Journal of Colloid and Interface Science 340 (2009) 230–236

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Journal of Colloid and Interface Science

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Fabrication of mesoporous SiO2–C–Fe3O4/c–Fe2O3 and SiO2–C–Femagnetic composites

Marta Sevilla a, Teresa Valdés-Solís a, Pedro Tartaj b, Antonio B. Fuertes a,*

a Instituto Nacional del Carbón (CSIC), Apartado 73, 33080-Oviedo, Spainb Instituto de Ciencia de Materiales de Madrid (CSIC), Cantoblanco, 28049-Madrid, Spain

a r t i c l e i n f o a b s t r a c t

Article history:Received 16 June 2009Accepted 1 September 2009Available online 6 September 2009

Keywords:Mesoporous materialsMagnetic nanoparticlesCarbon–silica compositesImmobilization of biomolecules

0021-9797/$ - see front matter � 2009 Elsevier Inc. Adoi:10.1016/j.jcis.2009.09.003

* Corresponding author.E-mail address: abefu@incar.csic.es (A.B. Fuertes).

A synthetic method for the fabrication of silica-based mesoporous magnetic (Fe or iron oxide spinel)nanocomposites with enhanced adsorption and magnetic capabilities is presented. The successfulin situ synthesis of magnetic nanoparticles is a consequence of the incorporation of a small amount ofcarbon into the pores of the silica, this step being essential for the generation of relatively large iron oxidemagnetic nanocrystals (�10 ± 3 nm) and for the formation of iron nanoparticles. These composites com-bine good magnetic properties (superparamagnetic behaviour in the case of SiO2–C–Fe3O4/c–Fe2O3

samples) with a large and accessible porosity made up of wide mesopores (>9 nm). In the present work,we have demonstrated the usefulness of this kind of composite for the adsorption of a globular protein(hemoglobin). The results obtained show that a significant amount of hemoglobin can be immobilizedwithin the pores of these materials (up to 180 mg g�1 for some of the samples). Moreover, we haveproved that the composite loaded with hemoglobin can be easily manipulated by means of an externalmagnetic field.

� 2009 Elsevier Inc. All rights reserved.

1. Introduction

The encapsulation of biomolecules inside inorganic or organicmatrixes has recently attracted widespread interest because ofits importance in numerous emergent areas such as drug delivery,enzyme immobilization or the recovery of biomacromolecules. Inparticular, considerable attention is being focused on differenttypes of mesoporous materials (i.e. polymer, carbon or silica), asthey are considered good candidates for this kind of applications[1–4]. Mesoporous silica materials doped with magnetic nanopar-ticles (magnetite or maghemite) are highly suitable for this endbecause they combine good textural properties (i.e. a large specificsurface area, a high pore volumes and a porosity made up ofuniform mesopores with sizes that can be tuned over a wide range,from 2 nm to >10 nm) with easy separability [5–7]. In fact, theintroduction of magnetic functionalities enables this hybrid systemto be easily separated/transported from/through the liquidmedium, where they are normally dispersed [8–11].

Most mesoporous silica magnetic composites are formed byinserting magnetic nanoparticles previously synthesised by refinedprocedures into the pores of the silica matrix [6,12–15]. This syn-thetic methodology is costly and complex in terms of scalability.For this reason, it is important to develop low-cost facile synthetic

ll rights reserved.

routes to fabricate magnetic porous silica materials. A simple syn-thetic strategy for preparing such composites consists in growingmagnetic nanoparticles in situ (i.e. inside the pores of porous silicamaterials). This approach has been investigated by differentresearchers [16–23]. It has been observed that it is difficult toobtain magnetic mesoporous silica composites that combine a highmagnetic saturation with a superparamagnetic behaviour. Thus, inmost of the cases, the iron oxide ferrites generated inside theporosity of the silica are too small (<7 nm), resulting in compositeswith poor magnetic properties [21,22]. In other cases, the compos-ites exhibit a high magnetization but magnetic memory (no super-paramagnetic behaviour) [23]. However, we have proved that inthe case of carbon matrixes, it results easy the in situ incorporationof iron oxide magnetic nanoparticles with large and easily tunablesizes in the 8–15 nm range, which give composites with excellentmagnetic capabilities (a high magnetic saturation and a superpara-magnetic behaviour) [24–28]. It is not clear why the silica or car-bon framework should affect the growth of iron oxide spinelnanoparticles. One possible explanation might be that the chemicalcharacteristics of carbon facilitate the reduction-diffusion-growthof iron species during the formation of iron oxide magnetic nano-crystals (ferrites). In this study, we propose a simple syntheticstrategy for the preparation of magnetic porous silica compositeswith good magnetic properties, similar to those of the magneticcarbon composites. The key to our synthetic methodology is toincorporate a small amount of carbon within the silica porosity

M. Sevilla et al. / Journal of Colloid and Interface Science 340 (2009) 230–236 231

in order to create an environment similar to that of the porosity ofcarbonaceous matrixes and then to facilitate the growth of ironoxide nanoparticles. This is accomplished by introducing into thepores of the silica (together with the precursor – iron nitrate – ofthe magnetic iron oxide nanoparticles) a small amount of sucrose,which is converted into carbon during the in situ synthesis of themagnetic nanoparticles (thermal treatment under an inert atmo-sphere). An additional advantage of this synthetic scheme withrespect to the undoped carbon strategy is that the nature of themagnetic nanoparticles inserted into the silica matrix can be mod-ulated; iron oxide spinel nanoparticles (temperature of synthesis�450 �C) or iron nanoparticles (temperature of synthesis�800 �C). In the present work, we assessed the ability of thesemagnetic composites to immobilize large biomolecules. In particu-lar, we choosed the bovine hemoglobin as a model and we investi-gated its adsorption on this kind of materials.

Finally, it is worth noting that these magnetic composites mayalso be important as catalysts. Indeed, taking advantage of the ver-satility of this procedure to insert iron oxide or iron within thepores of mesoporosu silica, this synthetic strategy constitutes aeasy way to prepare two different mesoporous catalytic systems(Fe2O3–silica or Fe–silica), which are active in several proccessesincluding Friedel–Crafts alkylations [29], the heterogeneous Fen-ton reaction [30] or propylene epoxidation [31].

2. Experimental section

2.1. Preparation of the mesoporous silica samples

Three types of mesoporous silica materials were selected for theinsertion of magnetic nanoparticles: (a) an ordered mesostructuredSBA-15 silica (denoted as S1), (b) a bimodal mesoporous silica witha spherical morphology (denoted as S2) and (c) a commercial mes-oporous silica gel (Aldrich, Cat No. 28,851-9, denoted as S3). Theordered mesostructured SBA-15 silica was synthesized accordingto a procedure reported in the literature [32]. Briefly, 1.3 g of sur-factant Pluronic P123 was dissolved in a mixture of 10 g water and40 g 2 M HCl, after which 2.8 g TEOS was added dropwise. The mix-ture was stirred for 5 min and then maintained at 35 �C for 20 h,after which it was transferred to an autoclave and heated at130 �C for 24 h. Finally, the sample was recovered by filtration,washed with abundant distilled water, dried and calcined at550 �C for 4 h. The spherical bimodal mesoporous silica was pre-pared as reported by Schulz-Ekloff et al. [33]. In a typical synthesis,2.5 g of CTAB (Cetyltrimethylammonium bromide, Aldrich) and1.28 g of the silica source (Sodium Silicate, Aldrich) were dissolvedin 45 g water, and this was followed by the addition of 3.95 g ofethyl acetate. The mixture was stirred for 30 s and then maintainedat room temperature for 5 h. Then, it was transferred to a closedTeflon vessel and maintained at a temperature of 90 �C for 2 days.The solid product was filtered, washed with deionised water, driedand calcined in air at 550 �C for 4 h.

2.2. Synthesis of iron oxide or iron magnetic nanoparticles within thesilica porosity

To incorporate the carbon and the magnetic iron oxide spinelnanoparticles into the pores of the silica materials, the latter wereimpregnated with a mixture of: 1 g H2O, 0.1 g H2SO4 98% (Prolabo),0.23 g sucrose (Aldrich) and 1.5 g Fe(NO3)3�9H2O (Aldrich). Thissolution was added dropwise until incipient wetness and thenthe impregnated sample was dried at 120 �C. This process was re-peated several times until the amount of iron nitrate infiltratedwas equivalent to 24 wt.% of the iron oxide present in the compos-ite. The nanoparticles of iron oxide spinel (FexOy) were generated

following a procedure recently reported by our group [24,27].Briefly, the porosity of the impregnated samples was filled withethylene glycol (Aldrich) and then thermally treated under N2 at450 �C for 2 h. The composition of the resulting composites was:4.5 wt.% carbon, 24 wt.% FexOy and SiO2 (�71 wt.%). They were de-noted as SX-FexOy, X being 1, 2 or 3 depending on the type of silica.The formula FexOy denoted the magnetic iron oxides with an in-verse cubic spinel structure, comprising both magnetite (Fe3O4)and maghemite (c–Fe2O3). The magnetic properties of these ironoxides are very similar. Maghemite only differs from magnetitein that all the Fe cations are in the trivalent state. Because, the ex-act nature of the crystalline phase (magnetite or maghemite) pres-ent in the iron oxide ferrite nanoparticles is difficult to discernfrom the XRD patterns, we used the general formula FexOy.

The procedure employed to incorporate the iron nanoparticleswas similar to that used for the FexOy nanoparticles except thatthe composition of the impregnation mixture was slightly different(1 g H2O, 0.1 g H2SO4, 0.7 g sucrose and 0.9 g Fe(NO3)3�9H2O) andthe impregnated sample was heat treated under N2 at 800 �C for2 h. The resulting composites contained 8 wt.% carbon, 15 wt.% Feand SiO2 (�77 wt.%). They were denoted as SX-Fe, X being 1, 2 or3 depending on the type of silica employed.

2.3. Adsorption of hemoglobin

Around 10 mg of the nanocomposite was dispersed at roomtemperature in 10 mL of bovine hemoglobin (Aldrich) solution (ini-tial concentration: 0.2 mg mL�1, pH 6 buffer solution) in a closedvessel to avoid evaporation. To evaluate the amount of protein ad-sorbed, the concentration of hemoglobin in the solution was peri-odically monitored by means of a UV–vis spectrophotometer(Shimadzu UV-2401PC) applying UV absorption at 410 nm.

2.4. Characterization

X-ray diffraction (XRD) patterns were obtained on a SiemensD5000 instrument operating at 40 kV and 20 mA, using CuKa radi-ation. The morphology of the powders was examined by scanning(SEM, Zeiss DSM 942) and transmission (TEM, JEOL-2000 FXII)electron microscopy. Nitrogen sorption isotherms were performedat �196 �C in a Micromeritics ASAP 2020 volumetric physisorptionsystem. The BET surface area was deduced by analyzing the iso-therm in the relative pressure range of 0.04 to 0.20. The total porevolume was calculated from the amount adsorbed at a relativepressure of 0.99. The pore size distribution (PSD) was calculatedby means of the Kruk-Jaroniec-Sayari method [34]. Fourier trans-form infrared (FT-IR) spectra for proteins loaded on magnetic com-posite were recorded on a Nicolet 8700 spectrometer fitted with adiffuse reflection attachment.

The saturation magnetization (Ms) and coercivity field values(Hc) were obtained from the magnetization curves recorded up toa field of 5T. For temperatures at which the magnetization curvesarer unblocked, the Ms values were obtained from 1/H extrapola-tion at high fields (Langevin approach). For the blocked regimesthe Ms values were obtained from the law of approach tosaturation.

3. Results and discussion

3.1. Structural characteristics of the magnetic mesoporous C–Fe/FexOy–SiO2 composites

The nanocomposites with FexOy or Fe nanoparticles exhibitplatelet-like, spherical or irregular morphologies, similar to thoseof the original silica materials. This is illustrated by the SEM images

232 M. Sevilla et al. / Journal of Colloid and Interface Science 340 (2009) 230–236

shown in Fig. 1 (insets). Evidence for the presence of iron oxide spi-nel (FexOy) or iron nanoparticles was obtained by X-ray diffractionanalysis (Fig. 2). For the SiO2–C–FexOy samples, the XRD patterns inFig. 2a contain well-defined diffraction peaks characteristic of ironoxide spinel, which evidences that the deposited nanoparticles ofFexOy are well crystallized. The sizes of the crystallites deducedby applying the Scherrer equation to the (311) peak of the XRD

Fig. 1. TEM images obtained for the SiO2–C–FexOy (a: S1-FexOy, c: S2-FexOy and e: S3-Fex

(e) show typical SEM images for the S1-, S2- and S3-FexOy composites, respectively.

pattern are in the 9.5–11.5 nm range (see Table 1). In order toassess the influence of the infiltrated carbon on the growth ofthe FexOy nanocrystals, we prepared silica–FexOy composites inthe absence of sucrose (no carbon in the composite). The XRDtraces for two composites obtained from the S1 silica in the pres-ence and in the absence of sucrose are compared in Fig. 2a.Whereas the sample prepared with sucrose (S1-FexOy) exhibits

Oy) and SiO2–C–Fe (b: S1-Fe; d: S2-Fe and f: S3-Fe) composites. Insets in (a), (c) and

20 30 40 50 60 702 Theta (º)

Inte

nsity

(a. u

.)

S3-FexOy

S2-FexOy

S1-FexOy

S1-FexOy (No Carbon)

(220)

(311)

(400) (422)

(511) (440)

a

b

S3-Fe

S2-Fe

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α-Fe (110)

α-Fe (200) α-Fe (211)

40 50 60 70 80

Inte

nsity

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.)

2 Theta (º)

* *

* Fe3C

Fig. 2. XRD patterns obtained for the: (a) SiO2–C–FexOy and (b) SiO2–C–Fecomposites.

M. Sevilla et al. / Journal of Colloid and Interface Science 340 (2009) 230–236 233

well-resolved, sharp peaks, the material synthesised without theaid of sucrose (S1-FexOy(no carbon)) shows weak XRD reflections,suggesting that the inserted FexOy nanoparticles have very smallsizes. Similar results were obtained for the other composites pre-pared with the S2 and S3 silica materials. The sizes of the FexOy

Table 1Physical properties of the porous silica and the magnetic nanocomposites.

Code Sample SBET (m2 g�1) Vp (cm3 g�1)a Pore si

S1 690 1.21 9.6S1-FexOy 390 0.70 (0.78) 8.9S1-Fe 410 0.70 (0.78) 9.0S2 860 2.06 2.8, 27S2-FexOy 474 1.04 (1.38) 2.8, 23S2-Fe 440 0.90 (1.51) 2.3, 22S3 340 0.84 12S3-FexOy 240 0.48 (0.76) 11S3-Fe 155 0.43 (0.57) 13

a In parentheses are indicated the theoretical values for the pore volumes taking intob Maximum/a of the pore size distribution.c The values in parentheses correspond to the crystallite’s sizes in the composites pred Saturation magnetization (MS) was measured at room temperature. The values in br

nanocrystals inserted into the samples prepared without sucroseare all below 7 nm (see Table 1). These results confirm our hypoth-esis and show that the presence of carbon in the pores of the silicais essential for the in situ synthesis of relatively large iron oxidespinel nanocrystals and, in consequence, essential also in order toensure that the silica-based mesoporous composites have goodmagnetic properties. This last point will be discussed in moredetail in Section 3.2.

The XRD patterns in Fig. 2b for the SiO2–C–Fe materials showvery sharp peaks associated with a-Fe and weak XRD peaks asso-ciated with Fe3C. The sizes of the Fe crystallites estimated byapplying the Scherrer equation to the (110) peak of the XRD pat-terns are in the 23–27 nm range. These sizes are remarkably largerthan those obtained for FexOy. This suggests that the formation oflarge clusters of Fe results from the diffusion of the iron nanocrys-tals formed from the reduction of FexOy nanoparticles through thepores of the silica.

Fig. 1 shows TEM micrographs of the magnetic composites thatcontain FexOy (Fig. 1a, c and e) and Fe (Fig. 1b, d and f) nanoparti-cles. It can be seen that the nanoparticles (dark points) arewell-distributed throughout the silica matrix. In the case of theSBA15-based composites, it can be observed that, whereas theFexOy nanocrystals are uniformly dispersed over the ordered poros-ity (Fig. 1a), the iron nanoparticles present in the compositesappear to form large clusters, suggesting that some of the Fe nano-crystals are occupying adjacent pores (Fig. 1b). In the case of the S2and S3-based nanocomposites, the corresponding TEM images inFig. 1 reveal that the FexOy and Fe nanoparticles are uniformly dis-tributed throughout the silica matrix. Although the TEM images donot allow the diameter of these nanoparticles to be determinedaccurately, it can be inferred that their sizes are within the rangeof those obtained from X-ray diffraction analysis (see Table 1).Finally, with respect to the distribution of the magnetic nanoparti-cles in this type of composites, it should be mentioned TEM inspec-tion did not detect any nanoparticles on the outer surface of thesilica particles.

The textural properties of the magnetic materials containingFexOy and Fe, as well as those of the parent silica samples are listedin Table 1. Both types of materials (i.e. silica and magnetic compos-ites) exhibit a porosity that is made up almost exclusively of mes-opores as is evident from the nitrogen sorption isotherms and thepore size distributions (see Fig. 3). The insertion of carbon andmagnetic nanoparticles gives rise to a reduction in the porosityof the composite in relation to that of the silica (see Table 1 andFig. 3). This reduction in textural characteristics is a consequenceof the incorporation of non-porous substances (carbon and FexOy

or Fe) which fill up and block some of the pores of the silica. Weperformed theoretical calculations of the pore volume of the com-posites, taking into account only the filling effect. Such calculated

ze (nm)b dXRD (nm)c MS (emu g�1)d HC (Oe)

– – –9.5 (5.6) 8.5 (5.3) 027 8.8 190– – –11.5 (5.3) 7.4 (3.4) 026 9.8 155– – –11.5 (7) 11.0 (7.4) 023 12.6 90

account only the filling effect.

pared in absence of carbon.ackets correspond to the MS values of the samples obtained in absence of carbon.

S1S1-FexOyS1-Fe

10 nm

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0

4

8

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16

20

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a

b

c

Fig. 3. Nitrogen sorption isotherm and pore size distributions (inset) for themesoporous silica and the corresponding magnetic composites.

Fig. 4. Magnetization curves at room temperature of the magnetic nanocomposites.Insets show low zoom fields.

234 M. Sevilla et al. / Journal of Colloid and Interface Science 340 (2009) 230–236

values were found to be larger than the experimental values (seeTable 1). This shows that a significant fraction of the porosity ofthe silica is inaccessible (closed pores), probably because a certain

number of pores are blocked by deposited carbon and nanosizedparticles. The results in Table 1 indicate that the proportion ofclosed porosity affects the composites based on the S2 and S3 silicasamples more than those obtained from the SBA-15 silica (S1).Nevertheless, although the deposited nanoparticles induce acertain collapse of the silica porosity, a large number of pores stillremain unobstructed, as can be deduced from the data listed inTable 1 and the pore size distributions (see insets in Fig. 3). Thus,a significant fraction of the silica porosity in the composite remainsavailable for adsorbing substances.

3.2. Magnetic properties of the mesoporous composites

The magnetization reversal process of the nanocomposites wasrecorded at room temperature (Table 1 and Fig. 4). The SiO2–C–FexOy

nanocomposites show signatures of superparamagnetic behaviour(zero coercivity field). Similar superparamagnetic behaviour wasalso observed for the nanocomposites prepared in the absence of su-crose but in this case the magnetic moment was significantly lower(see Table 1). It appears, therefore, that the addition of sucrose givesrise to nanocomposites that have a higher magnetic moment (nano-composites respond better to an external magnetic field) and nomagnetic memory (superparamagnetic behaviour). This is becausethe better control resulting from the addition of sucrose which givesrise to nanocomposites with iron oxide ferrites that are of a relativelylarge size and still display superparamagnetic behaviour. The SiO2–C–Fe nanocomposites display magnetization curves that appearblocked in accordance with the presence of a-Fe clusters with sizes>20 nm (Table 1 and Fig. 4). Despite the presence of a-Fe, the nano-composites have a similar magnetic moment to the nanocompositescontaining iron oxide ferrites. This might reflect the presence ofhighly disordered iron oxide passivation layers that are not normallyobserved by XRD as was reported for the Fe/FexOy nanosized parti-cles [35,36].

0

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Fig. 5. Variation with time in the amount of hemoglobin adsorbed by the silicamaterials and the composites with inserted FexOy or Fe nanoparticles (pH = 6.0,C0 = 0.2 mg mL�1, 10 mg support, 10 g solution). Inset in Fig. 5a: Example ofmagnetic separation using a S1-FexOy composite loaded with hemoglobin.

M. Sevilla et al. / Journal of Colloid and Interface Science 340 (2009) 230–236 235

3.3. Hemoglobin adsorption

In order to evaluate the capability of the SiO2–C–FexOy andSiO2–C–Fe magnetic nanocomposites for manipulating biomole-cules, we investigated their ability to immobilize a large protein(bovine hemoglobin). This is a globular heme protein that has ahigh molecular weight (MW = 64500) and a large size (Dimen-sions: 5.3 � 5.4 � 6.5 nm3). Hemoglobin was chosen because itsimmobilization on inorganic supports (carbon nanofibers, carbonnanotubes, gold nanowires, mesoporous carbon, mesoporous silica,mesoporous alumina, etc.) has generated great interest for the fab-rication of NO, NO�2 and H2O2 sensors [37–41] and also as an elect-rocatalyst [42]. The immobilization experiments were performedat a pH � 6 which is slightly below the isoelectric point of hemo-globin (pI = 6.8). This pH value was chosen because it will allowelectrostatic interactions between the positively charged hemoglo-bin and the negatively charged support (Isoelectric point ofSiO2 � 3). For purposes of comparison we also analysed the immo-bilization of hemoglobin over the mesoporous silica materials em-ployed for preparing the magnetic nanocomposites. Fig. 5 showsthe change with time in the amount of hemoglobin immobilizedfor the different materials. In relation to the adsorption of hemo-globin in the silica samples, it can be seen that the S2 bimodal silicashows higher adsorption rates than S1 (SBA-15) and S2 (Silica–gel).This result is caused by the difference in the pore sizes of the S2 sil-ica (around 25 nm) with respect to S1 (�10 nm) or S3 (�12 nm).Obviously, the large size of the S2 pores offer higher diffusion rates,which is relevant in the case of a bulky biomolecule such as hemo-globine. By comparing the immobilization curves for the silicasamples and the corresponding composites, it is evident that theinsertion of carbon and magnetic nanoparticles causes a reductionin the immobilization rate. This is especially true of the silica mate-rials with large pores (i.e. S2 and S3 based nanocomposites). Thus,the data represented in Fig. 5b and c clearly point to a significantreduction in the adsorption rate of the composites in relation tothose of the parent silica samples. Although the S1-Fe and S2-Fecomposites show lower adsorption rates in relation to the parentsilica samples, they exhibit similar adsorption uptakes at equilib-rium (for long durations of time), which indicates that the insertionof carbon and iron nanoparticles hardly affects the adsorptioncapacities of these composites. In contrast, the adsorption uptakesfor the S1-FexOy and S2-FexOy samples (�135 mg g�1 support) arearound 30% lower than those measured for the silica samplesand iron composites (�190 mg g�1 support). The results show thathigh hemoglobin loadings (amount adsorbed over long periods oftime – equilibrium conditions: �100–200 mg g�1 support, 10–20wt.%) are obtained for both the silica samples and the magneticcomposites. These values are substantially larger than those re-cently reported by Choi et al. [43] for hemoglobin immobilizationover a SBA-15 silica (pore size �7.7 nm, amount adsorbed:68 mg g�1 support) and over a SBA-15/polystyrene composite(pore size �6.9 nm, amount adsorbed: 94 mg g�1 support). Animportant advantage of employing magnetic composites for theimmobilization of biomolecules is that they can be easily recoveredfrom the reaction media by means of an external magnetic field(see inset in Fig. 5a).

In order to examine the structural stability of the immobilizedhemoglobin, FTIR analysis of native hemoglobin and hemoglobin-loaded samples were performed. The characteristic IR amide Iand amide II bands provide detailed information about the second-ary structure of proteins. The amide I band (1700–1600 cm�1) isrelated to the C@O stretching vibration of peptide linkages andthe amide II band (1620–1500 cm�1) is attributed to the bendingand stretching mode of the N–H and C–N vibrations, respectively[44,45]. At this point, it is important to indicate that the structuralchanges of proteins can be followed by the infrared spectroscopy

because the protein denaturation modifies the amide vibrationalfrequencies [46]. In Fig. 6 are represented the FTIR spectrum ofthe native hemoglobin and those for the samples (S2-Fe, S3-Feand S3) loaded with hemoglobin. By comparison, the amide I andamide II bands of immobilized hemoglobin are essentially thesame as those of the native hemoglobin, indicating that this

130018002300280033003800Wavenumber (cm-1)

Tran

smita

nce

(a. u

.)

Amide I

Amide II

c

a

d

b

Fig. 6. FTIR spectra of the native hemoglobin (a) and the samples loaded withhemoglobin (b: S2-Fe; c: S3-Fe and d: S3).

236 M. Sevilla et al. / Journal of Colloid and Interface Science 340 (2009) 230–236

protein retains the secondary structure after immobilization inboth, silica support and magnetic composites.

4. Conclusions

In summary, a methodology for inserting magnetic nanoparticlesof magnetite/maghemite or iron within the pores of mesoporous sil-ica matrixes has been developed. The key to this synthetic strategy isto incorporate inside the pores of the silica a small amount of carbon,which is essential for the growth of iron oxide nanocrystals and alsofor the formation of iron nanoparticles. In this way, magnetic meso-porous SiO2–C–Fe3O4/c–Fe2O3 and SiO2–C–Fe composites have beensuccessfully synthesized. These materials possess good magneticproperties, in particular the SiO2–C–Fe3O4/c–Fe2O3 composites thatexhibit a superparamagnetic behaviour. Although the incorporatedcarbon and magnetic nanoparticles partially fill and block the poresof silica, the resulting composites still retain a large surface area, ahigh pore volume and a porosity made up of wide mesopores withsizes in the 9–25 nm range (depending on the type of parent silica).The usefulness of this kind of composites for the immobilization andmagnetic separation of biomolecules has been clearly demonstratedin the case of hemoglobin. It was observed that these magnetic com-posites are able to immobilize large amounts of hemoglobin (up to180 mg g�1). The FTIR spectra obtained for the magnetic compositesloaded with hemoglobin show that the structure of this protein ispreserved after its immobilization.

Acknowledgments

The support for this research provided by the MEC (MAT2008-00407, MAT2005-03179, NANO2004-08805-C04-01) and CSIC-I3(200660I192) is gratefully acknowledged. M.S. acknowledges theassistance of the Spanish MCyT for the award of a FPU grant.

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