Ion-Specific Conformational Behavior of Polyzwitterionic Brushes

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Ion-Specific Conformational Behavior of Polyzwitterionic Brushes: Exploiting It for Protein Adsorption/Desorption Control Tao Wang,† Xiaowen Wang,† Yunchao Long,† Guangming Liu,*,† and Guangzhao Zhang*,‡ †

Department of Chemical Physics, Hefei National Laboratory for Physical Sciences at the Microscale, University of Science and Technology of China, Hefei, P. R. China 230026 ‡ Faculty of Materials Science and Engineering, South China University of Technology, Guangzhou, P. R. China 510640 S Supporting Information *

ABSTRACT: The conformation of polyzwitterionic brushes plays a crucial role in the adsorption/desorption of proteins on solid surfaces. By use of quartz crystal microbalance with dissipation (QCM-D) and surface plasmon resonance (SPR), we have systematically investigated the conformational behavior of poly(sulfobetaine methacrylate) (PSBMA) brushes as a function of ionic strength in the presence of different ions. The frequency change demonstrates that the effectiveness of anions to weaken the inter/intrachain association and to enhance the hydration of the grafted chains increases from kosmotrope to chaotrope in the low ionic strength regime, but the ordering of anions is almost reversed at the high ionic strengths. The dissipation change indicates that some heterogeneous structures are formed inside the brushes in the presence of chaotropic anions with the increase of ionic strength. In SPR studies, the change of resonance unit (ΔRU) with ionic strength is determined by the balance between the increase of thickness and the decrease of refractive index of the brushes. No anion specificity is observed in the SPR measurements because ΔRU is insensitive to the coupled water molecules inside the brushes. For the control of protein adsorption/desorption, our studies show that the brushes can more effectively resist the protein adsorption in the presence of a more chaotropic anion and a more chaotropic anion can also more effectively induce the protein desorption from the surface of the brushes. In addition, no obvious cation specificity can be observed in the conformational change of the brushes in either QCM-D or SPR measurements.

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hydrated ions are called chaotropes.20 A few previous studies have demonstrated that the solution properties such as viscosity and solubility of polyzwitterions are strongly influenced by ion type.17,18,21−23 However, very few investigations have been conducted to study the ion specificity in the conformational behavior of polyzwitterionic brushes though the ion-specific conformation of the brushes is crucial for their interfacial properties.24,25 The mechanism of how ion type influences the conformational behavior of polyzwitterionic brushes is still unclear. Recently, Chang et al. showed that polyzwitterionic brushes would lose their ability to resist the protein adsorption at low ionic strengths, even though the brushes have good protein resistance properties at high ionic strengths.26 This fact indicates that the protein adsorption/desorption could be controlled by the conformation of polyzwitterionic brushes. On the other hand, Jiang et al. reveal that the strength of interaction between the ions and the zwitterions is significantly dependent on ion type, which means that the conformation of polyzwitterionic brushes could be modulated by ion type.27,28 Thus, it is anticipated that the protein adsorption/desorption

INTRODUCTION It is well-known that polyzwitterionic brush-modified solid surfaces can effectively resist protein adsorption.1−3 Generally, the hydration layer of polyzwitterionic brushes formed via electrostatic interactions or hydrogen bonds is thought to be responsible for their excellent protein resistance properties.4,5 On the other hand, polyzwitterions usually have the so-called antipolyelectrolyte effect.6−8 For example, polyzwitterionic brushes would adopt a collapsed conformation at a low ionic strength with a low extent of hydration due to the electrostatic inter/intrachain association.9 In contrast, polyzwitterionic brushes would exhibit an extended conformation at a high ionic strength with a relatively high extent of hydration because the electrostatic inter/intrachain dipole−dipole interaction is weakened by the increase of ionic strength.10 Considering that the hydration of polyzwitterionic brushes is related to their conformation, their protein resistance properties should also be correlated with the conformation of the brushes. It has been widely recognized that the conformation of polyzwitterionic brushes is affected by ionic strength,10−12 pH,13,14 and temperature.15,16 Indeed, the conformation of polyzwitterions is also influenced by ion type.17,18 In general, ions can be categorized as kosmotropes and chaotropes in light of the strength of ionic hydration.19 The strongly hydrated ions are usually defined as kosmotropes, whereas the weakly © XXXX American Chemical Society

Received: March 20, 2013 Revised: April 29, 2013

A

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will not be influenced by the small change of pH induced by the variation of ionic strength. QCM-D Measurements. QCM-D and the AT-cut quartz crystals were from Q-sense AB.31 The quartz crystal resonator with a fundamental resonant frequency of 5 MHz was mounted in a fluid cell with one side exposed to the solution. The resonator has a mass sensitivity constant (C) of 17.7 ng cm−2 Hz−1.32 When a quartz crystal is excited to oscillate in the thickness shear mode at its fundamental resonant frequency (f 0) by applying a RF voltage across the electrodes near the resonant frequency, a small layer added to the electrodes induces a decrease in resonant frequency (Δf) which is proportional to the mass change (Δm) of the layer. In vacuum or air, if the added layer is rigid, evenly distributed and much thinner than the crystal, Δf is related to Δm and the overtone number (n = 1, 3, 5, ....) by the Sauerbrey equation33

on the surface of polyzwitterionic brushes could be controlled by ion type. In the present work, we have chosen poly(sulfobetaine methacrylate) (PSBMA) as a typical system for polyzwitterions and have systematically investigated the ion-specific conformational behavior of PSBMA brushes using quartz crystal microbalance with dissipation (QCM-D) and surface plasmon resonance (SPR). Furthermore, we have also exploited the ionspecific conformation to control the protein adsorption/ desorption on the surface of PSBMA brushes. Our aim is to understand the mechanism of how the conformational behavior of polyzwitterionic brushes is influenced by ion type and how the ion-specific conformation can be used to control the protein adsorption/desorption on the surface of polyzwitterionic brushes.

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Δm = −

EXPERIMENTAL SECTION

ρq lq Δf f0 n

= −C

Δf n

(1)

where f 0 is the fundamental frequency and ρq and lq are the specific density and thickness of the quartz crystal, respectively. The dissipation factor is defined by31

Materials. Sulfobetaine methacrylate (SBMA) (97%), 2,2′dipyridyl (≥97%), and methanol (HPLC, ≥99.9%) were purchased from Aldrich and used as received. ω-Mercaptoundecyl bromoisobutyrate was purchased from Beijing HRBio Biotechnology Co. and used as received. All the salts (99.99%, metals basis) were purchased from Aladdin and used as received. When studying anion specificity, we employed sodium salts so that the influence of cations was constant; similarly, chloride salts were used when investigating cation specificity. Copper(II) bromide (CuBr2, 99%) was purchased from Sinopharm and used as received. Copper(I) bromide (CuBr) was prepared from CuBr2 by reacting with sodium sulfite, then washed with glacial acetic acid, ethanol, and diethyl ether, and dried under vacuum for 12 h before use.29 Lysozyme (pI ∼ 11) purchased from Bio Basic Inc. and bovine serum albumin (BSA, pI ∼ 4.7) purchased from Hualvyuan Biotechnology Co. were purified by dialysis in water and by lyophilization to remove the inorganic salts. The water used was purified by filtration through a Millipore gradient system after distillation, giving a resistivity of 18.2 MΩ cm. Preparation of PSBMA Brushes. The gold-coated sensors were cleaned by using a piranha solution composed of 1 part H2O2 and 3 parts H2SO4 at 70 °C for ∼15 min, rinsed with Milli-Q water, and blown dry with N2 before use. The monolayer of ATRP initiator was prepared by placing the sensors in a 5.0 mM solution of ωmercaptoundecyl bromoisobutyrate in anhydrous ethanol for ∼24 h. Then the sensors were rinsed with ethanol, dried with flowing nitrogen, and used immediately for the following surface-initiated polymerization. PSBMA brushes were prepared by using surfaceinitiated atom transfer radical polymerization (SI-ATRP). Typically, SBMA (2.8 g, 10.0 mmol) and 2,2′-bipyridine (0.16 g, 1.0 mmol) were dissolved in 30.0 mL of water/methanol mixture (1:1, v/v). After the solution was stirred at 25 °C under N2 for 30 min, CuBr (0.07 g, 0.5 mmol) and CuBr2 (0.01 g, 0.05 mmol) were added under N2 and then continued for another 30 min stirring. Afterward, the initiatormodified sensors were placed inside the flask under the protection of N2 for polymerization at 25 °C for ∼24 h, followed by washing with water and methanol, and then soaked in 0.5 M NaBr solution overnight to remove ligand and unreacted monomer. The detailed characterization of the preparation of PSBAM brushes by X-ray photoelectron spectroscopy (XPS) and contact angle is shown in Figure S1 (Supporting Information). According to the previous study, the distance (d) between two grafting sites was estimated to be ∼0.5 nm on the basis of packing density of the self-assembled initiator monolayer on the gold surface.30 The thickness (t) of grafted PSBMA layer was ∼18 nm obtained from the ellipsometry measurements in the aqueous solution at a high ionic strength (Figure S2). Therefore, the grafted PSBAM chains should form brushes since t is much larger than d. Note that the repeat unit of PSBMA is composited of positively charged quaternary ammonium group and negatively charged sulfonate group. These two charged groups completely disassociate in aqueous solutions; that is, the overall net charge of PSBMA brushes

D=

Ed 2πEs

(2)

where Ed is the energy dissipated during one oscillation and Es is the energy stored in the oscillating system. The measurement of ΔD is based on the fact that the voltage over the crystal decays exponentially as a damped sinusoidal when the driving power of a piezoelectric oscillator is switched off.31 By switching the driving voltage on and off periodically, we can simultaneously obtain a series of changes of the resonant frequency and the dissipation factor. In the present study, all the results obtained were from the measurements of frequency and dissipation at the third overtone (n = 3). For the investigation of conformational behavior of PSBMA brushes, the changes in Δf and ΔD induced by the conformational change can be extracted by subtracting the background response of the blank resonator in corresponding salt solutions. In the protein adsorption/desorption measurements, the concentration of protein was fixed at 1.0 mg mL−1. All the experiments were performed at 25 ± 0.02 °C. SPR Measurements. When light crosses an interface from one media with a higher refractive index (e.g., glass) to another one with a lower refractive index (e.g., water), the light would be totally reflected from the interface (i.e., total internal reflection) if the angle of incidence is larger than a critical angle.34 Although the incident light is totally reflected, an evanescent wave will penetrate into the media with the lower refractive index.35 When the interface is coated with a thin layer of gold, the surface plasmon resonance will be generated at a certain incident angle (SPR angle or θSPR) by coupling the incident light to the freely oscillating electrons within the gold surface, which is detected as a minimum intensity of the reflected light beam.35 If the gold surface is coated with a polymer layer whose thickness (df) is less than the effective penetration depth of the evanescent wave, the change of θSPR is related to df and the refractive index of polymer layer (nf) by36−38

⎛ ε ε ⎞2 ⎛ ε ⎞⎛ ε − εm ⎞ ⎛ 2π ⎞ 1 ⎜ ⎟d Δ(sin θSPR ) ≈ ⎜ m s ⎟ ⎜1 − s ⎟⎜ f ⎟ f εf ⎠⎝ εs − εm ⎠ εs − εm ⎝ λ ⎠ ⎝ εm + εs ⎠ ⎝ (3) where εm, εs, εf, and λ are the real part of dielectric constant of gold film, the dielectric constant of solution, the dielectric constant of polymer layer, and the wavelength of incident light, respectively. Here, εf equals the square of nf (i.e., εf = nf2). In the present study, SPR measurements were carried out on a Biacore X (Biacore AB). The gold-coated sensor chip is attached to a glass prism with a silicone opto-interface between the sensor chip and the prism to ensure good matching of their refractive indices. Light from a near-infrared light-emitting diode (λ = 760 nm, p-polarized) is focused through the prism onto the sensor chip surface in a wedgeB

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shaped beam to give a fixed range of incident light angles. Light reflected from the sensor chip is monitored by a linear array of lightsensitive diodes covering the range of incident light angles. The response of θSPR is measured in resonance unit (RU), where 1000 RU correspond to an angle change of ∼0.1°. The change in ΔRU induced by the conformational change can be extracted by subtracting the background response of the blank resonator in corresponding salt solutions. The concentration of protein was fixed at 1.0 mg mL−1 in the protein adsorption/desorption measurements. All the experiments were performed at ∼25 °C. Other Measurements. The thickness of PSBMA brushes was measured by a spectroscopic ellipsometry (M-2000V, J.A. Woollam) at ∼25 °C. The ellipsometric data were acquired with an incident angle of 70° in air and with an incident angle of 65° in salt solutions. All the ellipsometric data were fit using Cauchy layer model to get the thickness of the brushes.39 X-ray photoelectron spectroscopy (XPS) measurements were conducted on an ESCALAB-250 spectrometer with a monochromatic Al Kα X-ray source (hν = 1486.6 eV), and all binding energies were obtained by setting the C1s contamination line at 284.6−285.0 eV as the reference. All contact angle measurements were performed using a KSV (Helsinki, Finland) CAM 200 contact angle goniometer at ∼25 °C.

solution due to the partial disassociation of the inter/intrachain dipole−dipole associates. Thus, some counterions of the positively or negatively charged groups are trapped inside the brushes. In the range of I < 1.0 × 10−3 M, the ion concentration outside the brushes might be lower than that inside the brushes. The added ions are difficult to enter into the brushes because of the relatively high osmotic pressure inside the brushes. Therefore, only the inter/intrachain association at the outer part of the brushes are weakened by the increase of I, and the extent of hydration of the grafted chains only slightly increases with I accompanied by a limited conformational change of the brushes.40 This is why Δf only exhibits a slight decrease with log I at I < 1.0 × 10−3 M. As I increases further from 1.0 × 10−3 M, the ion concentration may be larger than that inside the brushes. Thus, the added ions could enter into the brushes and the ionic strength inside the brushes would increase as I of the bulk solution increases. As a result, the electrostatic inter/intrachain dipole−dipole interaction inside the brushes is weakened and is gradually dominated by the interaction between the charged groups and water molecules with the increase of I. In other words, the extent of hydration of the grafted chains should increase with I, giving rise to a decrease of Δf with log I. Interestingly, Δf for SO42− exhibits a quite different change compared with other anions. Specifically, Δf only slightly decreases with log I at I < 1.0 M, and then it increases with the further increase of log I. The initially slight decrease of Δf with log I indicates a small increase in the extent of hydration of the grafted chains due to the weakening of inter/intrachain association and the strengthening of water-charged group interaction. When I is higher than 1.0 M, the increase of Δf with log I indicates the occurrence of dehydration of the grafted chains, which is opposite to the observation for other anions. However, this is understandable because the divalent SO42− is a strong kosmotrope, and it would compete for the water molecules in the hydration layer of the grafted chains.20 More specifically, the strongly hydrated SO42− anions would insert in the hydration layer of the grafted chains and compete for water molecules, making the water molecules unavailable in solvating the grafted chains.20 Thus, the amount of coupled water molecules of the grafted chains may decrease with I at the high ionic strengths in the presence of SO42−. The increase of Δf with log I at the high ionic strengths suggests that the hydration of the grafted chains induced by the weakening of inter/ intrachain association is dominated by the dehydration of the grafted chains due to the competing effect of SO42−. Likewise, Δf exhibits a similar change in the presence of another divalent kosmotropic anion (i.e., CO32−) (Figure S3). More interestingly, anion specificity is observed in the change of Δf. Figure 2a shows the slope of the plot of Δf versus log I decreases following the series SO42− > Cl− > Br− > ClO3− > SCN− > ClO4− in the range of I between 0.01 and 0.1 M, indicating that Δf exhibits a more rapid decrease as the anion changes from SO42− to ClO4− along the series in the low ionic strength regime. However, the slope of the plot of Δf versus log I decreases following an almost reverse series SCN− > ClO4− > ClO3− > Br− in the high ionic strength regime between 1.0 and 5.0 M (Figure 2b), which suggests that Δf decreases more rapidly as the anion changes from SCN− to Br− along the series. SO42− and Cl− are not included in the series in the high ionic strength regime as Δf increases with log I in the presence of SO42−, and no linear relationship exists between Δf and log I in the presence of Cl−.

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RESULTS AND DISCUSSION QCM-D Studies. To understand the mechanism of how ion type influences the conformational change of PSBMA brushes, we have first investigated the ion-specific conformational behavior of the brushes using QCM-D. Figure 1 shows the

Figure 1. Shift in frequency (Δf) for PSBMA brushes as a function of logarithmic ionic strength (log I) in the presence of different anions with Na+ as the common cation.

shift in frequency (Δf) for PSBMA brushes as a function of logarithmic ionic strength (log I) in the presence of different anions with Na+ as the common cation. It is known that Δf is indicative of mass change of the brushes during the conformational change, e.g., the increase/decrease of amount of the coupled water molecules inside the brushes induced by the hydration/dehydration of the grafted chains. For all the anions with the exception of SO42−, Δf only slightly decreases with log I in the range of I < 1.0 × 10−3 M, and then it exhibits a gradual decrease with log I at I > 1.0 × 10−3 M. There have been two competing factors to influence the conformation of PSBMA brushes. One is the electrostatic inter/ intrachain dipole−dipole interaction, which favors the collapse of PSBMA brushes and reduces the extent of chain hydration. The other is the interaction between water molecules and the charged groups on the zwitterionic units, which counteracts the inter/intrachain association and enhances the extent of hydration of the grafted chains. The dry thickness of PSBMA brushes is ∼10 nm in air. The thickness increases to ∼14 nm when immersing the brushes in water. This fact implies that the brushes adopt a partially swollen structure in the salt-free C

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The polarizability of the anions increases following the series SO42− < Cl− < Br− < ClO3− < SCN− < ClO4−, so that the ionic dispersion interaction between the quaternary ammonium group and the anions should also increase from SO42− to ClO4− along this series. This would lead to a similar conclusion with that on the basis of the concept of matching water affinities. In Figure 3, the change of ΔD exhibits a more complicated behavior in comparison with that of Δf. It is well-known that

Figure 2. Slope of the plot of Δf versus log I for the different anions obtained from Figure 1: (a) in the ionic strength regime between 0.01 and 0.1 M and (b) in the ionic strength regime between 1.0 and 5.0 M.

As mentioned above, the change of Δf is related to the hydration/dehydration of the grafted chains, which is influenced by the electrostatic inter/intrachain association. If an anion can more effectively weaken the inter/intrachain association, a faster hydration and a more rapid decrease of Δf would be resulted. It is suggested that the quaternary ammonium on the zwitterionic unit is a weakly hydrated group.41,42 According to Collins’ concept of matching water affinities, the strength of interaction between the quaternary ammonium group and the anions should increase following the series SO42− < Cl− < Br− < ClO3− < SCN− < ClO4− because the extent of hydration of the anions decreases from SO42− to ClO4− along this series.42 Hence, as the anion changes from SO42− to ClO4− along the series, it can more easily form ion pair with the quaternary ammonium group. A stronger interaction between the quaternary ammonium group and the anion would cause a weaker inter/intrachain dipole−dipole interaction, thereby leading to a stronger interaction between the negatively charged sulfonate group and the water molecules, resulting in a higher extent of hydration of the grafted chains. Thus, Δf exhibits a more rapid decrease with log I in the low ionic strength regime as the anion changes from SO42− to ClO4−. In the high ionic strength regime, almost all the inter/ intrachain dipole−dipole associates are disassociated. Meanwhile, the electrostatic attraction between the quaternary ammonium group and the anions is also strongly weakened. In other words, the ion pairs might gradually disassociate with the increase of I and the free dipoles may not form the inter/ intrachain associates again due to the high ionic strength. Therefore, the disassociation of ion pairs is favorable for the interaction between the quaternary ammonium group and the water molecules and is thereby favorable for the hydration of the grafted chains. A stronger ion pair is more difficult to disassociate accompanied by a slower hydration, causing a slower decrease of Δf with log I. This is why the slope of the plot of Δf versus log I decreases following the series SCN− > ClO4− > ClO3− > Br− in the high ionic strength regime. From the discussion above, the specific anion effect on the conformational change and on the hydration of the grafted chains is dominated by the formation and the disassociation of the ion pairs in the low and high ionic strength regimes, respectively. In addition, the ionic dispersion force may also have some influences on the observed specific anion effect.43−46

Figure 3. Shift in dissipation (ΔD) for PSBMA brushes as a function of logarithmic ionic strength (log I) in the presence of different anions with Na+ as the common cation.

ΔD reflects the structure of polymer brushes, e.g., the collapse/ swelling, the homogeneity/heterogeneity, and the compactness/looseness of polymer brushes.40 For all the anions, ΔD keeps almost constant with log I in the range of I < 1.0 × 10−3 M, indicating that no obvious structural or conformational change of the brushes occurs in this low ionic strength regime. This is because the added ions are difficult to enter into the brushes at the low ionic strengths. As I increases further, ΔD gradually increases with log I in the presence of Cl−, but it exhibits an initial increase and a subsequent decrease with log I in the presence of Br−, ClO3−, SCN−, and ClO4−. For SO42−, ΔD only slightly increases with log I in the range of I from 1.0 × 10−3 to 1.0 M, and then it decreases with the further increase of log I. The increase of ionic strength would weaken the inter/ intrachain association, leading to a swelling of the brushes. Thus, ΔD should increase with log I, like the change of ΔD in the presence of Cl−. As I increases from 1.0 × 10−3 to 1.0 M, the small increase in ΔD for SO42− is also attributed to the slight swelling of the brushes induced by the weakening of inter/intrachain association. However, the extent of hydration of the grafted chains decreases with I in the high ionic strength regime in the presence of SO42− (see Figure 1), suggesting that the strength of interaction between the grafted chains and water molecules decreases with I. Therefore, the energy loss due to the friction between the brushes and the water molecules should decrease with I during the oscillation of resonator.31 This explains why ΔD decreases with log I in the range of I > 1.0 M in the presence of SO42−. Also, the decrease of ΔD with log I in the high ionic strength regime indicates that the effect of swelling of the brushes on the shift of ΔD is dominated by the decrease of friction energy. Similar to the Δf, ΔD also exhibits a similar change in the presence of CO32− (Figure S3). In the cases of Br−, ClO3−, SCN−, and ClO4−, ΔD has a similar maximum value of ∼4 × 10−6, but the ionic strength at which ΔD has the maximum value increases following the series ClO4− < SCN− < ClO3− ≈ Br−. That is, ΔD increases more rapidly following the series Br− ≈ ClO3− < SCN− < ClO4−. It is known that SO42− is a kosmotropic anion, whereas Br−, ClO3−, D

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Scheme 1. Illustration for the Anion-Specific Conformational Behavior of PSBMA Brushes with Increasing Ionic Strength

SCN−, and ClO4− are chaotropic anions.20 Cl− locates between kosmotropes and chaotropes in the Hofmeister series.20 According to the law of matching water affinities, the chaotropic anions can form ion pair with the weakly hydrated quaternary ammonium group. In contrast, the counterion Na+ for the anions may not form ion pair with the negatively charged sulfonate group because Na+ locates between kosmotropes and chaotropes in the Hofmeister series.41 Consequently, as I increases, some zwitterionic chain segments in the brushes may become temporary negatively charged chain segments due to the formation of ion pairs between the quaternary ammonium groups and the chaotropic anions. This would cause the grafted chains to form some locally heterogeneous structures inside the brushes induced by the local electrostatic repulsion between the negatively charged sulfonate groups. The formation of heterogeneous structures would result in a rapid increase of ΔD, as reflected by the fact that ΔD for the chaotropic anions increases more rapidly with log I compared with that of Cl−. Because the extent of hydration of the anions decreases following the series Br− > ClO3− > SCN− > ClO4−, the anions can more easily form ion pair with the quaternary ammonium group from Br− to ClO4− along the series. Thus, the grafted chains are easier to form the heterogeneous structures as the anion changes from Br− to ClO4−, as reflected by the result that ΔD exhibits a more rapid increase as the anion changes from Br− to ClO4−. Above a certain ionic strength, the association of ion pair between the quaternary ammonium group and the chaotropic anions would be gradually weakened with the increase of I. As a result, the temporary negatively charged chain segments might gradually become zwitterionic chain segments again with the increasing I, which would produce more homogeneous brushes. Therefore, ΔD decreases again with log I after the ionic strength reaches the maximum value. In the presence of NaCl, both Na+ and Cl− cannot form ion pair with the charged groups on the zwitterionic unit. Thus, the change of ΔD should be

dominated by the swelling of the brushes, as reflected by the monotonous increase of ΔD with log I. It should be noted that the decrease of ΔD with log I in the presence of chaotropic anions and SO42− in the high ionic strength regime is not attributed to the collapse of the brushes because the thickness of the brushes monotonously increases with log I in the whole range of ionic strength (Figure S2). The anion-specific conformational behavior of PSBMA brushes with increasing ionic strength is illustrated in Scheme 1. The plot of ΔD vs −Δf in the presence of different anions is shown in Figure S4, which can also provide some information about the conformational behavior of PSBMA brushes. The anion-specific conformation of the brushes will lead to an anion-specific hydration of the grafted chains, which can be used to control protein adsorption/desorption on the surface. To exploit the anion-specific conformation to control the protein adsorption on the surface of PSBMA brushes, we have investigated the adsorption of lysozyme on the surface of the brushes as a function of ionic strength in the presence of two typical anions, i.e., ClO4− and Cl− (Figure 4). For all the monovalent anions used in this study, ClO4− has the weakest strength of hydration, whereas Cl− has the strongest strength of hydration. Therefore, ClO4− and Cl− would exhibit very different interactions with the PSBMA brushes, leading to different extents of hydration of the grafted chains (Figure 1) and a big difference in the conformational change of the brushes (Figure 3). Consequently, it is expected that the brushes should also exhibit different protein resistance properties between ClO4− and Cl−. We did not choose to use SO42− to investigate the protein adsorption as it might bridge two positively charged groups between the brushes and the protein molecules.47,48 In Figure 4, the amounts of adsorbed lysozyme at different ionic strengths are normalized by the amount of saturated adsorption of lysozyme in the saltfree solution based on the frequency change. For both Cl− and ClO4−, the brushes can effectively resist the protein adsorption E

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in the range of I between 0.1 and 3.0 M, but they can adsorb on the surface at I < 0.1 M. This fact indicates that the solubility of lysozyme induced by the variation of ionic strength may not have an obvious influence on the protein adsorption. The salt type may also have an influence on the solubility of lysozyme (i.e., Hofmeister effect). As lysozyme is a positively charged protein in water, ClO4− should be more effective in salting out of the protein molecules than Cl−.49,50 That is, the solubility of lysozyme in the presence of ClO4− might be lower than that in the presence of Cl−. However, Figure 4 shows the amount of adsorbed lysozyme in the presence of ClO4− is lower than that in the presence of Cl− at the same ionic strength in the range of I between 1.0 × 10−3 and 0.1 M, suggesting that the solubility of lysozyme induced by varying salt type may not influence the protein adsorption. Actually, Cl− and ClO4− also have different abilities to induce the protein desorption from the surface. In Figure 5, lysozyme

Figure 4. Relative adsorption of lysozyme on the surface of PSBMA brushes as a function of logarithmic ionic strength (log I) in the presence of ClO4− and Cl−. Here, the amounts of adsorbed lysozyme at different ionic strengths are normalized by the amount of saturated adsorption of lysozyme in the salt-free solution based on the frequency change.

in the range of I > ∼0.1 M, whereas the protein molecules can adsorb onto the surface at I < ∼0.1 M. Particularly, the brushes almost lose their protein resistance properties at I < ∼1.0 × 10−3 M. Moreover, the brushes in the presence of ClO4− can more effectively resist the protein adsorption than Cl− at the same ionic strength in the range of I between 1.0 × 10−3 and 0.1 M. As can be seen from Figure 1, for both Cl− and ClO4−, the grafted chains are highly hydrated at I > 0.1 M, but the extent of hydration of the grafted chains at I < 1.0 × 10−3 M is much lower than that at the high ionic strengths. Therefore, the brushes can effectively resist the protein adsorption at the high ionic strengths because the hydration layer around the brushes will create a barrier to prevent the adsorption of protein molecules. In contrast, the brushes would lose the protein resistance properties at the low ionic strengths due to the low extent of hydration of the grafted chains. Without the protection of the hydration layer, the protein molecules may adsorb onto the surface driven by hydrophobic interactions. In the range of I between 1.0 × 10−3 and 0.1 M, the extent of hydration of the grafted chains in the presence of ClO4− is higher than that of Cl− at the same ionic strength. As a result, the brushes can more effectively resist the protein adsorption in the presence of ClO4− than that of Cl−. In short, if an anion can induce a weaker inter/intrachain association of the brushes, it would lead the brushes to have a higher effectiveness to resist the protein adsorption due to the higher extent of hydration of the grafted chains. Thus, the effect of anion on the protein adsorption should follow the trend of the anions shown in Figure 1 (Figure S5). Besides, the brushes can also more effectively resist the BSA adsorption in the presence of ClO4− than that in the presence of Cl− (Figure S6), indicating that the protein adsorption is dominated by the hydration of the grafted chains instead of the electrostatic interactions since lysozyme and BSA are respectively positively and negatively charged protein in water. On the other hand, the solubility of lysozyme may also influence the protein adsorption. We found that lysozyme becomes insoluble at an ionic strength of 5.0 M, which may significantly increase the protein adsorption on the surface. To avoid the influence of solubility of lysozyme on the protein adsorption, the highest ionic strength used in the protein adsorption experiments was 3.0 M. At this ionic strength, the protein molecules can well dissolve in the solution. Figure 4 shows that the protein molecules cannot adsorb on the surface

Figure 5. Time dependence of shift in frequency (Δf) for the adsorption of lysozyme on the surface of PSBMA brushes and for the following desorption of lysozyme from the surface induced by the addition of 1.0 M salt solutions: (a) NaCl and (b) NaClO4.

is adsorbed onto the surface of PSBMA brushes first in the saltfree solution for ∼10 min, followed by a rinse with water. Clearly, the rinse with water cannot induce the protein desorption from the surface. Afterward, a 1.0 M NaCl (Figure 5a) or NaClO4 (Figure 5b) solution is injected into the QCMD cell for ∼10 min, followed by a rinse with water again. Obviously, the addition of the salt solutions can induce the protein desorption from the surface. The addition of salt solution enhances the extent of hydration of the grafted chains by strengthening the interaction between the zwitterionic chains and the water molecules, which would weaken the binding strength of protein molecules on the surface due to the competition between the protein molecules and the water molecules, thereby causing the protein molecules to leave the surface induced by Brownian motion. Nevertheless, the addition of NaCl solution cannot remove all the protein molecules from the surface, as indicated by the fact that Δf cannot return to the baseline in water. In contrast, the protein molecules can be removed completely from the surface by F

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using the NaClO4 solution. Namely, ClO4− can more effectively induce the protein desorption than Cl−. This result is consistent with the observation that ClO4− can lead to a higher extent of hydration of the grafted chains than Cl− at the same ionic strength (Figure 1). The higher extent of hydration is indicative of the stronger interaction between the grafted chains and the water molecules, which can more effectively lead the protein molecules to leave the surface. Additionally, Cl− and ClO4− have a similar effectiveness to induce the BSA desorption from the surface of PSBMA brushes (Figure S7), which might be due to the weak adsorption of BSA on the surface. We have also investigated the specific cation effect on the conformational behavior of PSBMA brushes using QCM-D (Figure 6). For all the cations, the decrease of Δf and the

tures would not be formed inside the brushes in the presence of different cations, as reflected by the monotonous increase of ΔD with log I. Also, the small difference in the strength of the interaction between the sulfonate group and the cations explains the weak cation specificity in Figure 6. The plot of ΔD vs −Δf in the presence of different cations is shown in Figure S8. SPR Studies. In comparison with the QCM-D measurements, the response of SPR is sensitive to the changes in refractive index and thickness of PSBMA brushes (eq 3), and the coupled water molecules inside the brushes are not included in the change of ΔRU.51 Figure 7 shows the change of ΔRU as a function of logarithmic ionic strength in the presence of different ions. In Figure 7a, for all the anions, ΔRU keeps almost constant with log I at I < 1.0 × 10−3 M, and then it gradually increases with log I as I increases from 1.0 × 10−3 to 0.5 M, followed by a rapid decrease of ΔRU with log I as I increases further. According to eq 3, ΔRU is determined by the combined effect of the changes in thickness and refractive index of the brushes. Figure S2 shows the thickness of the brushes increases with I (Supporting Information). However, the polymer volume fraction of the brushes should decrease with the increasing thickness; that is, the refractive index of the brushes should decrease with I. Therefore, the change of ΔRU with log I should be determined by the balance between the increase of thickness and the decrease of refractive index of the brushes. At I < 1.0 × 10−3 M, the added ions are difficult to enter into the brushes and the conformational change of the brushes is limited. This is why ΔRU keeps almost constant in the low ionic strength regime. As I increases from 1.0 × 10−3 to 0.5 M, the gradual increase of ΔRU indicates that the effect of decrease of refractive index on the response of SPR is dominated by that of the increase of thickness of the brushes. The decrease of ΔRU with the further increase of I implies that the contribution of the decrease of refractive index to the response of SPR dominates over that induced by the increase of thickness of the brushes at the high ionic strengths. Unlike the case of Δf in Figure 1, the change of ΔRU does not exhibit obvious anion specificity. The anion specificity observed in the change of Δf is attributed to the different extents of hydration of the grafted chains induced by the anions. Here, ΔRU is insensitive to the coupled water molecules of the grafted chains. Hence, no obvious anion specificity can be observed in the change of ΔRU. The change of ΔRU with log I in the presence of different cations has a similar result with that of anions (Figure 7b). Also, no obvious cation specificity in the change of ΔRU can be observed.

Figure 6. Shifts in frequency (Δf) and dissipation (ΔD) for PSBMA brushes as a function of logarithmic ionic strength (log I) in the presence of different cations with Cl− as the common anion.

increase of ΔD with log I are indicative of the enhancement of hydration of the grafted chains and the swelling of the brushes. No obvious cation specificity can be observed in the changes of Δf and ΔD. On the basis of the molecular dynamic simulations, Jiang et al. demonstrate that Li+, Na+, K+, and Cs+ weakly interact with the sulfonate group on the zwitterionic unit, and the strength of the interaction only exhibits a small difference between the cations.27 The weak interaction between the cations and the sulfonate group means that the cations may not form ion pair with the sulfonate group. Meanwhile, the counterion Cl− for the cations would also not form ion pair with the quaternary ammonium group. Thus, it is expected that the local electrostatic repulsion-induced heterogeneous struc-

Figure 7. Shift in resonance unit (ΔRU) for PSBMA brushes as a function of logarithmic ionic strength (log I) in the presence of different ions: (a) for the anions and (b) for the cations. G

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In addition, no obvious cation specificity can be observed in the conformational change of the brushes in either QCM-D or SPR measurements.

We have also studied the protein desorption from the surface of PSBMA brushes induced by the addition of NaCl and NaClO4 solutions using SPR (Figure 8). Similar to the

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ASSOCIATED CONTENT

S Supporting Information *

Additional data of the characterization of the preparation of PSBMA brushes, the change in thickness of PSBMA brushes, and the SPR measurements of protein desorption from the surface. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (G.M.); [email protected] (G.Z.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The financial support of National Program on Key Basic Research Project (2012CB933800), the National Natural Science Foundation of China (21004058, 91127042, 21234003), Scientific Research Startup Foundation of the Chinese Academy of Sciences, and the Fundamental Research Funds for the Central Universities (WK2060030008) is acknowledged.

Figure 8. Time dependence of shift in resonance unit (ΔRU) for the adsorption of lysozyme on the surface of PSBMA brushes and for the following desorption of lysozyme from the surface induced by the addition of 1.0 M salt solutions: (a) NaCl and (b) NaClO4.

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REFERENCES

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procedure in QCM-D measurements, lysozyme is adsorbed onto the surface first in the salt-free solution for ∼10 min to form a stable protein layer. Then, the protein molecules are desorbed from the surface by using 1.0 M NaCl or NaClO4 solution. For the NaClO4 solution, all the protein molecules can be removed from the surface. But, a part of protein molecules still adsorb on the surface after the protein desorption by NaCl solution, as indicated by the fact that ΔRU does not return to the baseline in water. This result suggests that ClO4− can more effectively induce the protein desorption than Cl−. Clearly, the results observed in the SPR measurements agree with that in the QCM-D studies. Additionally, the remained strongly adsorbed protein molecules after rinsing with 1.0 M NaCl solution can be removed from the surface by a further rinse with 1.0 M NaClO4 solution (Figure S9), which further indicates that ClO4− has a higher efficiency to induce the protein desorption than Cl−.

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CONCLUSION We have systematically investigated the ion-specific conformational behavior of PSBMA brushes by using QCM-D and SPR. Our studies demonstrate that the anions respectively follow a direct and a reverse Hofmeister series in the low and high ionic strength regimes according to the effectiveness of anions to weaken the inter/intrachain association and to enhance the hydration of the grafted chains. Also, some heterogeneous structures are formed inside the brushes in the presence of chaotropic anions. Our studies also show that the anion-specific conformation can be used to control the protein adsorption/ desorption on the surface of PSBMA brushes. The brushes can more effectively resist the protein adsorption in the presence of a more chaotropic anion and a more chaotropic anion can also more effectively induce the protein desorption from the surface. H

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