Novel Sequence for Generating Glycopolymer Tethered on a

Yan Li , Jing Quan , Christopher Branford-White , Gareth R. Williams , Jin-Xian Wu , Li-Min Zhu. Journal of Molecular Catalysis B: Enzymatic 2012 76, ...
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Langmuir 2005, 21, 10717-10723

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Novel Sequence for Generating Glycopolymer Tethered on a Membrane Surface Qian Yang,† Zhi-Kang Xu,*,† Meng-Xin Hu,† Jun-Jie Li,‡ and Jian Wu‡ Institute of Polymer Science, and Department of Chemistry, Zhejiang University, Hangzhou 310027, P.R. China Received July 5, 2005. In Final Form: August 25, 2005 Cell surface carbohydrates, usually binding with other biomacromolecules (such as lipids and proteins), are involved in numerous biological functions, including cellular recognition, adhesion, cell growth regulation, and inflammation. Synthetic carbohydrate-based polymers, so-called glycopolymers, are emerging as important well-defined tools for investigating carbohydrate-based biological processes and for simulating various functions of carbohydrates. In this study, a novel two-step sequence for the generation of a glycopolymer layer tethered on a polypropylene microporous membrane is described. First, a UV-induced graft polymerization of 2-aminoethyl methacrylate hydrochloride (AEMA) was carried out on the membrane to generate an amino-functionalized surface, and the effects of polymerization factors (monomer/initiator concentration and UV irradiation time) on the grafting density were studied. Second, sugar moieties were bound with the grafted functional layer to form glycopolymer by the reaction between the amino groups on the membrane surface and carbohydrate lactones. Chemical analysis by Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy combined with surface morphology observation by scanning electron microscopy confirmed the graft polymerization of AEMA and the formation of glycopolymer. The decreases of water contact angle and protein adsorption on the membrane revealed the enhancement of hydrophilicity and protein resistance due to the typical characteristics of the glycopolymer tethered on the surface. These results indicated that the novel sequence reported in this work is a facile process to form glycopolymer-modified surfaces.

Introduction Carbohydrates, ubiquitous in living things, play essential roles in many functions. Allowing almost unlimited structural variations, carbohydrates are featured as glycocode and are unrivalled in the density of information they can convey.1,2 It is now clear that carbohydrates play a major role in many recognition events. Recognition is key to a variety of biological processes and the first step in numerous phenomena based on cell-cell interactions, such as blood coagulation, immune response, viral infection, inflammation, embryogenesis and cellular signal transfer.3 These recognition processes are thought to proceed by specific carbohydrate-protein interaction. The proteins involved, generically named lectins, are most frequently found on cell surfaces. They have the ability to bind specifically and noncovalently to carbohydrates.4,5 Concurrently with the realization of their potential for coding biological information, carbohydrates are found on the surface of nearly every cell surface in forms of polysaccharides, glycoproteins, glycolipids, or other glycoconjugates.3,6 They, located on the external surface of the cell membrane, dominate many important biological processes. Membrane carbohydrates, especially glycocalyx, direct the specific interactions and contribute to the steric repulsion that prevents undesirable nonspecific adhesion * To whom all correspondence should be addressed. Fax: ++ 86 571 8795 1773. E-mail: [email protected]. † Institute of Polymer Science. ‡ Department of Chemistry. (1) Gruner, S. A. W.; Locardi, E.; Lohof, E.; Kessler, H. Chem. Rev. 2002, 102, 491. (2) Ladmiral, V.; Melia, E.; Haddleton, D. M. Eur. Polym. J. 2004, 40, 431. (3) Dwek, R. A. Chem. Rev. 1996, 96, 683. (4) Sharon, N.; Lis, H. Science 1989, 246, 227. (5) Bertozzi, C. R.; Kiessling, L. L. Science 2001, 291, 2357. (6) Wang, Q.; Dordick, J. S.; Linhardt, R. J. Chem. Mater. 2002, 14, 3232.

of other molecules and cells.7,8 Vividly, carbohydrates are entitled “antenna on the cell surface”. As a consequence, increasing interest has been paid on synthetic sugarcontaining polymers (glycopolymer) for the study of the specific recognition ability and for the “glycomimics”. There are four methods to synthesize artificial glycopolymers: additional polymerization of vinyl sugars, cationic polymerization of anhydro sugars, enzymemediated synthesis of carbohydrate polymers, and tethering of sugars onto functionalized synthetic polymers by polymer analogous reactions.9 Among these strategies, the first may be the most widely used because it provides more multiplicity and relatively convenient experimental processes. In our previous work,10 polymers containing linear and cyclic monosaccharides (glucose) were synthesized and used to develop the surface properties of different polymer materials. However, there is a vital disadvantage for this direct monomer synthesis method in that most vinyl sugars must be synthesized in a protected way to avoid unwanted side reactions in the hydroxyl groups. Also, these vinyl sugars, or so-called glycomonomers, are difficult to separate and purify from other chemicals, especially if the sugar moiety is a disaccharide or oligosaccharide. On the other hand, the separation and purification of glycomonomer can be avoided by tethering sugar moieties directly onto functionalized synthetic polymers through polymer analogous reactions. Although (7) Holland, N. B.; Qiu, Y. X.; Ruegsegger, M.; Marchant, R. E. Nature 1998, 392, 799. (8) Faucher, K. M.; Sun, X. L.; Chaikof, E. L. Langmuir 2003, 19, 1664. (9) Varma, A. J.; Kennedy, J. F.; Galgali, P. Carbohydr. Polym. 2004, 56, 429. (10) (a) Kou, R.-Q.; Xu, Z.-K.; Deng, H.-T.; Liu, Z.-M.; Seta, P.; Xu, Y.-Y. Langmuir 2003, 19, 6869. (b) Xu, Z.-K.; Kou, R.-Q.; Liu, Z.-M.; Nie, F.-Q.; Xu, Y.-Y. Macromolecules 2003, 36, 2441. (c) Yang, Q.; Xu, Z.-K.; Dai, Z.-W.; Wang, J.-L.; Ulbricht, M. Chem. Mater. 2005, 17, 3050.

10.1021/la051797g CCC: $30.25 © 2005 American Chemical Society Published on Web 09/29/2005

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Figure 1. Schematic diagram illuatrating the UV-induced graft polymerization of AEMA and the formation of glycopolymer.

it has obvious advantages, such as the fact that protected as well as unprotected sugars can be used, mild reaction condition, ease in controlling the number of sugars being incorporated, and facility in gaining a well-defined backbone structure,9 there are, however, few reports on the synthesis of glycopolymers by polymer analogous reactions. Herein, we report a novel strategy for the synthesis of surface-tethered glycopolymer. This method includes the UV-induced graft polymerization of functional monomers and the binding of sugar moieties. First, a functional layer containing the amino groups was generated on a porous support. Then, sugars in the lactone form were reacted with the amino groups and bound to the polymer backbone to generate glycopolymer. With the sugar moieties (glycopolymer) on the surface, we conceive this porous support (membrane) can serve as a lectin or antibody-binding assay,11-13 chromatographic support for affinity chromatography, and for the isolation of proteins with specificity for different sugar residues,14,15 molecularly imprinted polymer,16,17 and enzyme immobilization support.18-20 In this study we focused on the generation of the functional layer and its reaction with a monosaccharide. One can extend this strategy to other kinds of monosaccharides, oligosaccharides, and even polysaccharides expediently. Experimental Section Materials. The porous support, polypropylene microporous membrane (PPMM), was purchased from Membrana GmbH (11) Kitano, H.; Ishino, Y.; Yabe, K. Langmuir 2001, 17, 2312. (12) Kitano, H.; Maehara, Y.; Matano, M.; Sugimura, M.; Shigemori, K. Langmuir 1997, 13, 5041. (13) Kitano, H.; Ohno, K. Langmiur 1994, 10, 4131. (14) Roy, R.; Tropper, F. D. J. Chem. Soc., Chem. Commun. 1988, 1058. (15) Kobayashi, K.; Sumitomo, H.; Ina, Y. Polym. J. 1985, 17, 567. (16) Wulff, G.; Schauhoff, S. J. Org. Chem. 1991, 56, 395. (17) Nagahori, N.; Nishimura, S. Biomacromolecules 2001, 2, 22. (18) Deng, H.-T.; Xu, Z.-K.; Dai, Z.-W.; Wu, H.; Seta, P. Enzyme Microb. Technol. 2005, 36, 996. (19) Deng, H.-T.; Xu, Z.-K.; Wu, H.; Ye, P.; Liu, Z.-M.; Seta, P. J. Mol. Catal. B: Enzym. 2004, 28, 95. (20) Wang, P.; Hill, T. G.; Chaw, C. A. W.; Huston, M. E.; Oehler, L. M.; Smith, M. B.; Bednarski, M. D.; Callstrom, M. R. J. Am. Chem. Soc. 1992, 114, 378.

(Germany), which was prepared by the thermally induced phase separation (TIPS) method with an average pore size of 0.20 µm and a relatively high porosity of about 75-80%. All the membranes used in this study were cut into rotundity with a diameter of 3.95 cm (area ) 12.25 cm2). Before the graft polymerization, the membranes were dipped in acetone for 0.5 h and then rinsed with acetone several times to removal any impurities adsorbed on the surfaces. After being dried in a vacuum oven at 40 °C for 1 h, these membranes were stored in a desiccator. 2-Aminoethyl methacrylate hydrochloride (AEMA) was synthesized as described elsewhere.21,22 Triethylamine (TEA) was a commercial product and was distilled before use. Benzophenone (BP), heptane, and D-gluconolactone were analytical grade and were used without further purification. The water used in all syntheses and measurements was deionized to 18 MΩ. Graft Polymerization of AEMA. The functional layer with the amino groups was generated by the UV-induced graft polymerization of AEMA on the membrane surface. PPMM was dipped in 10 mL of photoinitiator solution (benzophenone in heptane) for 60 min and then dried in air for 30 min, whereafter the membrane was washed with acetone and quickly wiped with filter paper. The membrane with the pore still wetted by acetone was fixed between two filter papers (No. 593, Schleicher & Schuell) immediately and immersed into 10 mL of monomer solution in a reaction chamber. Then, UV irradiation was done for a predetermined time under an argon gas environment in an ultraviolet processor. The processor is equipped with a 500 W high-pressure mercury lamp. The distance between the lamp and the reaction chamber is 25 cm. Finally, the membrane was washed with water drastically using a vibrator. After being dried in a vacuum oven at 50 °C to constant weight, the grafting density (GD, µg/cm2) was calculated by the following equation:

GD )

W1 - W0 Am

where W0 is the mass of the nascent membrane and W1 is the mass of the membrane after graft polymerization and drying. Am represents the area of the membrane. Each result was the average of three parallel experiments. Formation of Glypolymer on the PPMM. Sugar moieties were bound onto the membrane surface by the reaction between (21) Artursson, P.; Brown, L.; Dix, J.; Goddard, P.; Petrak, K. J. Polym. Sci., Part A: Polym. Chem. 1990, 28, 2651. (22) Narain, R.; Armes, S. P. Biomacromolecules 2003, 4, 1746.

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the amino groups of the functional layer and D-gluconolactone as presented in Figure 1. PAEMA-grafted PPMM (PPMM-gPAEMA) was put in a D-gluconolactone water solution, and TEA was added. The mixture was shaken in a flask for 24 h at 20 °C. After rinsing with ethanol, the membrane was washed with water drastically using a vibrator and dried under vacuum at 50 °C. The binding density (BD, µg/cm2) was defined as

BD )

W2 - W1 Am

where W2 is the mass of the membrane tethered with glycopolymer (PPMM-g-GP). Reaction Ratio of the Amino Groups. The binding of sugar moieties and/or the formation of glycopolymer on the membrane surface was based on the reaction shown in Figure 1. The amount of amino groups on the membrane surface, introduced by the graft polymerization of AEMA, was calculated as

MNH2 )

W1 - W0 165.5

The amount of reacted amino groups, which was also equal to the amount of sugar moieties in the glycopolymer, was obtained as

Msugar )

W2 - W1 105

Thus, the reaction ratio of the amino group was

RNH2 )

Msugar MNH2

where 165.5 is the molecular weight of the repeat unit in the PAEMA chains and 105 is the molecular weight of the sugar residue that bound. Here, for the convenience of calculation, we ignored the weight loss caused by the neutralization of HCl. Characterization. To investigate the varieties in surface chemical structure and morphology before and after the modification and to confirm the grafting and the glycopolymer formation, surface characterization techniques were used (attenuated total reflectance Fourier transform infrared spectroscopy (FT-IR/ATR), X-ray photoelectron spectroscopy (XPS), and scanning electron microscopy (SEM)). FT-IR/ATR measurement was carried out on a Vector 22 FTIR (Brucker Optics, Switzerland) equipped with an ATR cell (KRS-5 crystal, 45°). Sixteen scans were taken for each spectrum at a resolution of 4 cm-1. XPS measurements of the original and modified membranes were performed on a PHI-5000C ESCA system (Perkin-Elmer, U.S.A.) with Al KR radiation (hν ) 1486.6 eV). In general, the X-ray anode was run at 250 W and the high voltage was kept at 14.0 kV with a detection angle at 45°. The pass energy was fixed at 93.9 eV to ensure sufficient sensitivity. The base pressure of the analyzer chamber was about 5 × 10-7 Pa. The survey spectra (from 0 to ∼1200 eV) and the core-level spectra with much higher resolution were both recorded. Binding energies were calibrated using the containment carbon (C1S ) 284.7 eV). The data analysis was carried out on the PHI-MATLAB software provided by PHI Corporation. No radiation damage was observed during the data collection time. Scanning electron microscopy (SEM) images were taken on a field emission SEM (SIRION, FEI, U.S.A.). For this purpose, samples were washed with a water-ethanol-hexane sequence, dried at room temperature, and then coated with a 20 nm gold layer before SEM analysis. Surface Properties Measurement. An OCA20 contact angle system (Dataphysics, Germany) was used for the determination of air/water contact angles at room temperature. The static contact angle was measured by the sessile drop method as follows. First, a water drop (∼5 µL) was lowered onto the membrane surface from a needle tip. Then, the images of the droplet were recorded in equal time intervals (10 s) for 1800 s (sometimes until the droplet disappeared). Contact angles were calculated

Figure 2. Effects of AEMA concentration (a), UV irradiation time (b), and initiator concentration (c) on grafting densities. from these images with software. Each value was an average of at least five measurements. Bovine serum albumin (BSA) was used as a model protein for the evaluation of the ability of the glycopolymer tethered PPMMs in reducing the nonspecific protein adsorption. Membranes with external surface area of 49 (12.25 × 4) cm2 were immersed in ethanol for 10 min and then in phosphate-buffered saline solution (PBS, pH ) 7.4) for 30 min to prewet. Then, each sample was put into a tube containing 10 mL of BSA solution with various concentrations whose pH was adjusted to 7.4 with PBS. The mixture was incubated at 30 °C for 24 h to reach an adsorptiondesorption equilibrium. The amount of adsorbed BSA was determined by measuring spectrophotometrically the difference between the concentrations of BSA in the solution before and after contact with the membranes. The spectroscopic analytical method utilized in this work for protein dosage was based on the reaction of albumin with Coomassie brilliant blue (Fluka) dyestuff to record the absorbance of the albumin-Coomassie brilliant blue complex according to Bradford’s method.23 A calibration curve between the spectrophotometrical absorbance and the BSA concentration was established to reduce the effect of protein adsorption on the surface of the experimental device for the adsorption measurement. The reported data were the mean value of triplicate measurements for each sample.

Results and Discussion Graft polymerization of AEMA. Figure 2 shows the effect of AEMA concentration on the GD. In this experiment, we used a 1.82 g/L photoinitiator concentration at all different AEMA concentrations. As can be seen from Figure 2a, the GD increased with the increase of AEMA concentration remarkably. However, the optimum AEMA concentration was 120 g/L, and the grafting density turned (23) Bradford, M. M. Anal. Biochem. 1976, 72, 248.

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Table 1. Chemical Composition PAEMA-Grafted and Sugar-Immobilized PPMMs Surfaces and the Reaction Ratio of the Amino Groups surface composition (mol %)

amino group reaction ratio (%)

sample

GD or BD (µg/cm2)

C

N

O

Cl

XPS

weight

PPMM-g-PAEMA PPMM-g-GP PPMM-g-AEMA PPMM-g-GP

285.71 136.06 174.15 84.36

87.89 84.45 94.47 91.37

2.98 2.20 1.71 1.91

7.12 13.35 3.02 6.72

2.01 0 0.79 0

67.80

75.0

25.30

76.0

to decrease when the concentration exceeded this value. With the increase of AEMA concentration, the living center of the graft polymerization reaction had more chance to contact with monomers and higher GD was achieved. However, the viscosity of the monomer solution also increased with the monomer concentration. The solution became viscous with high monomer concentration, and it was difficult for the monomer molecules to diffuse from the solution to the membrane surface. At the same time, extensive homopolymerization proceeded at the higher monomer concentration cases and consumed much more monomer than that of the lower monomer concentration cases. When the AEMA concentration exceeded 120 g/L, homopolymerization dominated the polymerization process and graft polymerization was depressed. Thus, the GD turned to decrease. The effect of UV irradiation time on the GD is shown in Figure 2b. It was found that the GD increased with the UV irradiation time in the range of 5-40 min. We intended to extend the UV irradiation time to 80 min, but the UV chamber was getting very hot and might have caused damage to the porous structure of the membrane. With the increase of UV irradiation time, more active sites were generated on the membrane surface and high GD was obtained. However, all the PPMMs were dipped into the photoinitiator solution for the same interval (60 min), and the adsorbed photoinitiator was approximately definite. Consequently, the active sites on the membrane surface could not increase infinitely, and it was reasonable to assume that the GD reaches a constant value at an irradiation time longer than 40 min. Figure 2 also shows the relationship between GD and BP concentration. A series of initiator solutions (BP in n-heptane) with different concentrations were used, and the monomer concentration and UV irradiation time were fixed as 60 g/L and 25 min, respectively, for this case. As can be seen from Figure 2c, increasing the initiator concentration could bring significant enhancement of GD because more active sites were generated, and subsequently, more polymer chains were tethered on the membrane surface. However, the adsorbed layer of initiator at the high concentration cases also disturbed the contact of monomer and the substrate surface and accelerated the homopolymerization. Therefore, the GD turned to decrease at high initiator concentration. Binding of Sugar Moieties and Formation of Glycopolymer. Sugar moieties were bound to the membrane surface by the reaction between the amino groups and D-gluconolactone to form the corresponding glycopolymer. As we know, AEMA is unstable22,24 because the free amino group may attack the carbonyl group in an internal rearrangement that generates 2-hydroxyethyl methacrylamide (see Figure 1). Taking this into consideration, we used a hydrochloride form of AEMA to protect the amino group. For the further reaction with Dgluconolactone, TEA, without which no reaction occurred, was used to neutralization the hydrochloride, and this in (24) Smith, D. A.; Cunningham, R. H.; Coulter, B. J. Polym. Sci., Part A: Polym. Chem. 1970, 8, 783.

Figure 3. Effects of PAEMA grafting density on sugar binding density (9) and the reaction ratio of the amino groups (O).

situ neutralization was preferred to reduce the rearrangement reaction. Figure 3 shows the influences of the GD of PAEMA on the BD of sugar moieties and the reaction ratio of the amino group, respectively. As expected, the amount of attached sugar moieties increased with the GD of PAEMA. The reaction ratio of the amino group remained in the range of ∼75% to ∼85% with the increase of GD. In this study, membranes with different GDs of PAEMA were reacted with the same amount of D-gluconolactone in the same reaction condition (time, temperature, and amount of TEA). The reaction between the amino groups and D-gluconolactone competed with the rearrangement reaction of the AEMA unit. With the increase of GD, these couple of reactions promoted comparably. Thus, more sugar moieties were bound to the PAEMA chains, but the reaction ratio of the amino group showed relatively stable. Data from Figure 3 also indicated that, in the reaction condition we chose, a considerable amount of the amino groups (15-25%) rearranged to carbonyl. Temperature might be the key factor for the competition of the two reactions. In addition to the data obtained by weighing the membranes before and after the binding of sugar moieties, XPS could also be used to determine the reaction ratio of amino groups (see the Supporting Information). As shown in Table 1, two samples with relatively low (174.15 µg/cm2) and high (285.71 µg/cm2) GDs of PAEMA were examined by XPS, and the reaction ratio of the amino groups was calculated from the peak intensity of C1S and O1S. As can be seen, the reaction ratios of the amino groups calculated from XPS were lower than those from weight data. The graft polymerization of AEMA and the attachment of sugar moieties took place not only on the membrane surface but also in the membrane pores. At the same time, XPS is a highly surface-selective analytical method, which supplies information of the most external surface (∼10 nm). Thus, sugar moieties in membrane pores could not be detected by XPS, and consequently, the mass weighing method always gave results larger than those of XPS. Furthermore, the results of the higher GD sample were more accordant than those of the lower PAEMA GD

Generation of Membrane-Tethered Glycopolymer

Figure 4. IR spectra of (a) nascent PPMM, (b) PPMM-gPAEMA before (GD ) 285.71 µg/cm2), and (c) PPMM-g-GP (BD ) 136.10 µg/cm2).

sample. For the lower GD cases, a majority of grafting and binding took place in the membrane pores, and the membrane surface might be exposed. However, in the case of higher GD, the membrane surface was covered with the PAEMA chains and subsequently the glycopolymer chains, which brought the approximating of the results obtained by the XPS and weighing methods. Characterization of the Surface. FT-IR/ATR was used to confirm the grafting of PAEMA and the formation of glycopolymer. Figure 4 shows the spectra of unmodified, PAEMA-functionalized, and glycopolymer tethered PPMM surfaces. The PAEMA-functionalized PPMM (PPMM-g-PAEMA) exhibited an absorption band at 1721 cm-1, which can be assigned to the CdO stretching in the carbonyl group of AEMA. However, for the glycopolymer tethered PPMM (PPMM-g-GP), three bands at 1721, 1646, and 1543 cm-1, ascribed to ester carbonyl, amide I, and amide II, respectively, were found. In addition, an adsorption band at 3387 cm-1, assigned to the -OH stretching vibration, could also been found for the PPMMg-GP. All these data indicated the grafting of PAEMA, the binding of sugar moieties, and the formation of glycopolymer. XPS has been used for characterizing polymer surfaces, especially new chemical structures introduced by plasma,25,26 UV,27,28 blending,29 and grafting of functionalization layers.30-32 Polymers having pendant saccharides were also investigated by XPS.33-35 XPS spectra for unmodified PPMM, PPMM-g-PAEMA, and PPMM-g-GP surfaces are shown in Figure 5 along with the analyses described in the Experimental Section. (25) Steen, M. L.; Jordan, A. C.; Fisher, E. R. J. Membr. Sci. 2002, 204, 341. (26) Chai, J. N.; Lu, F. Z.; Li, B. M.; Kwok, D. Y. Langmuir 2004, 20, 10919. (27) Hozumi, A.; Masuda, T.; Sugimura, H.; Kameyama, T. Langmuir 2003, 19, 7573. (28) Ji, L. Y.; Kang, E. T.; Neoh, K. G.; Tan, K. L. Langmuir 2002, 18, 9035. (29) Affrossman, S.; Kiff, T.; O’Neill, S. A.; Pethrick, R. A.; Richards, R. W. Macromolecules 1999, 32, 2721. (30) Ingall, M. D. K.; Honeyman, C. H.; Mercure, J. V.; Bianconi, P. A.; Kunz, R. R. J. Am. Chem. Soc. 1999, 121, 3607. (31) Herrera-Alonso, M.; McCarthy, T. J. Langmuir 2004, 20, 9184. (32) Wavhal, D. S.; Fisher, E. R. Langmuir 2003, 19, 79. (33) Nakamae, K.; Miyata, T.; Ootsuki, N.; Okumura, M.; Kinomura, K. Macromol. Chem. Phys. 1994, 195, 1953. (34) Nakamae, K.; Miyata, T.; Ootsuki, N.; Okumura, M.; Kinomura, K. Macromol. Chem. Phys. 1994, 195, 2663. (35) Wulff, G.; Schmidt, H.; Zhu, L.-M. Macromol. Chem. Phys. 1999, 200, 774.

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Figure 5. XPS spectra of the nascent and modified PPMM surfaces: (a) nascent PPMM, (b) PPMM-g-PAEMA (GD ) 285.71 µg/cm2), and (c) PPMM-g-GP (BD ) 136.10 µg/cm2).

Figure 6. C1S (left) and O1S (right) core-level spectra of the PPMM-g-PAEMA surface (top, GD ) 285.71 µg/cm2) and PPMM-g-GP surface (bottom, BD ) 136.10 µg/cm2).

For the unmodified PPMM surface, a major emission peak at 284.7 eV ascribed to the binding energy of C1S was found. However, additional peaks at 200.0, 402.8, and 534.0 eV, which were corresponding to the binding energies of Cl2P, N1S, and O1S, respectively, were detected from the spectrum of the PPMM-g-PAEMA surface. For the PPMMg-GP surface, the peak of Cl2P disappeared because of the neutralization process, which was essential for further reaction. The peak area of O1S for the PPMM-g-GP was almost 2 times that of the PAEMA-grafted ones (Table 1). This could be ascribed to the sugar moieties which contain many hydroxyl groups. The high-resolution spectra of the PPMM-g-PAEMA and PPMM-g-GP surfaces corresponding to C1S and O1S are shown in Figure 6 to distinguish the different types of functional groups on the surface. For the PPMM-g-PAEMA surface, as shown in Figure 6a, the C1S spectrum could be resolved into three peaks. The peak at a binding energy of 284.7 eV was assigned to C-H and C-C. Peaks at binding energies of 286.2 and 288.5 eV were ascribed to the C atoms bonded to the hydroxyl group (C-OH) and the ester group (OdC-O). As can be seen from Figure 6c, the spectrum of the PPMM-g-GP surface showed obvious dissymmetry. Four peaks at 284.7, 286.2, 287.2, and 288.5 eV binding energies could be observed. The additional peak at 287.2 eV was signal from the C atom associated with amide group (OdC-NH). Consistent results could also be concluded from the resulting O1S spectra. As shown in Figure 6b, the PPMM-

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Figure 8. Time dependence of water contact angles: (a) PPMMg-PAEMA (GD ) 193.20 µg/cm2); (b) PPMM-g-PAEMA (GD ) 239.46 µg/cm2); (c) PPMM-g-GP (BD ) 84.36 µg/cm2); (d) PPMMg-GP (BD ) 136.10 µg/cm2).

Figure 7. SEM images of (a) nascent PPMM, (b) PPMM-gPAEMA (GD ) 310.20 µg/cm2), and (c) PPMM-g-GP (BD ) 136.10 µg/cm2) surfaces.

g-PAEMA surface exhibited two separated peaks at 532.2 and 533.6 eV binding energies which are assigned to the double bond oxygen (OdC-O) and single bond oxygen (OdC-O) in the ester group, respectively. However, in the case of PPMM-g-PAEMA-S (Figure 6d), an additional peak located at 532.9 eV binding energy could be found. This peak was the emission bond of the oxygen in the hydroxyl group (C-OH) which was brought by the sugar moieties. The morphological change of the modified surface was detected by SEM. Typical SEM images are shown in Figure 7. As can be seen, the nascent PPMM used in this study shows relatively high porosity and small pore size (Figure 7a). However, after the graft polymerization of PAEMA, the surface was covered by PAEMA chains and the pores were blocked (Figure 7b). From the images of PPMM-gGP (Figure 7c), it can be observed that a dense glycopolyme layer was established on the membrane surface. Surface Properties. To study the hydrophilicity changes on the surface, contact angle measurements were carried out. The average contact angle of the virgin PPMM was ∼142°. After PAEMA grafting, all samples with different GD exhibit a decrease of hydrophilicity to some extent. However, ranging between ∼122° and 132°, the contact angles showed no distinct differences with various GDs (see the Supporting Information). The water drops took ∼2000, 2400, and 2800 s to soak into the membrane pores and disappeared completely from the surface of the virgin, 193.20, and 239.46 µg/cm2 PAEMA-grafted membranes (Figure 8, traces a and b), respectively. Commonly, water soaks into the pores more quickly for the hydrophilic surface than that of the hydrophobic one. However, compared with the nascent PPMM, PAEMA-grafted membranes showed blocked pores as can be seen in SEM

Figure 9. BSA adsorption at (a) nascent PPMM, (b) PPMMg-PAEMA (GD ) 310.20 µg/cm2), and (c) PPMM-g-GP (BD ) 117.0 µg/cm2) surfaces.

images. Despite the increase of hydrophilicity, water soaked slowly for the pore-clogged PPMM-g-PAEMA surfaces. For the same reason, water drops took more time (∼1500 s) to disappear from the glycopolymer tethered membrane surface with high BD (Figure 8d), which was more hydrophilic, than that of the low BD one (∼1250 s, Figure 8c). Digital images of water drops on the membrane surfaces along the experimental time are also shown in the Supporting Information. For various applications, nonspecific adsorptions of proteins and cells are expected to be reduced. Usually, a hydrophilic surface exhibits less interaction with biomacromolecules and cells. Also, saccharides on the external region of the cell membrane play a key role in preventing nonspecific adsorption of proteins and other cells. We used BSA as model protein to evaluate the protein adsorption characteristic of the nascent PPMM, the PPMM-gPAEMA, and the PPMM-g-GP. Typical results are shown in Figure 9. It was found that at low BSA concentrations (1 and 2 g/L) almost the same amount of BSA was adsorbed on these three different surfaces. However, at the higher BSA concentration cases (5 and 10 g/L), PPMM-g-GP showed the lowest amount of adsorbed BSA, and a higher BSA concentration could lead to a slight increase of BSA adsorption. Moreover, it was found that the PPMM-gPAEMA showed the highest amount of BSA adsorption. It could be ascribed that the grafted PAEMA chains were

Generation of Membrane-Tethered Glycopolymer

in the hydrochloride form and might enhance the interaction between the surface and the protein. Conclusions A functional layer containing amino groups was generated by UV-induced grafting polymerization of AEMA onto the porous support, polypropylene microporous membrane, surface. The grafting density could be adjusted by monomer concentration and UV irradiation time as well as photoinitiator concentration. Linear glucose was bound to these functional chains by the reaction between the amino groups and D-gluconolactone. SEM images revealed the morphological changes on the surface. FT-IR/ATR and XPS measurements confirmed the establishment of the sugar-containing layer and indicated that it was an effective way to tether glycopolymer chains to the support surface. Results in the water contact angle implied that a hydrophilic surface was achieved by the incorporation of sugar moieties. The BSA adsorption experiment proved

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that a glycopolymer tethered surface could reduce nonspecific protein adsorption obviously. Further work about the binding of biofunctional di- and oligosaccharides, which can be selectively recognized by proteins (such as lectins), on this PAEMA-functionalized membrane surface are being performed in our lab. These series of glycopolymer tethered membranes will be used to immobilize enzymes and to separate proteins. Acknowledgment. This work was financed by the National Natural Science Foundation of China (Grant No. 20474054). Supporting Information Available: Digital images of water drops, effect of the GD of PAEMA on the static contact angle, and reaction ratio of the amino groups calculated from XPS data. This material is available free of charge via the Internet at http://pubs.acs.org. LA051797G