Simple Synthesis of Functionalized Superparamagnetic Magnetite/Silica Core/Shell Nanoparticles and their Application as Magnetically Separable High-Performance Biocatalysts

Biocompatible materials

DOI: 10.1002/smll.200700456

Simple Synthesis of Functionalized SuperparamagneticMagnetite/Silica Core/Shell Nanoparticles and their Applicationas Magnetically Separable High-Performance Biocatalysts**Jinwoo Lee, Youjin Lee, Jong Kyu Youn, Hyon Bin Na, Taekyung Yu, Hwan Kim,Sang-Mok Lee, Yoon-Mo Koo, Ja Hun Kwak, Hyun Gyu Park, Ho Nam Chang,Misun Hwang, Je-Geun Park, Jungbae Kim,* and Taeghwan Hyeon*

Uniformly sized silica-coated magnetic nanoparticles (magnetite@silica) aresynthesized in a simple one-pot process using reverse micelles as nanoreac-tors. The core diameter of the magnetic nanoparticles is easily controlled byadjusting the w value ([polar solvent]/[surfactant]) in the reverse-micellesolution, and the thickness of the silica shell is easily controlled by varyingthe amount of tetraethyl orthosilicate added after the synthesis of themagnetite cores. Several grams of monodisperse magnetite@silica nanoparti-cles can be synthesized without going through any size-selection process.When crosslinked enzyme molecules form clusters on the surfaces of themagnetite@silica nanoparticles, the resulting hybrid composites are magneti-cally separable, highly active, and stable under harsh shaking conditions formore than 15 days. Conversely, covalently attached enzymes on the surface ofthe magnetite@silica nanoparticles are deactivated under the same conditions.

Keywords:· core/shell materials· iron oxides· silica· superparamagnetism· surface functionalization

1. Introduction

Magnetic nanoparticles have received a great deal of at-tention because of their potential use in various biomedicalapplications, including contrast agents in magnetic reso-nance imaging (MRI), the magnetic separation and sortingof cells and proteins, immunoassay in pathology laborato-ries, hyperthermia treatment for cancerous tumors, and thecontrolled and targeted delivery of pharmaceuticals andtherapeutic genes.[1] In recent years, much attention hasbeen focused on the synthesis of uniformly sized magneticnanoparticles.[2] However, most of these nanoparticles havebeen synthesized in organic solvents using hydrophobic cap-ping reagents. The resulting magnetic nanoparticles are dis-persible in hydrophobic organic solvents. However, the bio-logical applications of these nanoparticles are greatly re-stricted because of their poor dispersibility in aqueousmedia. Very recently, there have been several reports on thesynthesis of water-dispersible magnetic nanoparticles,[3] inwhich magnetic nanoparticles are functionalized with water-compatible chemical reagents.[4]

Silica surfaces are biocompatible and can be easily func-tionalized for bioconjugation purposes. Because of these ad-vantages, magnetic–silica composite nanoparticles with vari-ous shapes (spheres, rods, and plates) have been synthesized

[*] Dr. J. Lee, Y. Lee, H. B. Na, T. Yu, H. Kim, Prof. T. HyeonNational Creative Research Initiative Center forOxide Nanocrystalline Materials andSchool of Chemical EngineeringSeoul National UniversitySeoul 151-744 (Korea)Fax: (+82) 2-888-1604E-mail: [email protected]

Dr. J. H. Kwak, Dr. J. Kim+

Pacific Northwest National LaboratoryRichland, WA 99352 (USA)Fax: (+1) 509-375-5106E-mail: [email protected]

J. K. Youn, Prof. H. G. Park, Prof. H. N. ChangDepartment of Chemical and Biomolecular EngineeringKorea Advanced Institute of Science and TechnologyDaejeon 305-701 (Korea)

Dr. S.-M. Lee, Prof. Y.-M. KooCenter for Advanced Bioseparation TechnologyInha UniversityIncheon 402-751 (Korea)

M. Hwang, Prof. J.-G. ParkDepartment of Physics, Sungkyunkwan UniversitySuwon 440-746 (Korea)

[+] Current address: Department of Chemical and Biological Engineering

[**] J. L. and Y. L. contributed equally to this work.

Supporting Information is available on the WWW under http://www.small-journal.com or from the author.

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Superparamagnetic Core/Shell Nanoparticles as Biocatalysts

using reverse block-copolymer mesophases.[5] Although thissynthetic process is simple and novel, the magnetic nanopar-ticles that are obtained show ferromagnetic behavior, whichcauses a remanent magnetization problem when the appliedmagnetic field is removed. For biological applications, super-paramagnetic nanoparticles are desirable as the magnetichosts need to be easily dispersed after each magnetic sepa-ration.

Several methods have been developed for the prepara-tion of silica-coated magnetic nanoparticles with core/shellstructures by coating the silica shell on the preformed nano-particles.[6,7] To obtain silica-coated superparamagneticnanoparticles with more versatile biological applications, itwould be desirable to synthe-size uniformly sized magneti-te@silica nanoparticles via asimple and easy syntheticroute. To our knowledge,there have been no reportson the simple one-pot syn-thesis of discrete, uniformlysized magnetic-nanoparticle-core/silica-shell superpara-magnetic nanoparticles with-out using preformed nano-particles. Herein, we reporton the facile large-scale syn-thesis of magnetite@silicananoparticles by the simpleaddition of tetraethyl ortho-silicate (TEOS) in reversemicelles during the formationof uniformly sized magnetitenanoparticles. The two keyfeatures of the current syn-thetic procedure are the maintenance of the micelle struc-tures during the formation of the nanoparticles, which isachieved by conducting the synthesis at a low temperatureof 908C, and the controlled sol–gel reaction inside the re-verse micelles. Furthermore, the functionalization of themagnetite@silica nanoparticles could be easily achieved bymixing TEOS with silane agents such as 3-aminopropyltrie-thoxysilane (APS).

As a biological application of the silica-coated magneticnanoparticles, we fabricated highly stable crosslinkedenzyme clusters (CECs) on the surface of the magnetite@-silica nanoparticles. Lipase and a-chymotrypsin were usedas the model enzymes in this study. Recently, we demon-strated that the crosslinking of enzyme molecules in variousnanomaterials results in synergetic enzyme stabilization andimproves the apparent enzyme activity because of the highenzyme loading.[8] The magnetic nanoparticle and enzymewere simultaneously entrapped in the sol–gel silica matrixor hierarchically mesocellular mesoporous silica (HMMS)to allow magnetically separable heterogeneous biocataly-sis.[8b,9]

By using amino-functionalized magnetite@silica nano-particles, we demonstrate that the hybrid materials consist-ing of CECs and the magnetic nanoparticles also exhibit

high stability and activity, in addition to their facile magnet-ic separation.

2. Result and Discussion

2.1. Synthesis and Characterization of Magnetite/SilicaCore/Shell Nanoparticles

Magnetite nanoparticles coated with amorphous silicashells were produced by simply adding TEOS during theformation of the magnetite nanoparticles. This one-pot syn-thetic procedure is shown in Scheme 1. Recently, we report-

ed the synthesis of size-controllable and highly crystallinemagnetite nanoparticles using a reverse-micelle approach.[10]

Two minor modifications were made for the synthesis of themagnetite@silica nanoparticles. We replaced ethanol withdeionized water and increased the amount of hydrazine(N2H4) used for the hydrolysis of TEOS. By the simple addi-tion of TEOS, we could obtain monodisperse magnetitenanoparticles coated with silica shells. These magnetite@sili-ca nanoparticles could be readily dispersed in polar solventssuch as ethanol and water, suggesting that their surface wassuccessfully coated with a hydrophilic silica shell.

The transmission electron microscopy (TEM) images ofthe magnetite@silica nanoparticles are shown in Figure 1.The size distribution of the magnetite@silica nanoparticlesis very narrow over the wide range of the TEM grid area.The nanoparticles have a discrete core/shell structure (Fig-ure 1b), and their uniform magnetic core with a diameter of7 nm is surrounded by a 5-nm-thick silica shell (Figure 1a,inset). This size uniformity of the magnetite@silica stemsfrom the formation of uniformly sized core magnetite parti-cles and the subsequent uniform coating with the silicashells. The TEOS molecules, initially residing in the hydro-phobic xylene phase, start to hydrolyze at the interface withthe reverse micelles because of the presence of the water

Scheme 1. Schematic diagram of the one-pot synthesis of uniformly sized silica-coated magnetite nano-particles (see Experimental Section for full details).

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full papers J. Kim, T. Hyeon, et al.

molecules, and form a condensed, amorphous silica shell onthe surface of the magnetite nanoparticles. Interestingly, thissimple process resulted in the formation of silica shells ofuniform thickness. The temperature was not increased to re-fluxing temperature during the formation of the magnetitecore to prevent complete evaporation of the water presentin the hydrophilic phase, which contained the magnetitenanoparticles. The residual water remaining after formationof the magnetite core is critical for the hydrolysis of TEOS.

Even though the synthesis was achieved in a simple one-pot reaction, most of the magnetic/silica nanoparticles hadone magnetite nanoparticle per composite nanoparticle. Wealso found no silica nanoparticles (Figure 1b), indicatingthat all of the reverse micelles are occupied by magneticnanoparticles and that the secondary nucleation for a silicananoparticle did not occur. The broad peak at around 2q =

208 in the X-ray diffraction (XRD) pattern is due to theamorphous silica shell on the surface of the magnetite nano-particles (Figure 2a). The characteristic peaks of the mag-netite nanoparticles were also clearly identified in the XRDpattern.[10] The magnetite@silica nanoparticles were well-dispersed in distilled water, whereas we were unable to dis-perse them in hydrophobic solvents such as hexane (Fig-

ure 2b). This result shows that the magnetite@silica nano-particles can be easily used for bioapplications.

To assess the porosity of the silica shells in the magneti-te@silica nanoparticles with 7-nm cores and 5-nm shells, ni-trogen adsorption/desorption experiments were conducted.The Brunauer–Emmett–Teller surface area and microporoussurface area are 41 m2 g�1 and 13 m2 g�1, respectively (mi-cropore surface area was derived from t-plot anaysis). Themicropores are generated during silicate sol–gel reactionunder the basic reaction conditions.

The core diameter of the magnetic nanoparticles waseasily controlled by adjusting the w value ([polar solvent]/[surfactant]) in the reverse-micelle solution. For example,reducing the w value from 6.5 to 5.8 changed the averagecore diameter from 7 to 5 nm. On the other hand, the thick-ness of the silica shell could be easily controlled by varyingthe amount of TEOS added after the synthesis of the mag-netite cores. By combining these two parameters, we couldsynthesize magnetite@silica nanoparticles with various com-binations of core diameters and shell thicknesses (Figure 3).When the amount of TEOS added was further reduced to0.3 mL, no distinct silica shell could be observed in the low-resolution TEM image, but the final magnetite@silica nano-particles could be dispersed well in water.

For application of the magnetite@silica nanoparticles ashost materials for biomolecules, the grafting of functionalgroups onto their surface is critical. We prepared core/shellnanoparticles functionalized with the aminopropyl group bytwo different methods. The reagent used for the amino-group functionalization of the surface of the magnetite

Figure 2. a) X-ray diffraction pattern of magnetite@silica nanoparti-cles with core diameter of 7 nm and shell thickness of 5 nm. Thebottom row of tick marks indicates the reflection positions for a stan-dard magnetite pattern (Joint Committee on Powder DiffractionStandards (JCPDS) file no. 19-0629). b) Picture showing the dispersi-bility of silica-coated magnetite nanoparticles in a hydrophobic sol-vent (hexane) and in water.

Figure 1. TEM images of silica-coated magnetite nanoparticles withcore diameter of 7 nm and shell thickness of 5 nm (inset).

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nanoparticles was APS, which is commonly adopted for thefunctionalization of silica surfaces. At first, we preparedsilica-coated magnetite using a conventional, standardmethod, in which we synthesized magnetite nanoparticlescoated with amorphous silica shells and performed the post-treatment reaction of the hydroxyl functional group of thesilica layer with hydrolyzed APS molecules. The post-treat-ment was done in hexane by dispersing the magnetite nano-particles coated with amorphous silica shown in Figure 1.We confirmed that there was no remarkable change in theparticle shapes (core dimension and thickness) and that thenanoparticles were readily dispersible in polar solvents. TheFourier transform IR (FTIR) spectrum (Supporting Infor-mation, Figure S2) confirms the existence of amino groups(–NH2 bending) on the surface of the particles and the silicalayer from the characteristic Si–O–Si vibration.

We also synthesized magnetite nanoparticles functional-ized with amino groups directly by mixing two differentsilica precursors—TEOS and APS. Figure 4a and b showsthe TEM images of the magnetite nanoparticles coated withsilica shells functionalized by amino groups through theone-step reaction. Since the functionalized silica shell isvery thin, we could not observe the silica layer by TEManalysis. Even though the amount of silica precursors usedfor the synthesis of the core/shell nanoparticles with thepure silica shells and the aminopropyl-functionalized silicashells is nearly the same, the shell thickness of the amino-

propyl-functionalized silica-coated magnetite nanoparti-cles is far smaller than thatof the pure silica-coatedmagnetite nanoparticles. Thiscan be explained by the largesteric hindrance of APS. Inother words, if aminopropylgroups are incorporated intothe silica shell, further con-densation on the surface canbe hindered by the amino-propyl groups exposed onthe surface of the nanoparti-cles, leading to a thin coatingof the silica shell. The amine-functionalized silica@magne-tite particles are well-dis-persed in water but aggre-gate in hexane (Figure 4c),suggesting that the surface ofthe magnetite nanoparticlesis well-silanized. The NH2

bending vibration in the IRspectrum (Figure 4e) con-firms the existence of theamino groups in the directlyfunctionalized magneticnanoparticles. Elementalanalysis reveals that2.8 mmol g�1 of the aminogroup are present in the

amine-functionalized magnetite@silica nanoparticles.The field-dependent magnetization curve of the core/

shell nanoparticles with a 5-nm magnetite core and 5-nm-thick silica shell (Figure 5a) measured at 300 K exhibits nohysteresis, demonstrating superparamagnetic characteristics,which is extremely important for practical applications be-cause the magnetic supports or carriers should not retainany residual magnetism after the magnetic field is removed.On the other hand, the magnetization curve obtained at 2 Kshows hysteresis with non-zero remanence and coercivity,demonstrating ferrimagnetic behavior. This transition fromsuperparamagnetic behavior at high temperature to ferro-or ferrimagnetic behavior below the so-called blocking tem-perature is typically observed in magnetic nanoparticles.[2]

The temperature-dependent magnetization under zero-fieldcooled and field-cooled conditions under an applied field of100 Oe shows that the blocking temperature of the core/shell nanoparticles is 37 K (Figure 5c). The saturation mag-netization value of the silica@magnetite nanoparticles is ap-proximately 20 emu g�1, while the saturation magnetizationvalue of magnetite nanoparticles is 39.6 emu g�1.[10]

Figure 5b compares the field-dependent magnetizationcurves at 2 K of the two different kinds of magnetite nano-particles, namely, those with a 5-nm-thick silica shell andthose with the dodecylbenzene sulfonate surfactant coating,having the same core diameter of 5 nm. These two kinds ofnanoparticles were synthesized under similar reaction condi-

Figure 3. TEM images of silica-coated magnetite nanoparticles with different core diameters and shellthicknesses. The dimensions of each particle are 7 nm (core diameter)–5 nm (shell thickness) (a), 5 nm–5 nm (b), 5 nm–2 nm (c), and 5 nm with a very thin layer (d). The particle size distributions for (a–c) arepresented in Table S1 of the Supporting Information.



tions, with and without the addition of the silica precursor,respectively. Both magnetization curves show ferrimagneticbehavior with remanence and coercivity. However, the coer-civity (Hc) of the silica-coated magnetite nanoparticles ismuch lower than that of the surfactant-stabilized nanoparti-cles. Furthermore, the blocking temperature of the surfac-tant-stabilized nanoparticles is 85 K, which is much higherthan that of the 5-nm-thick silica-coated magnetite nanopar-ticles (37 K) with the same core diameter.[10] These resultsseem to be caused by a reduction of the surface anisotropycaused by the silica coating. It is well-known that the sur-face anisotropy of magnetic nanoparticles is reduced uponcoating.[11] For example, the coercivity of MnFe2O4 nanopar-ticles was reduced upon coating with silica[7a] or benzene-thiol,[12] and the blocking temperature of Co nanoparticlesdecreased from 350 to 240 K upon coating with CdSeshells.[13]

2.2. Crosslinked Enzyme Clusters on Magnetic Nanoparticles

Figure 6 schematically shows the covalent attachment(CA) of the enzyme molecules and the fabrication of cross-

linked enzyme clusters(CECs) on the surface of theamino-functionalized mag-netic nanoparticles. Themajor difference between theCA and CEC approaches isthe sequence of glutaralde-hyde (GA) treatment andenzyme addition. In the CAapproach, the amino-func-tionalized magnetic nanopar-ticles are treated with GA,and the unreacted excess GAis washed away. Then, theenzyme solution is added forthe covalent attachment ofthe enzyme molecules via theGA linker. On the otherhand, in the CEC approach,we first added the enzymesolution, and then GA wasadded to the mixture of en-zymes and magnetic nano-particles to form the enzymeclusters immobilized on themagnetic nanoparticles.

We prepared the CA andCEC samples of two modelenzymes: lipase (LP) and a-chymotrypsin (CT). Themagnetic-nanoparticle con-tent was determined by theFe content from inductivelycoupled plasma atomic emis-sion spectroscopy (ICP-AES)measurements. The enzyme

loading was calculated from the elemental analysis of nitro-gen because the enzyme was the only source of nitrogen inthe final samples and the GA treatment results in insolubleforms of crosslinked enzymes. As a control experiment, theelemental analysis of the magnetic nanoparticles and en-zymes was also performed. As shown in Table 1, the enzymeloadings in the CECs were always higher than those in theirCA counterparts. For example, the enzyme loadings of CA-LP and CEC-LP were 18% (w/w) and 24% (w/w), respec-tively. In the case of CT, the loadings of the CA and CECsamples were 6% (w/w) and 18% (w/w), respectively. TheGA treatment in the CEC approach is expected to formlarge and multiple-layered enzyme clusters on the surfacesof the magnetic nanoparticles via intermolecular crosslink-ing,[8] which is manifested in the high enzyme loadings inthe CECs. On the other hand, the CA approach cannot im-mobilize the enzyme molecules to produce more than amonolayer of coverage, leading to a limited enzyme loadingper unit surface area of the magnetic nanoparticle.

Very interestingly, the specific activities of the CEC sam-ples, which are normalized per unit weight of enzyme, aresignificantly higher than those of the CAs (Table 1). Themultiple layers of enzymes on the CEC samples are expect-

Figure 4. a,b) TEM images of magnetite nanoparticles coated with a silica shell functionalized by aminogroups through a one-step reaction. c) Picture of the solution prepared by dissolving magnetite nanopar-ticles coated with aminopropyl-functionalized silica shells in a polar (water) and an organic solvent(hexane). d) Pictures showing that aminopropyl-functionalized iron oxide nanoparticles can be easilyattracted by an applied magnetic field. e) IR analysis of silica-coated magnetite nanoparticles functional-ized with amino groups.



ed to place more limitations on the substrate transfer thanthe CA samples with monolayer coverage. In addition, theGA crosslinking in the CEC approach can also reduce the

enzyme activity. We presume that the improvement of thespecific activities for the CEC samples may be explained bythe reduction in enzyme inactivation during the enzyme im-mobilization.[14] Shear stress and collisions caused by thesevere shaking during the immobilization and subsequentwashing processes could damage the enzyme molecules. Themultiple-point covalent linkages and multilayered structureof the CECs seem to protect the enzyme molecules fromthis shear stress and collision damage. On the other hand,nearly all of the enzyme molecules of the CA samples aredirectly exposed to the solution and can therefore be easilydamaged. The specific enzyme activities of the CA andCEC samples were 15–34% of those of the free enzymes.This high recovery of enzyme activity could be achieved bythe careful optimization of the various immobilization pa-rameters, such as the GA concentration, incubation time,shaking condition, temperature, and washing. This suggeststhat the inactivation of the enzyme during enzyme immobi-lization plays a key role in determining the specific activityas well as the activity recovery, supporting the above hy-pothesis that the improved specific activities of the CECsamples are due to the prevention of enzyme inactivationduring immobilization. The apparent enzyme activities perunit weight of magnetic nanoparticles show a bigger differ-ence between the CA and CEC samples because bothenzyme loadings and specific enzyme activities of the CECsamples are higher than those of CAs (Table 1).

Figure 7 shows the stabilization of the enzyme activity inthe form of the CEC samples. Regardless of the enzymes,the CEC samples show negligible enzyme inactivationunder the vigorous shaking at 200 rpm (vials in a horizontalposition) for 16 days. This shaking condition is very harsh,when compared to most previous reports, in which the sta-bility was generally checked under a static condition. Forpractical industrial applications, it is critical to preserve theinitial activity under harsh shaking conditions, since most ofthe reactions using immobilized enzymes are conductedunder shaking to reduce the mass-transfer limitations of thesubstrates and products. The CA samples showed a marginalimprovement of their enzyme stability when compared tothe free enzymes, but were inactivated by one order of mag-nitude after 16 days of incubation under the same condi-tions. The stabilization of enzyme activity in the CEC ap-proach can be explained by the multiple-point attachmentof the enzyme molecules in the form of crosslinked clusters,which effectively prevents the denaturation of the enzymemolecules.[15]

To demonstrate the usefulness of magnetic separation,both CA and CEC samples on magnetic nanoparticles wererecycled by using a simple magnet-capture step (Figure 8).

Figure 5. a) Magnetization versus applied magnetic field for silica-coated magnetite nanoparticles (5-nm core/5-nm shell) at 2 and300 K. b) Magnetization curves at 2 K for 5-nm magnetite (circles)and silica-coated 5-nm magnetite (squares), showing remnant mag-netizations. c) Temperature dependence of magnetization for 5-nmmagnetite with a 5-nm silica shell. We measured the magnetizationunder zero-field cooled and field-cooled under an applied field of100 Oe.

Table 1. The enzyme loadings and activities of CAs and CECs on magnetic nanoparticles.

Samples CA-LP CEC-LP CA-CT CEC-CT

Enzyme loading [%, w/w] 18 24 6 18Specific activity per unit weight of enzyme [mm h�1 mg–1] 1.20 1.89 0.10 0.16Specific-activity ratio of sample-to-free enzyme [%] 22 34 15 23Activity per unit weight of magnetic nanoparticles [mm h�1 mg–1] 0.27 0.60 0.007 0.033



The enzyme reaction was initiated by adding an aliquot ofsubstrate solution into a suspension of each sample. Thesample was shaken at 200 rpm for the enzyme reaction, gen-

erating a yellow-coloredproduct as shown in Fig-ure 8a, and the initial ratewas determined from thetime-dependent increase ofabsorbance at 410 nm. Aftereach activity measurement,the sample was washed atleast six times by aqueousbuffer to remove all the sub-strate and products from thesample, before its use in thenext run. The CA sampleshows a continuous loss ofenzyme activity, which resultsfrom continuous inactivationof enzymes under the shearstress of shaking. On theother hand, the CEC samplesclearly stabilize the enzymeactivity for many iterativecycles of enzymatic reaction,magnetic separation, andwashing. For example, after

their recycled use at 30-time iterative cycles, CEC-LP andCEC-CT showed a marginal inactivation by maintaining90% and 88% of initial activities, respectively (Figure 8band c). The GA crosslinking between enzyme molecules sta-bilizes the enzyme activity during the iterative recycling ofCEC samples.

3. Conclusions

Uniformly sized silica-coated magnetic nanoparticleswere synthesized in a simple one-pot process using reversemicelles as nanoreactors. The synthesis was achieved bysimply adding a silica source (TEOS) to the reverse micellescontaining the magnetite nanoparticles. Several grams ofmonodisperse silica-coated magnetic nanoparticles could besynthesized without going through any size-selection pro-cess. This simple synthesis for silica-coated magnetic nano-particles should pave the way to the active use of silica-coated magnetic nanoparticles in enzyme technology andother bioapplications, such as drug targeting, magnetic reso-nance imaging, and protein immobilization. As an exampleof bioapplication, enzyme molecules were crosslinked toform clusters on the surface of the magnetic nanoparticles.The resulting hybrid CEC–magnetite composites were mag-netically separable, highly active, and stable under harshshaking conditions (200 rpm) for more than 15 days andunder many recycled uses. This enzyme system will make agreat impact in various enzyme applications such as fine-chemical synthesis, pharmaceuticals, commodity catalysts infood processing, and bioremediation. In particular, the mag-netic separation using a magnet or electricity-driven mag-netic field will make the recycling of enzymes much easier,and the automation of biocatalysis can be realized with aneconomical advantage.

Figure 6. Schematic image of the covalent attachment (CA) of enzymes and crosslinked enzyme clusters(CECs) on magnetic nanoparticles.

Figure 7. a) The stabilities of free LP, CA-LP, and CEC-LP on magneticnanoparticles in an aqueous buffer solution (100 mm sodium phos-phate, pH 7.4) under shaking (200 rpm and vials in a horizontalposition). b) The stabilities of free CT, CA-CT, and CEC-CT on nanopar-ticles in an aqueous buffer solution (100 mm sodium phosphate,pH 7.8) under shaking (200 rpm and vials in a horizontal position).



4. Experimental Section

Materials: Sodium dodecylbenzenesulfonic acid (NaDBS),xylene (isomers plus ethylbenzene, 98.5+%), hydrazine (N2H4,35 wt% solution in water), and iron(II) chloride tetrahydrate(FeCl2·4H2O, 99%) were purchased from Aldrich (Milwaukee, WI).Iron ACHTUNGTRENNUNG(III) nitrate nonahydrate (Fe ACHTUNGTRENNUNG(NO3)3·9H2O, 98%) was purchasedfrom Shinyo Pure Chemicals, and TEOS (98%) was purchasedfrom ACROS. CT, 4-nitrophenyl butyrate (4-NB), N-succinyl-Ala–Ala–Pro–Phe p-nitroanilide (TP), GA, and N,N-dimethylformamide(DMF) were purchased from Sigma (St Louis, MO), while LP waspurchased from Fluka. All other reagents and solvents were pur-chased from Aldrich (Milwaukee, WI, USA), and were of the high-est grade commercially available.

Characterization of the materials: The silica-coated magneticnanoparticles were characterized by TEM and XRD. TEM was per-

formed on a Jeol EM-2010 microscope. The XRD patterns wereobtained with a Rigaku D/Max2500 diffractometer equippedwith a rotating anode and a CuKa radiation source (l =

0.15418 nm). The IR spectra were collected on Mettler Toledo Re-actIR 4000 and Bomem DA 8 spectrometers. To estimate the ele-ment contents of the enzymes and magnetic nanoparticles in thefinal composite materials, elemental analysis (C, H, N, S, Fe)was performed using an elemental analyzer (EA1110, CE Instru-ment, Italy) and ICP-AES (SHIMADZU, Japan).

Synthesis of silica-coated magnetic nanoparticles: A micro-emulsion was prepared by dissolving 1.75 g of sodium dodecyl-benzenesulfonate in 15 mL of xylene by sonication. Metal saltswere dissolved in deionized (DI) water to enhance the hydrolysisreaction of TEOS. The salt solution was composed of FeCl2·4H2O,Fe ACHTUNGTRENNUNG(NO3)3·9H2O ([Fe3+]/ACHTUNGTRENNUNG[Fe2+] = 2) and DI water. In the case of the7-nm magnetite core, 1 mmol of FeCl2·4H2O, 2 mmol of Fe-ACHTUNGTRENNUNG(NO3)3·9H2O, and 0.9 mL of DI water were used, while 0.75 mmolof FeCl2·4H2O, 1.5 mmol of Fe ACHTUNGTRENNUNG(NO3)3·9H2O, and 0.75 mL DI waterwere used for the preparation of the 5-nm magnetite core. Thissalt solution was added to the microemulsion under vigorousstirring, and the solution was kept at room temperature forabout 12 h to allow for its stabilization. Then, the reverse-micellesolution was slowly heated to 90 8C under continuously flowingargon gas for an hour. Then, 1 mL of hydrazine (34 wt% aqueoussolution) was injected into the solution. These particles wereaged at 90 8C for 3 h, and then cooled down to 40 8C within anhour. The temperature was maintained at 40 8C to prevent thecomplete evaporation of the water in the hydrophilic phase con-taining the magnetite nanoparticles. After the temperature of themagnetite solution reached 40 8C and became stabilized, TEOSwas injected into the mixture of the as-synthesized magnetiteand xylene under vigorous stirring. At this point, the as-synthe-sized magnetite nanoparticles were stabilized by the surfactantmolecules and readily dispersible in organic solvents such ashexane and toluene. The TEOS molecules, which were initiallymixed with the organic xylene phase, started to hydrolyze in thewater region of the reverse micelles to form amorphous silicashells on the surface of the magnetite nanoparticles. The silica-coated magnetite nanoparticles were segregated from the xylenephase and dispersed well in water. The amount of TEOS injectedat the final step determined the thickness of the silica shells.For example, 2 mL of TEOS injected into 5 nm of magnetite solu-tion yielded a shell thickness of 5 nm, whereas the injection of0.7 mL of TEOS produced a shell thickness of 2 nm, and the in-jection of 0.3 mL of TEOS produced a very thin silica shell layer,which could not be observed by TEM.

Silica coating with amine functional groups on the surface ofthe magnetic nanoparticles: Two different methods were adopt-ed to functionalize the surface of the silica with amine groups:single-step functionalization or post-grafting of amino groupsonto the magnetite@silica nanoparticle surfaces. The first wassuccessfully accomplished by the simple injection of a mixtureof TEOS and APS in a one-to-one volume ratio. The secondmethod was accomplished as follows: 1 mL of magnetite@silicananoparticle solution in ethanol was mixed with 5 mL of tolueneto produce a homogeneously mixed solution; the ethanol wasvaporized in a vacuum. Then, 0.2 mL of APS was added, and themixture was slowly heated to 60 8C and kept at this temperaturefor one hour. The particles were made to settle by centrifugation

Figure 8. a) An iterative cycle of CEC-CT suspension, enzyme reaction,magnetic capture, and excessive washing using a magnet. Stabilitiesof b) CA-LP and CEC-LP and c) CA-CT and CEC-CT under recycled use.



after adding octane. The final samples were rigorously washedwith aqueous buffer (100 mm sodium phosphate, pH 7.4).

Preparation of CECs on magnetic nanoparticles: To developcrosslinked clusters of LP on the magnetic nanoparticles, 4 mgof LP was mixed well with 4 mg of the amino-functionalized mag-netic nanoparticles in 2 mL of aqueous buffer (100 mm sodiumphosphate, pH 7.4). Then, concentrated GA solution was addedto the mixture to obtain a final GA concentration of 0.1% (w/v).The mixture was incubated at 4 8C on a rocking chair (60 rpm) for17 h. The crosslinked clusters of LP on the magnetic nanoparti-cles were captured using a magnet, the solution was decanted,and fresh buffer solution was added. This washing process wasiterated five times. The unreacted aldehyde groups were cappedby incubating the solution in 100 mm Tris-HCl buffer (pH 7.4) for30 min. Finally, the samples were excessively washed until noenzyme was observed in the washing solution. The final sampleswere stored in a buffer at 4 8C until they were used for the activi-ty and stability measurements. The crosslinked clusters of CT onthe magnetic nanoparticles were prepared in a similar way.

As a control sample, magnetic nanoparticles with covalentlyattached enzymes were prepared. The amino-functionalized mag-netic nanoparticles were incubated in 0.1% GA solution for30 min. After the GA was excessively washed in buffer solution,the sample was incubated in 2 mg mL�1 of enzyme solution forthe covalent attachment of the enzyme molecules onto the mag-netic-nanoparticle surface. After 17 h of incubation, the unreact-ed aldehyde groups were capped in Tris buffer, and the finalsample was excessively washed, as described above. Elementalanalysis (C, H, N, S, Fe) was performed to measure the loadingof enzyme on the magnetite@silica nanoparticles. For elementalanalysis, the resulting samples were freeze-dried.

Measurement of enzyme activity and stability : The activitiesof LP and CT were measured by the hydrolysis of 4-NB and TP inaqueous buffer (100 mm sodium phosphate; pH 7.4 for LP whilepH 7.8 for CT), respectively. The formation of the products wasevaluated by measuring the increase in absorbance at 410 nm(A410) with a Cary 5G UV-vis-NIR spectrophotometer (Varian,Palo Alto, CA), and the enzyme activity was calculated from thetime-dependent increase of A410.

The enzyme stability was determined in aqueous bufferunder vigorous horizontal shaking (200 rpm, side by side) atroom temperature. The residual activity at each time point wasmeasured by the method described above. After each measure-ment of the residual activity, the sample was washed threetimes with aqueous buffer to remove all of the substrate andproducts from the sample. The excessively washed sample wasreused for the subsequent measurement of the residual activityafter incubation under vigorous shaking. The relative activity isdefined as the ratio of the residual activity to the initial activity.

Acknowledgements

T.H. would like to thank the financial support of the KoreanMinistry of Science and Technology through the National Cre-ative Research Initiative Program of the Korea Science andEngineering Foundation (KOSEF). J.K. would like to thank the

U.S. Department of Energy (DOE) LDRD funds administered bythe Pacific Northwest National Laboratory. The research wasperformed in part at the W. R. Wiley Environmental MolecularSciences Laboratory, a national scientific-user facility spon-sored by the DOE’s Office of Biological and Environmental Re-search and located at the Pacific Northwest National Labora-tory.

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Received: June 28, 2007



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Simple Synthesis of Functionalized Superparamagnetic Magnetite/Silica Core/Shell Nanoparticles and their Application as Magnetically Separable High-Performance Biocatalysts