Materials Research Bulletin 45 (2010) 1603–1607

Surface modification of superparamagnetic iron nanoparticles with calciumsalt of poly(g-glutamic acid) as coating material

Ramesh Kumar a, B. Stephen Inbaraj a, B.H. Chen a,b,*a Department of Food Science, Fu Jen University, Taipei 242, Taiwanb Graduate Institute of Medicine, Fu Jen University, Taipei 242, Taiwan

A R T I C L E I N F O

Article history:

Received 9 March 2010

Received in revised form 21 June 2010

Accepted 21 July 2010

Available online 30 July 2010

Keywords:

A. Polymers

B. Chemical synthesis

C. X-ray diffraction

C. Thermogravimetric analysis (TGA)

D. Magnetic properties

A B S T R A C T

Surface-modified magnetite nanoparticles (MNPs) were synthesized by co-precipitation of aqueous

solution of ferrous and ferric salts (molar ratio 1:2) upon adding a base followed by calcium salt of

poly(g-glutamic acid) (Ca-g-PGA) for uniform coating on the surface of MNPs. Both uncoated and Ca-g-

PGA-coated MNPs were characterized using various techniques including Fourier transform infrared

spectroscopy (FTIR), X-ray diffraction (XRD), transmission electron microscopy (TEM), thermogravi-

metric analysis (TGA) and vibrating sample magnetometric (VSM) studies. Compared with bare MNPs,

the IR spectra of coated MNPs showed characteristic peaks of g-PGA, implying the g-PGA coating on

MNPs did occur. The TEM images depicted an average size of 8–10 nm for bare MNPs and 14 nm for

coated MNPs, with their shape being spherical in nature. In the presence of applied magnetic field, a

superparamagnetic behavior was observed at room temperature for both bare and Ca-g-PGA-coated

MNPs, with no magnetism left upon magnetic-field removal.

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1. Introduction

Functionalized superparamagnetic iron nanoparticles haveattracted considerable attention because of their potentialapplications in various fields such as physics, medicine, biologyand materials science [1–5], which are attributed by theirmultifunctional properties such as superparamagnetism, smallsize, high magnetic susceptibility and non-toxic nature [6,7]. Iron-oxide nanoparticles such as magnetite (Fe3O4) and maghemite (g-Fe2O3) are mostly used as magnetic materials in ferrofluids, withthe former being preferred due to greater saturation magnetiza-tion. Magnetite is a common magnetic iron oxide that has a cubicspinel structure with oxygen forming a FCC closed packing and Fecations occupying the interstitial tetrahedral and octahedral sites[8]. Various routes of synthesis such as co-precipitation [9–11],sol–gel [12,13], emulsion technique [14], hydrothermal [15],mechanochemical [16] and DC thermal arc-plasma [17] havebeen developed to prepare magnetite nanoparticles (MNPs) and, ofwhich, co-precipitation is widely used owing to its ease and largevolume capability [18]. However, the MNPs have a tendency toagglomerate due to dipole–dipole attraction and/or undergo

Abbreviations: TEM, transmission electron microscopy; TGA, thermogravimetric

analysis; FTIR, Fourier transform infrared; Ca-g-PGA, calcium salt of poly(g-

glutamic acid); MW, molecular weight; XRD, X-ray diffraction.

* Corresponding author at: Department of Food Science, Fu Jen University, Taipei

242, Taiwan. Tel.: +886 2 29053626; fax: +886 2 29021215.

E-mail address: 002622@mail.fju.edu.tw (B.H. Chen).

0025-5408/$ – see front matter � 2010 Elsevier Ltd. All rights reserved.

doi:10.1016/j.materresbull.2010.07.017

oxidation in air, which can be remedied by surface modificationof MNPs with appropriate surfactant or polymer as a coatingmaterial [6,18]. A wide variety of coating materials employed tomodify the surface of MNPs and their biomedical applications havebeen well reviewed [6].

Polymeric materials have been frequently used for coatingMNPs, probably because of the cross-linked polymer matrix thatcan prevent coagulation and yield monodisperse particles. Severalsynthetic polymers used to coat MNPs include poly(vinylpyrro-lidone), poly(lactic-co-glycolic acid), polyethylene glycol andpoly(vinyl alcohol) [6], while gelatin [19], dextran [20], starch[21], arabic gum [22] and chitosan and/or its derivatives [1,23] areamong the natural polymers widely employed as coatingmaterials. Several surfactants such as sodium oleate, dodecyla-mine and sodium carboxymethylcellulose are used as well toenhance dispersibility in aqueous solution [6,24]. Coating materi-als with carboxyl functional groups have been particularlypromising as carboxyl groups can significantly enhance thedispersion of MNPs in a biological system by shifting the isoelectricpoint [25,26].

Poly(g-glutamic acid) (g-PGA), a natural and biocompatiblebiopolymer produced by common Bacillus microbes throughfermentation, is an unusual polypeptide containing g-amidelinkages formed between repetitive glutamic acid units to facilitateits conjugation with diverse compounds through numerous side-chain a-carboxyl groups [27,28]. The objectives of this study wereto prepare Ca-g-PGA-modified superparamagnetic iron-oxidenanoparticles by the conventional co-precipitation method under

[(Fig._1)TD$FIG]

Fig. 1. Coated magnetite nanoparticles in the dried state being attracted by a permanent magnet (a) and re-dispersed in water (b).

R. Kumar et al. / Materials Research Bulletin 45 (2010) 1603–16071604

non-oxidizing conditions. In addition, both uncoated and coatedMNPs were characterized with Fourier transform infrared spec-troscopy (FTIR), X-ray diffraction (XRD), transmission electronmicroscopy (TEM) and thermogravimetric analysis (TGA) as well asthe magnetization study by vibrating sample magnetometer(VSM).

2. Experimental

2.1. Materials

The coating material Ca-g-PGA with low MW (�250 kDa)synthesized from Bacillus subtilis var. Natto by salvage bioconver-sion pathway was supplied by Vedan Enterprise Corporation(Taichung, Taiwan). For synthesis of MNPs, the analytical-gradechemicals ferric chloride (FeCl3�6H2O) and ferrous sulfate (FeS-O4�7H2O) were obtained from Nacalai Tesque (Kyoto, Japan) andammonium hydroxide (28%) from J. T. Baker (Phillipsburg, USA).An IKA1 RW 20 overhead mechanical stirrer (Staufen, Germany)was used for vigorous stirring during synthesis of MNPs. Potassiumbromide (KBr) used for pelletizing MNPs to measure IR spectra wasalso from Nacalai and hydrochloric acid incorporated duringsynthesis for complete dissolution of iron salts was from Riedel deHaen (Seelze, Germany). The milli-Q deionized water was obtainedfrom Millipore (Bedford, MA) and deoxygenated prior to use bybubbling with nitrogen gas.

2.2. Synthesis of nanoparticles

Synthesis of both bare and Ca-g-PGA-coated MNPs by theconventional co-precipitation method was based on the conditionsreported by Liu et al. [26], which basically involved co-precipita-tion of ferrous and ferric iron salts in aqueous solution after addinga base. In short, 6.1 g of ferric chloride and 4.2 g of ferrous sulfatesalts were dissolved in a 100 ml of deoxygenated deionized water.Then, a few drops of hydrochloric acid were added and the mixturewas vigorously stirred at 2000 rpm with nitrogen gas beingbubbled to prevent oxidation of Fe2+. After the mixture was heatedto 85 8C, 14 ml of ammonium hydroxide was added rapidly to co-precipitate the iron salts and the color of solution eventuallychanged from light orange to black. Next, 50 ml of 0.5 g Ca-g-PGAsolution in deoxygenated deionized water was added and thevigorous stirring was continued for 1 h at 85 8C, with simultaneousbubbling of nitrogen gas to prevent atmospheric oxidation of MNPsand facilitate reduction in their size. Upon completion of thereaction, the precipitated MNPs were allowed to cool at roomtemperature, followed by magnetic decantation of supernatantfluid using a permanent magnet. After washing five times with

deoxygenated deionized water, the synthesized MNPs werevacuum-dried at 0 mm Hg and 40 8C for 24 h and used forcharacterization. Bare MNPs were also prepared and dried byadopting the same procedure but without adding Ca-g-PGA, andthe chemical reaction [18,29] involved may be represented asgiven below:

Fe2þ þ2Fe3þ þ8OH� ! Fe3O4 # þ 4H2O

Fig. 1a and b shows the as-synthesized dried sample of Ca-g-PGA-coated MNPs being strongly attracted by a permanent magnetat room temperature and the same being re-dispersed in deionizedwater, respectively.

2.3. Structural and magnetic characterization

The synthesized MNPs, both before and after modification, werecharacterized for surface morphology and magnetic properties.The Fourier transform infrared spectra of bare and coated MNPs aswell as pure Ca-g-PGA were recorded on a Horiba FTIRspectrophotometer (model FT 730, Kyoto, Japan) in the range of4000–400 cm�1 using KBr pellets prepared by mixing a smallamount of nanoparticles and KBr crystals. The X-ray diffraction(XRD) pattern was recorded on a Multiflex model of Rigakudiffractometer (Tokyo, Japan) using Cu-Ka radiation of wavelength1.540556 A at 40 kv/40 mA in the scan range (2u) of 20–708. Theaverage diameter of coated and uncoated MNPs was calculatedaccording to the Debye–Scherrer equation [24,32] by measuringXRD line-width. The average size and morphology of the magnetitenanoparticles were determined with a JEOL Transmission FieldEmission Electron Microscope (JEM 2100F, Tokyo, Japan). Thermo-gravimetric analysis (TGA) was carried out by using a Versa ThermHS model Cahn TGA analyzer (Thermo Fisher Scientific, USA) todetermine the weight percentage coating of Ca-g-PGA on MNPs.Samples weighing between 5 and 15 mg were heated from 30 to600 8C at a heating rate of 10 8C/min in air. The magnetizationmeasurements of the synthesized nanoparticles were determinedat room temperature (301 K) in a magnetic field up to 13,500 Oeusing a vibrating sample magnetometer (VSM DMS model 1660,ADE Technologies Inc., MA, USA).

3. Results and discussion

3.1. Infrared spectra

Fig. 2a and b shows FTIR spectrum of uncoated MNPs and Ca-g-PGA-coated MNPs, respectively, with the characteristic absorptionband of magnetite being found at 578 and 586 cm�1 for both and

[(Fig._2)TD$FIG]

Fig. 2. FTIR spectrum of uncoated (a) and Ca-g-PGA-coated (b) magnetite

nanoparticles.

[(Fig._3)TD$FIG]

Fig. 3. X-ray diffractogram of Ca-g-PGA-coated (a) and uncoated (b) magnetite

nanoparticles.

R. Kumar et al. / Materials Research Bulletin 45 (2010) 1603–1607 1605

corresponded to vibration of Fe–O bond [33,34]. The presence ofpeaks at 3432 and 1627 cm�1 in the IR spectrum of Fe3O4

nanopaticles may be assigned due to O–H stretching vibration ofabsorbed water [18]. Compared to bare MNPs, a large number ofpeaks in the spectrum of Ca-g-PGA-modified MNPs confirmed thesuccessful coating of polymer on iron-oxide surface (Fig. 2b) [25].Also, the characteristic band at 2931 cm�1 resulting fromasymmetric and symmetric stretching modes of C–H groups alsoconfirmed the successful coating of Ca-g-PGA on MNPs [30].However, the peak intensity was low which should be caused bythe overlap with the broad band in the frequency region of 2800–3800 cm�1. A broad band at 3432 cm�1 may be related to N–Hstretching vibration of amide groups. Besides, two characteristicabsorption bands of pure Ca-g-PGA appearing at 1635 and1562 cm�1 may be attributed to asymmetric stretching vibrationof carboxylate anion and bending vibration of N–H, respectively[28,31]. In addition, a peak at 1400 cm�1 may be due to symmetricstretching vibration of carboxylate anion [28,31]. The IR spectrumof pure Ca-g-PGA showed absorption bands at 3409, 2927, 1639–1585, 1411, 1126 and 1029 cm�1 (spectrum not shown), whichwere similar to that observed for Ca-g-PGA-coated MNPs. Theseresults suggested that the surface of MNPs was successfully coatedwith Ca-g-PGA.

3.2. X-ray diffraction studies

Fig. 3 shows the X-ray diffractograms of uncoated and coatedMNPs recorded in a 2u range of 20–708, both of which exhibited sixcharacteristic diffraction peaks of Fe3O4 by their indices (2 2 0),(3 1 1), (4 0 0), (4 2 2), (5 1 1), (4 4 0), indicating the preparedMNPs to be pure magnetite with inverse spinel structure [7,29].Moreover, no peak was observed corresponding to goethite,hematite and some other iron oxides like a-Fe2O3 or g-Fe2O3 inthe X-ray diffractograms for both types of MNPs, demonstratingthe synthesized product to be pure Fe3O4 nanoparticles. The meansize of nanoparticles was calculated from XRD peak broadeningusing the Scherrer equation [24,32]: d = (kl/b cos u); where k is theScherrer constant (0.9), l is X-ray wavelength (1.540556 A), b isfull-width at half maximum and u is Bragg angle in degree. Thecrystal size was calculated from broadening of the (3 1 1) reflection

and the mean diameter of uncoated and coated MNPs wasdetermined to be 12.8 and 17.2 nm, respectively, which furtherproved the high crystalline nature of the Ca-g-PGA-coated MNPs.

3.3. Size and morphology of particles

Fig. 4a and b illustrates the TEM images of uncoated andcoated MNPs, respectively, at a magnification of 100,000 alongwith the particle size distribution histogram. The averagediameter of bare MNPs and Ca-g-PGA-coated MNPs weredetermined to be 8–10 and 14 nm, respectively. This outcomeconformed well with the mean diameter (12.8 and 17.2 nm)calculated from XRD pattern using the Scherrer equation. It isdifficult to observe the coating of Ca-g-PGA directly on the TEMimage, yet the coating formation can be confirmed by the TGAresults. The morphology of both uncoated and coated MNPs wasof roughly spherical in shape.

3.4. Thermal analysis

The TGA curves of both bare and Ca-g-PGA-coated MNPs areshown in Fig. 5. The initial weight loss before 220 8C for bothsamples may be attributed to the desorption of surface hydroxylsand/or adsorbed water [25]. A marked one-step weight lossobserved above 250 8C for surface-modified MNPs may beaccounted for by the decomposition and subsequent evaporationof coating layer [35]. Additionally, the TGA curves indicated aweight percentage Ca-g-PGA coating of 11.8% based on the weightloss for coated MNPs, which was similar to that reported for humicacid-modified magnetite (11.4%) [26] or methacrylic acid-modifiedmaghemite nanoparticles (12.0%) [25].

3.5. Magnetic properties of magnetite particles

The plots of magnetization (M, emu/g) versus magnetic field (H,Oe) for uncoated and coated MNPs measured at room temperature inthe applied magnetic field ranging from +13,500 to �13,500 Oe areshown in Fig. 6. No coercivity or remanence was observed in themagnetic loop suggesting the magnetic nanoparticles to be super-paramagnetic in nature [29]. Apparently, the thermal fluctuationsbecome dominant over spontaneous magnetization at a given fieldand the net magnetization without external field is zero [36]. Themagnetic saturation (Ms) values were obtained by extrapolating the1/H value to zero in a graph of M versus 1/H (inset plot in Fig. 6). The

[(Fig._4)TD$FIG]

Fig. 4. TEM image of uncoated (a) and Ca-g-PGA-coated (b) magnetite nanoparticles along with their particle size distribution histogram.

[(Fig._5)TD$FIG]

Fig. 5. Thermogravimetric curves of uncoated (a) and Ca-g-PGA-coated (b)

magnetite nanoparticles.

[(Fig._6)TD$FIG]

Fig. 6. Measured magnetization moments (M) of uncoated (a) and Ca-g-PGA-coated

(b) magnetite nanoparticles as a function of applied magnetic field (H) at room

temperature. The inset picture shows the plot of M against 1/H for the

determination of saturation magnetization.

R. Kumar et al. / Materials Research Bulletin 45 (2010) 1603–16071606

R. Kumar et al. / Materials Research Bulletin 45 (2010) 1603–1607 1607

Ms value obtained at 15 kOe for uncoated MNPs was 71.2 emu/g,which was comparable to those reported in the literature [29,34].Nevertheless, this was lower than that reported for its bulkmagnetite (89–92 emu/g) [29,34], mainly due to the saturationmagnetization for single-domain superparamagnetic nanoparticlesbeing size-dependent, which could be explained by the surface effect[37,38]. The surface spins of magnetic particles lack completecoordination and are likely disordered resulting in less susceptibilityto changes in the strength of the external field [39]. Thisphenomenon became more pronounced for the nanosized particlesowing to their large surface-to-volume ratio and thereby it was quiterational to observe a lower saturation value for synthesized barenanoparticles even at 15 kOe as compared to bulk magnetite.Additionally, the crystallinity of the nanoparticles can also affect themagnetic properties [40]. Accordingly, the decrease in magneticmoment may be attributed to the amorphous coating beingundetectable by XRD at a grain boundary [41].

The Ms value of coated magnetic nanoparticles was 56.6 emu/g at 15 kOe, which is relatively lower than that for the bare MNPs.This may be accounted for by a coating of non-magnetic Ca-g-PGA on MNPs and possibly the exchange of electrons betweenthe surface Fe atoms and the functional groups on Ca-g-PGA[41,42]. Nonetheless, a slight difference in saturation magneti-zation values of uncoated and coated MNPs implied that coatingwith Ca-g-PGA did not significantly reduce the magneticproperty of bare MNPs. A comparison of the Ms value (emu/g)of uncoated (71.2) and Ca-g-PGA-coated (56.6) MNPs suggestedthe content of Ca-g-PGA in coated MNPs was about 14.6%,which was close to the value (11.8%) as determined by thermalanalysis.

4. Conclusions

The Fe3O4 nanoparticles with a size of 8–10 nm and Ca-g-PGA-coated Fe3O4 nanoparticles with an average diameter of 14 nmwere synthesized using the co-precipitation method. Both bareand coated magnetite particles were spherical in shape andpossessed superparamagnetic property. The value of saturationmagnetization of coated magnetite nanoparticles was slightlylower than that of bare magnetite nanoparticles.

Acknowledgements

We acknowledge the help of Mr. Y.H. Lin, a graduate student ofPhysics Department at Fu Jen University in carrying out the XRDstudies.

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