hydrophilic characteristics of magnetic microspheres on the immobilization of BSA

Colloids and Surfaces B: Biointerfaces 82 (2011) 302–306

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Effect of hydrophobic/hydrophilic characteristics of magnetic microspheres on the immobilization of BSA Dong-Hao Zhang a,b,∗ , Ya-Fang Zhang a , Gao-Ying Zhi c , Yu-Lei Xie a a

College of Pharmacy, Hebei University, Baoding 071002, China Key Laboratory of Pharmaceutical Quality Control of Hebei Province, College of Pharmacy, Hebei University, Baoding 071002, China c Computer Center, Hebei University, Baoding 071002, China b

a r t i c l e

i n f o

Article history: Received 13 July 2010 Received in revised form 27 August 2010 Accepted 1 September 2010 Available online 15 September 2010 Keywords: Magnetic microsphere Bovine serum albumin Immobilization Hydrophobicity Hydrophilicity

a b s t r a c t Core-shell magnetic poly(styrene-acrylamide-acrylic acid) microspheres with carboxyl groups were successfully synthesized via dispersion copolymerization in the presence of nano-particle of Fe3 O4 . The microspheres were characterized by FTIR spectra. They were used as carrier to immobilize bovine serum albumin (BSA). To investigate the effect of the microsphere surface properties on the immobilization of BSA, a series of microspheres with different hydrophobic/hydrophilic surface characteristics were prepared by adjusting molar percentages of monomers. The results showed that microspheres with different hydrophobicities/hydrophilicities had different immobilized ratios of BSA. In comparison with microspheres having hydrophilic characteristics those with hydrophobic characteristics had a much higher immobilized ratio. The possible reasons for these observations are discussed. In addition, ester activation and coupling times were optimized with respect to immobilized ratio. © 2010 Elsevier B.V. All rights reserved.

1. Introduction Magnetic microsphere has been fully developed as a new functional material in recent years, which is composed of polymer shell and inorganic magnetic particle core. These microspheres are widely used in biomacromolecules purification, cell separation [1], enzyme immobilzation [2], biosensors [3,4] and controlled drug delivery [5]. For example, dextran-coated magnetite was used as a drug carrier which allowed a targetable delivery with particle localization in a specific area [6], silane coated ferrite particles was utilized as radioimmunoassay [7]. In fact, the preparation of micron-size microspheres has been already widely studied, especially for polystyrene (PSt) as well as poly(Methyl Methacrylate) (PMMA) systems. Numerous literatures have reported the preparation of functionalized polystyrene microspheres by using other vinyl comonomers [8–12]. For all these applications, the magnetic microspheres not only were biocompatible but also possessed some advantages such as re-usable, easy separation and so on. Besides, the microspheres for immobilized protein studies must meet some

∗ Corresponding author at: Key Laboratory of Pharmaceutical Quality Control of Hebei Province, College of Pharmacy, Hebei University, Wusidong Road, 180#, Baoding 071002, China. Tel.: +86 312 5971107; fax: +86 312 5971107. E-mail address: [email protected] (D.-H. Zhang). 0927-7765/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2010.09.001

more requirements, which spurs the research of their preparation with desired particle size, surface properties, and functionality. Particularly, it is generally recognized that the surface properties of magnetic microsphere are very important because they can affect not only the interaction between carriers and proteins but also the conformational change of proteins and thus their denaturation and bioactivity loss. So far, some efforts have already been made to gain an insight of protein adsorption behaviors on polymer microspheres [13–16]. However, the information of protein immobilization onto magnetic microspheres with different surface characteristics, especially the hydrophobicity/hydrophilicity, is still rare. Here, P(St/AA/AM) magnetic microspheres with carboxyl groups were prepared via dispersion copolymerization. Bovine serum albumin (BSA) was selected as model protein to be covalently immobilized on the magnetic microspheres. In order to establish the optimum conditions of BSA immobilization, the influence of reaction time was studied. To further investigate the effect of microsphere surface hydrophobic property on BSA immobilization, a series of microspheres with different hydrophobic/hydrophilic surface characteristics were prepared by adjusting molar percentages of monomers. This work is believed to be of both academic interest and practical importance. Knowledge about these results may be utilized for rational design of carriers used in controlled drug delivery, protein immobilization, and so on.

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2. Materials and methods 2.1. Materials Bovine serum albumin (BSA) was obtained from Sigma (St. Louis, MO). Acrylic acid (AA) (99%) was from Kemiou Chemical Co. (China). Acrylamide (AM) (98%) and styrene (St) (98%) were purchased from Fuchen Chemical Co. (China). N-hydroxysuccinimide (NHS) (97%) and N,N -dicyclohexylcarbodiimide (DCC) (96%) were from Sinopharm Chemical Reagent Co., Ltd. While Ammonium persulfate, FeCl3 (99%), FeSO4 (99%) and other reagents were of analytical grade and purchased from local sources. Milli-Q ultra pure water was used in this study.

2.2. Methods 2.2.1. Preparation of Fe3 O4 magnetic particles Magnetic particles were prepared by chemical co-precipitation method [17,18]. A complete precipitation of Fe3 O4 was achieved under alkaline condition (25% NH3 H2 O) maintaining a molar ratio of Fe2+ :Fe3+ = 1:1.6. The resulting suspension was cooled down to room temperature and then washed with Milli-Q water to remove any unreacted chemicals. The particles obtained were black in colour and exhibited a strong magnetic response. Finally, the wet magnetic particles were modified by PEG-4000 to prevent the aggregation and used for the following experiments.

2.2.2. Preparation of microspheres Microspheres with micron size were prepared via dispersion polymerization method, which was performed with styrene, acrylamide, acrylic acid and Fe3 O4 magnetic particle. The pH was maintained at 7.0 and monomers’ total amount was maintained at 20 mmol during all the experiments. The general procedure can be described as follows: after the addition of acrylic acid, acrylamide, styrene and emulsifier, 1 mL of Fe3 O4 particles (magnetic fluid in water) was dispersed in 20 mL of 80% ethanol–water solution and ultrasonicated for 5 min. And then the polymerization reaction was started by adding ammonium persulfate as initiator at 70 ◦ C at 200 rpm. After 2 h, magnetic polymer microspheres were separated from the supernatant under the help of magnet. Then the microspheres were washed by ethanol and Milli-Q Water for several times. The resulted microspheres were dried at 55 ◦ C under vacuum for the following experiment and FTIR analysis.

2.2.3. Reaction of activated ester The magnetic polymer microspheres were dispersed in 4 mL ethanol with an excess of DCC and NHS dissolved in advance. The mixture was ultrasonicated for 5 min and then was left in a temperature-controlled incubator shaker at 160 rpm at 37 ◦ C for 1 h. The activated microspheres were washed with water for several times to remove any unreacted chemicals.

2.2.4. Reaction of BSA immobilization Immobilization of BSA on microspheres was carried out by mixing 2.5 mL of BSA buffer solution (0.4 mg/mL, pH = 7.0) with activated magnetic microspheres in a shaker at 160 rpm at 37 ◦ C for 2 h. BSA concentration in supernatant was measured by UVspectrometer (PGENERAL) at 595 nm using Bradford’s dye binding assay [19]. The amount of BSA immobilized on the microspheres was calculated from mass balance. All experiments were conducted in triplicate. The mean values were presented, and standard deviations were given as error bars in figures.

Fig. 1. Transmission electron micrographs of magnetic particles.

3. Results and discussion 3.1. Characterization of magnetic particles and magnetic microspheres Magnetite (Fe3 O4 ) nanoparticles were prepared by coprecipitation method. A typical transmission electron microscopy (TEM) photo of the particles was shown in Fig. 1. It showed that the size of magnetic particles was about 10–15 nm, which was similar to the previous reports [17,18]. It is known that magnetic particles less than 30 nm will exhibit superparamagnetism [18]. Therefore, the prepared Fe3 O4 particles had superparamagnetic properties and were expected to respond well to magnetic fields without any permanent magnetization. Dispersion polymerization is a very attractive method for preparing micron-size polymeric microspheres because of its simplicity and single-step process [20]. It is especially suitable for preparing microspheres of 0.1–15 ␮m in diameter, often of fairly good monodispersity [21]. Therefore, magnetic polymer microsphere with carboxyl groups was prepared by dispersion copolymerization. Fig. 2 showed Fourier transform IR spectra of different samples, including magnetic Fe3 O4 particles, P(St/AA) magnetic microsphere and P(St/AA/AM) magnetic microsphere. As observed, the spectra a, b and c had several common features since the components of all those samples contained Fe3 O4 . For example, the spectrum of Fe3 O4 had adsorption peaks at 600 cm−1 . However, the spectra of samples b and c presented some new adsorption peaks compared with sample a, which indicated that styrene and acrylic acid were successfully enfolded on the surface of the magnetic particles. For example, in the spectrum of sample b, the adsorption peak at 1700 cm−1 , 3430 cm−1 and 1050 cm−1 should be attached to the stretching vibrations of C O, O–H and C–O of carboxyl groups [22,23]. The peak at 1450 cm−1 and 2900 cm−1 was due to the benzene ring in-plane bending vibration and the C–H stretching. As can be seen from spectrum c, after the addition of acrylamide, the characteristic absorption peaks of acrylamide at 1640 cm−1 (NH2 scissors vibration), 1100–1400 cm−1 (C–N stretching) and 3400 cm−1 (N–H stretching) were present compared with spectrum b. Besides, comparing spectra a, b, and c, it was found that the chemical interaction between Fe3 O4 and monomers was not significant since no noticeable shift of the IR band due to the binding was observed. 3.2. Effect of reaction time on BSA immobilization It is well known that BSA cannot be coupled with carboxyl groups easily under mild conditions. Thus the immobilization reac-

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Fig. 3. The immobilized ratio of BSA (0.4 mg/mL) in buffers (pH = 7.00) containing 50 mM Na2 HPO4 and 50 mM NaH2 PO4 at different activation times at 37 ◦ C. The coupling time was fixed at 2 h.

Wavenumber (cm )

tion was divided into two steps, i.e. forming activated ester and then coupling with protein (Scheme 1). Firstly, carboxyl groups could be converted to activated ester, which means that the magnetic microspheres with carboxyl groups were treated by NHS in the presence of DCC. After that, the activated carboxyl groups could easily and directly couple with BSA by the formation of –CO–NH– covalent bond under mild condition. In order to establish the optimum conditions of BSA immobilization, the influences of the activation time and coupling time were studied, respectively. Fig. 3 showed the effect of activation time on the immobilized ratio of BSA when fixing the coupling time at 2 h. As observed, when the activation time was increased from 1 h to 5 h, there was no significant change on the immobilized ratio of BSA. These results indicated that the carboxyl groups on the microspheres’ surface could be easily activated with excess NHS in a short time. Fig. 4 showed the effect of coupling time on the immobilization ratio of BSA. As can be seen, with the increase of coupling time from 1 h to 2.5 h, there was a significant increase for immobilized ratio, approximately 25%. However, as coupling time was continuously increased from 2.5 to 3 h, a slight increase was observed, from 77 to 81%. This result indicated that the coupling reaction between microspheres and BSA could reach equilibrium within 3 h. 3.3. Effect of hydrophobicity/hydrophilicity on the immobilization of BSA Styrene and acrylamide were used as hydrophobic and hydrophilic groups, respectively, on the microspheres’ surface so as to modulate the hydrophobicity/hydrophilicity. Scheme 2 showed the conceptions of the surface structures of hydrophobic/hydrophilic microspheres. The carboxyl was used as functional

O COOH + HO N O

Microsphere

NHS

group (as well as hydrophilic group) to bond with BSA molecule and its amount was fixed. However, the amounts of styrene and acrylamide were changed to adjust the microspheres’ hydrophobicity/hydrophilicity. As presented in Scheme 2, type 1 was characterized by amide group, which ruled a high hydrophilicity. From types 2 to 7, the benzene ring amount was increased gradually (the molar percentages of styrene were 20%, 50%, 66.7%, 75%, 80%, and 85%, respectively), which meant the increase of hydrophobicity. Due to a lot of hydrophobic groups (benzene ring) on the surface of type 8, its hydrophobicity was the highest. The BSA immobilization experiments were conducted by using magnetic polymer microspheres of types 1–8 as carriers, and the results were shown in Fig. 5. As can be seen, when microspheres’ surface was occupied by amide group (type 1), the immobilized ratio of BSA was lower, approximately 60%. With the increase of styrene amount (types 2–7), the immobilized ratio increased subsequently. When the molar percentage of styrene was enhanced to 91% (type 8), a significant increase on the immobilization ratio was observed, over 20%. These results indicated that, during the process of immobilization, the type 8 microsphere was more easy to touch and subsequently covalently bind BSA molecule than other types of microsphere.

100

80

Immobilized ratio (%)

Fig. 2. FTIR speetra of (a) Fe3 O4 particles, (b) poly(styrene-acrylic acid) microspheres, and (c) poly(styrene-acrylic acid-acrylamide) microspheres.

60

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O DCC

O C O

N

BSA

O C BSA

1

1.5

2

2.5

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O

Activated ester

0 0.5

Immobilized BSA

Scheme 1. The procedure of the coupling reaction of BSA with microsphere.

Fig. 4. The immobilized ratio of BSA (0.4 mg/mL) in buffers (pH = 7.00) containing 50 mM Na2 HPO4 and 50 mM NaH2 PO4 at different coupling times at 37 ◦ C. The activation time was fixed at 1 h.

COOH

COOH

CO NH

2

COOH

2

COOH

CO NH

2

COOH 2

COOH

NH CO

NH CO

2

2

CO NH

CO NH

COOH

2

CO NH

COOH

2

COOH

CO

NH

COOH

2

CO NH

COOH

Type 8

Type 2-7

Type 1

305

COOH

2

2

NH CO

CO NH

2 NH CO

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Scheme 2. Surface structure of different magnetic polymer microspheres studied. The functional groups (–COOH) are highlighted in blue color. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

It is generally believed that the microspheres’ surface may represent three main interactions between microspheres and protein molecules [20]: hydrophobic interaction, hydrogen bond, electrostatic force. It should be noticed that the net charge on the surface of all types of microspheres is zero since there is no ionization for the activated polymer microspheres. As well as, the zeta potential analysis showed that the value of zeta potential of activated microspheres is zero at pH 7.0. Thus the electrostatic force between microspheres and BSA molecules can be ignored. As for type 1, hydrogen bond could be formed between amide group and aminoacid residue owing to the presence of acrylamide [14], which might be the main interaction between the hydrophilic microsphere and BSA protein. However, from types 2 to 8, the hydrophobicity was enhanced gradually due to the increase of benzene ring amount, as well as the hydrogen bond interaction was weakened gradually due to the decrease of acrylamide amount. Not surprisingly, hydrophobic interaction therefore should become the prevailing interaction between microsphere and BSA protein, which resulted in more BSA molecules approaching microspheres and subsequently coupling with them. As a consequence, the immobilized ratio increased drastically. Apparently, type 8 microsphere had the highest immobilized ratio. Therefore, our results seemed to suggest that hydrophobic interaction played an important role in increasing the immobilized ratio of BSA, which was also pointed out in Hou et al.’s work [20]. They even suggested that hydrophilic amide group might resist BSA molecules from approaching through the hydrophobic interaction [20]. In addition, Gruüttner et al. also pro-

posed that the surface of magnetic polystyrene microspheres was very hydrophobic, which resulted in a high amount binding of proteins on the microspheres’ surface [24]. 3.4. Effect of acrylic acid content on the immobilization of BSA The amount of added acrylic acid has important effect on the immobilization of BSA because it provides the functional group (–COOH). In order to study the effect of acrylic acid amount on the immobilized ratio, the microspheres were prepared by adding different amounts of acrylic acid from 0.23 mmol to 10.01 mmol and controlling the total amount of all monomers at 20 mmol. As known, increase of acrylic acid amount could result in the increase of not only functional group amount but also hydrophilic group amount. To keep hydrophobicity constant, the amount of acrylamide is decreased accordingly when the amount of acrylic acid is increased, regardless of the slight difference in hydrophilicity between acrylic acid and acrylamide. The results were presented in Fig. 6, which showed the relationship between immobilized ratio and the amount of acrylic acid. The amount of –COOH group on the microsphere surface was estimated by titration, and the result showed that the microsphere possessed more –COOH group on the surface with increase of acrylic acid amount added (data not shown). In Fig. 6, the observation showed more BSA coupling to the microspheres with increase of acrylic acid amount till 3.64 mmol, which proved that the immobilized ratio was highly dependent on the amount of carboxyl. We attributed this to that more acrylic acid caused more carboxyl to be coated on the 100

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Immobilized ratio (%)

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66.7 75 Percent of St (%)

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Fig. 5. The immobilization ratio of BSA at the different percentages of styrene. All those microspheres were prepared by controlling the total amount of all monomers at 20 mmol and acrylic acid amount at 1.82 mmol.

0

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Acrylic acid (mmol) Fig. 6. The immobilization ratio of BSA at the different amounts of acrylic acid. All those microspheres were prepared by controlling the total amount of all monomers at 20 mmol and the molar percentages of styrene at 50%.

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microspheres and thus increased the immobilized ratio. However, the BSA immobilization ratio did not increase continuously with higher amount of acrylic acid used though more acrylic acid than 3.64 mmol could cause more carboxyl groups to be attached on the microspheres, which was coincident with the result in Fig. 5. The possible reason was that when certain amount of BSA was coupled on one of the microsphere, there is no enough surface space to hold more BSA molecules. That is, the steric hindrance may affect the furthermore immobilization of more BSA molecules. 4. Conclusions To immobilize BSA protein, magnetic polymer microsphere with carboxyl groups was prepared by dispersion copolymerization. FTIR spectra analysis indicated that the monomers were successfully enfolded on the microspheres’ surface. In order to investigate the effect of microspheres’ hydrophobicity on the immobilized ratio of BSA, we synthesized a series of microspheres with different hydrophobic/hydrophilic characteristics. Both hydrophilic microspheres and hydrophobic microspheres have shown effective coupling to BSA. However, these results furthermore suggested that the introduction of hydrophobic benzene ring onto the surface of microspheres could observably increase the immobilized ratio of BSA, which indicated the importance of hydrophobic interaction between microspheres and protein. In addition, the conditions of BSA immobilization were optimized. It has been found that immobilized time had an observable effect on BSA immobilization, while the effect of activation time was comparatively insignificant above 1 h. The information gained from the present work could be utilized for rational design of polystyrene carriers for enzyme and antibodies/receptors used in solidphase immunoassay, especially SPA in HTS.

Acknowledgments This work was supported by the project sponsored by the Scientific Research Foundation for the returned overseas Chinese scholars, State Education Ministry (the project-sponsored by SRF for ROCS, SEM) and the project supported by Science Foundation of Hebei University, China (No. 2008-139). References [1] H. Honda, A. Kawabe, M. Shinkai, T. Kobayashi, J. Ferment. Bioeng. 86 (1998) 191. [2] A. Kondo, H. Fukuda, J. Ferment. Bioeng. 84 (1997) 337. [3] L. Viveros, S. Paliwal, D. McCrae, J. Wild, A. Simonian, Sens. Actuators B: Chem. 115 (2006) 150. [4] A. Galperin, D. Margel, S. Margel, J. Biomed. Mater. Res. A 79 (2006) 544. [5] S. Slomkowski, T. Basinska, B. Miksa, Polym. Adv. Technol. 13 (2002) 906. [6] A.N. Rusetski, E.K. Ruuge, J. Magn. Magn. Mater. 85 (1990) 299. [7] L.R. Witherspoon, S.E. Shuler, S. Gilbert, Clin. Chem. 31 (1985) 413. [8] C. Chen, W. Lee, J. Polym. Sci. A: Polym. Chem. 37 (1999) 1457. [9] Z. Tao, W. Yang, H. Zhou, C. Wang, S. Fu, Colloid Polym. Sci. 278 (2000) 509. [10] W. Yang, J. Hu, Z. Tao, L. Li, C. Wang, S. Fu, Colloid Polym. Sci. 277 (1999) 446. [11] W. Yang, D. Yang, J. Hu, C. Wang, S. Fu, J. Polym. Sci. A: Polym. Chem. 39 (2001) 555. [12] V.L. Covolan, Macromolecules 33 (2000) 6685. [13] J. Yoon, J. Kim, W. Kim, Colloids Surf. A 153 (1999) 413. [14] J. Yoon, H. Park, J. Kim, W. Kim, J. Colloid Interface Sci. 177 (1996) 613. [15] A. Gessner, A. Lieske, B.-R. Paulke, R.H. Muller, J. Biomed. Mater. Res. A 65 (2003) 319. [16] M. Luck, B.R. Paulke, W. Schroder, T. Blunk, R.H. Muller, J. Biomed. Mater. Res. 39 (1998) 478. [17] Z.G. Peng, K. Hidajat, M.S. Uddin, J. Colloid Interface Sci. 271 (2004) 277. [18] Z.G. Peng, K. Hidajat, M.S. Uddin, Colloids Surf. B 33 (2004) 15. [19] M.M. Bradford, Anal. Biochem. 72 (1976) 248. [20] X. Hou, B. Liu, X. Deng, B. Zhang, J. Yan, J. Biomed. Mater. Res. A 83 (2007) 280. [21] S. Kawaguchi, K. Ito, Adv. Polym. Sci. 175 (2005) 299. [22] Y. Yong, Y.-X. Bai, Y.-F. Li, L. Lin, Y.-J. Cui, C.-G. Xia, Process Biochem. 43 (2008) 1179. [23] B. Hu, J. Pan, H.-L. Yu, J.-W. Liu, J.-H. Xu, Process Biochem. 44 (2009) 1019. [24] C. Gruüttner, S. Rudershausen, J. Teller, J. Magn. Magn. Mater. 225 (2001) 1.