Preparation of Bovine Serum Albumin Surface-Imprinted

Magnetically susceptible molecularly imprinted polymers using free ... LLS (BIC Particle Sizing Software 90 Plus, Brookhaven Instruments Corp.). ...
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Anal. Chem. 2008, 80, 683-692

Preparation of Bovine Serum Albumin Surface-Imprinted Submicrometer Particles with Magnetic Susceptibility through Core-Shell Miniemulsion Polymerization Chau Jin Tan,† Hong Gap Chua,† Kwee Hong Ker,† and Yen Wah Tong*,†,‡

Department of Chemical & Biomolecular Engineering, and Division of Bioengineering, National University of Singapore, 21 Lower Kent Ridge Road, Singapore 119077

Molecular imprinting is a state-of-the-art technique for preparing mimics of natural, biological receptors. Nevertheless, the imprinting of macromolecules like proteins remains a challenge due to their bulkiness and sensitivity to denaturation. In this work, a surface imprinting strategy based on covalently immobilized template molecules was adopted for protein imprinting. Bovine serum albumin (BSA) surface-imprinted submicrometer particles (500600 nm) with magnetic susceptibility were prepared through a two-stage core-shell miniemulsion polymerization system using methyl methacrylate and ethylene glycol dimethacrylate as functional and cross-linking monomers, respectively. The particles possessed a novel red blood cell-like structure and exhibited a very favorable recognition property toward the template BSA molecules in aqueous medium. In a two-protein system, the particles had shown a very high specific recognition of the template proteins over the nontemplate proteins. The magnetic susceptibility was imparted through the successful encapsulation of Fe3O4 nanoparticles. Their superparamagnetic nature increases their potential applications in the fields such as magnetic bioseparation, cell labeling, and bioimaging. In addition, the importance of template immobilization for successful protein imprinting had also been illustrated to demonstrate the potential of this approach as a general methodology for protein imprinting. Molecular imprinting has been widely recognized as a promising technique for imparting a predetermined molecular recognition property onto synthetic materials such as polymers. For the past few decades, the interest and attention shown toward this field has been increasing at an amazing pace.1 This is mainly attributed to the potential wide applications of molecularly imprinted polymers (MIPs) in the fields of separation,2-7 catalysis,8,9 analyti* To whom correspondence should be addressed. E-mail: chetyw@ nus.edu.sg. Tel: +65-6516 8467. † Department of Chemical & Biomolecular Engineering. ‡ Division of Bioengineering. (1) Andersson, H. S.; Nicholls, I. A. In Molecularly Imprinted Polymers: Manmade mimics of antibodies and their application in analytical chemistry; Sellergren, B.m Ed.; Elsevier: Amsterdam, The Netherlands, 2001; Vol. 23, pp 1-2. (2) Ulbricht, M. J. Chromatogr., B 2004, 804, 113-125. 10.1021/ac701824u CCC: $40.75 Published on Web 01/09/2008

© 2008 American Chemical Society

cal chemistry,10,11 and biosensing.12-15 Compared to its biological counterparts, enzymes and antibodies, MIPs can not only display comparable molecular selectivity, they are also more robust, reusable, and most of all, easy and inexpensive to prepare. Therefore, MIPs represent a new class of materials that could mimic and possibly replace their biological equivalents. However, to date, with the conventional approach, most of the successes in molecular imprinting have been based on small target ligands whereas the imprinting of macromolecules like proteins has been limited. One of the major difficulties faced by these large molecules for the imprinting application lies in the diffusion limitations, which restrict the ease of template removal as well as uptake. In response to such limitation, surface imprinting has been proposed as a viable strategy for protein imprinting. Some early illustrations of the application of surface imprinting for proteins are those by Kempe et al.16 and Shi et al.17 Subsequent works include those by Yan et al.,18 who coated protein-imprinted thin films over polystyrene microspheres using 3-aminophenylboronic acid (APBA) as the functional monomer. This strategy proved viable for the imprinting of lysozyme and hemoglobin. Chou et (3) Da Costa Silva, R. G.; Augusto, F. J. Chromatogr., A 2006, 1114, 216-223. (4) Theodoridis, G.; Lasa´kova´, M.; Sˇ kerˇ´ıkova´, V.; Tegou, A.; Giantsiou, N.; Jandera, P. J. Sep. Sci. 2006, 29, 2310-2321. (5) Wu, G.; Wang, Z.; Wang, J.; He, C. Anal. Chim. Acta 2007, 582, 304-310. (6) Baggiani, C.; Baravalle, P.; Giraudi, G.; Tozzi, C. J. Chromatogr., A 2007, 1141, 158-164. (7) Baggiani, C.; Anfossi, L.; Giovannoli, C. Anal. Chim. Acta 2007, 591, 2939. (8) Cheng, Z.; Li, Y. J. Mol. Catal. A: Chem. 2006, 256, 9-15. (9) Pasetto, P.; Maddock, S. C.; Resmini, M. Anal. Chim. Acta 2005, 542, 6675. (10) Hawkins, D. M.; Trache, A.; Ellis, E. A.; Stevenson, D.; Holzenburg, A.; Meininger, G. A.; Reddy, S. M. Biomacromolecules 2006, 7, 2560-2564. (11) Bossi, A.; Piletsky, S. A.; Piletska, E. V.; Righetti, P. G.; Turner, A. P. F. Anal. Chem. 2001, 73, 5281-5286. (12) Li, X.; Husson, S. M. Langmuir 2006, 22, 9658-9663. (13) Liu, K.; Wei, W. Z.; Zeng, J. X.; Liu, X. Y.; Gao, Y. P. Anal. Bioanal. Chem. 2006, 385, 724-729. (14) Lakshmi, D.; Prasad, B. B.; Sharma, P. S. Talanta 2006, 70, 272-280. (15) Tappura, K.; Vikholm-Lundin, I.; Albers, W. M. Biosens. Bioelectron. 2007, 22, 912-919. (16) Kempe, M.; Glad, M.; Mosbach, K. J. Mol. Recognit. 1995, 8, 35-39. (17) Shi, H.; Tsai, W. B.; Garrison, M. D.; Ferrari, S.; Ratner, B. D. Nature 1999, 398, 593-597. (18) Yan, C. L.; Lu, Y.; Gao, S. Y. J. Polym. Sci. Polym. Chem. 2007, 45, 19111919.

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al.19,20 successfully applied surface imprinting through the microcontact printing approach where thin films of protein-imprinted polymers were created on glass cover slides through the use of protein stamps. In addition, surface imprinting has often been applied as a convenient strategy for the coating of sensor electrodes with protein-imprinted films for applications in biosensing. This strategy has been successfully applied to quartz crystal microbalance21,22 and surface plasmon resonance.23 Other than surface imprinting, alternative strategies that had been employed for the imprinting of proteins include the preparation of protein-imprinted hydrogel24,25 and the application of the epitope approach.26-28 More work on protein imprinting could be found in some recent reviews.29-31 For beads, surface-imprinted microspheres for hemoglobin were first prepared by Shiomi et al.32 using silanes based on template covalent immobilization. Bonini et al.33 modified the approach to fabricate silica-based imprinted beads for human serum albumin using APBA instead of silanes. Such template immobilization strategy allows the imprinting of proteins that may not be soluble in the polymerization mixture and can potentially be employed as a generally applicable methodology for protein imprinting. In this work, using a two-stage core-shell miniemulsion polymerization inspired by the above work of Bonini et al., bovine serum albumin (BSA) surface-imprinted particles were prepared with the immobilization of the template protein molecules on polymeric (instead of silica) support beads. The sizes of the particles were significantly reduced to increase the available surface area for a higher template BSA loading. In addition, magnetic susceptibility was imparted to the particles to widen their scope of potential applications. For example, the magnetically susceptible imprinted particles can be applied as an affinity adsorbent in protein purification to recognize and preferentially adsorb the protein of interest. The particles can then be easily isolated by the use of an external magnetic field. Besides that, the imprinted particles can also be prepared with predetermined affinity toward proteins that are abundantly expressed on tumor tissues. With such affinity, the superparamagnetic imprinted particles will be concentrated at the tumor site, thus (19) Chou, P. C.; Rick, J.; Chou, T. C. Anal. Chim. Acta 2005, 542, 20-25. (20) Lin, H. Y.; Hsu, C. Y.; Thomas, J. L.; Wang, S. E.; Chen, H. C.; Chou, T. C. Biosens. Bioelectron. 2006, 22, 534-543. (21) Rick, J.; Chou, T. C. Anal. Chim. Acta 2005, 542, 26-31. (22) Hayden, O.; Haderspo ¨ck, C.; Krassnig, S.; Chen, X.; Dickert, F. L. Analyst 2006, 131, 1044-1050. (23) Matsunaga, T.; Hishiya, T.; Takeuchi, T. Anal. Chim. Acta 2007, 591, 6367. (24) Demirel, G.; O ¨ zc¸ etin, G.; Turan, E.; C¸ aykara, T. Macromol. Biosci. 2005, 5, 1032-1037. (25) Hawkins, D. M.; Stevenson, D.; Reddy, S. M. Anal. Chim. Acta 2005, 542, 61-65. (26) Rachkov, A.; Minoura, N. Bichim. Biophys. Acta 2001, 1544, 255-266. (27) Tai, D. F.; Lin, C. Y.; Wu, T. Z.; Chen, L. K. Anal. Chem. 2005, 77, 51405143. (28) Nishino, H.; Huang, C. S.; Shea, K. J. Angew. Chem., Int. Ed. 2006, 45, 2392-2396. (29) Bossi, A.; Bonini, F.; Turner, A. P. F.; Piletsky, S. A. Biosens. Bioelectron. 2007, 22, 1131-1137. (30) Turner, N. W.; Jeans, C. W.; Brain, K. R.; Allender, C. J.; Hlady, V.; Britt, D. W. Biotechnol. Prog. 2006, 22, 1474-1489. (31) Janiak, D. S.; Kofinas, P. Anal. Bioanal. Chem. In press. (32) Shiomi, T.; Matsui, M.; Mizukami, F.; Sakaguchi, K. Biomaterials 2005, 26, 5564-5571. (33) Bonini, F.; Piletsky, S.; Turner, A. P. F.; Speghini, A.; Bossi, A. Biosens. Bioelectron. 2007, 22, 2322-2328.

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Scheme 1. Surface Functionalization Reactions of the Support Particles for Template BSA Immobilization in the Two-Stage Miniemulsion Polymerization Imprinting Process

allowing the imaging of the tumor through magnetic resonance imaging. Methyl methacrylate (MMA) and ethylene glycol dimethacrylate (EGDMA) were employed as the functional and cross-linking monomers, respectively. Nanosized Fe3O4 magnetite was first prepared using a coprecipitation method and then encapsulated into poly(MMA-co-EGDMA) beads in the first-stage core-shell miniemulsion polymerization to fabricate the support polymeric beads. Following that, a series of surface functionalization reactions were performed to immobilize BSA molecules onto the support particle surface, and X-ray photoelectron spectroscopy (XPS) was used to monitor each functionalization step. Subsequently, a second-stage core-shell miniemulsion polymerization was carried out to create a poly(MMA-co-EGDMA) shell over the support particles. Finally, the template BSA molecules were removed through base hydrolysis. Complementary binding sites were thus created for the BSA on the particle surface (Scheme 1). The imprinted particles were characterized by field-emission scanning electron microscopy (FESEM), transmission electron microscopy (TEM), laser light scattering (LLS), swelling ratio (SR), and nitrogen gas sorption measurements. Additionally, the magnetic property of the particles was measured with vibrating sample magnetometer (VSM), and the encapsulation efficiency of Fe3O4 was determined by thermogravimetric analysis (TGA). Most importantly, the adsorption characteristics of the polymeric particle were elucidated through batch rebinding tests, competitive rebinding tests, and adsorption kinetics studies. EXPERIMENTAL SECTION Materials. Bovine serum albumin was used as the template protein, while lysozyme (Lys) from chicken egg white was used as the nontemplate (control) protein. Both proteins, sodium bisulfite (minimum 99%), and glutaraldehyde (50%) were purchased from Sigma. MMA (99%), EGDMA (98%), oleic acid (90%), sodium dodecyl sulfate (SDS; minimum 98.5% GC), sodium bicarbonate (99.7 - 100.3%), sodium bisulfite (minimum 99%),

ammonium persulfate (APS, 98%), hydrochloric acid, cetyl alcohol (CA, 95%), ethylene diamine (EDA, 99%), and trifluoroacetic acid (TFA, 99%) were purchased from Aldrich . Ammonia solution (25%), ethanol, N,N-dimethylformamide (DMF), iron(II) chloride tetrahydrate (FeCl2‚4H2O), and iron(III) chloride hexahydrate (FeCl3‚6H2O) were obtained from Merck. Sodium hydroxide pellets were from J.T. Baker, acetic acid from Fisher Chemicals (UK), and HPLC-grade acetonitrile from Tedia. All chemicals were used directly without further purification. Preparation of Fe3O4 Magnetite. Fe3O4 magnetite was prepared by the coprecipitation method.34 A 25-mL mixture containing 0.8 M FeCl3·6H2O, 0.4 M FeCl2·4H2O, and 3 vol % concentrated hydrochloric acid was prepared in deionized (DI) water. The resulting clear yellowish green solution was then added into 250 mL of a 5.23 vol % ammonia solution. Upon addition, the solution turned black and was then stirred magnetically at 1000 rpm for 1 h. The Fe3O4 magnetite was then washed three times with DI water before being suspended in the water. Preparation of Superparamagnetic Support Particles. One gram of the Fe3O4 magnetite prepared above was mixed with 1.0 mL of oleic acid to obtain a black viscous gel. MMA (1.28 mL) and EGDMA (9.05 mL) in the molar ratio of 1:4 were then added to the oleic acid-coated magnetite and mixed thoroughly. The mixture was then ultrasonicated at 65% power level for 80 s (Sonics Vibracell VCX 130, Sonics & Materials Inc., Newtown, CT). After homogeneity was achieved, the resulting mixture was added dropwise into a 50-mL solution of 0.01 M SDS and 0.03 M CA, which was magnetically stirred at 300 rpm. The mixture was further ultrasonicated at 65% power level for 90 s to create a miniemulsion. The miniemulsion was then added dropwise into 600 mL of a 0.05 w/v % SDS solution. This reaction mixture was transferred to a 1-L, three-neck, round-bottom flask and purged with nitrogen gas for 15 min to displace oxygen while maintaining the temperature at 80 °C. Subsequently, APS (0.5 g) was added to the reaction mixture to initiate the polymerization reaction. The reaction was allowed to proceed for 24 h. Upon completion, the polymeric support beads were washed three times with DI water, three times with 50 vol % ethanol, and finally, three times with DI water. Aminolysis. One gram of the polymeric support particles prepared above was washed twice with DMF and redispersed in 20 mL of DMF. Subsequently, 20 mL of EDA was added to the mixture and magnetically stirred at 400 rpm for a 12-h reaction under reflux at 110 °C. The amine-functionalized core particles were then washed once with DI water, twice with 50 vol % ethanol, and finally, twice with DI water. Aldehyde Functionalization. A buffer solution of pH 5 was prepared using acetic acid and NaOH. One gram of the aminefunctionalized polymeric support particles prepared above was soaked in 10 mL of buffer solution and degassed for 10 min at room temperature. The buffer was then removed, and the particles were redispersed in 10 mL of fresh buffer with 5 vol % glutaraldehyde. This mixture was allowed to react with magnetic stirring at 400 rpm for 12 h at room temperature. The aldehydefunctionalized particles were then washed three times with DI water posttreatment. (34) Liu, X.; Guan, Y.; Liu, H.; Ma, Z.; Yang, Y.; Wu, X. J. Magn. Magn. Mater. 2005, 293, 111-118.

Immobilization of Template BSA. The aldehyde-functionalized polymeric particles prepared above were washed once with 0.01 M phosphate buffer saline (PBS). A 10-mL aliquot of BSA solution (2.5 mg/mL) was then added to 1.0 g of the particles. The mixture was magnetically stirred at 300 rpm for 3 h at 4 °C for the coupling to occur. The BSA-immobilized core particles were then washed three times with DI water upon completion of the reaction. Shell Layer Synthesis. MMA (1.28 mL) and EGDMA (9.05 mL) were mixed with 1.0 g of the surface-modified superparamagnetic core particles. The mixture was then ultrasonicated at 45% power level for 90 s to ensure that it was thoroughly mixed. It was then added dropwise into a 50-mL solution of 0.01 M SDS and 0.03 M CA, which was stirred at 300 rpm. The mixture was ultrasonicated again at 45% power level for 110 s to generate the miniemulsion. The resulting miniemulsion was then added dropwise into 600 mL of a 0.05 w/v % SDS solution and was stirred at 300 rpm. This mixture was subsequently transferred to a 1-L, three-neck, round-bottom reactor and purged with nitrogen gas for 15 min at 40 °C to displace oxygen. Sodium bisulfite (0.25 g) followed by APS (0.25 g) was then added into the mixture to initiate the polymerization reaction, which was allowed to proceed for 24 h. Upon completion, the polymeric core-shell particles were washed three times with DI water, three times with 50 vol % ethanol, and finally, three times with DI water. Template Removal. After the formation of the shell layer, the immobilized template BSA molecules were removed by hydrolysis. A 10-mL aliquot of a 1.0 M NaOH solution was added to 1.0 g of the core-shell particles. The hydrolysis mixture was stirred at 300 rpm and allowed to react for 5 h under reflux at 35 °C. These surface-imprinted particles (iMIP) were washed three times with DI water and resuspended in DI water for characterization and adsorption studies. Preparation of Nonimprinted Particles from SurfaceModified Support Beads (iNIP). The corresponding nonimprinted particles to the above iMIP were prepared using steps similar to those above, except without the surface immobilization of BSA templates before polymerization of the external shell layer. These particles were used as control samples for comparison in the characterization studies. Preparation of Molecularly Imprinted Particles from Unmodified Core Beads Using Free Template (fMIP). Magnetically susceptible molecularly imprinted polymers using free (nonimmobilized) BSA template were also prepared. The magnetically susceptible polymeric support beads were prepared as above. However, no further surface modification reactions were carried out except for the following shell polymerization. Twenty-five milligram of BSA was first dissolved in 10 mL of DI water. MMA (1.278 mL), EGDMA (9.054 mL), and 10 mL of the prepared BSA solution were then added to 1.0 g of the superparamagnetic core particles. Subsequently, the resulting mixture was ultrasonicated at 45% power level for 90 s. A brown viscous mixture was obtained, which was then added dropwise to a 50-mL solution of 0.1 M SDS and 0.3 M CA. The mixture was then ultrasonicated at 45% power level for 110 s to produce the miniemulsion. The miniemulsion was added dropwise to a 600 mL of 0.05 w/v % SDS solution before being transferred to a 1-L, three-neck, round-bottom flask. This mixture was purged with nitrogen to displace oxygen and was Analytical Chemistry, Vol. 80, No. 3, February 1, 2008

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heated to 40 °C. Sodium bisulfite (0.25 g) followed by APS (0.25 g) was then added to initiate the reaction. With the temperature maintained at 40 °C, the mixture was mechanically stirred at 300 rpm and the polymerization reaction was allowed to proceed for 24 h. Upon completion of the reaction, the fMIP were washed twice with DI water, three times with a solution of 10 w/v % SDS to 10 v/v % acetic acid, three times with 50 vol % ethanol, and finally, three times with DI water for template removal. Preparation of Nonimprinted Particles from Unmodified Core Beads (fNIP). A corresponding control sample to the above fMIP was prepared using a similar method except without the addition of the template BSA protein in the miniemulsion. Analysis and Measurement. XPS (AXIS His-165 Ultra, Kratos Analytical, Shimadzu) was employed to determine the surface elementary composition of the support particles at each stage of surface modification. The sizes of the polymeric particles were determined using LLS (BIC Particle Sizing Software 90 Plus, Brookhaven Instruments Corp.). Morphological observation of the polymeric particles was performed with a FESEM (JSM-6700F, JEOL) and a TEM (JEM-2010, JEOL). TGA (TGA 2050, TA Instrument) was employed to determine the efficiency of the magnetite encapsulation within the polymeric particles. Lastly, the magnetic property of the polymeric particles was measured using a VSM (Lakeshore, OH). As part of the morphological characterization, the specific surface area, pore volume, and pore diameter measurements were carried out through a nitrogen sorption method with a 7-point BET (NOVA 3000 series, Quantachrome Instruments) and a 20-point isotherm calculations. Determination of Estimated Swelling Ratio. The polymeric particles that were initially dispersed in DI water were first isolated by centrifugation at 9000 rpm for 40 min. Measurement of the particle swollen weight (Ww) was made after the supernatant was removed. Subsequently, the particles were freeze-dried for 24 h and weighed again to obtain the dry weight (Wd). The swelling ratio of the polymer was then calculated as follows:35

SR )

Ww - W d Wd

The final concentration, Cf, was determined by using an Agilent 1100 series HPLC unit with an Agilent Zorbax 300SB-C18, 4.6 × 150 mm, 5-µm reversed-phase column. At the end of 24 h, the samples were centrifuged (Universal 32R, Hettich Zentrifugen) at 9000 rpm for 40 min in order to extract the supernatants, which were prefiltered using sterile 0.2-µm filter units and subsequently analyzed by HPLC. Two mobile phases, (A) ultrapure water with 0.1 vol % TFA and (B) 80 vol % acetonitrile and 20 vol % water with 0.09 vol % TFA, were used for the linear gradient elution. The solvent flow rate was set at 1 mL/min with solvent B increasing from 25 to 70 vol % in 40 min. The analyte injection volume was 50 µL, and the column temperature was set at 60 °C. The samples were analyzed by an UV detector at a wavelength of 220 nm. For a comparative assay, the iNIP, fMIP, and fNIP were also subjected to the batch rebinding test. Similar tests had also been carried out with the nontemplate Lys. All tests were conducted in triplicates. Competitive Batch Rebinding Tests. In the competitive batch rebinding tests, the selectivity of the imprinted particles was studied in a binary BSA-Lys solution. The polymeric particles were subjected to a binary protein mixture of BSA and Lys with individual initial concentrations of 1.8 mg/mL. The adsorption mixture was rotary mixed for 24 h and analyzed similarly as in the batch adsorption experiments above. All of the competitive batch rebinding tests were conducted in triplicate. Adsorption Kinetics Study. The adsorption kinetics of the particles prepared was studied with an initial BSA concentration of 1.8 mg/mL. The adsorption runs were performed similarly to the single-protein batch rebinding tests. To determine the adsorption profiles of the samples, analytes were drawn at regular intervals for HPLC analysis to determine the BSA concentrations. The tests were conducted in triplicate. Statistical Analysis. Standard deviation calculations and Student’s t-test were carried out using Microsoft Excel (Seattle, WA) for statistical comparisons between pairs of samples. The groups were considered statistically different when p < 0.05.

where Q (mg of protein/g of polymer) is the mass of protein adsorbed per gram of polymer, Ci (mg/mL) is the initial protein concentration, Cf (mg/mL) is the final protein concentration, V (mL) is the total volume of the adsorption mixture, and m is the mass of polymer in each rebinding mixture.

RESULTS AND DISCUSSION Preparation of the Magnetically Susceptible Polymeric Support Beads. The superparamagnetic Fe3O4 magnetite nanoparticles were first prepared using the coprecipitation method. Previous measurements36 of the particles by FESEM showed that their sizes were ∼18 nm. The magnetite gel was made hydrophobic with a coating of oleic acid, which helped to enhance the penetration of the magnetite into the hydrophobic interior of micelles during the first-stage core-shell miniemulsion polymerization. This strategy was successful in the fabrication of the magnetically susceptible support polymeric beads. MMA has been chosen as the monomer for this preparation. It is a common monomer used for the oil-in-water (o/w) miniemulsion polymerization and also is commonly used in molecular imprinting through hydrophobic interactions.36,41 In addition, it is able to provide ester and methoxy groups for subsequent surface functionalization. Being a weak electron donor, the esters groups are susceptible to nucleophilic attack during the aminolysis substitution reaction. The addition of EGDMA as a cross-linker maintained the stability

(35) Lu, S.; Cheng, G.; Pang, Z. J. Appl. Polym. Sci. 2006, 100, 684-694.

(36) Tan, C. J.; Tong, Y. W. Anal. Chem. 2007, 79, 299-306.

Batch Rebinding Tests. The initial BSA concentrations of the adsorption samples varied from 1.2 to 2.0 mg/mL. The samples were affixed onto a Rotamix (RKVS, ATR Inc.) and agitated by end-to-end rotary mixing for 24 h at room temperature. The amount of protein adsorbed by the polymeric particles at the end of each run was determined by the following formula:

Q)

686

(Ci - Cf)V m

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Table 1. Surface Atomic Compositions of the Support Particles from the XPS Widescan Spectra elemental atomic composition stage

C

O

N

core surface NH2 functionalization CHO functionalization protein immobilization after alkaline hydrolyzis

80.04 75.52 79.63 76.05 69.11

19.88 23.51 19.66 21.00 29.33

0.00 0.87 0.60 2.73 0.95

of the imprinting sites while making the product polymeric beads easier to be handled and processed. Surface Immobilization of the Template BSA Molecules. Upon completing the postsynthesis processing of the support particles, they were then subjected to a series of surface functionalization reactions as illustrated in Scheme 1. XPS was employed as the primary tool to monitor the reactions. It is routinely applied for characterizing surface modifications and has been found to be able to provide insights on the surface information of a material. Elemental wide scans were conducted and the surface atomic compositions are reported in Table 1. Fourier transform infrared (FT-IR) spectroscopy was also considered as an alternative probe for the purpose but was found to be not suitable because FT-IR examined the bulk composition of the particles rather than just the surface. The success of each surface modification reaction could be associated with changes in the surface composition of nitrogen atoms. The increase in the nitrogen atomic composition from 0.00 to 0.87% after the first aminolysis functionalization suggested that amine groups were successfully introduced onto the surfaces of the polymeric support particles. An activated carboxylic acid derivative is usually required for the aminolysis reaction, which involves nucleophilic acyl substitution. Nevertheless, MMA and EGDMA are capable of providing activated yet thermally more stable surface ester groups for the nucleophilic acyl substitution reaction. This approach of introducing amine groups to methacrylate surfaces was adopted from work of Liu et al.34 The amine-modified surface was subjected to further reactions to introduce aldehyde groups. Glutaraldehyde was chosen as the bridging agent as it possesses two terminal aldehyde groups. As one of the aldehyde groups was reacted with the amine groups on the polymeric support particle surface, the other was left free. Subsequently, under the suitable conditions, the free surface aldehyde was reacted with free amine groups in the template BSA molecules (from lysine,33 for example), thus successfully immobilizing BSA molecules onto the support particle surfaces. Both reactions involved nucleophilic addition that allowed the formation of imine bonds between an aldehyde and an amine groups. The reactions are reversible and acid catalyzed at an optimum pH of 5.37 After the aldehyde functionalization, the decrease in the nitrogen composition from 0.87 to 0.60% and an accompanying (37) Organic Chemistry, 5th ed.; McMurry, J., Ed.; Brooks/Cole: Pacific Grove, CA, 2000. (38) Surface analysis by electron spectroscopy : measurement and interpretation; Smith, G. C., Ed.; Plenum Press: New York, 1994. (39) Handbook of monochromatic XPS spectra; Crist, B. V., Ed.; Wiley: New York, 2000.

increase of carbon composition from 75.52 to 79.63% might be indicative of the relative increase in carbon and oxygen content from glutaraldehyde. The successful introduction of aldehyde groups was substantiated by the success of the subsequent protein immobilization process, which required the presence of anchored, free aldehyde groups and was further ascertained by the deconvolution of the C1s spectrum (Figure 1). Although the deconvolution procedure is not exact, it provided some insights on the type of functional groups that could be found on the particle surface. Thus, the success of BSA immobilization can be seen by the significant increase in the nitrogen composition from 0.60 to 2.73% through XPS analysis. This increase was attributed to the abundant peptide bonds from the protein molecules. In this series of surface functionalization reactions, after modifying the particle surface with amine moieties, the template BSA molecules was coupled to the particle surfaces through bridging glutaraldehyde molecules instead of direct coupling via an amide bond. This is to prevent any couplings between the protein molecules. To ensure the success of the functionalization reactions, the products of each modification step were analyzed by XPS with the C1s and O1s spectrums deconvoluted for further analysis. The observed carbon ratio obtained for the unmodified support beads that contain surface ester groups is in good agreement with the theoretical ratio. The C-N peak from the deconvoluted C1s spectrum in Figure 1a indicates successful reaction between the surface methacrylate groups and EDA while the deconvoluted *CdO peak in Figure 1b suggests the presence of free aldehyde groups, which were available for template BSA immobilization. Nevertheless, the experimental carbon ratios for the amine- and aldehyde-functionalized support particles were lower than the expected ratio for 100% conversion, and these experimental ratios were unable to provide conclusive evidence on the conversion yield (see Supporting Information Table S1 for carbon ratio values). Synthesis of the BSA Surface-Imprinted Particles. An external imprinted polymeric shell was created over the BSAimmobilized support beads during the second-stage miniemulsion polymerization with MMA and EGDMA as the functional and cross-linking monomers, respectively. Subsequently, the BSAsurface imine linkage was hydrolyzed to remove the template BSA, leaving behind complementary binding sites on the particle outer shell. The ease of hydrolyzing the imine bond was the primary reason for its use in this work32 with oxalic acid and sodium hydroxide being the common catalysts used for this reaction.32,40 An initial attempt was made to remove the template by acid hydrolysis; however, this resulted in the dissolution of the iron oxide in the core particles; hence alkaline hydrolysis was employed instead. The successful removal of the BSA molecules was verified by the significant reduction of nitrogen composition (Table 1) and the disappearance of the N1s peak from the XPS wide scan spectra (see Supporting Information Figure S1a and S1b for the spectra). A corresponding change was not observed for the nonimprinted particles (iNIP). Furthermore, there were no significant differences between the surface elemental composition of the iMIP and iNIP (see Supporting Information Figure S1b and S1c for comparison). This further verified the success of the template removal. Other than the imprinted particles based on (40) Dash, A. C.; Dash, B.; Panda, D. J. Org. Chem. 1985, 50, 2905-2910.

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Figure 1. Deconvoluted C1s peaks of (a) amine-functionalized and (b) aldehyde-functionalized support particles. Peaks had their width (fwhm) kept below 1.8 eV beyond which there is an indication of a further component.38 χ2 values were between 1 and 2, which indicated a good curve fit.39 Table 2. Morphological Features of the Polymeric Particles Prepared polymer

mean effective diameter (nm)a

polydispersity

swelling ratio

support beads iMIP iNIP fMIP fNIP

352.8 535.2 580.9 603.4 489.6

0.141 0.005 0.006 0.005 0.060

3.58 ( 0.78 2.73 ( 0.53 2.47 ( 0.32 2.41 ( 0.56

a

Results obtained from LLS measurements.

immobilized template molecules (iMIP), three other types of particles, namely, nonimprinted particles with similar surface functionalization (iNIP), imprinted particles with non-immobilized (or free) template molecules (fMIP), and nonimprinted particles without the surface modification (fNIP), had also been prepared. These particles were used as control samples for subsequent characterization studies. Size Measurements. The sizes of the support beads, iMIP, iNIP, fMIP, and fNIP, were determined using LLS and the results are as tabulated in Table 2. From the measurement, it was found that the particles were monodispersed in size. The support beads sized ∼350 nm while the mean effective diameters of the other four particles ranged from 500 to 600 nm. The larger sizes of the particles suggested a successful shell formation over the core beads. Morphological Observations. FESEM and TEM were employed to observe the morphological features of the particles. From the FESEM images (Figure 2a), the polymeric support particles appear to be spherical in shape. Through TEM observation, due to a difference in the densities of the copolymer and the iron oxide, the magnetite is seen as the darker spots inside the support beads (Figure 2d). This illustrates the successful encapsulation of the magnetite into the core particles. Being different from the support core particles, the iMIP and iNIP were monodispersed with a unique “red blood cell” (RBC)-like morphology (Figure 2b and c) and there were no significant morphological differences between all of the particles (fMIP and fNIP also had similar morphological features, results not shown). A reduced amount of monomers had been used in the second-stage polymerization reaction for the fabrication of the RBC-like core-shell 688

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particles. With this structure, the external shell created (the concave morphology) would be close to the core particle surface and, hence, enabling the formation of imprinted binding sites near the product core-shell particle surface. After the second-stage miniemulsion polymerization, the immobilized template BSA molecules could have been covered by the polymeric shell layer. Even so, with the unique concave morphology, the binding sites created for BSA would still be very close to the surface and thus the template removal through base hydrolysis would not face any hindrances. In addition to that, as seen from Table 2, the particles sizes did not change significantly after the second-stage polymerization and this further substantiates the presence of the BSA binding site near the surface. It is well-known that the concave shapes of red blood cells provide maximum surface area per unit volume, thus facilitating gas transfer into and out of the cells. Similarly with the RBC-like morphology, the core-shell imprinted particles possessed high specific surface area for effective template uptake during adsorption processes (see discussion below). In fact, the thickness of a polymeric shell layer can also be controlled through the application of a controlled polymerization technique such as surface-initiated atom-transfer radical polymerization. However, the strategy used here to produce RBC-like particles proved to be relatively simpler and is a more convenient alternative as fewer complications were involved. With similar effective diameters and morphology between the pairs of imprinted and nonimprinted particles (iMIP with iNIP and fMIP with fNIP), any differential protein uptake between the particles in the subsequent study would be due to the molecular imprinting effect rather than morphological differences. Swelling Ratio Measurements. The SR values of the particles in water were determined based on the amount of water uptake. It was found that the SR values were comparable to the particles obtained by Lu et al.,35 which averaged between three and five. As shown in Table 2, when compared to iNIP, the iMIP have a significantly higher SR value (p < 0.05). The exact reason for this was not known, but it could possibly be due to the formation of binding cavities on the surfaces of the iMIP, which enhanced water penetration and thus results in higher water uptake and measured SR value. On the other hand, there were no significant differences (p > 0.05) between the SR values of fNIP and fMIP. Additionally, this could be an indication of poor imprinting

Figure 2. Microscopic observation of the prepared particles. FESEM images of (a) support particles, (b) iMIP, and (c) iNIP. (d) TEM images illustrating the successful encapsulation of the Fe3O4 magnetite. Table 3. Results from the Nitrogen Gas Sorption Measurements particles

specific surfac area (m2/g)

total pore volume (cm3/g)

mean pore diameter (nm)

iNIP iMIP fNIP fMIP

18.9 ( 7.1 13.7 ( 3.3 16.1 ( 2.9 23.9 ( 1.8

0.027 ( 0.006 0.030 ( 0.007 0.038 ( 0.011 0.049 ( 0.005

8.57 ( 0.34 8.70 ( 0.00 9.25 ( 1.07 8.11 ( 0.28

efficiency with the use of nonimmobilized template BSA molecules for surface imprinting, as illustrated in the subsequent adsorption studies. Nitrogen Sorption Measurements. Nitrogen sorption measurements were performed for all of the particles prepared, and the results are shown in Table 3. The particles did not differ significantly in terms of their specific surface areas, pore volumes, and pore diameters (p > 0.05). The specific surface areas of the particles averaged ∼18.0 m2/g. Compared to previous work41 where protein surface-imprinted nanoparticles of sizes around 40 nm had been prepared, the specific surface areas of the nanoparticles (∼25 m2/g) did not differ significantly from the submicrometer particles obtained here despite the size difference of more than 10 times. This showed that, with the RBC-like morphology, the particles possessed high specific surface areas for their sizes and this would allow high template protein loading. The pores of the particles were measured of relatively small sizes although they were not observable in the SEM images. By combining the results obtained from the XPS and nitrogen sorption measurements, a rough calculation was performed to (41) Tan, C. J.; Tong, Y. W. Langmuir 2007, 23, 2722-2730.

estimate the surface density of immobilized template BSA molecules on the iMIP to be ∼0.1 µmol/g of particles. Thermogravimetric Analysis. The amount of magnetite encapsulated in the particles was measured through TGA. In this analysis, the temperature was slowly increased from 25 °C and when it reached ∼200 °C, the poly(MMA-co-EGDMA) particles started to degrade thermally, which accounted for the significant mass loss. The onset temperature of thermal decomposition was relatively close to that obtained in similar studies done by Yu et al.42 Eventually, all of the copolymer was degraded, leaving behind the thermally stable Fe3O4 magnetite. From this study, it was found that the magnetite encapsulation efficiency for the core support particles was 13.28 wt %. The achieved encapsulation efficiency was considerably high and satisfactory when compared to those from similar works,43-44 where the content of Fe3O4 averaged only 2-4 wt %. On the other hand, both iMIP and iNIP displayed a lower magnetite mass percentage of 4.44 and 4.46 wt %, respectively, than the support beads (see Supporting Information Figure S2 for the TGA thermogram). This was well within expectation as the encapsulated magnetite mass percentage relative to the overall mass of the particle should decrease after the formation of the external shell layer over the support core beads. Vibrating Sample Magnetometer Measurements. VSM had been employed as the probe to characterize the magnetic properties of the particles (see Supporting Information Figure S3 for VSM magnetization curves). From the VSM data, the support (42) Yu, D.; An, J. H.; Bae, J. Y.; Kim, S.; Lee, Y. E.; Ahn, S. D.; Kang, S.; Suh, K. S. Colloid Surf., A 2004, 245, 29-34. (43) Lu, S.; Cheng, G.; Pang, X. J. Appl. Polym. 2006, 99, 2401-2407. (44) Lu, S.; Cheng, G.; Pang, X. J. Appl. Polym. 2003, 89, 3790-3796.

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Figure 3. Results of (a) BSA batch rebinding tests, +, p < 0.05; -, p < 0.08; (b) Lys batch rebinding tests in water (0, iNIP; 9, iMIP; crosshatch, fNIP; broken crosshatch, fMIP).

core beads exhibited a saturation magnetization value (S) of 16.6 emu/g. This value was very close to that obtained in our previous work.36 It was observed that the shapes of the magnetization curves for the particles were symmetrical about the origin. This is a typical superparamagnetism property, which will be essential for future applications of the material as it enables reusability and the ease of particle control and monitoring. The iMIP and iNIP retained their superparamagnetic properties even after the formation of the shell layer as shown by the decrease in their respective saturation magnetization values (8.0 emu/g for iNIP and 7.6 emu/g for iMIP). This was expected due to the shielding effect of the additional polymeric shell layer. Batch Rebinding Tests. In characterizing the adsorption behaviors of the core-shell particles, they were subjected to batch rebinding, competitive rebinding, and adsorption kinetics studies. The batch rebinding tests were performed at different initial protein concentrations, ranging from 1.2 to 2.0 mg/mL. In addition to the template BSA, the tests were also conducted using Lys as the control nontemplate protein because Lys is much smaller size than BSA. The imprinted sites created for BSA will thus not be able to keep the competitor Lys out based on size exclusion. Hence, any preferential uptake of BSA over Lys will be a strong indication of the molecular imprinting effect. Nevertheless, there may be concerns over the suitability of Lys as a competing protein due to its significantly different isoelectric point from that of BSA. In many cases, monomers such as MAA and acrylamide have been applied for protein imprinting due to their favorable hydrogen bond formation and electrostatic interactions with template protein molecules. However, in this work, in order to achieve the desired molecular affinity for the template BSA in an aqueous environment, the synergistic effect of hydrophobic interactions and shape complementarity were used instead. Thus, the rather hydrophobic MMA was used for particle fabrication and thus will result in reducing the effects of the acidity/basicity of proteins on the recognition and rebinding processes in this system. It is hypothesized, as in a previous work,36 that protein-imprinting is thus due solely to these hydrophobic interactions. As shown in Figure 3a, the iMIP exhibited significantly higher BSA loadings than the counterpart control iNIP for all different initial concentrations, with the highest loading of 854 nmol/g at the initial concentration of 1.8 mg/mL. This is a proof of the successful creation of imprinted cavities on the iMIP. From related works,43,45-47 where BSA had 690 Analytical Chemistry, Vol. 80, No. 3, February 1, 2008

been used as the template for the preparation of BSA-imprinted microspheres through suspension polymerization, the BSA adsorption capacity ranged from 40 to 100 nmol/g. It can be seen that the iMIP obtained here displayed a significantly higher BSA loadings. It is hypothesized that this is due to the RBC-like morphology of the imprinted particles with its high surface area to volume ratio. Furthermore, the BSA loadings of iMIP were also generally higher than that for the fNIP and fMIP. Although the shell layers of fMIP had been created in the presence of nonimmobilized BSA templates, they did not consistently adsorb more BSA than the control fNIP in the batch rebinding tests. In fact, the BSA loadings for fNIP and fMIP were not significantly different, illustrating the poor imprinting efficiency with the use of the free template strategy. When the test was conducted with Lys (Figure 3b), the Lys uptake of all the particles was random with no conclusive trend to be drawn. This was expected since Lys was the nontemplate protein and its adsorption was attributed to be from nonspecific interactions. Similarly, as Lys is smaller than BSA, more Lys molecules could thus adsorb nonspecifically onto the material causing the Lys loadings of the particles as generally higher than BSA as observed. Despite this, the significantly higher BSA uptake by iMIP compared to other particles (iNIP, fNIP, fMIP), which was not observed for the case of the nontemplate Lys, is a convincing indication of the recognition property imparted through molecular imprinting. Based on the amount of BSA adsorbed (Q) at the initial concentration of 1.8 mg/mL, the imprinting efficiency had been calculated and the results are presented in Table 4. It is shown that the iMIP achieved an imprinting efficiency of 6.51 while the fMIP imprinting efficiency is only 0.94. This demonstrated the recognition property of the iMIP and the importance of template immobilization for the imprinting process. Competitive Batch Rebinding Tests. To further illustrate the recognition property of the iMIP, the core-shell particles were subjected to binary protein competitive assay where, similarly, Lys had been employed as the competitor protein. The results are shown in Figure 4. The iNIP adsorb more Lys than BSA while (45) Pang, X.; Cheng, G.; Li, R.; Lu, S.; Zhang, Y. Anal. Chim. Acta 2005, 550, 13-17. (46) Pang, X.; Cheng, G.; Zhang, Y.; Lu, S. React. Funct. Polym. 2006, 66, 11821188. (47) Pang, X.; Cheng, G.; Lu, S.; Tang, E. Anal. Bioanal. Chem. 2006, 384, 225230.

Table 4. Results Obtained from the Batch Rebinding Tests polymer

Q at 1.8 mg/mL (nmol/g)

imprinting efficiencya

iNIP iMIP fNIP fMIP

131.98 859.21 358.26 335.29

6.51 0.94

a Imprinting efficiency ) Q (for imprinted particles)/Q (for nonimprinted particles)

Figure 5. Rebinding kinetic behavior of the particles (9, iNIP; b, iMIP; 2, fNIP; 1, fMIP) in water.

Figure 4. Results of the competitive rebinding tests for iNIP and iMIP (0, Lys; 9, BSA) at the initial concentration of 1.8 mg/mL; +, p < 0.01; *, no significant adsorption observed.

iMIP had not only exhibited a higher uptake of BSA than Lys in the competitive system, the adsorption of the nontemplate Lys had been effectively suppressed. In a competitive environment of protein adsorption, the adsorbent surface is usually first occupied by smaller proteins, which have higher diffusion coefficients. Nevertheless, at later stages, the already adsorbed proteins will be displaced by proteins (in this case, BSA) that have greater affinity toward the adsorbent surface. This is known as the Vroman48 effect and is probably responsible for the effective suppression of Lys adsorption observed. This indicated the molecular affinity of iMIP for the template BSA molecules. In addition, the iMIP displayed a significantly higher BSA loading (595 nmol/g) than the iNIP (273 nmol/g) in the binary protein system. It was noteworthy that, in this case, the BSA uptake of the iMIP was significantly reduced as compared to that observed in the single-protein adsorption systems (batch rebinding tests). This was nevertheless expected and was attributed to the adsorption competition from the second protein. When fNIP and fMIP were subjected to a similar competitive assay (results not shown), the fMIP did not exhibit a preferential uptake of the template BSA over its corresponding nonimprinted iNIP . Instead, the two types of particles displayed similar BSA and Lys loadings. This further illustrated the poor imprinting efficiency for the fMIP where nonimmobilized BSA had been employed in the molecular imprinting process. Rebinding Kinetics Study. The adsorption kinetics of proteins is one of the important considerations for the practical application of molecularly imprinted particles. The rebinding (48) Lutanie, E.; Voegel, J. C.; Schaaf, P.; Freund, M.; Cazenave, J. P.; Schmitt, A. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 9890-9894.

kinetics of BSA to the particles was therefore studied in this investigation. The results obtained in terms of percentage completion are shown in Figure 5. In general, the observed rebinding curves for all samples are typical as in most adsorption processes, having a relatively high initial adsorption rate that decreases slowly over time to finally achieve equilibrium. It was observed that there was no significant variation between the rebinding kinetics for the fMIP and fNIP. This showed that there were no differences in the particles as adsorbents for BSA, thus indicating that the use of free template molecules for surface imprinting was inefficient. For the iNIP, the adsorption kinetics was favorable, reaching equilibrium (>95% completion) in ∼150 min. On the other hand, despite a display of significant molecular selectivity in the batch and competitive rebinding tests, the iMIP had surprisingly slower kinetics as compared to iNIP. For the iNIP, the template adsorption could be nonspecific, while for the iMIP, more time would probably be required for the template molecules to orient themselves to specifically fit into the imprinted cavities. This hypothesis provided a possible explanation for the slower rebinding kinetics observed in the iMIP. Furthermore, the BSA loadings of the nonimprinted particles were less than their imprinted counterparts, thus probably enabling the equilibrium to be achieved within a shorter period of time. CONCLUSIONS BSA surface-imprinted particles had been successfully synthesized with a two-stage core-shell miniemulsion polymerization. The imprinting strategy was based on the surface immobilization of template BSA molecules with a series of surface modification of the support beads. The product particles had sizes of ∼500600 nm. Characterization of the magnetic properties showed that sufficient magnetite was encapsulated and the particles displayed the desired superparamagnetic susceptibility. In the rebinding characterizations, an excellent template recognition property was displayed by the iMIP in the batch and competitive adsorption tests. On the other hand, fMIP, which were fabricated based on nonimmobilized template molecules, did not display the expected molecular affinity. This illustrated the importance of template immobilization for the success of surface imprinting. In the competitive adsorption environment, it can be seen that the iMIP prepared using this strategy can display highly specific recognition Analytical Chemistry, Vol. 80, No. 3, February 1, 2008

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of BSA with no adsorption of the non-template proteins. Although only BSA had been employed as the target ligand in this investigation, this method of protein imprinting based on immobilized template core-shell miniemulsion polymerization shows great promise to be a general approach for the molecular imprinting of protein macromolecules.

SUPPORTING INFORMATION AVAILABLE XPS analysis of the deconvoluted C1s peaks at each stage of surface modification; XPS wide scan spectra of the modified particles; TGA thermogram of the core support, iNIP and iMIP particles; VSM magnetization data for the core support, iNIP and iMIP particles. This material is available free of charge via the Internet at http://pubs.acs.org.

ACKNOWLEDGMENT

Received for review August 30, 2007. Accepted November 6, 2007.

The authors acknowledge funding from the National University of Singapore under grant R279000228112.

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