range D < 50 nm so that it can cancel out the attractive bridging force in the overall force profile. Indeed, both effects lead to forces that are small compared with the noise level in our system. It is for these reasons that we believe that no net bridging attraction is observed on approach of the surfaces. On the other hand, on separation/retraction, the hydrodynamic force is attractive as bridging force and would be a component of the overall adhesion in the out-profile Figure 6. Nonetheless, its contribution is at most ca. 15 μN/m (for D = 10 nm), which is small compared with the total measured value (F/R ∼ 0.1−0.2 mN/m) induced by the stretching and detachment of polymers, as explained below. Other effects such as van der Waals attraction have been ignored as most of them are negligible at the onset of bridging. Because of the weakness of bridging force on surface compression, it is usually difficult to measure specifically, excluding effects from other forces. But it may be relevant to the dramatic effect of divalent ions on condensating the grafted HA layer as observed by Albersdorfer et al.25

reasonable value for this HA in pure water,51,52 the mobile counterion concentration in the HA layer is n ≈ f [(MHA/Mds)/ (ARg)] ≈ 8.4 × 1021−16.5 × 1021 m−3 (equivalent to a counterion concentration of 1.5 × 10−5−3 × 10−5 M, comparable with the effective ion concentration in ultrapure water) where f is the degree of ionization (f ≈ 0.2 from literature47,48) and Mds the molecular weight of disaccharide unit. Therefore, the expected counterion osmotic pressure is Πc/i ≈ nkBT ≈ 35−68 N/m2. As shown in the Supporting Information calculation, a compression of ΔD = 50 nm from 100 to 50 nm results in F/R ≈ 0.01−0.02 mN/m, which is considerably less than the double-layer repulsion F/R(D=50 nm) ≈ 0.1 mN/m in water as in Figure 3a. This explains why the force profiles with HA in pure water are so similar to those without HA. The effect of 1 mM Mg2+ on the HA layer thickness is also considered. This salt concentration is more than 30-fold higher than the counterion concentration (1.5 × 10−5−3 × 10−5 M) in the in-water HA layer so that it could osmotically compress the HA into a thinner layer. The thickness L of surface-attached polyelectrolyte layers has been previously measured to vary with ion concentration c in solution as L ∼ c−α,57 where the exponent α is in the range ∼0.2−0.3.41,42 A value of α = 0.2 could result in a ∼50% reduction in the HA layer thickness L. Applying this equation to our model system, a HA layer of 100 nm thickness in water will be “squeezed” to 50 nm on addition of the 1 mM salt. This 50 nm thickness in salt is consistent with the measured layer thickness in both approach and retraction force profiles in Mg2+ solution, as in Figures 4a and 5, where double-layer repulsion is largely screened and the polymerinduced repulsion becomes measurable, that is, F/R > 0.01 mN/m, from the separation D ∼ 50 nm. The proposed model system in Figure 7a not only explains the water profiles as above but may also account for why, as in Figure 5, the F/R is ∼0.01 mN/m at D = 50 nm for HA layer in 1 mM MgCl2 salt solution: here the double-layer repulsion is very short ranged, so only the compression of the HA layer, dominated by the counterion osmotic pressure, contributes to F/R. The calculation above has given F/R within the range of 0.01−0.02 mN/m for Rg ≈ 200−250 nm, which fits the measured observation 0.01 mN/m MgCl2 in-profile in Figure 5. This applies to ΔD = 50 nm from separation 100 to 50 nm with the assumption that the compression of counterions commences from 100 nm separation, the same as the layer thickness LHA. If taking into account the increasing value of n as the HA layer is compressed, n would increase with a factor of 2, within which the F/R would still be in line with the measured value. Hydrodynamic Force. In approach and retraction profiles, the hydrodynamic force can be estimated. Such a hydrodynamic force between the two surfaces results from the flow of liquid in and out of the gap. Previous studies of the influence of hydrodynamic forces indicate a distance-dependent repulsion during the probe approach as fluid is squeezed out of the gap, while on retraction an attraction can be induced as the surrounding fluid is drawn into the gap.58 The magnitude of the hydrodynamic force, either attractive or repulsive, depends on the nature of the surface, surface separation, and especially the relative velocity.59,60 The hydrodynamic force FH during approach and retraction between a sphere of radius R and a flat surface can be simplified as FH/R = 6πηRν/D, where η is the solution viscosity and ν the approach (ν > 0) or retraction (ν < 0) velocity.61,62 It may be estimated that at D = 50 nm, for 12199

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The retraction profiles in Figure 6 demonstrate the adhesion/adsorption of HA to the opposing silica surface, which we believe is due to the bridging effect of divalent counterions Mg2+. This bridging effect is manifested as several continual wells when HA polymers are forced to peel off the silica surface during surface separation. The wells can be associated with the stretching and detachment of polymer chains, as illustrated in upper right inset of Figure 6. The depth of the four main wells, marked as shaded regions A, B, C, and D, indicate the maximum adhesion (in a given separation run) in the continuous pulling, and it is instructive to consider this below. Well A occurs at separation D = 50−75 nm, somewhat larger than the ∼50 nm thickness of the HA layer in the 1 mM salt. Compared to wells B, C, and D, it is the deepest, probably because most stretched HA bridges, composed mostly of loops and tails as in Figure 7a, between two surfaces are broken here. On retraction, work is done stretching the polymer loops and tails until the elastic energy stored in the stretched segments exceeds the total adsorption energy of the adsorbed segments at which point the adsorbed segments are pulled away from the bare silica probe, as the chain relaxes elastically to its original conformation. It has been argued theoretically that when the retraction rate is faster than the monomer exchange rate at a monomer−surface contact site, the magnitude of the pulling force determines the detachment of monomer from the surface.63 The associated force profile will be discontinuous, comprising individual detachment events and that, on average, the magnitude of the detachment force decreases with consecutive detachments as fewer loops make contact with the opposing surface. This could be the reason why depths of wells A, B, C, and D decrease in a single out-profile, and the irregular sawtooth pattern is a consequence of the sequential stretching of the HA loops. These wells were also observed in the force measurements between HA layer and tartrate crystals in similar divalent counterion conditions.26 Based on these measured bridging forces, broader research is needed to examine the influence of possible specific interactions between divalent ions and silica on bridging. Although many literatures have reported on such interactions, there is still no consensus on quantitative determination of the mechanism, partly due to the challenging experimental conditions. Meanwhile, further work needs to be explored to understand the effect of divalent ion type, counterion concentration, and other parameters with a view to determine the underpinning mechanism of the divalent ion bridging.

Article

ASSOCIATED CONTENT

S Supporting Information *

Figures S1 and S2. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail [email protected] (L.J.). *E-mail [email protected] (J.K.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Dr. Robert Jacobs for the ellipsometric measurements and Dr. Robert Thomas for useful discussions. We thank the EPSRC and the Physical and Theoretical Chemistry Laboratory (Oxford University) for providing support for this work and the Royal Society for a University Research Fellowship (Simon Titmuss). This research is supported by the Petroleum Research Fund (grant 45964-AC7), European Union sixth Framework program Specific Targeted Research Project Nanointeract, National Natural Science Foundation of China (grants 21204102 and U1262102), and the Fundamental Research Funds for the Central Universities (13CX02061A and 13CX05018A). Jacob Klein thanks the Israel Science Foundation and the European Research Council for support of this work (ERC Advanced Grant HydrationLube).

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REFERENCES

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CONCLUSIONS In summary, the normal interactions between negatively charged polyelectrolyte HA and the negatively charged silica surfaces, with and without the divalent counterions, were directly measured. It was found that the divalent ion Mg2+ can mediate the adsorption of HA to a similarly charged opposing surface, resulting in the change of net interaction from an osmotic repulsion to an adhesive attraction. These force measurements thus provide direct experimental support for the previous simulation and modeling of divalent ions’ role in modulating polyelectrolyte’s interaction with similarly charged surfaces. To our knowledge, this is also the first time the bridging role of divalent counterions between grafted HA and similarly charged surface are measured quantitatively in surface force experiment. 12200

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