Comparison and Functionalization Study of Microemulsion-Prepared

Article pubs.acs.org/Langmuir

Comparison and Functionalization Study of Microemulsion-Prepared Magnetic Iron Oxide Nanoparticles Chuka Okoli,†,‡ Margarita Sanchez-Dominguez,§,∥ Magali Boutonnet,*,‡ Sven Jar̈ ås,‡ Concepción Civera,⊥ Conxita Solans,∥ and Gunaratna Rajarao Kuttuva*,† †

Environmental Microbiology, Royal Institute of Technology (KTH), 106 91 Stockholm, Sweden Chemical Technology, Royal Institute of Technology (KTH), 100 44 Stockholm, Sweden § Group of Embedded Nanomaterials for Energy Scavenging (GENES), Centro de Investigación en Materiales Avanzados, S. C. (CIMAV), Unidad Monterrey, Alianza Norte 202, 66600 Apodaca, Nuevo León, Mexico ∥ Instituto de Química Avanzada de Cataluña, Consejo Superior de Investigaciones Científicas (IQAC−CSIC), CIBER en Biotecnología, Biomateriales y Nanomedicina (CIBER BBN), Jordi Girona 18-26, 08034 Barcelona, SPAIN ⊥ Departamento de Química Física II, Facultad de Farmacia, Universidad Complutense de Madrid, 28040 Madrid, Spain ‡

S Supporting Information *

ABSTRACT: Magnetic iron oxide nanoparticles (MION) for protein binding and separation were obtained from water-in-oil (w/o) and oil-in-water (o/w) microemulsions. Characterization of the prepared nanoparticles have been performed by TEM, XRD, SQUID magnetometry, and BET. Microemulsion-prepared magnetic iron oxide nanoparticles (ME-MION) with sizes ranging from 2 to 10 nm were obtained. Study on the magnetic properties at 300 K shows a large increase of the magnetization ∼35 emu/g for w/o-ME-MION with superparamagnetic behavior and nanoscale dimensions in comparison with o/w-MEMION (10 emu/g) due to larger particle size and anisotropic property. Moringa oleifera coagulation protein (MOCP) bound w/ o- and o/w-ME-MION showed an enhanced performance in terms of coagulation activity. A significant interaction between the magnetic nanoparticles and the protein can be described by changes in fluorescence emission spectra. Adsorbed protein from MOCP is still retaining its functionality even after binding to the nanoparticles, thus implying the extension of this technique for various applications. monolayer of surfactant molecules.9 Depending on the ratio of oil and water and on the hydrophilic−lipophilic balance (HLB) of the surfactant (Scheme 1), microemulsion can exist as oilswollen micelles disperse in water (o/w microemulsion) or water-swollen inverse micelles dispersed in oil (w/o microemulsion); at intermediate compositions and HLBs, bicontinuous structures can exist. Specifically, the preparation of magnetic iron oxide has been carried out in w/o microemulsion.10 Although many interesting magnetic oxides, such as mixed ferrites, have been prepared for various applications,1,12,13 the

1. INTRODUCTION The preparation of nanoscale magnetic particles has great importance for both fundamental studies and applications.1−3 These magnetic nanoparticles show characteristic properties such as high field reversibility, superparamagnetism, and high saturation field.1 Since the magnetic nanoparticle behavior strongly depends on size, surface chemistry, and state of aggregation of the particles, preparation methods to produce nanoparticles with unique properties are required. For this purpose, several methods have been used.4−6 Among them, the microemulsion-based method seems to be a powerful tool for the preparation of magnetic nanoparticles.1,7,8 Microemulsions are transparent and thermodynamically stable colloidal dispersions in which two liquids initially immiscible (typically, water and oil) coexist in one phase due to the presence of a © 2012 American Chemical Society

Received: February 10, 2012 Revised: May 10, 2012 Published: May 11, 2012 8479

dx.doi.org/10.1021/la300599q | Langmuir 2012, 28, 8479−8485

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nanoparticle efficacy in protein purification and activity assay of the protein-bound magnetic nanoparticles are reported.

Scheme 1. Microstructure of a Microemulsion Ternary Mixture at a Given Concentration of Water, Oil (Organic Liquid), and Surfactants As a Function of Temperaturea

2. EXPERIMENTAL SECTION 2.1. Magnetic Iron Oxide Preparation. The magnetic iron oxide nanoparticles of the present study was prepared from two different microemulsion systems, (o/w) and (w/o) microemulsions. For synthesis via the o/w microemulsion reaction method, the microemulsion system consists of a nonionic surfactant (21.5 wt %, Synperonic 10/6, which was a gift from Croda); oil phase containing the iron precursor (14 wt %, hexane plus iron(III) 2-ethylhexanoate, with a 2 wt % concentration of Fe in the solution); and aqueous phase (64.5 wt %, Milli-Q water). The microemulsion containing the corresponding organometallic precursor was prepared by mixing the three components in the aforementioned proportion and stirring at 30 °C until a homogeneous, transparent brownish isotropic phase was obtained. The precipitating agent (30 wt % NH3) was added directly (in a continuous, steady stream slightly faster than dropwise addition) while stirring the mixture at medium speed, in order to increase the pH and precipitate the magnetic iron oxide nanoparticles. NH3 was added until pH 11 was reached (this occurred within a few minutes of addition). The reaction mixture was kept stirring at ∼30 °C for ∼48 h. The obtained magnetic nanoparticles were separated by centrifugation. It must be noted that, upon separation of nanoparticles, the supernatant was a stable microemulsion. The nanoparticles were washed by several cycles of centrifugation and resuspended in ethanol−water (1:1) and finally ethanol. The magnetic nanoparticles were either dried at 70 °C or suspended in Milli-Q water and kept at 4 °C until further use. In our previous work, we have reported room temperature preparation of the w/o microemulsion system.10 Briefly, the studied microemulsion consists of a cationic surfactant (CTAB), cosurfactant (1-butanol), oil phase (n-octane), and aqueous phase. The aqueous phase contains the Fe salt precursors in a mole ratio of 2:1 [FeCl3/ FeCl2]. The addition of precursor solution to the mixture of CTAB/1butanol/n-octane will give rise to the formation of a microemulsion. Formation of magnetic nanoparticles was achieved by adding the precipitating agent (NH3) to the microemulsion containing the precursor upon vigorous stirring until pH 11 was achieved. The obtained magnetic nanoparticles were separated by centrifugation and washed by several cycles of precipitation and resuspension in ethanol− water (1:1) and, finally, ethanol. The obtained magnetic nanoparticles were either dried at 70 °C or suspended in Milli-Q water and kept at 4 °C until further use. 2.2. Nanoparticle Characterization. X-ray diffraction data of the microemulsion prepared nanoparticles were performed between 20° and 70° on Siemens D5000 diffractometer using Cu Kα radiation. The size and morphology of the nanoparticles were examined by highresolution transmission electron microscopy (HRTEM), which was carried out using a field emission transmission electron microscope, JEM-2200FS, 200 kV, with 0.19 nm resolution in TEM mode and 0.1 nm resolution in STEM mode and spherical aberration correction in STEM. For this purpose, a dry powder sample was dispersed in isopropanol and deposited onto a Formvar carbon copper grid. Selected area electron diffraction patterns (SAED) of the nanoparticles were also investigated from the electron micrographs. Room temperature magnetic hysteresis loops of the nanoparticles were measured using a superconducting quantum interference device (SQUID) magnetometer, quantum design MPMS XL. The magnetic moments of the particles were determined in a field over a range of ±60 kOe. Surface area and porosimetry of the nanoparticles were calculated by the Brunauer−Emmet−Teller (BET) method using micromeritics ASAP 2010. Specific surface area as well as the specific pore volume was detected after degassing the sample overnight at 110 °C prior to the measurement. Attenuated total reflectance infrared (ATR-IR) spectroscopy was carried out using a Thermo Nicolet 6700 FTIR spectrometer with ATR accesory, from 4000 to 400 cm−1, a resolution of 4 cm−1, employing 50 scans.

a

A region of isotropic single-phase solution is observed, which extends from the water-rich to the oil-rich side at constant surfactant concentration. At increased oil concentration, a bicontinuous phase with undefined shape is formed, which further transforms into a structure of small water droplets in a continuous oil phase (reverse micelles) at a higher oil concentration. Adapted with permission from ref 11. Copyright 1994 Elsevier.

present study is focused on magnetic iron oxide nanoparticles (magnetite or maghemite) prepared from microemulsion systems since these are nontoxic, superparamagnetic, and less susceptible to change when prepared in a controlled environment.14,15 They can also be tailor-made such that a desired size with more uniform nanoparticles and surface specificity are obtained in order to enhance their efficiency.16 These specific characteristics give microemulsion-prepared iron oxide nanoparticles an edge over other magnetic nanoparticles; moreover, they are the most studied materials for different applications such as magnetic separation, protein purification, water treatment, drug delivery, and catalyst, among others.1,10 The present work demonstrates advancement in protein purification as well as development of a suitable surface for protein attachment for various applications; this is based on the use of inorganic nanoparticles prepared from oil-in-water (o/w) microemulsions,17 in contrast to the typically reported water-inoil (w/o) microemulsion method.18,19 The methods of synthesis are based on both microemulsion routes to obtain small and uniform nanoparticles with a high degree of crystallization. The w/o and o/w microemulsion prepared iron oxide nanoparticles have been characterized with different techniques. The potential of both approaches for the production of nanocrystalline magnetic iron oxide with high surface area for protein binding/protein purification is investigated and compared. Physical properties as well as 8480


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Scheme 2. Proposed Overall Reaction of Iron Organometallic Precursor with Ammonia in Order to Form Iron Oxide Nanoparticles o/w-ME-MION

2.3. Protein Purification. For this study, Moringa oleifera coagulation protein (MOCP) seed extract4 was used as the model protein. A batch system was employed in the purification of the coagulant protein. The w/o and o/w microemulsion prepared iron oxide nanoparticle (ME-MION) suspensions were used as obtained. The nanoparticles (2.5 mg, w/v) were washed three times with 10 mM ammonium acetate buffer, pH 6.7, to equilibrate the particles and then suspended in buffer. Binding of MOCP onto the particles was performed in 10 mM ammonium acetate buffer, pH 6.7. The samples were incubated for 60 min at room temperature, and the unbound protein was separated by the help of an external magnet. The protein was then eluted with 0.8 M NaCl in the buffer solution. The protein content was determined by the Bradford method.20 Protein profile, molecular weight of the purified protein, and that of the crude extract were determined and compared by 10% SDS-PAGE mini gels. 2.4. Preparation of Protein-Bound Magnetic Nanoparticles and Activity Test. The preparation of protein-bound nanoparticles was performed by binding MOCP onto the nanoparticles according to the earlier described method4 except that the protein was not eluted from the nanoparticles. Briefly, the w/o and o/w microemulsion prepared iron oxide nanoparticle were equilibrated with 10 mM ammonium acetate buffer, pH 6.7, followed by binding of MOCP onto the nanoparticles. After room temperature incubation for 1 h, the protein-bound magnetic nanoparticle was washed with ammonium acetate buffer and then separated with an external magnet. The separated protein-bound nanoparticle was suspended in the same buffer or Milli-Q water and kept at 4 °C prior to use. The functionality of the prepared protein-bound magnetic nanoparticles was tested in clay suspension according to the protocol reported in our previous work.10 2.5. Fluorescence Study of Protein-Bound Magnetic Nanoparticles. Emission fluorescence spectra were obtained on a modular PTI Spectrometer with an excitation wavelength of 255 nm. Emission was recorded at λ = 285 to 475 nm. The excitation and emission slits were adjusted to 2 nm. Temperature was controlled at 22 °C with a thermoelectric temperature controller, TLC 50. Cells of path length 1 cm were used, and all the experiments were performed at least twice. The average spectra were subtracted from appropriate blanks before analysis.

consists in the use of organometallic precursors, dissolved in nanometer scale oil droplets of o/w microemulsions, and stabilized by a monolayer of hydrophilic surfactant. If the usual water-soluble precipitating agents are used, these can be added directly or as aqueous solutions, without compromising microemulsion stability and droplet size; alternatively, if oilsoluble precipitating agents are available, then a two-microemulsion approach can be used. In the present investigation, for the sake of comparison and simplicity and due to ecological and economical reasons, only one o/w microemulsion was employed, and the precipitating agent (concentrated ammonia) was added directly, increasing the concentration of the aqueous phase and the pH. The microemulsion remained stable despite this dilution. Although the nucleation and growth stages of nanoparticle formation may be dependent on the formation of transient dimers, collisions, and coalescence of droplets, the reaction itself is believed to be interfacial (i.e., o/w interface) since the precursor is dissolved in the oil droplets and the precipitating agent is dissolved in the continuous aqueous phase. Eastoe et al.21 observed that cobalt(II) 2-ethylhexanoate precursor in AOT w/o microemulsions resided at the oil/water interface. In the present system, the same type of organometallic precursor is used; hence it is reasonable to assume that its most likely location is at the o/w interface, at least partially. However, the chemistry of metal 2-ethylhexanoates is still largely unexplored; typically, reaction mechanisms involve either thermal decomposition or photolysis.22 However, neither of these conditions is provided in the present method, and we may postulate that the reaction in Scheme 2 takes place. In fact, NH4OH was added in large excess, which, combined with the extremely large interfacial area of the microemulsion environment, may catalyze such reaction, in a similar way to organic reactions that take place due to micellar catalysis.23 Furthermore, Ristić et al. reported the formation of ZnO nanoparticles from ethanolic solutions of zinc(II) 2-ethylhexanoate induced by the addition of a base (tetramethylammonium hydroxide).24 Additional detailed mechanistic studies, out of the scope of the present work, are necessary in order to fully understand the formation of nanoparticles in o/w microemulsions comprising such organometallic precursors. 3.2. Characterization of Iron Oxide Nanoparticles. Diffraction patterns of the dry powders indicate the presence of a magnetic phase of either Fe3O4 or γ-Fe2O3; which is quite typical (Figure 1) at 35° for (311) and 63° for (440). The XRD pattern of w/o-ME-MION (Figure 1a) shows that the nanoparticles correspond to crystalline magnetite, which reflects to the well matching of the diffraction peaks with magnetite pattern.10,25 However, we cannot rule out the existence of γ-Fe2O3. The o/w-ME-MION X-ray diffraction pattern (Figure 1b) showed wide peaks characteristic of small nanoparticles, which imply that the original size of the nanoparticles is very small and fits well to a typical spinel

3. RESULTS AND DISCUSSION 3.1. Iron Oxide Nanoparticle Synthesis in w/o and o/w Microemulsions. The formation of nanoparticles in w/o microemulsions can be achieved by mixing two w/o microemulsions of identical composition, one comprising the inorganic salt precursor and another one comprising the precipitating agent in the aqueous phase nanodroplets. The one-microemulsion approach is less common; in such case, the precipitating agent is added directly as an aqueous solution to the microemulsion comprising the metallic precursor, thereby increasing the water/surfactant ratio and hence increasing the droplet size. This approach can be used as long as the microemulsion remains monophasic and stable upon this increase in aqueous phase, in order to maintain the confinement of the reaction to the aqueous phase droplets. The latter approach was chosen in this work due to economical reasons. However, the oil-in-water microemulsion reaction method 8481


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Table 1. Surface Area and Porosimetry of w/o- and o/w-MEMION magnetic nanoparticles w/o-MEMION o/w-MEMION

size (nm)

specific surface area (m2/g)

pore size (nm)

pore volume (cm3/g)

9.2

147

18

0.61

2

304

5

0.39

the large surface energy of the particles. The agglomeration of the nanoparticles has already been observed during the synthesis;10 this depends on experimental parameters like temperature, pH, reaction environment, and surface condition. These parameters may influence the composition of the obtained iron oxide nanoparticles if not properly controlled. Selected area electron diffraction patterns (SAED) of nanoparticles prepared from w/o-ME-MION and o/w-MEMION are presented as insets (Figure 2b,d, respectively). The area selected for the electron diffraction pattern of the agglomerates of w/o-ME-MION shown in Figure 2b could be indexed to the cubic-type cell of magnetite, which further confirms that w/o-ME-MION particles were composed of nanocrystalline Fe3O4.27 Regarding o/w-ME-MION, the diffraction rings shown in the inset of Figure 2d corresponds to d-spacings equal to 0.147 nm (hkl, 440) and 0.251 nm (hkl, 311), which, in good agreement with XRD results, confirms the presence of maghemite. However, a small contribution from maghemite or hematite in w/o-ME-MION and o/w-ME-MION cannot be neglected, which may result from oxidation of magnetite during synthesis and washing cycles; nevertheless, the contribution is too little to be detected. As shown by XRD studies (Figure 1b) and HRTEM (Figure 2c,d), the nanoparticles obtained by the o/w microemulsion approach were rather small, around 3 nm, and homogeneous, which indicates that the reaction was indeed confined and probably very fast, favoring nucleation over growth. Furthermore, in addition to the possibility of Synperonic 10/6 surfactant molecules capping the freshly formed Fe2O 3 nanoparticles, 2-ethylhexanoate ion may also interact strongly with the surface of nanoparticles, as suggested by Li et al.,28 thereby providing additional stability and an efficient barrier toward particle growth. Infrared analysis shown in Supporting Information indeed suggests the presence of organic material attached to the as-obtained nanoparticles. Magnetic moments of the samples were determined in a field over a range of ± 60 kOe. Figure 3b shows the results from magnetization (M) versus applied field (H) measurement at room temperature (300 K) for w/o and o/w microemulsion prepared iron oxide nanoparticles. It can be seen that the magnetization curve for w/o-ME-MION in Figure 3b (□-shape) displayed no magnetic remanence and coercivity, confirming a superparamagnetic behavior and nanoscale dimensions of the particles. The recorded saturation magnetization (Ms) value for w/o-ME-MION is 30 emu/g. However, the magnetic moment of o/w-ME-MION (Figure 3b, △-shape) exhibited a weak or incomplete saturation magnetization of 10 emu/g indicating a decrease in mean particle size; the specific magnetization of iron oxide nanoparticles is known to depend on their size.29 Zero-field-cooled (ZFC) data of the nanoparticles were recorded. The temperature at which the ZFC plot is at the maximum is largely modified in the case of w/o-ME-MION in Figure 3a (□-shape). The curve becomes

Figure 1. X-ray diffraction patterns of iron oxide nanoparticles synthesized in microemulsions: (a) w/o-ME-MION and (b) o/w-MEMION.

phase of either magnetite (Fe3O4) or maghemite (γ-Fe2O3) or a combination of both.8,26 The size, morphology, agglomeration, and diffraction patterns of the magnetic nanoparticles were examined by HRTEM and SAED. The influence of w/o and o/w microemulsion types with respect to the particle size and morphology was investigated. As seen in Figure 2a,b, the

Figure 2. TEM micrographs of iron oxide nanoparticles synthesized in microemulsions: (a,b) w/o-ME-MION and (c,d) o/w-ME-MION. Insets to panels a and c are HRTEM; insets to panels b and d are diffraction patterns, respectively.

observed w/o-ME-MION nanoparticles consisted mainly of elongated, rod-like morphologies with widths ranging from 5 to 10 nm and lengths from 20 to 50 nm, coexisting with globular particles in the order of 5−8 nm. As for o/w-ME-MION nanoparticles, these were globular and in the order of 3 nm, which is in agreement with the broad XRD peaks observed in Figure 1b and the high specific surface area and porosity results presented in Table 1. However, there is a considerable overlap and large agglomeration in both prepared nanoparticles due to 8482


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Figure 3. Magnetization data for w/o-ME-MION (□) and o/w-ME-MION (△): (a) zero-field-cooled (ZFC) data; (b) magnetization as a function of magnetic field at 300 K.

broader with an increase in temperature. This change reflects an increase in average blocking temperature, TB with increasing particle size; the behavior observed fits well with known blocking temperature of superparamagnetic magnetic particles system.7 Conversely, the temperature at the peak point of ZFC curve (TB) for o/w-ME-MION is seen at 50 K (Figure 3a, △-shape). Hence, from magnetization vs temperature curves, a superparamagnetic behavior above TB (50 K) is observed. Subsequently, above the blocking temperature, the magnetization of the particles decreases as the temperature increases;30 this behavior was evident in o/w-ME-MION. The Brunaeur−Emmett−Teller (BET) surface areas of the present study were calculated from an N2-sorption experiment with surface area and porosimetry system. The results from the BET method (Table 1) reveal that the average specific surface areas of the nanoparticles are 147 m2/g for w/o-ME-MION and 304 m2/g for o/w-ME-MION. These results show a relatively large specific surface area of the prepared nanoparticles when compared to 105 m2/g reported in a similar technique.29 A higher specific surface area and porosity seen in o/w-ME-MION is attributed to the small size of the nanoparticle. Table 1 presents a detailed result obtained from the surface area and porosimetry study. 3.3. Protein Purification. Results from the batch system purification of Moringa oleifera coagulation protein (MOCP) with w/o and o/w microemulsion prepared iron oxide nanoparticles are shown in Figure 4. The data from SDSPAGE analysis is in agreement with data published in our previous work,4,10 which clearly reveal that the purified form of MOCP can be obtained from the crude extract when eluted with 0.8 M NaCl in ammonium acetate buffer solution. However, characterization of the magnetic nanoparticles prepared in this work demonstrates the high surface area and mesoporosity of the obtained powder, which enhanced their performance. The molecular mass of the purified MOCP is ∼6.5 kDa as determined by SDS-PAGE, indicating that the coagulant protein was eluted from the fractions.10 It can be concluded that the purification efficacy of both systems (w/oand o/w-ME-MION) is comparable in terms of their performance. 3.4. Preparation of Protein-Bound Magnetic Nanoparticles and Activity Test. To further establish the enhanced property of the studied ME-MIONs, protein-bound nanoparticles were developed from the two microemulsionprepared systems. The coagulation activity of the protein-

Figure 4. SDS-PAGE analysis of MOCP protein. Lane 1 is a low molecular weight protein marker (Sigma); lane 2 represent crude MOCP extract; lanes 3 and 4 represent elution of purified protein from o/w- and w/o-ME-MION, respectively, using 0.8 M NaCl elution buffer. The corresponding purified MOCP presents a molecular mass of ∼6.5 kDa indicating that the coagulant protein was eluted from the fractions.

bound nanoparticles was compared with bare nanoparticles in the turbidity reduction of clay suspension (Figure 5). The protein-bound w/o- and o/w-ME-MION exhibited more than 80% turbidity reduction in clay suspension when compared to

Figure 5. Coagulation activity of protein-bound w/o- and o/w-MEMION in synthetic clay suspension after 1 h reaction time. An enhanced performance in terms of coagulation activity when compared to bare ME-MION is observed. The control samples are clay (gray) and crude protein (green). 8483


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characteristics of small nanoparticles, which imply that the original size of the nanoparticles is very small. The proteinbound ME-MIONs exhibited a significant reduction of clay particles in suspension as compared to bare nanoparticles, evidencing a significant interaction between the magnetic nanoparticles and the protein. Finally, this work demonstrates advancement in the preparation of magnetic iron oxide nanoparticles for protein separation as well as the development of suitable surface for protein attachment for various applications.

about 50% reduction of their counterparts (bare nanoparticles). These results demonstrate that there is a significant interaction between the magnetic nanoparticles and the protein;10 moreover, the adsorbed protein from MOCP is still retaining its functionality even after binding to the nanoparticles. However, as the coagulation activity is based on the sedimentation of flocs, a slight increase in activity of the protein-bound o/w -ME-MION was observed as compared to the protein-bound w/o-ME-MION. Emission fluorescence spectra of protein (MOCP), proteinbound w/o-ME-MION, and protein-bound o/w-ME-MION are presented in Figure 6. The MOCP has a broad peak with

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ASSOCIATED CONTENT

S Supporting Information *

Attenuated total reflectance infrared (ATR-IR) spectrum of o/ w ME-MION sample and Synperonic 10/6. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (G.R.K.); [email protected] (M.B.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful for the financial support of the Swedish Research Council, Formas, as well as the Cost Action D43, Colloid and Interface Chemistry for Nanotechnology. M.S.-D. acknowledges NaNoTeCh, the National Nanotechnology Laboratory of Mexico, and Cesar Leyva (CIMAV, S.C.) for HRTEM/STEM measurements and assistance, and A.T.T. (CIMAV, S.C.) for ATR-IR measurements and assistance. Financial support by Ministerio de Ciencia e Innovación (MICINN Spain, grant number CTQ2008-01979) and Generalitat de Catalunya (Agaur, grant number 2009SGR961) is also aknowledged. C.C. acknowledges financial support from Spanish Ministerio de Ciencia e Innovacion; MAT 200802542 and GR35/10-A-950247. We thank Professor Francisco ́ Garcia-Blanco for helpful comments on fluorescence data.

Figure 6. Fluorescence emission spectra of MOCP (red), proteinbound w/o- (blue) and o/w-ME-MION (green) at 255 nm excitation wavelength.

apparently two maxima at 310 and 326 nm. A shift to λmax of 295 nm is observed with protein-bound w/o-ME-MION, implying a modification of protein fluorophore environment. Similar behavior is observed in the absorption spectra of protein-bound o/w-ME-MION. Binding of nanoparticles in the ligand binding site of the protein makes the environment of the aromatic side chains in the binding site less polar.31 The decrease in the fluorescence intensity can be attributed to the incorporation of the MOCP onto the nanoparticles surface. In fact, the small particle size and large surface-to-volume ratio (Table 1) could be critical for this behavior; hence, the density of protein-bound ME-MIONs increased following the absorption of protein on the particles.

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REFERENCES

(1) Vidal-Vidal, J.; Rivas, J.; Lopez-Quintela, M. Synthesis of Monodisperse Maghemite Nanoparticles by Microemulsion Method. Colloids Surf. 2006, 288, 44−51. (2) Jonsson, T.; Mattson, J.; Djurberg, C.; Khan, F.; Nordblad, P.; Svedlindh, P. Aging in a Magnetic Particle System. Phys. Rev. Lett. 1995, 75, 4138−4141. (3) Goya, G.; Berquo, T.; Fonseca, F.; Morales, M. Static and Dynamic Magnetic Properties of Spherical Magnetic Nanoparticles. J. Appl. Phys. 2003, 94, 3520−3528. (4) Okoli, C.; Fornara, A.; Qin, J.; Toprak, M.; Dalhammar, G.; Muhammed, M.; Rajarao, G. Characterization of Superparamagnetic Iron Oxide Nanoparticles and Its Application in Protein Purification. J. Nanosci. Nanotechnol. 2011, 11, 10201−10206. (5) Lu, A.; Salabas, E.; Schuth, F. Magnetic Nanoparticles: Synthesis, Protection, Functionalization and Application. Angew. Chem., Int. Ed. 2007, 46, 1222−1244. (6) Iglesias-Silva, E.; Vilas-Vilela, J.; Lopez-Quintela, M.; Rivas, J.; Rodriguez, M.; Leon, L. Synthesis of Gold-Coated Iron Oxide Nanoparticles. J. Non-Cryst. Solids. 2010, 356, 1233−1235. (7) Lopez Perez, J.; Lopez Quintela, M. Advances in the Preparation of Magnetic Nanoparticles by Microemulsion Method. J. Phys. Chem. B 1997, 101, 8045−8047.

4. CONCLUSIONS The preparation of w/o and o/w magnetic iron oxide nanoparticles (ME-MION) was successfully carried out from two different microemulsion systems using a cationic surfactant (CTAB) and a nonionic surfactant (synperonic 10/6), respectively. CTAB-based w/o and synperonic 10/6-based o/ w microemulsions are well suited for the preparation of magnetic iron oxide nanoparticles. The magnetization value of the w/o-ME-MION is higher than that of o/w-ME-MION due to large particles size (9.2 nm) seen in the former, compared to the latter (2 nm). However, magnetic nanoparticles prepared from o/w microemulsion method showed larger surface area and porosity. It can be noted that, as the particle size decreases for instance in the case of o/w-ME-MION, the specific surface area and porosity increases while the magnetic behavior decreases. This correlates with the data obtained from SQUID magnetometry and BET study; in addition, X-ray diffraction pattern o/w-ME-MION indicates wide peak 8484


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Comparison and Functionalization Study of Microemulsion-Prepared

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