Research Article

Received: 29 October 2010 Revised: 10 May 2011 Accepted: 20 June 2011 Published online in Wiley Online Library: 6 September 2011

(wileyonlinelibrary.com) DOI 10.1002/jctb.2704

Application of magnetic iron oxide nanoparticles prepared from microemulsions for protein purication
b Chuka Okoli,a,b Magali Boutonnet,b Laurence Mariey,c Sven Jars and Gunaratna Rajaraoa
Abstract
BACKGROUND: Magnetic nanoparticles are of immense interest for their applications in biotechnology. This paper reports the synthesis of magnetic iron oxide nanoparticles from two different water-in-oil microemulsion systems (ME-MIONs), their characterization and also their use in purication of coagulant protein. RESULTS: ME-MIONs have demonstrated to be an efcient binder in the purication of Moringa oleifera protein when compared with the superparamagnetic iron oxide nanoparticles prepared from coprecipitation in aqueous media. The size and morphology of the ME-MIONs were studied by transmission electron microscopy (TEM) while the structural characteristics were studied by X-ray diffraction (XRD). The microemulsion magnetic iron oxide nanoparticles (ME 1-MION and ME 2-MION) obtained have a size range 710 nm. The protein and ME-MIONs interaction was investigated by Fourier transform infrared spectroscopy (FT-IR); the presence of three peaks at 2970, 2910 and 2870 cm1 respectively, conrms the binding of the protein. The purication and molecular weight of the coagulant protein was 6.5 kDa as analyzed by SDS-PAGE. CONCLUSION: The ME-MIONs have the advantage of being easily tailored in size, are highly efcient as well as magnetic, cost effective and versatile; they are, thus, very suitable for use in a novel purication technique for protein or biomolecules that possess similar characteristics to the Moringa oleifera coagulant protein. c 2011 Society of Chemical Industry Keywords: microemulsion; magnetic nanoparticles; iron oxide; protein purication; magnetic separation; Moringa oleifera

INTRODUCTION
The emergence of nanobiotechnology has contributed immensely in the development of new techniques for solving many health and environmental issues. Nanometer-sized magnetic nanoparticles have received considerable attention because of their unique physical and chemical properties owing to their extremely small size and large specic surface area.1 3 Magnetic nanoparticles have been used in many applications such as data storage devices,4 6 and skin care products.7 In recent years, nanometersized magnetic nanoparticles have been used in both medical and biological elds owing to their strong magnetic properties and virtually no toxicity for applications including drug delivery,8 11 biosensors,12,13 water purication systems14,15 and biomolecular separation/purication.10,16 19 Protein is the central dogma of cell function and study of proteins is essential to a better understanding of the cell function. Protein purication has been a fundamental requisite in advances made in biotechnology.18,20 Pure protein helps to resolve the basic questions about the functional characteristics of protein while the development of apt techniques is essential where more than one step is required for protein purication.1,16,18 Recombinant protein technologies are being developed in order to produce protein in large quantities; however, many problems remain unresolved, such as host contamination, solubility and structural integrity.20 In

biotechnology, afnity protein purication using antibody-based separation or a matrix with specic tags for binding target protein are commonly used methods.16 18 The challenge is to use a common matrix for purication of different proteins. Magnetic nanoparticles can be used as a simple and quick method for the efcient capture of selected target biomolecules in the presence of other suspended solids even for a small sample volume.21 The use of magnetic nanoparticles such as superparamagnetic iron oxide nanoparticles for purication and immobilization of biomolecules such as proteins/peptides, have the following advantages: (i) high specic surface area can be obtained to bind large amounts of protein samples; (ii) selective targeting of biomolecules with

Correspondence to: Gunaratna Rajarao, Royal Institute of Technology (KTH), Environmental Microbiology, Stockholm, 106 91 Stockholm, Sweden. E-mail: gunar@biotech.kth.se

a Royal Institute of Technology (KTH), Environmental Microbiology, Stockholm, 106 91 Stockholm, Sweden b Royal Institute of Technology (KTH), Chemical Technology, Stockholm, 100 44 Stockholm, Sweden c Laboratoire Catalyse et Spectrochimie, ENSICAEN, Universite de Caen, CNRS, 140 50 Caen-Cedex, France

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Magnetic iron oxide nanoparticles for protein purication desired surface characteristics and easy separation from the reaction mixture by magnetic eld; (iii) elimination of several steps in the purication process such as centrifugation, ltration, etc.1 Using magnetic separation offers a gentle and fast alternative; targets are captured on the surface of the desired magnetic particle and a rapid separation is achieved from the sample by the use of an external magnetic eld. With most conventional or commercially available protein purication/immobilization systems, the cost of protein purication on a large scale is still a major challenge. A promising approach is the use of magnetic iron oxide nanoparticles prepared from microemulsion (ME-MIONs) for the purication or immobilization of proteins. This technique is based on microemulsion synthesis, which allows tailormade nanoparticles in order to achieve the desired size.22,23 Microemulsions are optically transparent, thermodynamically stable solutions which consist of spherical aqueous nanodroplets, so-called reverse micelles, stabilized by surfactant molecules. In the aqueous core of the reverse micelle, iron metal precursor can be solubilized and then precipitated to form iron oxide nanoparticles.22 ME-MIONs are single domain magnetic dipoles that show no preferred directional ordering in the absence of an applied external magnetic eld due to weak dipoledipole interactions. On application of a sufciently high magnetic eld gradient, the nanoparticles exhibit a preferential ordering in the direction of this external eld.24 This paper reports a simple and fast method for protein purication and/or immobilization using ME-MIONs obtained from two different microemulsion compositions. The ME-MIONs were synthesized and their characteristic properties studied. The purication of coagulant protein is achieved by binding and elution of the Moringa oleifera protein. The coagulation efciency of ME-MIONs puried proteins was examined and compared with protein puried with superparamagnetic iron oxide nanoparticles (SPION) prepared by co-precipitation in aqueous media (Okoli et al., in press) and the results are discussed.

www.soci.org and is termed microemulsion 1-magnetic iron oxide nanoparticles (ME 1-MION) while microemulsion 2 consists of CTAB/1-butanol/noctane/water and is termed microemulsion 2-magnetic iron oxide nanoparticles (ME 2-MION). The aqueous phase contains either the Fe salt precursors in a mole ratio of 2 : 1 [FeCl3 : FeCl2 ] or aqueous ammonia (NH3 ). It has been demonstrated that particles prepared with this method exhibit a size range 650 nm with high surface area to volume ratio and a superparamagnetic behavior.24 Microemulsion system 1 for synthesis of ME 1-MION The ME 1-MION was synthesized by mixing two w/o microemulsion solutions; scheme 1 (a). Microemulsion 1 solution was obtained by adding the iron precursor solutions (1.2 mol L1 FeCl3 + 0.6 mol L1 FeCl2 ) to the mixture of the surfactant, co-surfactant and oil phase (molecular concentration of CTAB : 1butanol : hexanol : water = 1.5 : 1 : 1.4 : 1.6); for microemulsion 2, a precipitating agent solution (NH3 , 32%) was added to the mixture. Microemulsion 2 was then added to microemulsion 1 dropwise in order to obtain the formation of particles at 25 C upon vigorous stirring for 2 h. The pH of the mixture was adjusted to 11 with 32% ammonia solution. Formation of magnetic nanoparticles was indicated by black coloration. The magnetic nanoparticles obtained were separated using an external magnetic eld and then washed by several cycles of precipitation and resuspension in a mixture of chloroform and methanol (1 : 1), water and nally methanol to remove all the surfactant and oil left in the system. The magnetic nanoparticles were either dried at 70 C or suspended in Milli-Q water and kept at 4 C until further use. Microemulsion system 2 for synthesis of ME 2-MION Microemulsion system 2 consists of a single-step mode of preparation. In this experiment, only one type of microemulsion solution is needed for formation of the nanoparticles, scheme 1 (b). For ME 2-MION preparation, the following conditions were used: 0.67 mol L1 FeCl3 and 0.42 mol L1 FeCl2 at mole ratio (2 : 1) in Milli-Q water; the molar concentration of CTAB : 1-butanol : noctane : water = 0.6 : 0.5 : 3 : 1.3. The addition of precursor solution to the mixture of CTAB/1-butanol/n-octane will give rise to the formation of a microemulsion. Formation of magnetic nanoparticles was achieved by adding the precipitating agent (NH3 , 32%) dropwise to the microemulsion containing the precursor upon vigorous stirring until pH 11 was achieved. The reaction mixture was stirred for 2 h at 25 C. A dark brown coloration indicates the formation of ME 2-MION in the reaction mixture. The magnetic nanoparticles obtained were then washed with a mixture of chloroform and methanol (1 : 1), water and nally methanol to remove all the surfactant and oil left in the system. The magnetic nanoparticles were either dried at 70 C or suspended in Milli-Q water and kept at 4 C until further use. Synthesis of superparamagnetic iron oxide nanoparticles (SPION) SPIONs were prepared by co-precipitation in aqueous media (Okoli et al., in press). Briey, the iron source was prepared by dissolving FeCl3 and FeCl2 in Milli-Q water at a molar ratio of 2 : 1 followed by precipitation of nanoparticles with ammonia solution at 70 C under vigorous mechanical stirring. The reaction was allowed to proceed for 45 min under N2 atmosphere in a closed system. The black precipitated powder was collected by sedimentation with the help of an external magnetic eld and washed with Milli-Q water. The magnetite obtained was re-suspended in water prior to use.

EXPERIMENTAL
Materials All chemicals were of reagent grade and were used without further purication. Ferric chloride hexahydrate (FeCl3 .6H2 O, >99%), ferrous chloride tetrahydrate (FeCl2 .4H2 O, >99%), cetyl trimethyl ammonium bromide (C19 H42 BrN), 1-butanol (C4 H10 O), n-octane (C8 H18 ) and hexanol (C6 H13 OH) were purchased from SigmaAldrich, Sweden. Ammonia (NH3 , 32%), sodium chloride (NaCl), ammonium acetate (CH3 COONH4 ), and kaolin clay (Al2 Si2 O5 (OH)4 ) were purchased from Merck, Sweden. Ethanol (C2 H5 OH, >99.7%) was obtained from Solveco, Sweden. Moringa oleifera seeds were obtained from Kenya. Synthesis of magnetic iron oxide nanoparticles (ME-MIONs) by water-in-oil (w/o) microemulsion reaction method The magnetic iron oxide nanoparticles of the present study were prepared in a w/o microemulsion. In this work, two different types of microemulsion systems were investigated for the preparation of ME-MIONs. Each microemulsion system consists of a surfactant, cetyl trimethyl ammonium bromide (CTAB), a co-surfactant (1-butanol), oil phase (n-octane or hexanol), iron precursors (FeCl3 and FeCl2 ), precipitating agent (32% NH3 ) and water (Milli-Q water). Microemulsion system 1 consists of CTAB/1-butanol/hexanol/water

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Scheme 1. Preparation of ME 1-MION (a) and ME 2-MION (b).

Characterization of magnetic iron oxide nanoparticles from microemulsion The ME-MIONs were characterized by transmission electron microscopy (TEM; ZEISS, EM 906) with an accelerating voltage of 200 kV, X-ray diffraction (XRD; Siemens D5000, using Cu K radiation of wavelength; = 0.1540 nm). The investigated ME-MION samples were either dried at 70 C or calcined at 450 C. Fourier transform infrared spectroscopy (FTIR; Nicolet 5700 spectrometer) was used to investigate the protein interaction on the nanoparticle surfaces. The samples were analyzed as a disc with an area of 2 cm2 and a mass of 15 mg.

where T0 is the initial absorbance and Tf is the nal absorbance. Percentage activity (%) = T0 Tf 100 T0

Protein estimation and molecular weight determination The protein content was estimated using the Bradford method with bovine serum albumin (BSA, 1 mg mL1 ) as a standard.27 This method is based on binding of the dye Coomassie brilliant blue to protein, and is measured at 595 nm. The protein prole and the molecular weight of the puried protein and that of the crude extract were determined and compared using 10% SDS-PAGE mini gels. Purication of coagulant protein with magnetic iron oxide nanoparticles In this study, a batch system was employed in the purication of the coagulant protein. ME 1-MION, ME 2-MION and SPION suspensions were used as obtained without any heat treatment. The magnetic iron oxide nanoparticles were washed three times each with 10 mmol L1 ammonium acetate buffer, pH 6.7, to equilibrate the particles and then suspended in water or buffer with the following concentrations: ME 1-MION (36 mg mL1 ), ME 2-MION (44 mg mL1 ) and SPION (10 mg mL1 ). The volume of each solution was adjusted such that the total weight of magnetic particles was the same, 2.5 mg, in the resultant solution. The immobilization of MOCP (2.6 mg mL1 ) on the particles was performed in 400 L of 10 mmol L1 ammonium acetate buffer, pH 6.7. After 60 min incubation at room temperature, the unbound

Coagulant protein extraction and activity assay Moringa oleifera coagulation protein (MOCP) extraction was performed following the method described previously.25 The Moringa oleifera seeds were obtained from Kenya. Ethanol (95%) was used to extract the oil from the crushed seeds and the active components were extracted with Milli-Q water. Kaolin clay (1%) was added to 1 L of tap water, stirred for half an hour and allowed to settle for 24 h for complete hydration. Small-volume coagulation activity assay on clay suspension was performed.26 A sample volume of 10 L was added to the clay suspension to a nal volume of 1 mL in cuvettes and mixed instantly. Absorbance at 500 nm was measured with Thermo Spectronic UV-visible spectrophotometer at time 0 and after 60 min settling time. A decrease in absorbance relative to control denes coagulation activity. The percentage coagulation activity was calculated using the formula below;

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Magnetic iron oxide nanoparticles for protein purication protein was separated by applying an external magnetic eld and the pure protein eluted with 0.6 and 0.8 mol L1 NaCl solution. The specic coagulation activity of the puried protein was calculated by dividing the pure protein (% coagulation activity) by the total protein content/mg/ml. Adsorption kinetic studies Studies of adsorption kinetics were carried out in a batch system with a xed concentration of magnetic iron oxide nanoparticles (0.5 mg) at varying protein concentrations (0.52.1 mg mL1 ). The immobilization assay was performed in 1.5 mL of 10 mmol L1 ammonium acetate buffer. After 1 h of immobilization, the samples were separated and the supernatants collected to estimate the protein content. Based on results for the controls, a change in protein concentration was attributed to adsorption by the magnetic nanoparticles. The amount of adsorbed protein was estimated from the Langmuir equilibrium adsorption model:28 Ce 1 Ce = + qe b(Xm ) Xm (1)

www.soci.org are either magnetite (Fe3 O4 ) or maghemite ( -Fe2 O3 ) or a combination of both magnetite and maghemite as can be seen from XRD results showing similar patterns in their crystal structure.29,30 The ME-MION particle size obtained by TEM (Fig. 1) is in agreement with the particle size values obtained from XRD analysis (Fig. 2). The XRD patterns of ME 1-MION and ME 2-MION are shown in Fig. 2. It can be seen that the XRD patterns of both samples after calcination at 450 C appear quite similar, but somewhat different after drying at 70 C; this implies that both ME-MIONs have similar spinel crystal structure at 450 C in contrast to the samples dried at 70 C. It is difcult to conclude whether the nanoparticles consist of magnetite or maghemite. The broad diffraction peaks from the nanoparticles imply that the original particle size is very small; similar results have been reported by Li et al.31 From the XRD analysis of the particles dried at 70 C, a particle size of 9.2 nm for ME 1-MION and 7.8 nm for ME 2-MION was obtained. Coagulant protein extraction and activity assay Water was used to extract active coagulant protein extracted from Moringa oleifera. In order to estimate the actual activity of the coagulant protein, control samples are used and differences in control and coagulant protein are considered as real coagulation activity. It has been reported that protein activity with salt or buffer extracts show comparable activity but protein coagulation activity from water extract tends to be higher. It is believed that protein obtained by water extraction has a better binding environment with the clay solution.25,32 Purication of coagulant protein with magnetic iron oxide nanoparticles The coagulant protein was puried with ME 1-MION and ME 2-MION and their purication efciency was compared with SPION (15 nm size) prepared by a co-precipitation method. The prepared magnetic particles exhibit similar size and magnetic behavior (Okoli et al., in press). Purication was achieved using the same elution buffer and pH as described previously. In this work, the active coagulant protein was eluted from ME 1-MION, ME 2-MION and SPION with 0.6, 0.8 and 1 mol L1 NaCl concentrations, respectively (Fig. 3). By increasing the salt concentration of the elution buffer to 1 mol L1 , it was envisaged that the remaining protein molecules that were strongly bound to the particles would be detached. The protein puried with ME 1-MION and ME 2-MION showed coagulation activity similar to that of SPION in the rst elution step. In subsequent elution steps (2nd and 3rd), the coagulation activity of the ME-MIONs increased progressively compared with the SPION. When 1 mol L1 NaCl was used, the activity of

where Ce is the amount of protein in solution at equilibrium (mg L1 ), qe is the amount of protein adsorbed per weight of adsorbent (mg g1 ), b is a constant related to the heat of adsorption (mL g1 ) and Xm is the maximum adsorption capacity (mg g1 ).

RESULTS AND DISCUSSION

Preparation of magnetic iron oxide nanoparticles from microemulsion The microemulsion system is one approach to obtaining magnetic iron oxide nanoparticles with a uniform size distribution and identical chemical and physical properties. In the present work, magnetic iron oxide nanoparticles were obtained from two different w/o microemulsion systems (Scheme 1). The size and morphology of the ME-MIONs were determined using TEM. ME-MIONs were examined as prepared. The inuence of w/o ratio in the microemulsion on the particle size was investigated. Figure 1(a) and (b) presents TEM images of the prepared nanoparticles. A part of the ME-MIONs agglomerated due to the large surface energy of the nanoparticles. The prepared ME-MIONs are almost spherical in shape and the size is in the range of 710 nm. It is important to note that the oxidation of iron oxide strongly depends on experimental parameters such as temperature, reaction environment and surface conditions. These parameters inuence the composition of the iron oxide nanoparticles; our ndings suggest that the particles obtained

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Figure 1. TEM micrographs of ME 1-MION (a) and ME 2-MION (b).

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Figure 2. XRD patterns of ME 1-MION (a) and ME 2-MION (b) dried at 70 C and 450 C.

Table 1. Total protein concentration and specic coagulation activity Specic coagulation activity (%) mg1 mL1 24 84 91 90 Total protein content (%) mg1 mL1 100 74 83 88

Sample Crude extract SPION ME 1-MION ME 2-MION

Figure 3. Results showing the coagulation activity of MOCP puried with different nanoparticles after 60 min using different salt concentrations.

protein puried from SPION was reduced by 50% whereas the puried proteins from ME 1-MION and ME 2-MION retained high activity. This suggests that the binding with the latter magnetic particles is stronger compared with that to SPION; however, the probable binding mechanism is still under investigation (Okoli et al., in press). The non-adsorbed protein separated from the particles showed a signicant low activity of less than 5%, which conrms that more than 90% of the protein was attached to the magnetic nanoparticles. The specic coagulation activity conrms that purication with SPION showed an increase of 60% mg1 mL1 compared with the crude extract, whereas an increase of 66% mg1 mL1 was observed after purication with ME 1-MION and ME 2-MION (Table 1). On the other hand, the preparation of microemulsion system by two different methods presented similar particle size and protein binding. The presence of surfactant molecules on ME-MIONs was critical in reducing particle agglomeration while maintaining a small particle size which possibly enhances a large specic surface area. We envisage that the residual surfactant from microemulsion promotes better

Figure 4. SDS-PAGE analysis of MOCP protein samples eluted with two salt concentrations. Lane 1 is a low molecular weight protein marker (Sigma), lane 2 represents crude extract; lanes 3, 4 and 5 represent 0.6 mol L1 elution with ME 1-MION, ME 2-MION and SPION, respectively. Lanes 6 and 7 represent 0.8 mol L1 elution with ME 1-MION, ME 2-MION.

proteinparticle binding. The results presented in this work imply that the functionality of the protein eluted from ME-MIONs is as good as that of the SPION; however, the advantage of the former is that particle synthesis can be designed to achieve the desired size with the possibility to improve their efciency in adsorbing the protein (Fig. 3). With the magnetic nanoparticles, it was possible to selectively purify the active protein from a mixture of other proteins; this was evident in the SDS-PAGE (Fig. 4) and MS/MS peptide sequence analysis.

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Figure 5. FT-IR spectra of protein (a), ME 2-MION (b) and (c) ME 2-MION + protein in air, in vacuum I and vacuum II at RT, 50 and 90 C.

Protein content/specic coagulation activity and molecular weight determination The amounts of protein in crude extract and different fractions of puried samples were estimated. The crude extract has a protein content of 2.6 mg mL1 . After purication with magnetic particles, the total recovery of the puried protein was 1.92 mg mL1 , 2.15 mg mL1 and 2.30 mg mL1 for SPION, ME 1-MION and ME 2-MION, respectively. The high protein content in the crude extract is as a result of other proteins present in the sample; consequently, a reduction in concentration of the puried protein shows that the protein of interest has been eluted from the nanoparticles. The total protein concentration and specic coagulation activity measurements in (%) mg1 mL1 are shown in Table 1. The protein specic coagulation activity and total protein recovery increased progressively after purication with the particles. The extracts were further elucidated in 10% SDS gel electrophoresis. SDS-PAGE was performed in order to verify the purication efciency and molecular weight of the eluted coagulant protein. The molecular mass of the puried protein is approximately 6.5 kDa as determined by SDS-PAGE using a low range molecular weight marker from Sigma (Fig. 4).

Mass spectrometric analysis of the puried protein after SDSPAGE was carried out based on MS/MS peptide sequence analysis (data not shown). The peptide sequences obtained conrmed that the puried protein is identical or similar to the MOCP sequence already published.25 Protein and ME-MIONs interaction study FT-IR analysis was performed in order to understand the interaction between the protein molecules and the magnetic iron oxide nanoparticles prepared by microemulsion systems. FT-IR studies of the following samples were carried out: protein alone, ME 1-MION and ME 2-MION with and without protein interaction. For each sample, several IR spectra were taken: (i) spectrum in air; (ii) spectrum in primary vacuum (103 torr); (iii) spectrum in secondary vacuum RT (room temperature) (106 torr); (iv) spectrum in secondary vacuum at 50 C; and (v) spectrum in secondary vacuum at 90 C (above this temperature the protein starts to denature). The IR spectra of the protein show that the treatment in vacuum, at RT, 50 C or 90 C does not modify the structure of the protein. Treatment in primary vacuum modies the spectrum, with a decrease of the NH peaks

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www.soci.org at 3297 and 1655 cm1 . Peaks at 1655, 1538, 1238, 1023 cm1 correspond to NH vibrations while those at 1447 and 1405 may be attributes to CH (Fig. 5(a)).33 The ME 1-MION and ME 2-MION present similar peaks, and it is observed that the treatments in vacuum at RT, 50 C and 90 C affect the structure of the iron oxide particles. After treatment in primary vacuum the peaks at 3490 and 1634 cm1 , corresponding to the adsorbed water, decrease. Treatment in vacuum shows three peaks at 3690, 3660 and 3625 cm1 , which can be distinguished from the nanoparticles. These peaks correspond to the OH group. The peaks at 900 and 800 cm1 correspond to the iron oxide structure. FT-IR spectra of ME 2-MION are shown in Fig. 5(b). The IR spectra of ME 2-MION + protein were obtained after treatments in vacuum, at RT, 50 C and 90 C. Similar to the ME-MIONs, the treatments have no effect on the structure of the iron oxide/protein composite. After protein adsorption, the peaks at 3690, 3660 and 3625 cm1 disappeared in the spectrum; hence peaks at 2970, 2910, 2870 cm1 are characteristic of the adsorbed protein, corresponding to CH vibrations (Fig. 5(c)), this suggests strong binding between the protein and the solid support.33 In order to verify if the protein is denatured at temperatures higher than 90 C, the sample was heated at temperatures of 120 and 150 C. After 24 h at 150 C, new peaks at 2185 and 2091 cm1 appeared and the structural peaks at 900 and 800 cm1 disappeared (Fig. 5(c)). It was difcult to analyze the ME 1-MION + protein using the disc method. An effort to deposit the powder onto a silicon sheet was not successful. Adsorption kinetic studies In the present work, we compared the optimal protein adsorption of the ME-MIONs with that of SPION. The rate of protein adsorption onto the nanoparticles was rapid in the beginning and then decreased rapidly. The optimum equilibrium time can be up to 120 min. For monolayer coverage, the Langmuir isotherm model (Equation (1)) can be used to estimate the equilibrium adsorption parameters. The estimated optimal adsorption capacity of the protein on ME 1-MION and ME 2-MION was, on average, 400 mg protein g1 , which is similar to the value obtained for SPION (450 mg g1 ). The maximum capacity of carboxyl methyl cellulose (CMC), a commercial magnetic micro bead with similar composition to the present nanoparticles, has been reported to have a binding capacity 131 mg protein g1 beads.32 Similarly, Ghebremichael et al. reported that the conventional protein purication method using carboxymethyl (CM) Sepharose IEX matrix has a capacity of 21 mg g1 matrix for binding crude protein (six times less than the CMC beads).25 In contrast, results from our study show that with magnetic nanoparticles, the maximum adsorption of the crude extract protein is three times higher than that of CMC beads and twenty times higher than that of CM Sepharose matrix. The size and specic surface structure of the nanoparticles played a major role in their efcient binding with the crude extract; hence, this makes the use of microemulsion magnetic nanoparticles attractive for the purication of coagulant protein.

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efcient way and which could promote large-scale production. The Moringa oleifera coagulant protein (MOCP) is captured onto the magnetic nanoparticles supposedly via an ionic charge interaction mechanism. In order to study interaction of the ME-MIONs/protein, FT-IR analysis was used to investigate the attachment of the protein onto the ME-MIONs. Consumption of the OH groups of iron oxide (peak at 3660 cm1 ) was observed when the protein was added to the particles. This indicates that the protein is indeed attached to the surface of the oxide. In contrast to the conventional method, purication of MOCP with ME-MIONs suggests a simple and cost effective method for purifying coagulant proteins. The results from kinetic studies show that the optimal adsorption capacity of the protein on ME-MIONs is approximately three times higher than that of commercial beads. It is proposed that this method be extended to the purication of proteins with similar characteristics, and possibly other biomolecules.

ACKNOWLEDGEMENTS
The authors wish to thank the Swedish Research Council Formas for nancing this project. We would also like to extend our gratitude to Dr Gustav Sundqvist, School of Biotechnology, KTH for the mass spectrometric analysis.

REFERENCES
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CONCLUSIONS
A novel approach to purify Moringa oleifera coagulant protein using magnetic nanoparticles is reported. The method is based on magnetic nanoparticles prepared from microemulsions (MEMIONs) that are able to capture proteins in a simple and

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J Chem Technol Biotechnol 2011; 86: 13861393

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