Control of Lysozyme Adsorption by pH on Surfaces Modified with

Dec 30, 2013 - In the modeling of ellipsometry, the software supplied by the instrument producer was used, and the Cauchy model for polymer brushes wa...
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Control of Lysozyme Adsorption by pH on Surfaces Modified with Polyampholyte Brushes Hongyan Lei, Mengmeng Wang, Zengchao Tang, Yafei Luan, Wei Liu, Bo Song,* and Hong Chen* Jiangsu Key Laboratory of Advanced Functional Polymer Design and Application, Department of Polymer Science and Engineering, College of Chemistry, Chemical Engineering and Materials Science, Soochow University, Suzhou 215123, PR China S Supporting Information *

ABSTRACT: The adsorption of lysozyme is difficult to control by pH because of the relatively high isoelectric point of this protein (11.1). In this article, we demonstrate good control of lysozyme adsorption by pH in the range of 4−10 on silicon surfaces through modification with poly(2-(dimethylamino ethyl) methacrylate)-block-poly(methacrylic acid) (PDMAEMA-b-PMAA) diblock copolymer brushes. We show that the thickness of the outer PMAA block (lPMAA) is critical to the adsorption. When lPMAA was less than 10 nm, adsorption increased with increasing pH, and the difference in adsorption between high and low pH increased with lPMAA. The ratio of adsorption at pH 10 and pH 4 reached values as high as 16.4. When lPMAA was more than 10 nm, the adsorption tendency on the PDMAEMA-b-PMAA diblock copolymer brushes was similar to that on PMAA homopolymer brushes. These results indicate that the combination of PDMAEMA and PMAA gives adsorption behavior reflecting the properties of both polymers. However, if the outer PMAA block is thicker than a critical value, then the protein-resistant effect of the inner PDMAEMA block is screened.

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near the “neutral” region. For instance, de Vos et al.15 and Chen et al.16 investigated the adsorption of BSA on poly(acrylic acid) (PAA) brushes and fibrinogen on poly(methacrylic acid)(PMAA) brushes, respectively, and found that the proteins were adsorbed in different quantities at pH above and below their respective IEPs. However, this approach cannot be rationally applied to basic proteins such as lysozyme whose IEP is high (11.117). Because lysozyme is positively charged at pH < 11 and lacks any abrupt change in charge state in this pH range, we cannot rely on the variation of the pH to tune the adsorption. The work of Rauscher et al. has shown that the maximum difference in the adsorption of lysozyme on PAA brushes is a mere 0.11 μg/cm2 (0.16 to 0.27 μg/cm2) when varying the pH in the range of 4− 7.18 Because of the distinctive nature of lysozyme, it seems that controlling its adsorption on polyelectrolyte brushes is not easily accomplished using homopolymers. We hypothesize that a combination of weak base and weak acid polyelectrolyte brushes should give a wider response range for the control of lysozyme adsorption by pH. In the present work, PDMAEMA and PMAA were selected as the weak base and weak acid polyelectrolytes, respectively, and were used to modify silicon surfaces. It is demonstrated that lysozyme adsorption is low on PDMAEMA brushes and high on PMAA brushes over the pH range investigated.

INTRODUCTION Stimuli-responsive polymers, also referred to as “smart” polymers, undergo property changes upon the application of external stimuli such as temperature, pH, ionic strength, electrical potential, light, and solvent.1−3 When stimuliresponsive polymer brushes are introduced onto material surfaces, the wettability, roughness, and biomolecule adsorption properties can be adjusted by these same environmental stimuli. Surfaces modified with smart polymers can be used to control the adsorption of proteins.4−6 For example, by modifying silicon with thermoresponsive poly(N-isopropylacrylamide), Cheng et al. were able to “tune” the adsorption of bovine serum albumin (BSA), antiferritin antibody, and fibrinogen by varying the temperature.7 In attempts to control the adsorption of proteins by varying the salt concentration, we used the ionresponsive poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) to functionalize silicon nanowire arrays and found pronounced adsorption differences for both lysozyme and horseradish peroxidase at different sodium chloride concentrations.8 In addition to thermo- and ion-responsive polymer brushes,9−11 polymer brushes that undergo changes in charge and swelling with changing pH may also be used to control the adsorption of proteins. To date, research using pH to control the adsorption of proteins on polyelectrolyte brushes has relied mainly on the difference in charge of the protein above and below its isoelectric point (IEP). This strategy works very well for proteins such as fibrinogen (IEP = 5.512), BSA (IEP = 4.913), and human serum albumin (HSA, IEP = 4.714), whose IEPs are © XXXX American Chemical Society

Received: June 20, 2013 Revised: December 17, 2013

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Ltd.). The measurements were performed with a Vacuum Generator ESCALAB-MKII spectrometer equipped with an Al Kα excitation source (1486.6 eV) at 300 W under a vacuum of 2 × 10−6 Pa. The photoelectron spectra were acquired with a constant analyzer pass energy of 25.0 eV. The binding energy is estimated to be accurate to within 0.2 eV. The results of XPS are shown in the Supporting Information. Static water contact angles (WCAs) were measured via the sessile drop method using a C201 optical contact angle meter (Solon Information Technology Co., Ltd.). Prior to measurements, the samples were incubated in water with controlled pH values for 1 h and then taken out and blown dry in a stream of nitrogen. The measurements were carried out as quickly as possible after the above operations. In doing so, 3 μL of the probing liquid was injected on the surface, and the side-view images were taken. The water used in the measurement has the same pH value as that used in the incubation of the samples.21−24 Water with different pH values was prepared by adding HCl or NaOH and monitored with a pH meter (Sartorius AG). The ionic strengths of the pH solutions were kept at 1 mmol/L with compensation by NaCl. The thickness of the polymer brushes on the silicon was determined with a spectroscopic ellipsometer (M-88, J. A. Woollam Co., Inc.) between 400 and 1100 nm (both PDMAEMA and PMAA have no absorption in this range) with a detection angle of 70°. In the modeling of ellipsometry, the software supplied by the instrument producer was used, and the Cauchy model for polymer brushes was employed to evaluate the refractive indices of the homopolymer films. Silicon_JAW. was chosen as the substrate and added in layers in the order of APTES, BIBB, PDMAEMA, and PMAA (or PNaMA). The refractive indices used for the fitting were as follows: 1.439 for APTES, 1.507 for BIBB, 1.517 for PDMAEMA, 1.48 for PMAA, and 1.401 for PNaMA.25−27 The silicon oxide layer was about 1.8 ± 0.1 nm thick, the APTES layer was about 2.0 ± 0.4 nm thick, and the BIBB layer was 0.6 ± 0.1 nm thick. The in situ topography of the surface and the wet thickness of the brushes in different pH solutions were studied with a Multi-Mode Nanoscope8 atomic force microscope (AFM, Bruker Co., Santa Barbara, CA) with an NP-10 nonconductive silicon nitride tip (Bruker, nominal spring constant 0.58 N/m, resonance frequency 40−75 kHz) in quantitative nanomechanical mapping mode. The scanning areas are 5 μm × 5 μm or 20 μm × 20 μm. Before the measurement, the sensitivity of the cantilever was calibrated on a hard surface. During the measurement, an O-ring was used to seal the liquid. When changing the solutions with different pH, sufficient solution was flushed over the substrate to replace the former solution. Afterward, the whole system was kept still for 0.5 h until the start of scanning. The images were processed with first-order flattening, and no further treatment was applied. The thicknesses were determined by step-height analysis. In the step-height measurement, a scratch was made on the film, and then the AFM probe was carefully located at the edge of the scratch before scanning. Protein Adsorption. Lysozyme was dissolved in phosphatebuffered saline (PBS, pH 7.4) and then radio-labeled with 125I using the iodine monochloride (ICl) technique. The free radioactive iodide was removed by ion-exchange chromatography on AG-1-X4 resin. Labeled and unlabeled proteins were mixed (1/19 labeled/unlabeled) to a total concentration of 1 mg/mL. Silicon wafers were first immersed in solutions of different pH for 1 h to achieve equilibrium and then transferred to 96-well microtiter plates containing 250 μL of protein solution in each well. Adsorption was allowed to proceed for 3 h under static conditions at room temperature. After being removed from the protein solution, the wafers were immersed in a freshly prepared protein-free solution of the same pH for 10 min. Two additional immersions in protein-free solution were carried out to remove loosely attached protein molecules. The wafers were then wicked onto filter paper and transferred to clean counting vials for radioactivity determination (Wallac 2480 Wizard 3″ Automatic Gamma Counter, PerkinElmer Life Sciences).

Adsorption on the PDMAEMA-b-PMAA diblock copolymer brushes reflects the properties of both blocks when the thickness of the outer PMAA block is less than 10 nm: at low pH, adsorption is consistently low; at high pH, adsorption increases with pH and with the thickness of the outer PMAA block.

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EXPERIMENTAL SECTION

Materials. 2-(Dimethylamino ethyl) methacrylate (DMAEMA, 98%) and sodium methacrylate (NaMA, 99%) were from Aldrich and used without further treatment. Copper(I) bromide (CuBr, 98%) from Fluka was purified by stirring in acetic acid and then washed with methanol and dried under vacuum. The freshly prepared CuBr was stored in brown bottles and placed in a desiccator. Both 2,2′-bipyridine (Bpy, 99%) from Sigma and copper(II) bromide (CuBr2, 98%) from Aldrich were recrystallized before use. (3-Aminopropyl)triethoxysilane (APTES, 99%) from Aldrich and α-bromoisobutyryl bromide (BIBB, 98%) from Fluka were used as received. Triethylamine (TEA) and all other solvents were from Shanghai Chemical Reagent Co. and purified according to standard procedures. Silicon wafers (p-doped, (100)oriented, 0.45 mm thick, and 100 mm in diameter) were from Guangzhou Semiconductor Materials (Guangzhou, China). Milli-Q water with a minimum resistivity of 18.2 MΩ·cm was produced by a Millipore water-purification system (High-Tech Instruments Co.). Nitrogen gas was of high purity. Lysozyme (molecular weight 14.0 kDa) was purchased from Beijing Solarbio Science & Technology Co., Ltd. Na125I was from Chengdu Gaotong Isotope Co., Ltd. AG-1-X4 resin was from Bio-Rad Laboratories (Hercules, CA). Preparation of Polymer Brushes on Silicon Substrates. BIBB initiator-functionalized silicon surfaces were prepared as reported previously.9 The details of preparation of APTES and BIBB are described in the Supporting Information. PDMAEMA polymer brushes were grafted from Si surfaces via surface-initiated atomtransfer radical polymerization (SI-ATRP). DMAEMA (1.8 mL, 10.68 mmol), Bpy (33.3 mg, 0.21 mmol), and CuBr (15.4 mg, 0.11 mmol) were added to a mixed solvent of 2 mL of methanol and 2 mL of water. The mixture was sonicated for 2 min and then bubbled with nitrogen for at least 30 min to remove possibly dissolved oxygen. The solution was then transferred to a glovebox, purged with nitrogen, and transferred to a glass vessel in which the wafers functionalized with BIBB initiator were placed. The polymerization was carried out at room temperature under nitrogen. After the desired period of time (to control the thickness of the brush), the silicon wafers were taken out of the reactant, washed alternately with water and methanol, and then dried in a flow of nitrogen.19 The bromoisobutyryl residues terminating the PDMAEMA were used as initiators for further modification of PMAA. The SI-ATRP of NaMA was carried out as previously reported.20 NaMA (5.4 g, 50 mmol) and Bpy (390 mg, 2.5 mmol) were dissolved in water (12.5 mL) with stirring at 50 °C. After the mixture was cooled to room temperature, CuBr (143.5 mg, 1 mmol), CuBr2 (45 mg, 0.2 mmol), and NaOH (3 μL, 2 mol/L) were added. The polymerization procedure for NaMA was similar to that described above for DMAEMA. The resulting silicon wafers were thoroughly rinsed with water to remove the sodium ions and then blown dry with nitrogen. Silicon wafers modified with PDMAEMA-b-PMAA diblock copolymer brushes were thus obtained. In this procedure, if BIBB initiatorfunctionalized silicon surfaces instead of bromoisobutyryl residues terminating the PDMAEMA were used as initiators, then silicon wafers modified with PMAA homopolymer brushes could be obtained. Surface Characterization. Fourier transform infrared spectra (FTIR) of the polymer brushes were obtained using a Nicolet 6700 FTIR spectrometer (Thermo Fisher Scientific Inc., US) equipped with a mercury cadmium telluride detector. (Caution! This detector should be used only af ter being thoroughly cooled with liquid nitrogen.) The transmission mode was adopted, and the wavelength step was 4 cm−1. The chemical composition of the silicon surfaces modified with PDMAEMA, PMAA, and PDMAEMA-b-PMAA brushes was determined with an X-ray photoelectron spectrometer (XPS) (VG Scientific B

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Scheme 1. Preparation Protocol of PDMAEMA-b-PMAA Diblock Copolymer Brushes on Silicon Wafers

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RESULTS AND DISCUSSION

Preparation of Polymer Brushes. SI-ATRP was used to prepare PDMAEMA, PMAA, and PDMAEMA-b-PMAA polymer brushes on silicon wafers. Scheme 1 shows a typical protocol for the diblock copolymer brushes; the procedure for the two homopolymer brushes was analogous. To illustrate the preparation procedure, the diblock copolymer brushes are taken as an example. First, the silicon wafers were treated with piranha solution to hydroxylate the surface, and then they were reacted with APTES to introduce amino groups. BIBB initiator was conjugated to the surface amino groups via amidation.9 Second, DMAEMA and MAA were sequentially grafted from the initiator silicon surfaces by SI-ARTP. The film thickness was controlled by varying the reaction time. (The relationship between the thickness of the brush film and the reaction time is shown in Supporting Information Figure S1.) It should be noted that the direct ATRP of methacrylic acid may be hampered by the formation of catalytically ineffective metal carboxylates.28 In addition, because of the potential breakage of the ester bond in the PDMAEMA block during deprotection, the use of tert-butyl methacrylate is not appropriate either. Therefore, sodium methacrylate (NaMA) was chosen as the monomer for ATRP. After the reaction, the surfaces were treated with neutral or diluted acid solution to remove the sodium ions. Silicon wafers modified with PDMAEMA-bPMAA diblock copolymer brushes were thus obtained. The grafting density was 0.41 nm−2, which was estimated by the thickness of the brushes and the number-average molecular weight of polymers formed under the same conditions in solution.29,30 The measurement and calculation details are shown in the Supporting Information. Characterization with FTIR. FTIR spectra of the surfaces are shown in Figure 1. Spectrum a is the adsorption of the silicon wafer after modification with PDMAEMA. The peaks at around 1730, 1145, and 1270 cm−1 correspond to CO, C− O−C, and tertiary amine vibrations, respectively. The appearance of these peaks indicates the successful attachment of PDMAEMA to the silicon surface.31 Spectrum b is the adsorption of the silicon wafer after sequential graft polymerization of DMAEMA and NaMA; the peak at around 1550 cm−1 is assigned to COO−. The intensity of this peak is not as strong as expected, possibly because of the partial conversion of

Figure 1. FTIR spectra of (a) PDMAEMA, (b) PDMAEMA-bPNaMA, and (c) PDMAEMA-b-PMAA polymer brushes. The inset shows magnified spectra in the 1730 cm−1 region, where spectrum c is split into two peaks by Lorentzian−Gaussian fitting.

COO− to COOH by atmospheric CO2, as also suggested by the appearance of the shoulder peak at 1717 cm−1. Moreover, after treatment with water, the peak at around 1550 cm −1 diminishes; correspondingly, the peak at 1717 cm−1 (as shown in the inset in Figure 1, the peak in spectrum c is split into two peaks at around 1730 and 1717 cm−1) increases. This provides further evidence for the conversion of COO− to COOH. These results indicate the successful preparation of PDMAEMA-b-PMAA diblock copolymer brushes, which can also be evidenced by XPS (Figure S2 and Table 1 in Supporting Information). The C/O signal ratio of dibliock copolymer brush is 2.65, which is between the values for the two homopolymer brushes (PDMAEMA 2.86, PMAA 2.37). These results indicate that the modification of PMAA on PDMAEMA is successful. Wettability of Polymer Brushes: Dependence on pH. Figure 2 shows data on the pH responsiveness of the silicon wafers modified with different polyelectrolyte brushes as assessed by WCA. It can be seen clearly that the WCA of the PDMAEMA polymer brush increases with increasing pH, with a sharp increase at pH 4−6. The low WCA is mainly due to the protonation of the amine groups on PDMAEMA chains, of which the charged state increases the polarity of the film. The C

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investigate the thickness and surface morphology under different pH conditions. The thicknesses of the brush films were measured by in situ AFM (i.e., the measurement was carried out under aqueous solution conditions) step-height scanning. The scanning was maintained in approximately the same region throughout the measurement on the same sample. Figure 3 shows a typical AFM image and the step-height analysis. During the analysis, an average Z value was adopted at a specific location to decrease the system error. Figure 4 shows the swelling behavior of the polymer brushes upon changing the system pH. The PDMAEMA brush has a swelling ratio (i.e., the ratio of the thickness at a certain pH to that of the dry state) as high as 3.7 at pH 4, followed by a decrease in the thickness with increasing system pH. At pH 4, the swelling is associated with the adsorption of water and repulsive interactions caused by the net positive charges on the polymer chains. In particular, the latter makes the main contribution to the swelling. The swelling of the brush indicates that the PDMAEMA chains adopt a stretched conformation at pH 4. When the pH is increased to 7 and 10, the brush film “shrinks”, although it is still thicker than the thickness in the dry state. On the contrary, PMAA exhibits a high swelling ratio at pH 10, which is mainly caused by the net negative charges due to the deprotonation of carboxylic acid groups. In the case of the PDMAEMA-b-PMAA copolymer brush, the swelling ratio decreases and then increases with increasing pH from 4 to 7 to 10. The total thickness change is a result of the combined swelling behavior of the two blocks. As shown in Scheme 2, the PDMAEMA and PMAA sections swell most at pH 4 and 10, respectively, as a result of the charge repulsion. At pH 7, although these two blocks might be partially charged, the charge recombination between amino and carboxyl groups will decrease the repulsive interaction between the polymer chains. Thus, the whole film shows a relatively collapsed state. The conformational change of the polymer chains can also be reflected by the surface morphology of the brush films. As shown in Figure 5, taking PDMAEMA as an example, we found that the image at pH 4 becomes fuzzy when comparing the images in dry state and under other pH conditions, further confirming the swelling of the brushes. In case of the copolymer brushes, it may be clearly observed that the surface morphology of copolymer II (lPMAA = 13 nm) has the same tendency to change as the pure PMAA brush, whereas the surface morphology of copolymer I (lPMAA = 6 nm) does not change greatly at different pH values. As mentioned, the thickness of the PMAA block will determine the surface

Figure 2. pH dependence of WCAs of polymer brushes. PDMAEMA (■), PMAA (○), and PDMAEMA-b-PMAA with different thicknesses of PDMAEMA and PMAA (copolymer I, 13 and 6 nm (△); copolymer II, 8 and 17 nm (●). Data are mean ± standard error, with n = 3.

abrupt change at pH 4−6 is an indication of the charge-state conversion, suggesting that the amine groups are deprotonated as a result of the pH change. The PMAA polymer brush shows the reverse behavior. In the pH range of 3−11, the WCA decreases steadily from 50 to 30°. PMAA is known to be a hydrophilic polymer because of its dangling carboxylic acid groups, so it is easy to understand the steady decrease in the WCA from a relatively low value to a lower value. In the case of the PDMAEMA-b-PMAA diblock copolymer brushes, the wettability change depends on the thickness of the outer PMAA block (lPMAA). When lPMAA is small (lPMAA ≤ 10 nm, based on the adsorption data shown below), the WCA first increases at low pH and then decreases at high pH. At pH 3 to 4, PDMAEMA is expected to make the major contribution to hydrophilicity because of the protonation of amino groups, thus explaining the low WCA. Similarly, at pH 10 to 11, PMAA becomes dominant as a result of the deprotonation of carboxylic acid groups, leading to a low WCA. At pH 7, the WCA is relatively high as a result of the formation of polyelectrolyte complexes.27,32 When lPMAA is high, the wettability variation is similar to that of the PMAA homopolymer brush in the pH range of 3−11. This result suggests that the wettability of the PDMAEMA-b-PMAA diblock copolymer brushes is determined mainly by the thickness of the outer PMAA block. We were considering that the wettability changes depending on the system pH may imply a conformational change of the polymer chains. In the following section, we employed AFM to

Figure 3. (a) AFM image of the scratches on the polymer brush films. (b) Thickness measurement by the step-height analysis method. The Z direction is the average height at the X position in the dashed rectangle in image a. For a clear illustration, the whole curve is shifted by defining the lower horizontal part close to zero. D

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Figure 4. Investigation of swelling behaviors of PDMAEMA, PMAA, and PDMAEMA-b-PMAA copolymer brushes determined by the AFM stepheight method. (a) Thickness vs pH, (b) swelling ratio vs pH, and (c) reversible thickness change by recycling the system pH. The thicknesses for the dry states of PDMAEMA, PMAA, and PDMAEMA-b-PMAA brushes are 15 ± 1.8, 16 ± 2.1, 16 ± 1.0 nm, respectively.

the different surfaces. Adsorption on unmodified silicon was about 0.25 μg/cm2, which corresponds to a close-packed monolayer based on the dimensions of lysozyme (sphere of diameter ∼3 nm). Adsorption on the PDMAEMA brush was very low (< 0.08 μg/cm2) at all three pH values, 4, 7, and 10, suggesting that the PDMAEMA brush is protein-resistant, in agreement with data reported by Kusumo et al.33 In contrast to the PDMAEMA brush, the PMAA brush had a high adsorption at all pH values. The highest value on the PMAA brush with a thickness of 10 ± 0.5 nm at pH 7 is about 8.3 μg/cm2. The high adsorption amount indicates that lysozyme can be adsorbed not only on the surface but also inside the polymer brushes. This can be attributed to the swelling of the film as well as the electric attraction between lysozyme and the surface. It is worth noting that the bioactivity of the adsorbed lysozyme increases with increasing pH on different brushes (Supporting Information, Figure S3). Because lysozyme adsorbs strongly on PMAA, the effect of the brush thickness was investigated systematically. As shown in Figure 6b, the adsorbed amount at pH 7 tends to increase with the thickness. In addition, the adsorption at pH 7 shows the greatest value on the brushes with the same thickness when compared to the adsorption at other pH values. These results are in good agreement with that reported previously.18 Protein adsorption can be influenced by several factors, including surface wettability, 34 surface charge, 10 ionic strength,10 and protein concentration.35 Throughout our study, the ionic strength and concentration of the protein solutions were kept constant so that the effects of these variables could be excluded. The wettability of the surfaces modified with polyelectrolyte brushes is strongly dependent on pH as shown in Figure 2, but the adsorption of lysozyme does not correlate with wettability. For example, the WCA of the PDMAEMA polymer brush increases with increasing pH, and the adsorbed quantities of lysozyme show little pH dependence. Although we cannot completely exclude the influence of surface wettability on lysozyme adsorption, we are sure that it is not the dominant factor causing the differences in adsorption among different surfaces. Electrostatic interactions between the brushes and the protein provide a more likely explanation. When the pH is lower than the IEP of lysozyme (11.1),17 the protein is positively charged and its net charge decreases with increasing pH.36 The PDMAEMA polymer brush is positively charged at pH < 10.37 Electrostatic repulsion between the PDMAEMA brush and lysozyme leads to low adsorption. Therefore, PDMAEMA shows a lysozyme resistivity in the pH

Scheme 2. Illustration of the Swelling Behavior of PDMAEMA-b-PMAA Diblock Copolymer Brushes When Varying the pH from 4 to 7 to 10

Figure 5. AFM images of PDMAEMA, PMAA, copolymer I (PDMAEMA-b-PMAA with 11 nm PDMAEMA and 6 nm PMAA) and copolymer II (PDMAEMA-b-PMAA with 11 nm PDMAEMA and 13 nm PMAA) in the dry state and at pH 4, 7, and 10. All of the scanning areas of the images are 5 μm × 5 μm. The color scales are shown on the right side of the image rows.

wettability of the copolymer brushes. The surface morphology also implies such a tendency. Protein Adsorption. Lysozyme adsorption on the surfaces was measured using the radiolabeled protein. The ionic strength of all protein solutions was kept constant at 1 mmol/L. Figure 3 shows the adsorbed amount versus pH on E

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Figure 6. (a) pH-dependent adsorption of lysozyme (1.0 mg/mL) on unmodified silicon, silicon modified with APTES (2.0 ± 0.4 nm), a PDMAEMA polymer brush (11 ± 0.7 nm), and a PMAA polymer brush (10 ± 0.5 nm). The adsorbed quantities were measured with 125I radiolabeling. (b) pH-dependent adsorption of lysozyme on PMAA polymer brushes with different thicknesses. Data are mean ± standard error, n = 3.

ratio of adsorbed quantities at different pH values. For lPMAA = 10 nm, the adsorbed quantity was 2.4 μg/cm2 at pH 10 (i.e., 16.4-fold greater than at pH 4 on the polymer brushes with the same thickness). At pH 4, the PDMAEMA block is protonated and swells, which may lead to the populating of PDMAEMA on the surface; it is then the main determinant of lysozyme adsorption. Thus, the diblock copolymer brushes showed the lowest adsorption of lysozyme at this pH. At pH 7 and 10, deprotonation (or partial deprotonation) leads to the swelling of the PMAA block, which supplies more capacity for the adsorption of lysozyme. Therefore, the adsorbed amount of lysozyme increases with pH. In addition, the adsorbed quantity changes little as the thickness of the PDMAEMA varies from 7 to 14 nm, suggesting that the thickness of the PDMAEMA block has little effect on the adsorption of lysozyme. Increasing lPMAA enhances the contribution of the negative charges so that the adsorbed quantity increases with lPMAA. lPMAA > 10 nm. As shown in Figure 8, the adsorbed quantity of lysozyme on PDMAEMA-b-PMAA was much greater than lPMAA ≤ 10 nm. At pH 4, the diblock copolymer brush was

range of 3−10. In contrast, the PMAA brush is negatively charged at pH > 3,38 leading to a high adsorption of lysozyme on this surface. The adsorption of lysozyme at pH 7 is higher than that at pH 4, presumably because of the increasing net negative charge with increasing pH on the PMAA brush. At pH 10, adsorption is smaller again because the net positive charge of lysozyme decreases when the pH is close to the IEP. These trends in the adsorption of lysozyme on the PMAA brush agree with those seen for lysozyme on the PAA brush.18 The adsorption of lysozyme on the diblock copolymer brushes exhibits very different pH responsiveness depending on whether the thickness of the outer PMAA block is less or more than 10 nm. lPMAA ≤ 10 nm. Figure 7 shows data for the adsorption of lysozyme on PDMAEMA-b-PMAA diblock copolymer brushes

Figure 7. pH-dependent adsorption of lysozyme (1.0 mg/mL) on PDMAEMA-b-PMAA diblock copolymer brushes with different thicknesses of the outer PMAA block (lPMAA). The thicknesses of PDMAEMA and PMAA are 11 and 6 nm, 8 and 8 nm, and 14 and 10 nm, respectively. Data are mean ± standard error, with n = 3.

with different lPMAA at different pH values. On all three surfaces with different thicknesses, the adsorbed quantity increased stepwise with increasing pH. If we take the surface with lPMAA = 6 nm as an example, the adsorbed quantity increased from 0.13 to 0.57 μg/cm2 as the pH increased from 4 to 10. Increasing lPMAA resulted in an increase in the adsorbed quantity and in the

Figure 8. pH-dependent adsorption of lysozyme (1.0 mg/mL) on PDMAEMA-b-PMAA diblock copolymer brushes with different thicknesses of the outer PMAA block (lPMAA) for lPMAA > 10 nm. The thicknesses of PDMAEMA and PMAA are 11 and 13 nm and 8 and 17 nm, respectively. Data are mean ± standard error, with n = 3. F

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resistant to lysozyme for lPMAA ≤ 10 nm; for lPMAA > 10 nm, the diblock copolymer brush became adsorptive. For lPMAA = 17 nm, the trend of adsorption on the diblock copolymer brush with pH was similar to that on the homo PMAA brush, implying that the influence of the PDMAEMA layer is screened when the PMAA layer becomes too thick. Coincidentally, in previous work, we found a critical thickness (∼10 nm) of polymer brushes for the adsorption of protein (i.e., the adsorbed amount does not decrease any further as the thickness of the brushes reaches 10 nm39,40). It is possible that in general a critical thickness is required for polymer brushes to screen the effect of the underlying layer.

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CONCLUSIONS

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REFERENCES

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The adsorption of lysozyme at different pH values was investigated on PDMAEMA, PMAA, and PDMAEMA-bPMAA polymer brushes. The data indicate that lysozyme adsorption is low on PDMAEMA polymer brushes and high on PMAA polymer brushes in the pH range investigated. Adsorption to PDMAEMA-b-PMAA diblock copolymer brushes showed the influence of both polyelectrolytes. At low pH, the adsorption was low; at high pH, the adsorption increased with increasing pH and with increasing thickness of the outer PMAA block. At a PMAA block thickness of 10 nm, the adsorbed quantity at pH 10 was as high as 16.4-fold greater than at pH 4. Nevertheless, the thickness of the PMAA block in PDMAEMA-b-PMAA is critical. When this thickness was greater than 10 nm, the protein-resistant properties of PDMAEMA were screened and the diblock copolymer brush exhibited adsorption behavior similar to that of the PMAA homopolymer brush. Therefore, for purposes of tuning the adsorption of lysozyme on these surfaces by pH, the thickness of the PMAA block should be below a critical value; if the thickness is greater than the critical value, then the effect of the inner block PDMAEMA will be screened.

S Supporting Information *

Experimental procedures, increasing thickness of polymer brushes with increasing reaction time, estimation of the grafting density of PDMAEMA brushes, surface characterization with XPS, and bioactivity of adsorbed lysozyme. 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]. *E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Dr. J. Brash of McMaster University for helpful discussions. This work was supported by the National Science Fund for Distinguished Young Scholars (21125418), the National Natural Science Foundation of China (21204054 and 21334004), the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), and the Project of Scientific and Technologic Infrastructure of Suzhou (SZS201207). G

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