Article pubs.acs.org/Langmuir
Biofouling Resistance of Ultrafiltration Membranes Controlled by Surface Self-Assembled Coating with PEGylated Copolymers Yen-Che Chiag,† Yung Chang,*,† Wen-Yih Chen,‡ and Ruoh-chyu Ruaan*,†,‡ †
R&D Center for Membrane Technology and Department of Chemical Engineering, Chung Yuan Christian University, Jhong-Li, Taoyuan 320, Taiwan ‡ Department of Chemical and Materials Engineering, National Central University, Jhong-Li, Taoyuan 320, Taiwan ABSTRACT: Block and random PEGylated copolymers of poly(ethylene glycol) methacrylate (PEGMA) and polystyrene (PS) were synthesized with a controlled polydispersity using an atom transfer radical polymerization method and varying molar mass ratios of PS/PEGMA. Two types of PEGylated copolymers were self-assembly coated onto the surface of poly(vinylidene fluoride) (PVDF) ultrafiltration membranes for enhancing biofouling resistance. It was found that the adsorption capacities of random copolymers on PVDF membranes were all higher than those of block copolymers. However, the specific and overall protein resistance of bovine serum albumin (BSA) on PVDF membranes coated with block copolymers was much higher than that with random copolymers. The increase in styrene content in copolymer increased the amount of polymer coating on the membrane, and the increase in PEGMA content enhanced the protein resistance of membranes. The optimum PS/PEGMA ratio was found to be close to 2 for the best resistance of protein adsorption and bacterial adhesion on the PEGylated diblock copolymer-coated membranes. The PVDF membrane coated with such a copolymer owned excellent biofouling resistance to BSA, humic acid, negatively surface charged bacteria E. coli, and positively surface charged bacteria S. maltophilia.
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INTRODUCTION Control of protein adsorption and bacterial adhesion onto a solid substrate coating with a biofouling-resistant interface is crucial for the design of antifouling surfaces for biomedical materials that come in contact with the biological system.1,2 In general, biofouling surfaces with hydrophobic moieties will lead to the nonspecific adsorption of protein in cell- or bacteriacontaining solutions.3 Protein adsorption is an early event in biofoulant−material interactions. Even a small amount of protein on a surface can lead to propagation of unwanted biofouling reaction.4 It is believed that the increase of hydrophilic moieties on a hydrophobic material surface can effectively reduce its biofouling ability due to the reduced hydrophobic interactions between the biofoulants and the hydrophobic surface.3,5−7 In particular, an ordered layer structure of hydrophilic domain and hydrophobic domain is important to improve the biofouling resistance of the hydrophobic surface.3 Poly(ethylene glycol)- (PEG) or poly(ethylene oxide) (PEO)-based materials are the most commonly used biofouling-resistant materials.8 Many hydrophobic surfaces modified with PEG-based materials can greatly reduce protein adsorption. Several previous works proposed that the distribution density and chain length of PEG on the surfaces were two key parameters in determining protein repelling behavior.9−11 © 2011 American Chemical Society
Poly(vinylidene fluoride) (PVDF) is widely used to fabricate ultrafiltration (UF) membranes because of its outstanding properties: excellent chemical resistance and thermal stability.12 However, the biofouling problem of PVDF membranes was frequently criticized by the users.13−15 Several strategies have been adopted to improve the biofouling resistance of membranes, such as blending PEG-containing polymers before membrane formation or surface grafting of PEG moiety.11,16−21 Although surface grafting has been shown highly efficient in improving biofouling resistance, large-scale surface modification through this technique is still difficult.20,21 Polymer blending before membrane formation is a much more preferable method, but limited PEG moiety can be incorporated into the membrane so that improvement of biofouling resistance is also limited. Compared to surface grafting and polymer blending, surface coating provides several distinct advantages.16,22−26 Surface coating by physical adsorption is easy to operate and suitable for large-scale preparation. However, coating stability is always a major concern. Although the protein resistance of PEGylated PVDF membrane surfaces with varying grafting coverage of PEG brushes via the graf ting f rom approach has been studied,11,20,21 Received: October 13, 2011 Revised: December 4, 2011 Published: December 8, 2011 1399
dx.doi.org/10.1021/la204012n | Langmuir 2012, 28, 1399−1407
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Figure 1. Schematic illustration of the preparation process of the PEGylated membranes via surface self-assembled coatings with PS-b-PEGMA and PS-r-PEGMA. bromide (CuBr; 99.999%), methyl 2-bromopropionate (MBrP; 98%), 2,2′-bipyridine (bpy; 99%), tetrahydrofuran (THF; HPLC grad), diethyl ether, hexane, and ethanol (absolute 200 proof) were purchased from Sigma-Aldrich. PVDF powder having a molecular weight of 370 000 g/mol was obtained from the commercial products of Kynar 760 and washed with acetone before use. N,NDimethylacetamide (DMAc; 98%) for preparing the membrane casting solution was also obtained from Sigma-Aldrich. Bovine serum albumin (BSA) was purchased from Sigma Chemical Co. Deionized water (DI water) used in the experiments was purified using a Millipore water purification system to a minimum resistivity of 18.0 MΩ·m. Phosphate-buffered saline (PBS) was purchased from Sigma. Preparation of PEGylated Copolymers. Block and random PEGylated copolymers of PEGMA and polystyrene (PS) were synthesized by the atom transfer radical polymerization (ATRP) method, which used CuBr/bpy as the catalyst system and MBrP as the initiator. Poly(styrene-block-poly(ethylene glycol) methacrylate) (PSb-PEGMA) was synthesized under nitrogen in dry box. For polymerization of PS block with different molecular weights, styrene (42.2 mmol) was polymerized under bulk using [styrene]/[MBrP]/ [CuBr]/[bpy] = 30/1/1/2, 50/1/1/2, and 90/1/1/2 at 120 °C. After 24 h, 1H NMR analysis indicated that 99% of the styrene monomers had been polymerized. The resulting reaction solution diluted with 45 mL of THF was passed through an aluminum oxide column, precipitated into 300 mL of methanol, and redissolved into THF repeatedly three times to remove residue ATRP catalysts. This purification protocol resulted in the loss of up to 30% styrene homopolymer due to adsorption. After solvent evaporation, the copolymer was dried in a vacuum oven at room temperature to yield a white powder. THF GPC analysis indicated the prepared PS homopolymers with a set of MW around 3389 (PS31), 5529 (PS52), and 10 130 (PS96) and a controlled range of Mw/Mn between 1.20 and 1.30, as shown in Figure 2a. For the following polymerization of the second block of PEGMA, PEGMA (3.79 mmol) was polymerized in 5.5 mL of THF with PS31, PS52, and PS96 as the macroinitiators using [PEGMA]/[PS]/[CuBr]/[bpy] = 10/1/1/2−25/1/1/2 at 60 °C.
it is still unclear how molecular structures of PEGylated copolymers via the graf ting onto (surface coating) approach would influence the correlation between surface PEGylation and biofouling resistance. It is also important to understand the effects of hydrophobic−hydrophilic structure sequence and composition balance of PEGylated copolymers on the coating stability associated with thier antifouling properties. The results from such studies would directly enable the rational design of PEGylated membranes for use in biofouling-resistant applications. In this study, block and random copolymers synthesized by poly(ethylene glycol) methacrylate (PEGMA) and styrene were coated onto the PVDF ultrafiltration membranes, as shown in Figure 1. The hydrophobic polystyrene (PS) segment is needed for polymer anchoring onto the hydrophobic PVDF surface, and the PEGMA segment is needed for biofouling resistance. The increase in styrene content may enhance polymer adsorption but reduce biofouling resistance. The optimum PS/PEGMA ratio needs to be determined to optimize overall biofouling resistance. In addition, diblock and random copolymers were synthesized. Their coating stability and antifouling properties were compared. Protein, bovine serum albumin (BSA), and bacteria, Escherichia coli and Stenotrophomonas maltophilia, adhesion were used to discriminate the antifouling properties of various polymer-coated membranes. BSA and humic acid filtration tests were also performed to further evaluate the biofouling resistance.
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MATERIALS AND METHODS
Materials. Poly(ethylene glycol) methacrylate (PEGMA) macromonomers with a molecular weight of about 500 Da and an average number of ethylene glycol units of about 10 were purchased from Aldrich. Styrene (Fluka; 99%) was purchased and dried over CaH2, and the inhibitors were removed by vacuum transfer. Copper(I) 1400
dx.doi.org/10.1021/la204012n | Langmuir 2012, 28, 1399−1407
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solution diluted with 30 mL of THF was passed through an aluminum oxide column, precipitated into 250 mL of ether/hexane, and redissolved into THF repeatedly three times to remove residue ATRP catalysts. Finally, the copolymer was dried in a vacuum oven at room temperature to yield a white powder. The molar mass ratios of PS/PEGMA in the prepared diblock copolymers were controlled with a set of 0.8 (PS31-b-PEGMA39), 2.1 (PS52-b-PEGMA25), and 3.6 (PS96b-PEGMA27) and a range of Mw/Mn between 1.10 and 1.20, as summarized in Table 1. Poly(styrene-random-poly(ethylene glycol) methacrylate) (PS-rPEGMA) was also synthesized using the ATRP method under nitrogen in a dry box. To control the similar ratios of PS/PEGMA in the PS-r-PEGMA compared with PS-b-PEGMA, styrene (9.6 mmol) was polymerized in 10.0 mL of THF with different molar mass ratios of PEGMA as the comonomers using [styrene]/[PEGMA]/[MBrP]/ [CuBr]/[bpy] = 25/6/1/1/2, 30/15/1/1/2, and 50/50/1/1/2 at 60 °C. After 24 h, 1H NMR analysis of the reaction solution indicated that 60−65% of the total styrene and PEGMA monomers had been polymerized. The purification process of the prepared PS-r-PEGMA followed the same protocol of the previous description of the prepared PS-b-PEGMA. From the combination of THF GPC analysis, the molar mass ratios of PS/PEGMA in the prepared random copolymers were controlled with a set of 0.9 (PS47-r-PEGMA52), 1.7 (PS20-r-PEGMA12), and 3.7 (PS26-r-PEGMA7) and a range of Mw/Mn between 1.40 and 1.50, as shown in Figure 2b. Characterization of PEGylated Copolymers. The structure of block and random PEGylated copolymers, PS-b-PEGMA and PS-rPEGMA, was characterized by 1H NMR spectra using a 500 MHz spectrometer and D2O as solvent. A typical spectrum for the prepared PEGylated copolymers is shown in Figure 3. Results showed that the pure PS-b-PEGMA and PS-r-PEGMA copolymers were obtained. The composition of the PS-r-PEGMA copolymers was estimated by 1H NMR in D2O from the relative peak area of the aromatic proton resonance of the PS side groups in the range of δ between 6.3 and 7.2 ppm and that of the methoxyl proton resonance and methylene resonance of the PEGMA side groups in the range of δ between 3.38 and 3.8 ppm. Molecular weights (Mw) and molecular weight distribution (Mw/Mn) of prepared copolymers were determined by THF gel permeation chromatography (GPC; Viscotek GPCmax Module, USA), using 1 column of Jordi Gel CN15073 (the range of molecular weight was from 100 Da to 20 kDa) connected to a model VE3580 refractive index (RI) detector with a 1.0 mL/min flow rate and 25 °C column temperature. Polystyrene (PS) standards from
Figure 2. THF GPC curves for (a) three PS homopolymers and PS-bPEGMA diblock copolymers and (b) three PS-r-PEGMA random copolymers (determined with PS as a reference) prepared via ATRP at 60 °C. After 24 h, 1H NMR analysis indicated that 50−65% of the PEGMA macromonomers had been polymerized. The resulting reaction
Table 1. Characteristic Data of PS-b-PEGMA and PS-r-PEGMA Copolymers and PEGylated Membranes characterization of copolymersa
compositions of copolymersb
characteristics of copolymer coated membranes
sample ID
Mw (g/mol)
Mw/Mn
PS (mol %)
PEGMA (mol %)
PS/PEGMA
virgin PVDF PS31-b-PEGMA39 PS31-b-PEGMA46 PS31-b-PEGMA55 PS52-b-PEGMA25 PS96-b-PEGMA27 PS47-r-PEGMA52 PS20-r-PEGMA12 PS26-r-PEGMA7
21965 24575 29454 17423 23036 29753 8124 6077
1.17 1.18 1.07 1.13 1.17 1.43 1.48 1.41
44 40 36 68 78 47 63 79
56 60 64 32 22 53 38 21
0.8 0.7 0.6 2.1 3.6 0.9 1.7 3.7
c
d
Cmax (mg/cm2) 0.00 0.06 0.05 0.04 0.08 0.10 0.11 0.16 0.18
± ± ± ± ± ± ± ±
0.006 0.003 0.003 0.01 0.01 0.01 0.02 0.02
Pmine (μg/cm2)
RBSAf (μg/(mg PEGMA))
± ± ± ± ± ± ± ± ±
0 531 482 522 457 287 172 127 136
43 15 19 24 7 12 25 20 18
3 3 3 4 1 4 2 5 3
a
Molecular weights (Mw) and molecular weight distributions (Mw/Mn) were estimated by GPC and calibrated with PS. bComposition of the PS-rPEGMA copolymers was estimated by 1H NMR in D2O from the relative peak area of aromatic proton resonance of the PS side groups in the range of δ between 6.3 and 7.2 ppm and that of the methoxyl proton resonance and methylene resonance of the PEGMA side groups in the range of δ between 3.38 and 3.8 ppm. cValue of PS/PEGMA is defined as the molar mass ratio of PS segments to PEGMA segments for a PEGylated copolymer. dCmax is the maximum adsorption amount of each prepared copolymer on the PVDF membranes ePmin is the minimum adsorption amount of specific BSA on PEGylated membranes. fSpecific BSA resistance (RBSA) of PEGylated membranes is defined as the minimum adsorption amount of specific BSA on PEGylated membranes divided by the maximum adsorption amount of PEGMA in each prepared copolymer on the PVDF membranes. 1401
dx.doi.org/10.1021/la204012n | Langmuir 2012, 28, 1399−1407
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Figure 3. 1H NMR spectrum of the PS-r-PEGMA random copolymer in d-chloroform. ethanol for 30 min and transferred into a clean test tube, followed by addition of 20 mL of PBS solution for 30 min. Then, the membrane was soaked in 5 mL of 1 mg/mL BSA in 0.1 M PBS solution (pH 7.4) for 24 h at 37 °C. The membrane was then followed by addition of dye reagent containing Coomassie Brilliant Blue G-250 and incubated for 5 min. The absorbance at 595 nm was determined by a UV−vis spectrophotometer. Bacterial Adhesion Assay. Two bacterial species, E. coli and S. maltophilia, were used to investigate bacterial adhesion behavior on the surface of PEGylated copolymer-coated membranes. E. coli and S. maltophilia were cultured in a medium containing 3.0 mg/mL beef extract and with 5.0 mg/mL peptone. These cultures were incubated at 37 °C and shaken at 100 rpm until the stationary phase was reached at a final E. coli concentration of 106 cells/mL for 12 h and a final S. maltophilia concentration of 108 cells/mL for 15 h. The prepared membranes 0.28 cm2 in size were incubated with 75 wt % ethanol for 1 h at 25 °C and washed by PBS 3 times in a 24-well plate. One milliliter of bacteria suspension was added to each well. The bacteria were then incubated with the samples for 24 h at 37 °C. The sample was then washed with PBS 3 times at 37 °C to remove the loosely attached bacteria. Bacteria firmly adhering to the membrane surfaces were stained with 200 μL Live/Dead BacLight for 10 min. After washing with PBS 3 times, samples with stained bacteria were observed from confocal laser scanning microscopy (CLSM) images at a 200 magnification from five different places on the same membranes. During observation, the images were taken at λex = 488 nm/λem = 520 nm for detection of the fluorescent dye. This analysis was performed using a NIKON CLSM A1R instrument. Ultrafiltration Experiments. A dead-end cell filtration system connected with a nitrogen gas cylinder and solution reservoir was designed to characterize the filtration performance of the prepared membranes. The system consisted of a filtration cell (HP4750 stirred cells, Sterlitech Corp.) with a volume capacity of 300 mL and an inner diameter of 49 mm. Before the filtration experiments, the virgin or prepared membranes were incubated and pressurized with doubledistilled water for 30 min at 1.5 atm. All ultrafiltration experiments were operated at a pressure of 1.0 atm, a temperature of 25 °C, and a stirring speed of 300 rpm. The ith cycle permeation flux (Jwi or JPi) was checked from time to time until steady and calculated by the following equation
American Polymer Standards Corp. (APSC, USA) were used for calibration. Self-Assembled Coatings of PEGylated Copolymers on PVDF Membrane Surfaces. Two self-assembled coatings were formed on the PVDF ultrafiltration (UF) membranes: (1) PS-bPEGMA-coated membranes and (2) PS-r-PEGMA-coated membranes. The PVDF UF membranes were first prepared by the wet phase inversion from a DMAc solution containing 15 wt % PVDF powder with pore-forming agent poly(ethylene glycol) (PEG8000, PVDF/ PEG weight ratio = 5.7:1). The casting solutions were stirred for 24 h at 40 °C and left 6 h to allow complete release of bubbles. After casting the solutions with a casting knife of 300 μm on a glass plate, the plate was immediately immersed in a coagulation bath of double-distilled water. The UF membrane was formed via phase inversion at 4 °C. The formed UF membranes were washed with DI water for 24 h to completely remove the residual solvent and pore-forming agent. For the process of self-assembled coatings, the cleaned PVDF membrane was soaked in an ethanol solution and then incubated with various concentrations of PS-b-PEGMA and PS-r-PEGMA copolymer solution in the range of 0.1−10 mg/mL for 30 min. The membrane was rinsed with ethanol in 50% v/v water repeatedly three times to remove weakly adsorbed copolymers. Finally, the residual solvent was removed in a vacuum oven under reduced pressure for 1 day. In this study, all membranes after self-assembled coating treatment were cleaned under the same postwash procedures. The polymer-coated amount of PS-bPEGMA and PS-r-PEGMA on the PVDF membrane was determined by the extent of weight increase compared with the virgin PVDF membrane and normalized to the outer surface area of the membranes. Prior to weight measurements, the prepared PVDF UF membranes of about 20 cm2 in surface area were dried overnight in a vacuum oven at 50 °C. Weight measurements were performed using three independent membranes for each modified membrane, and the average value was reported. The dry weight of virgin membrane is about 190 mg. The copolymer-coated membrane had a 20 cm2 surface area after successively washing, and the remaining polymer on the membrane was measured every five washings. The 20 mL of cosolvent used in the first three washings was 50% ethanol/water solution. The solvent used in the following washings was pure water. The amount of BSA adsorption was measured after membrane washings at 3, 5, 15, and 30 times. Protein Adsorption on the Membranes. Adsorption of BSA (99%, purchased from Sigma-Aldrich) onto the PEGylated copolymercoated membranes was evaluated using the method of Bradford according to the standard protocol of the Bio-Rad protein assay. The membrane with a 20 cm2 surface area was rinsed with 20 mL of
Jwi = 1402
V Vwi or JPi = Pi AΔt AΔt dx.doi.org/10.1021/la204012n | Langmuir 2012, 28, 1399−1407
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where the parameters Vwi, VPi, A, and Δt denote the pure water and protein solution permeate volume in the ith cycle (in L), membrane area (in m2), and permeation time (in h). For each cyclic operation, the filtration cell was emptied and refilled with 1 mg/mL protein solution and the flux was checked from time to time until a steady flux was obtained.
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RESULTS AND DISCUSSION Polymerization of PEGylated Copolymers. To study the effects of surface coverage and structures of the surfaceanchored PEGylated copolymers on the biofouling characteristics, two types of PEGylated copolymers, PS-b-PEGMA and PS-r-PEGMA, were synthesized in this work. A summary of average molecular weights, various PS/PEGMA compositions, and copolymer coating characteristics of prepared copolymers is given in Table 1. The degree of polymerization was controlled by the initial molar mass ratio of monomer to initiator. Low polydispersities of the PS-b-PEGMA copolymers were obtained, less than 1.18, which confirms the good living character of the ATRP polymerization. The final PS-b-PEGMA copolymers obtained had a molecular weight (Mw) ranging from 17 to 30 kDa with a well-controlled PS/PEGMA ratio between 0.8 and 3.6. To study biofouling resistance associated with the physical chain structures of the PEGylated copolymers, three random copolymers with different PS/PEGMA ratios (0.9, 1.7, and 3.7) were prepared from various molar mass ratios of styrene to PEGMA in the reaction solutions. The compositions of the synthesized PS-r-PEGMA copolymers were calculated from integration of 1H NMR analysis and GPC data using the OmniSEC software from Viscotek and are shown in Table 1. The PS-r-PEGMA copolymers have similar molecular weight distributions (i.e., Mw/Mn = 1.41−1.48). The hydrodynamic diameter of all prepared copolymers in the soluble unimer state (
