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Journal of Magnetism and Magnetic Materials 265 (2003) 98105

Preparation and characterization of polymer-coated coreshell structured magnetic microbeads

Z.L. Liua, Z.H. Dinga,*, K.L. Yaoa, J. Taoa, G.H. Dua, Q.H. Lua, X. Wanga, F.L. Gongb, X. Chenc
a

Department of Physics and State Key Lab of Laser Technology, Huazhong University of Science and Technology, Wuhan 430074, China b School of Basic Medical Science, TongJi Medical College, Wuhan 430030, China c Institute of Electronics, Chinese Academy of Sciences, Beijing 100080, China Received 21 January 2003; received in revised form 18 February 2003

Abstract Composite microbeads consisting of polymer-coated iron oxide nanoparticles are prepared by the microemulsion polymerization of styrene, divinyl benzene and methacrylic acid in the presence of emulsiers. Fourier transform infrared spectrometer analysis indicates the presence of COOH groups and Fe3O4 of the microbeads. The amount of COOH groups localized on the surface, which is about 0.15 mmol/g, is determined by conductometric titration. Transmission electron microscope picture reveals that the microbeads have a coreshell structure. The dissolving experiments of microbeads in hydrochloric acid and toluene further identify the coreshell structure. Optical microscope indicates that the magnetic microbeads have uniform and spherical forms with the size of 15 mm. Magnetic sensitivity measurement indicates that the microbeads can be used conveniently. Magnetic property measurement shows very little residual magnetization and coercivity, which are below 0.5 emu/g and around 15 Oe, respectively. The magnetic properties are greatly related to the particle sizes. The thermal gravity analysis result shows the improvement of thermal stability. The experiment of immobilized antibody indicates that the functional groups on the surface are appropriate. r 2003 Elsevier Science B.V. All rights reserved.
Keywords: Coreshell; Microbeads; Magnetic property; Functional groups

1. Introduction Magnetic separation has been widely applied to various aspects in biotechnology and biomedical engineering, such as cell separation [1], immobilized enzyme [2], protein separations [3] and target
*Corresponding author. Tel.:+86-27-875-56262; fax:+8627-875-42425. E-mail address: zhdingmou@yahoo.com.cn (Z.H. Ding).

drug [4], etc. Magnetic separation is relatively rapid and easy, cost effective and highly efcient. To make full use of the magnetic separation technology, magnetic carriers with good properties are necessary and indispensable. The magnetic carriers are usually composed of two parts. One is the magnetic core, which is often inorganic magnetic nanoparticles, such as Fe3O4, nickel, etc. The other is the polymer shell around the magnetic core, which provides favorable

0304-8853/03/$ - see front matter r 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0304-8853(03)00230-0

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biocompatibility, high magnetic susceptibility, appropriate size distribution and abundant functional groups on the surfaces of covalently coupled ligands for selective binding. There are two main ways to prepare the magnetic polymer microbeads. One is coating or encapsulating of magnetic particles with performed polymer [5]. The other is using monomer polymerization, which is used more widely. Several kinds of polymerization can be used, such as emulsion polymerization [6], dispersion polymerization [7], suspension polymerization [8] and microemulsion polymerization [9], etc. This paper introduces the hybrid microbeads with Fe3O4-based core and strongly bound polymer coatings prepared by microemulsion copolymerization of styrene (St) and methacrylic acid. We analyze the functional groups on the surface, the size distribution and shape, the magnetic properties, which are three main factors for the application of magnetic microbeads. These polymer-coated magnetic microbeads have better properties than other ironpolymer materials, which lack tightly bound polymer coatings, tend to be sensitive to oxidation and corrosion, and lack enough functional groups on the surface [10,11].

2. Experiment and measuring method Magnetic microbeads are prepared by the microemulsion polymerization. Benzoyl peroxide (BPO) is dissolved in the mixed magnetic organic solution of surface-modied Fe3O4 nanoparticles [17], St and divinyl benzene (DVB), forming the magnetic liquid. The hydrophobic surface of modied Fe3O4 nanoparticles makes them disperse very evenly in organic solutions, which guarantees the ideal coating by polymer molecules. We usually disperse the solution by ultrasonic wave for 30 min before polymerization. This solution is called A. Secondly, sodium dodecyl sulfate (SDS) and cetyl alcohol (CA) are blended and dissolved in water. Sodium hydroxide is added to the mixture to adjust the pH value to the range of 9 11 [12]. The solution is called B. When B is agitated in a glass ask, A is dripped evenly. The stirring rate is 750 rpm. After 40 min, a mixed water solution of KPS (potassium persulphate) and methacrylic acid (MAA) is added. After 3 h of polymerization, we can obtain about 100 ml microemulsion, and then the emulsion is washed by ethanol and water three times with the help of magnetic eld. Finally the expected microbeads are obtained.

Fig. 1. FTIR spectrum of polymer microbeads (1) and magnetic polymer microbeads (2).

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2.4

Fourier transform infrared spectrometer (FTIR) spectral of the magnetic beads is obtained. Dry microbeads are completely mixed with analytical grade KBr and pressed into a form of tablet, and the spectrum is then recorded. Conductometric titration determines the amount of carboxyl groups on the surface, which is measured by a conductometer (model DDS-11A). TEM picture is taken to observe the structure. Optical microscope picture is taken to observe the morphology and size distribution of the magnetic microbeads. A vibrating-sample magnetometer (VSM) is used to measure the magnetic properties.

A
2.2

conductance (ms/cm-1)

2.0

1.8

1.6

1.4

1.2

1.0 0 5 10 15 20 25 30

NaOH volume (ml)

3. Results and discussions The presence of COOH groups on the surface of the microbeads is conrmed by FTIR spectra shown in Fig. 1. Comparison is made between polymer microbeads and magnetic polymer microbeads. The carboxyl band observed at 1697.99 cm1(C=O), 1452.77 cm1(CO), 1 1 904.68 cm (OH), 1601.32 cm (phenyl ring) indicates the presence of COOH groups on the surfaces of the microbeads. The strong peaks at 759.33, 701.19 cm1 and several characteristic absorbing peaks near 3000 cm1 are corresponding to St absorbed spectrum. The Fe3O4 weak absorbing peak at 564.15 cm1 conrms that these magnetic nanoparticles are located mainly in the interior part. The stretching vibration band of vinyl at 1630 cm does not appear, which suggests that the composite has been completely polymerized. The locations of the wave number are almost same for the nonmagnetic microbeads and magnetic beads, which suggests that the polymer coating is probably mainly mechanically and physically formed and not chemically bonded on the surface of the nanoparticles [13]. Otherwise the vibration absorbing bands will disappear, shift, or be transferred. The conductometric titration curve is shown in Fig. 2. About 10 ml HCL (0.01 mol/l) solution is added to 200 ml microbead suspension (solid content is 0.3 g) and stirred at 20 C for 20 min. Then a 0.01 mol/l NaOH aqueous solution is used as titrant. The amount of COOH groups on the

Fig. 2. Conductometric titration curve of COOH groups on the surface.

0.30

0.25

Surface-COOH, mmol/g

0.20

0.15

0.10

0.05

0.00 0.2 0.4 0.6 0.8 1.0

Monomer MAA, Vol%

Fig. 3. Relationship between amounts of monomer MAA and surface COOH groups.

surface is calculated from the break in curve and expressed in terms of milliequivalent of COOH groups per 1 g of the microbeads [14]: COOHmmol=g NV =W ; where N and V are the concentration and volume of NaOH titrant, respectively, and W is the solid content weight of the magnetic microbeads. The amount of COOH groups on the surface in Fig. 3 is about 0.15 mmol/g, which vary due to the ratio of MAA volume to the total volumes of all

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the monomers. The results are shown in Fig. 2. The amounts of COOH groups increase nonlinearly with the increment of monomer MAA. The microbeads have abundant functional COOH groups located on the surface, which can be tailored to suit specic applications especially to cell separation and antibody coupling. The structure of the composite materials is analyzed by TEM. Simple coreshell particles are observed. From Fig. 4, we know that most of the magnetic nanoparticles are dispersed evenly throughout the interior. The microbeads bear a strongly bound polymer thin shell, which is clearly visible in the TEM images. Most of the microbeads are polymer-coatings, which cover not only the surface but extends toward the central part. Fe3O4 is located mainly in the interior part, which enhances the absorbing ability, bicompatibility and resistance to oxidation and corrosion. In order to identify the coreshell structure further, the microbeads are dissolved with 0.1 mol/l HCL water solution and toluene, which are good solvents for Fe3O4 and polymer layers, respectively. There is no loss of magnetite after overnight incubation in HCL water solution. It is the reason that magnetic nanoparticles are so tightly coated by polymer coating that no H+ can enter the interior to dissolve them. There are also no residual clusters, bulks, visible particles after several overnight incubations in toluene. Only black magnetic liquid dispersed slightly stably is obtained. It is the reason that the outer polymer layer is dissolved and the interior magnetic nanoparticles are dispersed by toluene. These

indicate that the Fe3O4polymer microbeads have a coreshell structure. Fig. 5 is the optical microscope pictures of magnetic microbeads. Fig. 5 shows the size distribution of magnetic microbeads. From Figs. 5 and 6, we know that the magnetic microbeads have a narrow size distribution with a mean diameter of 2.8 mm. Most of the magnetic microbeads vary from 1.5 to 4.5 mm. The constituents of the mixed monomers and the stirring rate are two important parameters for the preparation of desirable magnetic beads. The two aspects control both formation and shape of particles, which enable higher surface area favoring better adsorption of antigens and proteins. The TGA results show that the thermal stability of magnetic composite microbeads is improved

Fig. 5. Optical microscope view of magnetic microbeads.

110 100 90 80

Normal distribution Gauss distribution

Percentage

70 60 50 40 30 20 10 0 500 1000 1500 2000 2500 3000 3500 4000 4500 5000

Particle Diameter (nm)

Fig. 4. TEM photograph of magnetic microbeads.

Fig. 6. Particle size and its distribution of magnetic microbeads.

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102 Z.L. Liu et al. / Journal of Magnetism and Magnetic Materials 265 (2003) 98105 Table 1 Magnetic sensitivities of microbeads Sample number Fe3O4 content (wt%) Time(s) 3000/G Time(s) 1200/G 1 1 100 160 2 5 70 100 3 10 50 60 4 15 30 40 5 20 20 30

greatly. The TGA is preformed under a nitrogen atmosphere to minimize mass increase due to oxidation. Fig. 7 shows that the hybrid polymers are decomposed at about 300 C, while the composites with 23% Fe3O4 particles are decomposed at about 400 C. The magnetic microbeads can disperse very well in water solutions and move directionally under outer eld. We nd that the microbeads move toward outer eld very quickly and obviously, and can be separated completely from water in 20 s under a 3000 G eld. We can nd an obviously directional movement of magnetic microbeads as long as outer eld is applied. The magnetic sensitivities increase with the increasing of Fe3O4 content. In order to compare the magnetic sensitivities qualitatively, we measure the required time when microbeads with different Fe3O4 contents are separated completely under 3000 and 1200 G eld, respectively. These elds are not too strong and are convenient and easy for various applications. The results are shown in Table 1.The magnetic microbeads move more quickly and have stronger magnetic sensitivities with the increasing of Fe3O4 content. We can nearly make the conclusion that the magnetization properties are large enough to be useful. Magnetic measurements are made with a VSM magnetometer. A typical hysteresis curve is shown in Fig. 8, and the saturated magnetization (Ms ), the residual magnetizations (Mr ) per gram of
100 90 80 70

Notes: The microbeads are dispersed evenly by 50 ml water in a 100 ml beaker. All the microbeads have the same size approximately of 15 mm. The average size is about 2.5 mm. The magnetic elds are about 3000 and 1200 G, respectively.

Grav:60.7mg V*Ms:4.235emu/g V*Mr:0.302emu/g Hc:20.874Oe

Magnetization (emu/g)

-2

-4

-3000

-2000

-1000

1000

2000

3000

Magnetic Field (Oe)

Fig. 8. Magnetization curve obtained by VSM at room temperature of magnetic microbeads.

hydrobrid polymer magnetic microbead

60 50 40 30 20 10 0 0 200 400 600 800

Temperature (c)

Fig. 7. The TGA of hybrid polymer and magnetic polymer microbeads.

samples and the coercivities (Hc ) are given in Table 2. Measurements are also made on samples which are dispersed evenly in various mediums. There are some remarkable differences between the two sets of samples. All the samples are composed of approximately the same content of nanoparticles per gram (about 20 wt% measured by thermogravimetry). The S0 sample is for the Fe3O4 nanoparticles and sample S1 is for the polymer magnetic microbeads. The L1, L3 and L4 samples are for the microbeads with the same concentration in different dispersion mediums, respectively. The dispersants are water (pH=7), phosphate buffer solution (pH=6.8), and carbonate buffer solution (pH=8.4), respectively. The L1 and L2 are for the same medium with different concentrations of microbeads. The concentration of L2 is two times of that of L1.

Weight (%)

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The hysteresis loop of magnetic microbeads, which is measured in powdered state, is shown in Fig. 8. We know that 3000 G magnetic eld is sufcient to excite all of the dipole moments. The typical characteristics of superparamagnetic behavior are observed showing immeasurable remanence and little coercivity. The saturated and residual magnetization, which is 4.235 and 0.302 emu/g, respectively, are signicantly less
Table 2 Properties of magnetic microbeads in different conditions Sample number S0 S1 L1 L2 L3 L4 Particle size (mm) 0.01 3.0 3.0 3.0 3.0 3.0 Ms (emu/g) 50.2 2.681 3.833 6.160 6.381 6.163 Mr (emu/g) 4.84 0.163 0.364 0.596 0.619 0.609 Hc (Oe) 15.78 13.184 31.494 39.551 40.382 41.084

Notes: The S0 sample is for the Fe3O4 nanoparticles and sample S1 is for the polymer magnetic microbeads. The L1, L3 and L4 samples are for the microbeads with the same concentration in different dispersion medium, respectively. The dispersants are water (pH=7), phosphate buffer solution (pH=6.8), carbonate buffer solution (pH=8.4), respectively. Sample L2 has a content of microbeads that is twice more than sample L1 but in the same dispersion medium.

than that of Fe3O4 magnetic nanoparticles, which is 50.2 and 4.84 emu/g. From Table 2, it is also shown that coercivities of the magnetic microbeads are a bit higher than that of Fe3O4 nanoparticles. The magnetization values of the microbeads are considerably lower than that of iron oxides. The agglomeration of Fe3O4 nanoparticles in the microbeads and the dipolar interactions between particles may cause hysteresis when anisotropic energy is increased, resulting in the increase of coercivity and causing the changes from single domain to multi-domains. As to the magnetization, magnetic microbeads have far less content of magnetic nanoparticles per gram than iron oxides. So the magnetic susceptibility of the magnetic microbeads is greatly reduced. A dead magnetic layer mainly made of the large fraction of low density polymer coating is formed over the surface layer due to the strong interaction between the atoms on the surface and the molecular chain, which prevents the coupled dipoles from aligning along the magnetic orientation. Thus there is a tendency toward lower magnetization values [15]. Fig. 9 shows the trend of the magnetization value toward lower values as the particle diameter increases, and the trend of coercivity value toward
30

4.5 4.0

Ms(emu/g) Mr(emu/g)
Coercivity(Oe)

28 26 24

Magnetization (emu/g)

3.5 3.0 22 2.5 20 2.0 18 1.5 16 1.0 14 0.5 12 0.0 0.5 1.0 1.5 2.0 2.5 3.0

Particle size (um)

Fig. 9. Effects of particle size on saturation magnetization (Ms ) residual magnetization (Mr ) and coercivities: (1) IgG antibody, (2) CD3 antibody.

Coercivity (Oe)

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Fig. 10. Fluoroscope view of magnetic microbeads after antibody immobilization.

anomalous changes. As to the magnetization, the trend may be caused due to interactions among nanoparticles, polymer molecules and nanoparticles, which give rise to spinning, sluggish, blocking and dangling bonds. The coercivity reaches the maximum for the least size and then shows irregular changes decreasing rst, increasing then, and then decreasing again. Comparisons of the magnetic properties between the powdered samples and the dispersed samples are given in Table 2. For the powdered samples, the magnetization value and the coercivity are much lower than that of the dispersed samples. The reason is as following: The relaxation time T1 depends on the height of the energy barrier as T1 T0 expkeff V =kB T; where keff is the anisotropy constant, V is the particle volume, kB the Boltzmann constant, and T is the absolute temperature, T0 is a constant and is usually taken as T0 D1010 s: For the dispersed sample, there is another relaxation time T2 3V Z0 =kB T; where V is the volume of the magnetic particles and Z0 the mucilaginous coefcient of the liquid medium. The total relaxation time of the dispersed sample is determined by T1 and T2; whereas that of the powdered sample is determined by T1 only, which leads to that the total relaxation time of the dispersed sample is larger than that of the powdered sample. Prolonging of the total relaxation time is attributed to the existence of the larger hysteresis of the dispersed sample [16]. It is found that for L1 and L2; the magnetization values are increased approximately twofold, which is due to the twofold magnetic content of

L2: As to L2; L3 and L4; it is obvious that the mucilaginous coefcient of water is lower than that of the phosphate buffer solution (pH=6.8) and that of carbonate buffer solution (pH=8.4). To investigate the feasibility of carboxyl groups on the surface of the magnetic microbeads, we perform the experiments of antibody adsorption. To covalently attach antibody IgG and CD3 to the magnetic microbeads, the carboxyl groups on the surface are activated with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC) solution. Then the microbeads are modied with IgG and CD3 antibody. The IgG-modied and CD3modied microbeads are observed, respectively, with a uorescence-labeled method. From Fig. 10, we can see almost all of the microbeads are linked with antibody, giving quite good properties in biological adsorption. The microbeads can also be used to attach other antibody, immobilize enzymes and to separate cells, which are in progress.

4. Summary Polymer-coated functional microbeads are prepared by the microemulsion polymerization. The constituents of the materials are analyzed by FTIR, which indicates that there are abundantly carboxyl function groups on the surface. So the microbeads can easily be linked by biological materials such as antibody and enzyme. TEM observation reveals the coreshell structure of the materials. Magnetic nanoparticles are tightly coated by external polymer, which increases the resistance ability to corrosion and oxidation. The

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high magnetic sensitivity makes the microbeads responding easily and quickly. The composites are ferromagnetic, and the magnetic properties are correlated with the particle size.

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