pubs.acs.org/Langmuir © 2009 American Chemical Society
Preparation of Monodisperse Magnetic Polymer Microspheres by Swelling and Thermolysis Technique Chengli Yang,* Qian Shao, Jie He, and Biwang Jiang* Nano-Micro Materials Research Center, Key Laboratory of Chemical Genomics, School of Chemical Biology & Biotechnology, Peking University Shenzhen Graduate School, Shenzhen 518055, China Received September 28, 2009. Revised Manuscript Received October 26, 2009 A novel process for the preparation of monodisperse magnetic polymer microspheres by uniquely combining swelling and thermolysis technique was reported. The monodisperse polystyrene microspheres were first prepared by dispersion polymerization and swelled in chloroform. Then, ferric oleate was dispersed in chloroform as a precursor and impregnated into the swollen polymer microspheres. Subsequently, the iron oxide nanoparticles were formed within the polymer matrix by thermal decomposition of ferric oleate. The morphology, inner structure, and magnetic properties of the magnetic polymer microspheres were studied with a field emission scanning electron microscope (SEM), transmission electron microscope (TEM), and superconducting quantum interference device (SQUID) magnetometer. The results showed that the average diameter of the magnetic polymer microspheres was 5.1 μm with a standard deviation of 0.106, and the magnetic polymer microspheres with saturation magnetization of 12.6 emu/g exhibited distinct superparamagnetic characteristics at room temperature. More interestingly, the magnetite nanoparticles with a spinel structure are evenly distributed over the whole area of the polymer microspheres. These magnetic polymer microspheres have potential applications in biotechnology.
*To whom correspondence should be addressed. E-mail: clyang@szpku. edu.cn (C.Y.);
[email protected] (B.J.).
formation approaches,9 core-shell processes,10 emulsion polymerization,11 miniemulsion polymerization,12 dispersion polymerization,13 suspension polymerization,14 and cross-linking.15 Despite the success of these methods in preparing magnetic microspheres, each has its own pros and cons with respect to the synthetic methods and the properties of the microspheres prepared. Currently, achieving micrometer scale magnetic polymer microspheres, some modified suspension polymerizations16,17 are widely used. However, the as-synthesized microspheres still have a very broad size distribution and a nonuniform magnetite fraction in each microsphere. The well-known commercial magnetic microspheres, Dynabeads, were prepared by a multistep procedure consisting of preparing porous polymer latex, precipitating magnetic nanoparticles within the pores, and capping the pores with a polymer layer to seal in the magnetic particles.9 However, the multistep reactions involved make the preparation tedious and time-consuming. Therefore, it is a new challenge to develop novel methods for the preparation of magnetic microspheres that satisfy all major requirements in biotechnology, in particular, achieving microspheres with a uniform size and high saturation magnetization.
(1) Qian, H.; Lin, Z.; Xu, H.; Chen, M. Biotechnol. Prog. 2009, 25, 376–383. (2) Yang, S.; Lien, K.; Huang, K.; Lei, H.; Lee, G. Biosens. Bioelectron. 2008, 24, 861–868. (3) Kang, K.; Choi, J.; Nam, J.; Lee, S.; Kim, K.; Lee, S.; Chang, J. J. Phys. Chem. B 2009, 113, 536–543. (4) Lea, T.; Vartdal, F.; Nustad, K.; Funderud, S.; Berge, A.; Ellingsen, T.; Schmid, R.; Stenstad, P.; Ugelstad, J. J. Mol. Recognit. 1988, 1, 9–18. (5) Kuhara, M.; Takeyama, H.; Tanaka, T.; Matsunaga, T. Anal. Chem. 2004, 76, 6207–6213. (6) Lei, H.; Wang, W.; Chen, L.; Li, X.; Yi, B.; Deng, L. Enzyme Microb. Technol. 2004, 35, 15–21. (7) De Palma, R.; Reekmans, G.; Liu, C.; Wirix-Speetjens, R.; Laureyn, W.; Nilsson, O.; Lagae, L. Anal. Chem. 2007, 79, 8669–8677. (8) Matsunaga, T.; Maruyama, K.; Takeyama, H.; Katoh, T. Biosens. Bioelectron. 2007, 22, 2315–2321. (9) (a) Ugelstad, J.; Ellingsen, T.; Berge, A.; Hellgee, O. B. U.S. Patent 4,654,267, 1987.(b) Ugelstad, J.; Berge, A.; Ellingsen, T.; Schmid, R.; Nilsen, T.-N.; Mork, P. C.; Sienstad, P.; Hornes, E.; Olsvik, O. Prog. Polym. Sci. 1992, 17, 87–161. (c) Lea, T.; Vartdal, F.; Nustad, K.; Funderud, S.; Berge, A.; Ellingsen, T.; Schmid, R.; Sienstad, P.; Ugelstad, J. J. Mol. Recognit. 1988, 1, 9–18.
(10) Caruso, F; Spasova, M.; Sucha, A.; Giersig, M.; Caruso, R. A. Chem. Mater. 2001, 13, 109–116. (11) (a) Kondo, A.; Kamura, H.; Higashitahi, K. Appl. Microbiol. Biotechnol. 1994, 41, 99–105. (b) Xu, X.; Friedman, G.; Humfeld, K.; Majetich, S.; Asher, S. Chem. Mater. 2002, 14, 1249–1256. (c) Xu, X.; Majetich, S.; Asher, S. J. Am. Chem. Soc. 2002, 124, 13864–13868. (12) Lu, S.; Forcada, J. J. Polym. Sci., Part A: Polym. Chem. 2006, 44, 4187– 4203. (13) Horak, D. J. Polym. Sci., Part A: Polym. Chem. 2001, 39, 3707–3715. (14) Cocker, T.; Fee, C.; Evans, R. Biotechnol. Bioeng. 1997, 53, 79–87. (15) (a) Muller-Schulte, H. U.S. Patent 6,204,033, 2001.(b) Lin, H.; Watanabe, Y.; Kimura, M.; Hanabusa, K.; Shirai, H. J. Appl. Polym. Sci. 2003, 87, 1239–1247. (16) (a) Yang, C.; Guan, Y.; Xing, J.; Liu, J.; An, Z.; Liu, H. AIChE J. 2005, 51, 2011–2015. (b) Yang, C.; Guan, Y.; Xing, J.; Liu, H. Langmuir 2008, 24, 9006–9010. (c) Yang, C.; Guan, Y.; Xing, J.; Liu, H. J. Polym. Sci., Part A: Polym. Chem. 2008, 46, 203–210. (d) Yang, C.; Guan, Y.; Xing, J.; Liu, H. Polymer 2006, 47, 2299–2304. (17) Lu, S.; Cheng, G.; Zhang, H.; Pang, X. J. Appl. Polym. Sci. 2006, 99, 3241– 3250.
Introduction Magnetic supports have attracted significant interest because of the unique superparamagnetic properties, which have found application in increasingly diverse areas of biotechnology, including purification of proteins,1 viruses2 and nucleic acids,3 cell sorting and isolation,4,5 enzyme immobilization,6 and biosensors.7 Compared with the conventional separation, the separation process based magnetic supports can be performed directly in crude samples containing suspended solid materials (e.g., fermentation broth) without any pretreatment. The targeted molecules adsorbed by magnetic supports are fast separated by applying an external magnetic field. Subsequently, the targeted molecules will be easily recovered by elution. Thus, the magnetic separation provides an effective high-throughput method for processing a large scale of samples,8 which is attractive for numerous applications in biotechnology. Over the past two decades, there have been various techniques for the preparation of magnetic microspheres, such as in situ
Langmuir 2010, 26(7), 5179–5183
Published on Web 11/12/2009
DOI: 10.1021/la903659z
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In this study, we developed a novel and simple process for the preparation of monodisperse magnetic polymer microspheres by uniquely combining swelling and thermolysis technique. The properties of the magnetic polymer microspheres obtained were characterized with a transmission electron microscope (TEM), field emission scanning electron microscope (SEM), and superconducting quantum interference device (SQUID) magnetometer and by thermogravimetric analysis (TGA) and powder X-ray diffraction (XRD).
Experimental Section
Figure 1. Schematic diagram of the preparation of monodisperse magnetic polymer microspheres by swelling and thermolysis technique. interference device (SQUID) magnetometer (MPMS XL, Quantum Design). The temperature dependence of magnetization was measured in zero field cooling (ZFC) and field cooling (FC) modes in the temperature range from 5 to 320 K and with an applied field of 100 Oe. The magnetization curves were obtained at 5 and 300 K in fields up to 10 kOe. The crystalline type of the iron oxide species present in the polymer microspheres was characterized by powder X-ray diffraction (XRD) on a Rigaku D/max 2500 v/pc diffractometer with Cu KR radiation at 40 kV and 200 mA.
Materials. Styrene and divinylbenzene (DVB) obtained from Sigma-Aldrich were distilled under reduced pressure to remove inhibitors. All these treated monomers were stored in a refrigerator prior to use. 2,20 -Azobisisobutyronitrile (AIBN) and polyvinylpyrrolidone (PVP K-30, Mw = 40 000) were purchased from Sigma-Aldrich and used as initiator and stabilizer, respectively. Ferric chloride hexahydrate (FeCl3 6H2O), sodium oleate, and ethanol were purchased from Shanghai Chemical Reagent Corp. (China). Hexane, chloroform, and 1-octadecene were purchased from Alfa Aesar. All other chemicals were of analytical grade and used as received. Water was purified by distillation followed by deionization using ion exchange resins. Synthesis of Polystyrene Microspheres. Polystyrene microspheres were synthesized by dispersion polymerization. Typically, 160 g of ethanol and 18 g of water were added to a 500 mL threenecked round-bottom flask equipped with a mechanical stirrer and a reflux condenser. Three grams of PVP, 10 g of styrene, and 0.5 g of DVB were added in the above solution for 0.5 h with nitrogen purging. After the temperature was elevated to 74 °C, 0.4 g of AIBN was added into the reactor and polymerization was continued at 120 rpm for 24 h. The polystyrene microspheres obtained were repeatedly washed by centrifugation (3000g for 10 min) with distilled water before being dried at 60 °C overnight. Synthesis of Ferric Oleate. The ferric oleate was synthesized by reacting ferric chloride and sodium oleate18 with some modifications. Typically, 10 g of ferric chloride hexahydrate and 35 g of sodium oleate were dissolved in a mixture of 90 mL of ethanol, 70 mL of water, and 130 mL of hexane. The mixed solution was heated to 70 °C for 4 h. The resulting ferric oleate was washed four times with 50 mL of distilled water and dried at 50 °C. Preparation of Magnetic Polymer Microspheres. The schematic illustration for the preparation of magnetic polymer microspheres by swelling and thermolysis technique is shown in Figure 1. Typically, 8 g of the ferric oleate was mixed with 30 mL of chloroform to form the ferric oleate complex. Three grams of polystyrene microspheres was added to the above solution, mixed, and incubated at room temperature overnight. Then, 50 mL of 1-octadecene was added, mixed, and heated to 310 °C with a constant heating rate of 4 °C min-1 under the environment of nitrogen gas and then kept at that temperature for 0.5 h. The resulting magnetic polymer microspheres were cooled to room temperature and washed six times with 100 mL each of hexane and ethanol. Characterization. The morphology and structure of the magnetic polymer microspheres were studied by using a field emission scanning electron microscope (FEI SIRION 200) and a transmission electron microscope (JEOL-2010 with 200 kV accelerated voltage). The particle hydrodynamic size was measured by using a Beckman Coulter Counter laser size analyzer. The mass fraction of iron oxide nanoparticles in the magnetic polymer microspheres was determined by thermogravimetric analysis (TGA-2050, Du Pont Instruments), with a temperature ramp of 10 °C min-1. Magnetic susceptibility and hysteresis loop measurements were obtained by using a superconducting quantum
Preparation of Magnetic Polymer Microspheres. The magnetic polymer microspheres were prepared by swelling and thermolysis technique as shown in Figure 1. The micrometer-sized polystyrene microspheres with 0.5% cross-linker were readily prepared by dispersion polymerization and used without any treatment. The ferric oleate prepared was fully hydrophobic and was homogenously mixed with chloroform to form the ferric oleate complex. Upon mixing the black oil phase of the ferric oleate complex with these hydrophobic polystyrene microspheres, the polystyrene microspheres would be swelled in the oil phase, as the chloroform was a good solvent for polystyrene. Meanwhile, the ferric oleate complex would impregnate into the swollen polymer microspheres due to the different ferric oleate concentration between the outer phase and inner phase of the polymer microspheres. It would be eventually absorbed into the polymer microspheres after a certain period of stirring coupled with ultrasonic assistance, forming ferric oleate-swollen polymer microspheres with high monodispersity (as confirmed by using an optical microscope). Following the increase of the temperature, the chloroform evaporated and the polymer microspheres would be deswelled gradually. When the system was heated to an appropriate temperature, the iron oxide nanoparticles were formed within the polymer microspheres by thermal decomposition of ferric oleate. It was found that the temperature played a very important role in the formation of the iron oxide nanoparticles in the polymer microspheres. When aged at 300 °C for 24 h, the polymer microspheres did not show any magnetism. That means the iron oxide nanoparticles are not produced within the polymer microspheres. When aged at 310 °C for 0.5 h, the polymer microspheres displayed very strong magnetism, and the iron oxide nanoparticles were formed within the polymer microspheres, which was confirmed by the TEM image as shown in Figure 2D. Upon decomposition of the ferric oleate,18,19 the formation of iron oxide nanoparticles can be attributed to the separation of nucleation and growth processes, which results from the different temperature dependence. One oleate group dissociates from the Fe(oleate)3 at 200-240 °C, and the two remaining oleate ligands start dissociating at 300 °C. However, the dissociation will completely end at around 310 °C, and the major growth occurs immediately, resulting in the formation of the iron oxide nanoparticles. Our results verified that the iron oxide
(18) Park, J.; An, K.; Hwang, Y.; Park, J. G.; Noh, H. J.; Kim, J. Y.; Park, J. H.; Hwang, N. M.; Hyeon, T. Nat. Mater. 2004, 3, 891–895.
(19) Bronstein, L.; Huang, X.; Retrum, J.; Schmucker, A.; Pink, M.; Stein, B.; Dragnea, B. Chem. Mater. 2007, 19, 3624–3632.
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Results and Discussion
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Figure 2. SEM of the (A) polymer seed microspheres prepared by dispersion polymerization, (B) overall appearance of magnetic polymer microspheres prepared by swelling and thermolysis technique, (C) outer surface of magnetic polymer microspheres, and (D) TEM image of ultramicrotomed magnetic polymer microspheres. Localization of iron oxide nanoparticles in polymer microspheres.
nanoparticles produced from ferric oleate within the polymer microspheres were similar to those produced in oleic acid.18,19 Scanning electron microscope (SEM) images of the polymer microspheres and the resultant magnetic polymer composite microspheres are presented in Figure 2. The particle size was determined by statistically measuring the size of more than 500 individual microspheres from different regions of the SEM micrographs. In comparison with the polymer microspheres (Figure 2A) having an average diameter of 5 μm (standard deviation of 0.104), the magnetic polymer composite microspheres (Figure 2B) prepared by the proposed novel method had the same spherical shape and good monodispersity. In particular, the mean diameter of the magnetic polymer microspheres is 5.1 μm with a standard deviation of 0.106, which was a little larger than that of the original polymer microspheres. A large amount of iron oxide nanoparticles were buried within the polymer matrix, so the size of the magnetic polymer microspheres increased somewhat. Student0 s t test confirmed that there was no statistical difference in size between the original polymer microspheres and magnetic polymer composite microspheres. The TEM image of the magnetic polymer microspheres, as shown in Figure 2D, further confirmed that the iron oxide nanoparticles with a mean diameter of 11 nm (standard deviation of 0.210) were distributed evenly over the whole area of the polymer microspheres. Since the ferric oleate was kept within the cross-linked polymer microspheres, the additional nucleation was inhibited during the growth of iron oxide nanoparticles, which is useful for the formation of the ultrafine iron oxide nanoparticles (as shown in Figure 2D). The surface morphology of the magnetic polymer microspheres was Langmuir 2010, 26(7), 5179–5183
investigated with the SEM, with images taken under higher magnification. A smooth spherical structure could be observed from the surfaces of magnetic polymer microspheres, as shown in Figure 2C. The resulting magnetic polymer microspheres were washed six times each with n-hexane and ethanol until no black nanoparticles in the supernatant were detected by the UV-vis spectrophotometer. It was concluded that the iron oxide nanoparticles on the surface of the magnetic polymer microspheres could be cleaned up completely. This is very crucial for the surface modification of the magnetic polymer microspheres, either by functional polymer coating or by surface chemical reaction. Magnetic Properties. The magnetic properties of the magnetic polymer microspheres were tested by using a SQUID magnetometer at temperature range from 5 to 320 K under different applied magnetic fields. Figure 3 shows the temperature dependence of magnetization for the magnetic polymer microspheres. The zero field cooling (ZFC) and field cooling (FC) of magnetization measurements displayed typical superparamagnetic behavior, showing the blocking temperature (TB) at which the magnetization curve with the ZFC exhibits a cusp. While the temperature was below about 171 K, MZFC decreased rapidly with decreasing temperature, revealing TB = 171 K. Below this temperature, the microspheres displayed ferromagnetic behavior, whereas with increasing temperature they turned to the superparamagnetic state.21 (20) Krı´ zovaa, J.; Spanov aa, A.; Ritticha, B.; Horak, D. J. Chromatogr., A 2005, 1064, 247–253. (21) Yang, C.; Xing, J.; Guan, Y.; Liu, J.; Liu, H. J. Alloys Compd. 2004, 385, 283–287.
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Figure 3. Temperature dependence of the magnetization for the magnetic polymer microspheres.
Figure 4 shows the magnetization versus field plots for the magnetic polymer microspheres at temperatures of 5 K (Figure 4A) and 300 K (Figure 4B). When the temperature was decreased to 5 K, the thermal agitations which tended to destabilize the magnetic moments of the single domain particles were reduced and a ferromagnetic behavior appeared. The curve recorded at 300 K exhibited no coercivity and remanence, in line with superparamagnetic properties, indicating the absence of a long-range magnetic dipole-dipole interaction among the iron oxide nanoparticles within the polymer microspheres. This clearly verified that the iron oxide nanoparticles were well separately distributed in the polymer microspheres as 11 nm nanoparticles (TEM image shown in Figure 2D), which was smaller than the superparamagnetic critical size of magnetite particles (DP = 25 nm). Thus, the magnetic polymer microspheres containing such ultrafine iron oxide nanoparticles were expected to be superparamagnetic. Their saturation magnetization at room temperature was found to be 12.6 emu g-1 (Figure 4B), which was higher than that reported previously.11,17,20 As a result, the magnetic polymer microspheres in their homogeneous dispersion were attracted to a permanent magnet (1000 Oe) in about 60 s and redispersed quickly with a gentle shake once the magnetic field was removed (Figure 5). It was verified that these magnetic polymer microspheres possessed excellent magnetic responsivity and redispersibility, which was an especially important property needed for their applications. Thermogravimetric analysis (TGA) was used to study the iron oxide content of the magnetic polymer microspheres, and the results are shown in Figure 6. The weight loss observed at temperatures higher than 380 °C was attributed to the slow decomposition/vaporization of the polymer species present in the microspheres. It was obviously noted that polymer species completely disappeared at about 450 °C, revealing that the amount of iron oxide nanoparticles in the microspheres was 15.9 wt %. Thus, it was estimated that around 4 106 iron oxide nanoparticles were buried in a single microsphere. X-ray Diffraction Pattern. To verify the phase transformation of the ferric oleate in the polymer composite microspheres upon treatment with thermal decomposition, powder X-ray diffraction (XRD) analysis was performed on the microsphere samples, and the XRD patterns are shown in Figure 7. XRD patterns a and b in Figure 7 correspond to the microsphere samples before and after thermal decomposition. The ferric oleate 5182 DOI: 10.1021/la903659z
Figure 4. Hysteresis loops measured at 5 K (A) and 300 K (B) for magnetic polymer microspheres.
Figure 5. Separation-redispersion process of magnetic polymer microspheres. Langmuir 2010, 26(7), 5179–5183
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Figure 6. TGA plot of magnetic polymer microspheres.
in the original polymer composite microspheres existed in poorly crystallized form. However, a remarkable increase in crystallinity was observed in the polymer microspheres after thermal decomposition, and their XRD patterns were in agreement with the standard spectra for magnetite (Fe3O4) with inverse cubic spinel structure, which had seven diffraction peaks: (220), (311), (222), (400), (422), (511), and (440). The mean crystallite diameter of the magnetite nanoparticles was 11.4 nm, as calculated from the halfwidth of the (311) reflection peaks of the XRD patterns using the Scherrer formula where the k value was taken as 0.94 for cubic shaped particles. The results were almost identical to those observed from TEM images (11 nm, as shown in Figure 2D). It is well-known that the ultrafine magnetite particles are very easily oxidized during thermal decomposition. Thus, they always display two forms of iron oxide such as magnetite (Fe3O4) and maghemite (γ-Fe2O3).18,19 However, from the powder XRD pattern (Figure 7), except for pure cubic magnetite (Fe3O4), no other phase such as R- or γ-Fe2O3 was detected, which might result from the protection of nitrogen gas during the reaction. Therefore, the magnetic polymer microspheres displayed high saturation magnetization as shown in Figure 4B. In addition, the broad peak appeared in the range from 15 to 25°, indicating the existence of amorphous polymer.
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Figure 7. XRD patterns of (A) ferric oleate/polymer microspheres and (B) magnetic polymer microspheres.
Conclusions In conclusion, a novel swelling and thermolysis technique has been developed for the preparation of monodisperse magnetic polymer microspheres. The process involved the impregnation of ferric oleate within the swollen polymer microspheres, and subsequently the iron oxide nanoparticles were formed within the polymer matrix by thermal decomposition of ferric oleate. More importantly, this novel method can be considered as an inexpensive way of preparing monodisperse magnetic polymer microspheres to be used in biotechnology, such as for cell separation, enzyme immobilization, and purification of proteins. Acknowledgment. This work was financially supported by the Shenzhen-Hong Kong Innovative Circle Project, China (Grant No. SG200810140023A), the Shenzhen Industry-Education-Research Cooperation Project, China (Grant No. SY200806300190A), and the Science and Technology Research Projects of Shenzhen, China (Grant No. JC200903160366A). We thank the Materials School of Shenzhen University for materials characterization.
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