PAPER www.rsc.org/materials | Journal of Materials Chemistry

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Tb-doped iron oxide: bifunctional fluorescent and magnetic nanocrystals†

Yunxia Zhang,‡ Gautom Kumar Das,‡ Rong Xu* and Timothy Thatt Yang Tan*

Received 30th September 2008, Accepted 16th March 2009

First published as an Advance Article on the web 20th April 2009

DOI: 10.1039/b817159a

A new class of fluorescent and superparamagnetic bifunctional nanocrystal has been successfully

prepared by a facile, non-hydrolytic method. The synthesized Tb-doped g-Fe2O3 nanocrystals are

highly monodispersed with a diameter of 13 nm, show superparamagnetic behaviour with saturation

magnetism strength of 30 emu g�1, and exhibit photoluminescence at room temperature. The

nanocrystals are amine-functionalized to render them water-dispersible and ease of further

functionalization by other biomolecules. In vitro cytotoxicity tests indicate that these nanocrystals are

non-toxic. Nanocrystals of such bifunctionality are envisioned to have potential as probes in integrated

imaging technique, of which at least two imaging modalities (for example magnetic resonance imaging

(MRI) and fluorescence microscopy) are combined to provide enhanced visualization at tissues and

cellular levels.

1. Introduction

Magnetic materials have been found with a variety of applica-

tions due to their interesting properties in many traditional areas

including magnetic data storage, ferrofluid technology, magne-

torheological polishing, and energy storage. Recent advances in

superparamagnetic colloidal synthesis has enhanced their

potential for many other fields related to biology, pharmacy, and

diagnostic, including magnetic fluids, magnetic bioseparation of

labelled cells and biological entities,1,2 magnetic resonance

imaging (MRI) contrast agents,3 targeted drug delivery,4 radio

frequency induced destruction of cells and tumours via hyper-

thermia5 and biosensing.6 For example, Zhao et al.7 have

demonstrated a highly efficient collection of a trace amount of

DNA/mRNA from a mixture, down to femtomolar concentra-

tions, by using such nanoparticles. In a targeting drug-delivery

system, the magnetic labelling of drugs could be easily adminis-

tered and transported to the tumour under the guidance of an

external magnetic field, leading to a safer and more effective

tissues-specific release of drugs.8

In bioanalysis, luminescence has been extensively exploited for

detection and sensing. There are several classes of materials that

are currently employed as fluorescent emitters/probes, including

organic, metal–organic dye molecules, fluorescent proteins,

semiconductor quantum dots (QDs), and polymer/dye-based

nanoparticles, etc.9 Usually, dye molecules suffer from photo-

bleaching and quenching due to interactions with solvent mole-

cules and reactive species such as oxygen or ions dissolved in

solution when they are exposed to a variety of harsh

School of Chemical and Biomedical Engineering, Nanyang TechnologicalUniversity, Singapore 637459, Singapore. E-mail: tytan@ntu.edu.sg;RXu@ntu.edu.sg; Fax: +65 6794 7553; Tel: +65 6316 8829

† Electronic supplementary information (ESI) available: Energydispersive X-ray spectroscopy (EDS) analysis of ZnS coated Tb-dopedg-Fe2O3 nanocrystals (Fig. S1). XRD pattern of undoped and dopediron oxide nanocrystals (Fig. S2). Percentage weight loss of theas-prepared Tb-doped iron oxide nanocrystal using TGA (Fig. S3). SeeDOI: 10.1039/b817159a

‡ Y. Z. and G. K. D. contributed equally to this article.

3696 | J. Mater. Chem., 2009, 19, 3696–3703

environments.10 Luminescent QDs exhibit higher photostability

and show narrower emission peaks compared to organic fluo-

rophores.11 However, they are generally composed of heavy

metal ions such as Pb2+ or Cd2+, so their use exposes researchers

and experimental systems to these toxic materials as well as

generating a toxic waste stream into the environment.12 To solve

these issues, it is important to develop new photostable and

nontoxic or less toxic labelling materials.

Multi- or bifunctional nanomaterials such as hybrid nano-

crystals or nanocomposites can provide a new platform for both

diagnostics and treatment of disease due to their enhanced

functionality and multifunctional properties in contrast to their

single counterparts. Efforts have been devoted to synthesize and

investigate nanocomposite materials which comprise fluorescent

nanocrystals incorporated into silica shells,13,14 magnetic nano-

crystals attached to fluorescent nanocrystals by means of ligand

molecules,15 or magnetic and luminescent nanoparticles encap-

sulated into silica spheres.16,17 Another strategy is to produce

hybrid nanostructures by colloidal syntheses, where each nano-

crystal could be made of any desired inorganic materials

purposely assembled together for tailored applications.18–20

These methods of tailoring multifunctional nanocomposites are

rather tedious in the sense that they involve synthesizing the

functional nanocrystals separately, and then putting them into

one entity.

In our previous work, we have achieved doping and co-doping

of rare-earth ions into oxide nanomaterials.21,22 The rare-earth

ions exhibit intense narrow-band intra-4f luminescence in a wide

range of hosts. The shielding provided by the 5s2 and 5p6 elec-

trons causes rare-earth radiative transition in solid hosts to

resemble those of the free ions. They are hence a good choice for

biological labelling and medical diagnostics due to their large

Stokes shift, sharp emission spectra, long lifetime, multiphoton

and up-conversion excitation, low toxicity and reduced photo-

bleaching over semiconductor nanocrystals like quantum dots

and organic phosphors.23–26 In this work, we demonstrate a new

strategy to fabricate novel Tb-doped g-Fe2O3 nanocrystals,

which combine two useful functions, superparamagnetism and

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luminescence into the same nanocrystal. We have further

improved the luminescence of the synthesized nanocrystals by

affording surface-passivation with a thin layer of ZnS coating,

demonstrated amine surface-functionalization which renders this

bifunctional nanocrystal suitable for further functionalization

for bio-conjugation, and conducted in vitro cytotoxicity studies

of the synthesized nanocrystals.

2. Experimental

2.1. Chemicals

All chemicals were used as received without further purification.

Terbium(III) chloride hexahydrate (99.9%), 1-octadecene (tech.

90%), diethylzinc (1.0 M solution in hexanes), tetramethy-

lammoniumhydroxide (25 wt% in methanol) (TMAH) and Igepal

CO-520 (polyoxyethylene(5)nonylphenylether) were purchased

from Aldrich. Iron(III) chloride hexahydrate (98%) and oleic acid

(tech. 90%) were purchased from Alfa Aesar. NaOH (reagent

grade, 97%, beads), 3-aminopropyltrimethoxysilane (APS) (97%)

trioctylphosphine (TOP), hexamethyl disilthiane were purchased

from Fluka. Ethanol, hexane, cyclohexane and chloroform were

of analytical reagent grade.

2.2. Tb-doped g-Fe2O3 nanocrystals

Magnetic-fluorescent Tb-doped g-Fe2O3 nanocrystals were

synthesized based on a modified method developed by Hyeon

et al.27 In a typical synthesis process, 2 mmol (0.54 g) of

FeCl3$6H2O and 0.2 mmol (0.075 g) of TbCl3$6H2O was dissolved

in 3 mL of deionized water. To this mixture, 1.9 mL (6.0 mmol) of

oleic acid, 7 mL of hexane and 4 mL of ethanol were added and

stirred at room temperature for 30 min. Then, 6.25 mmol (0.25 g) of

NaOH was added to the reaction mixture and heated with stirring

in a close vessel at 70 �C for 4 h. The resultant solution was allowed

to form two different layers in a separatory funnel. The top organic

layer containing Fe (Tb)-oleate complex was collected, washed with

30 mL of water and then heated at 70 �C overnight in order to

remove the hexane. The sticky Fe (Tb)-oleate precursor was

dispersed in 1.5 mL (4.7 mmol) of oleic acid and 12 mL (37.5 mmol)

of 1-octadecene. The mixture solution was degassed with N2 for 30

min at room temperature. After that, the mixture was heated to 320�C at 3 �C min�1 and maintained at that temperature for 30 min

under N2 flow. Afterwards, the solution was cooled to room

temperature and precipitated by excess ethanol. The precipitate was

collected by centrifugation and the supernatant decanted. The

isolated solid was re-dispersed in hexane and then precipitated with

ethanol. The precipitation–re-dispersion process was repeated for

several times to purify the as-prepared nanocrystals. The above

procedure was also carried out in the absence of Tb source for the

synthesis of undoped g-Fe2O3 nanocrystals.

2.3. ZnS coated Tb-doped g-Fe2O3 nanocrystals

The as-prepared oxide nanocrystals were dried at 70 �C overnight.

A method similar to literatures28,29 was adopted to form ZnS

coating onto Tb-doped g-Fe2O3 nanocrystals. 30 mg of dried

nanocrystals was dispersed in 2 g (5.4 mmol) of trioctylphosphine

(TOP). The solution mixture was then degassed with N2 for 30 min

at room temperature and slowly heated up to 280 �C under N2

This journal is ª The Royal Society of Chemistry 2009

flow. A solution of 250 mL (1.185 mmol) of hexamethyl disilathiane

and 1 mL (5.88 mmol) of diethylzinc pre-mixed in 2 g (5.4 mmol) of

TOP was injected very slowly into the hot reaction mixture and the

reaction was kept at that temperature for 1 h under N2.

2.4. Quantum yield (QY) measurement

QY was estimated by comparing the integrated emission inten-

sity of the nanocrystals to that of reference organic dye (Fluo-

rescein QY ¼ 95%) at the same optical density and excitation

wavelength.30

2.5 Amine functionalizations

Silanization and amine functionalization were carried out in

a similar process described by Selvan et al.31 In a typical process,

nanocrystals dispersed in hexane were separated by centrifuga-

tion and 4 mg of the nanocrystals were re-dispersed in chloro-

form. Micelles were prepared by dissolving 0.2 g (0.453 mmol) of

Igepal CO-520 in 4 mL of cyclohexane and the mixture was

stirred vigorously for 1 h. Next, 200 mL of nanocrystals in

chloroform was added to the mixture and stirred for 15 min.

Subsequently 50 mL (0.286 mmol) of aminopropyl trimethox-

ysilane (APS) was added and the mixture was stirred for another

1 h. Then, 20 mL (0.19 mmol) of tetramethylammonium

hydroxide (TMAH) in methanol was added. After 1 h of stirring,

10 mL of deionized water was added and stirred for another 30

min. At this stage, some globules were formed and settled at the

bottom of the flask leaving a transparent organic phase at the

top. The transparent organic phase was then discarded and

globules containing the nanocrystals were washed with chloro-

form and ethanol for the complete removal of excess surfactant

and other reactants from the surface. The silica-coated nano-

crystals were then dispersed in deionized water.

2.6. In vitro cytotoxicty studies

Three types of nanocrystals (CdSe, g-Fe2O3 and Tb-doped

g-Fe2O3) were synthesized and then amine-functionalized. CdSe

nanocrystals were synthesized using methods described in liter-

ature.32 Three types of sample solutions were prepared with the

synthesized nanocrystals: (1) 0.25 mg mL�1 dissolved nano-

materials in phosphate buffer solution (PBS), (2) leachate of

nanomaterials at 70 �C for 24 h in PBS, and (3) leachate of

nanomaterials at 121 �C for 1 h in PBS. Samples from (1) were

used in direct contact method of which the nanomaterials

were incubated directly with the cells. Samples from (2) and (3)

were prepared according to American National Standard ISO

10993-5 for quantification and identification of degradation

products from biomaterials. Two most extreme leaching condi-

tions (at 70 and 121 �C) were chosen out of the four prescribed in

the Standard using 0.25 mg mL�1 as the starting nanomaterial

concentrations. Cell viability test was performed to evaluate the

cytotoxcity of the samples. HepG2 cells were seeded at a seeding

density of 1 � 104 cells mL�1 and cultured with 125 mL well�1

medium in 96-well flat-bottom microassay plates for 24 h before

adding the sample solution. A volume of 25 mL of the sample

solution was added into each well and the cells were cultured at

37 �C for 24 and 48 h. To evaluate cell viability, 10 mL of alamar

blue was added to the culture media and incubated for 4 h at

J. Mater. Chem., 2009, 19, 3696–3703 | 3697

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37 �C. The optical fluorescence (excitation: 530 nm, emission:

590 nm) was obtained by a GENios Pro multilabel counter

(Tecan). The experiments were repeated three times to ensure

reproducibility.

2.7. Characterizations

The as-prepared samples were characterized by powder X-ray

diffraction (XRD) (D8 X-ray diffractometer), transmission

electron microscopy (TEM), high resolution TEM (HRTEM)

(JEOL JEM-3010) and Energy Dispersive X-ray Spectroscopy

(EDS) supported by TEM. X-Ray photoelectron spectroscopy

(XPS) investigation was conducted on a VGESCALAB 250

spectrometer using a monochromatic Al Ka X-ray source

(1486.6 eV). All binding energies (BEs) were referenced to the C

1s peak (BE ¼ 284.6 eV) arising from surface hydrocarbons.

Photoluminescence spectra were collected on a Shimadzu RF-

5301 PC Spectrofluorophotometer using 150 W xenon lamp as

an excitation source. The magnetic properties of these iron oxide

nanocrystals were studied using a vibrating sample magnetom-

eter (VSM) (Lake Shore, 7300). Concentrations of Cd, Fe, and

Tb ions in leachate solutions were determined using High

Dispersion Induction Coupled Plasma Optical Emission Spec-

troscopy ICP-OES (Teledyne Prodigy). Thermogravimetric

analysis was performed using a Diamond TG/DTA (Perkin

Elmer). Dynamic light scattering (DLS) experiments were per-

formed with a Brookhaven 05-LHP-928 laser light scattering

system (He–Ne laser, 35 mW).

3. Results and discussion

Fig. 1a shows a typical TEM image of as-synthesized Tb-doped

g-Fe2O3 samples. It is easily seen that almost all the nanocrystals

are spherical in shape and highly monodispersed with an average

diameter of 13 nm. The clear lattice fringes in the inset show their

highly crystalline nature of the g-Fe2O3 nanoparticles. The

distance between two adjacent planes is measured to be about

0.29 nm, corresponding to (220) lattice planes in the spinel-

structured g-Fe2O3. The composition of these nanocrystals was

also analyzed by energy dispersive X-ray spectroscopy (EDS).

Fig. 1b shows that these nanocrystals are composed of C (from

the surfactant and carbon film on TEM copper grid), Fe, O and

Tb. The peaks of Cu element are from the copper TEM grid.

Quantitative analysis shows that the atomic ratio of Fe to Tb is

about 10.5 : 1.

Fig. 1 (a) A typical TEM image of Tb-doped g-Fe2O3 nanocrystals; the

inset showing HRTEM image of a single nanocrystal. (b) Energy

dispersive X-ray spectroscopy (EDS) result of the nanocrystals.

3698 | J. Mater. Chem., 2009, 19, 3696–3703

The X-ray powder diffraction pattern of the as-prepared

sample shown in Fig. 2a confirms that all the peaks match very

well with the XRD pattern of g-Fe2O3 (maghemite) (PDF no. 04-

0755). The diffraction peaks at 30.3, 35.5, 43.1, 53.5 57.1 and

62.5� can be indexed as (220), (311), (400), (422), (511) and (440)

planes of g-Fe2O3. The broadening of the peaks indicates small

crystalline size of the resulting iron oxide particles. As calculated

using Sherrer’s formula,33 the average size of these iron oxide

nanoparticles is about 12.8 nm, which is quite consistent with the

result obtained from TEM analysis. The hydrodynamic size of

the colloidal suspension was also obtained using dynamic light

scattering (DLS) (as shown in Fig. 2b). According to the particle

size distribution plot, the average particle size is about 12.7 nm,

which is close to the results from TEM and XRD. XRD patterns

for Tb-doped and undoped g-Fe2O3 are also compared (ESI,

Fig. S2).† Comparing the two patterns, it can be observed that

there is a slight shift towards the lower angles for the Tb-doped

sample. Due to a larger ionic radius of Tb3+ cation compared to

that of Fe3+ cation, such a phenomenon indicates that solid

solution was likely formed by incorporating Tb3+ into the

structure of g-Fe2O3.

X-Ray photoelectron spectroscopy (XPS) is used to examine

the oxidation states of Fe and Tb on the surface of these nano-

crystals. The representative XPS survey spectrum is shown in

Fig. 3a. The peaks of the C 1s, O 2p and Fe 2p observed indicate

the organic coating (oleic acid) on the surface of iron oxide

nanoparticles. The binding energies at 710.9 and 724.7 eV are

attributed to Fe 2p3/2 and Fe 2p1/2 core-level electrons, respec-

tively, in good agreement with the values reported for g-Fe2O3

but not for Fe3O4.34,35 The XPS spectrum of Tb 4d5/2 of the as-

prepared sample is shown in Fig. 3d. A single peak at 149.5 eV

corresponds to Tb in the trivalent state.36

The room temperature photoluminescence (PL) spectra of Tb-

doped g-Fe2O3 nanocrystals dispersed in hexane are shown in

Fig. 4. The emission spectra at 235 nm excitation display four

sharp emission bands, which can be assigned to 4f / 4f transi-

tions within Tb3+ ions. The most intense peak at 545 nm corre-

sponds to the 5D4 / 7F5 transition. The other peaks correspond

to transitions from 5D4 to 7F6, 7F4 and 7F3, respectively.

Although the emission bands suggest the presence of Tb3+ ions

in g-Fe2O3 nanocrystals, the possibility of Tb3+ ions adsorbed at

the surface and bound to organic ligands rather than doped in

the g-Fe2O3 matrix cannot be ruled out completely. Considering

this possibility, a surface modification procedure was carried out:

the Tb-doped g-Fe2O3 nanocrystals were surface-coated with

a thin layer of ZnS. TEM image (Fig. 5) shows a thin layer of ZnS

shell forms over the nanocrystals while the inset shows uniform

dispersity of the ZnS coated nanocrystals in the solution. EDS

analysis (ESI Fig. S1)† of the ZnS coated nanocrystals was also

carried out which shows that nanocrystals are composed of C,

Fe, O, Tb, Zn, S and P. Carbon (C) and phosphorous (P) peaks

are due to the surfactants used (oleic acid and trioctylphosphine,

respectively) in the synthesis process.

The PL spectra of the nanocrystals (Fig. 6) before and after

ZnS coating suggest that ZnS-coating enhances the PL intensity

of the nanocrystals. The quantum yield (QY) was estimated to be

2.40% for Tb-doped g-Fe2O3 nanocrystals using 0.1 M NaOH-

solubilized fluorescein (QY ¼ 95%) as a reference dye, which is

similar to the reported value (2.5(�1) � 10�2 for CdS: Tb

This journal is ª The Royal Society of Chemistry 2009

Fig. 2 (a) XRD pattern of as-prepared Tb-doped g-Fe2O3 nanocrystals, showing the cubic spinel structures; (b) particle size distribution of Tb-doped

g-Fe2O3 nanocrystals.

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nanocrystals) by Chengelis et al.37 As expected, the QY of ZnS-

coated Tb-doped g-Fe2O3 nanocrystals was increased to 2.99%.

The difference in luminescence properties can be ascribed to

quantum confinement effect, various dimensions, morphologies

and crystal structure. Previous studies elucidated that surface

states play a key factor for the occurrence of band-gap states that

quench the excitation luminescence.38,39 ZnS is a higher bandgap

material which may help to enhance the fluorescence through the

passivation of the nanocrystalline surface.15 The ZnS shell can

reduce luminescence quenching at the nanocrystal surface and

increase the stability of nanocrystal to photochemical corrosion,

which improve the QY. Here, the increase of QY after ZnS

Fig. 3 XPS spectra of the as-prepared Tb-doped g-

This journal is ª The Royal Society of Chemistry 2009

coating is not very significant. The PL spectrum of ZnS-coated

Tb-doped g-Fe2O3 also strongly evidences the incorporation of

Tb3+ ions in the g-Fe2O3 matrix instead of present as adsorbates

on the nanocrystal surface.40 For comparison, the QY for ZnS

coated CdSe quantum dots was measured to be 24.5%.

Fig. 7 shows the room-temperature hysteresis magnetization

of undoped, Tb-doped and silanized g-Fe2O3 nanocrystals. The

coercivity values of undoped and Tb-doped g-Fe2O3 nano-

crystals at room temperature are almost negligible at 8.52 G and

3.53 G, respectively, which is a typical characteristic of super-

paramagnetic materials.41 This also indicates that the thermal

energy can overcome the anisotropy energy barrier of a single

Fe2O3: (a) survey; (b) Fe 2p; (c) O 1s; (d) Tb 4d.

J. Mater. Chem., 2009, 19, 3696–3703 | 3699

Fig. 4 Excitation and emission spectra of as-obtained Tb doped

g-Fe2O3 nanocrystals in hexane at room temperature.

Fig. 5 HRTEM image of a single Tb-doped g-Fe2O3 nanocrystal coated

with ZnS layer. Inset: uniform dispersity of the ZnS coated nanocrystals.

Fig. 6 PL spectra of Tb-doped g-Fe2O3 nanocrystals before and after

ZnS coating.

Fig. 7 Room temperature magnetic hysteresis curves for Tb-doped,

undoped and silanized Tb-doped g-Fe2O3. Insert: magnified curves at

low fields.

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particle at room temperature, and that the net magnetization of

the particle assemblies in the absence of an external magnetic

field is zero. The saturation magnetization of Tb-doped g-Fe2O3

3700 | J. Mater. Chem., 2009, 19, 3696–3703

was 30.51 emu g�1, which is slightly lower than those of similar

size nanoparticles prepared by other methods.42 The saturation

magnetization of undoped g-Fe2O3 was found to be similar at

30.39 emu g�1. The lower the saturation magnetization is most

likely attributed to the existence of surfactants on the surface of

g-Fe2O3 nanoparticles. Kodama et al’s studies suggested that

non-co-linear spin structure, which originated from the pinning

of the surface spins and coated surfactant at the interface of iron

oxide, results in the reduction of magnetic moment in such

nanoparticles.43 The amount of weight loss due to the surfactant

layer on our as-prepared nanocrystals was estimated. Based on

the TGA result (ESI Fig. S3)†, it can be estimated that the

percentage loss due to the removal of oleate was around 30%,

indicating that our nanocrystals are covered by oleate groups.

Silanization was carried in a reverse microemulsion to render

the bifunctional nanocrystals water dispersible and ease of

further functionalization for bio-conjugation. A thin layer of

silica was produced on Tb-doped g-Fe2O3 with surface NH2

groups after silanization using aminopropyl trimethoxysilane

(APS). The silanized nanoparticles were well-dispersed in

deionized water at pH 6.95. A proposed structure of the amine-

functionalized nanocrystal (NC) is shown in Fig. 8a. FTIR

spectroscopy was conducted to confirm the presence of silica

layer and surface amine (NH2) group. The FTIR spectrum in

Fig. 8b(i) was obtained for as-synthesized Tb-doped g-Fe2O3

capped with oleic acid. The peaks at 2923 cm�1 and 2852 cm�1 are

due to asymmetric and symmetric C–H stretches in oleic acid.

Peaks at 1709 cm�1, 1562 cm�1, 1450 cm�1 and 1132 cm�1 are

attributed to C]O stretching, C]C stretching, C–O–H in plane

bending and C–O stretching bands, respectively.44 A relatively

small peak at 580 cm�1 is due to Fe–O vibration band. The

spectrum for amine functionalized nanocrystals is shown in

Fig. 8b(ii). The incorporation of silica framework and amine

groups can be qualitatively confirmed by this spectrum. A rela-

tively strong peak at 630 cm�1 is due to bending of N–H bonds

indicating the existence of amine groups. The C–N stretching

vibration, normally observed in the range of 1000–1200 cm�1, is

This journal is ª The Royal Society of Chemistry 2009

Fig. 8 (a) Proposed silanized Tb-doped g-Fe2O3 with exposed amine functional groups; (b) FTIR spectra of (i) as-synthesized Tb-doped g-Fe2O3; (ii)

APS-modified Tb-doped g-Fe2O3 nanocrystals. (c) Particle size distribution of Tb-doped g-Fe2O3 nanocrystals after silica silanization. (d) HRTEM

image of amine functionalized Tb-doped g-Fe2O3 nanocrystals; inset: well-dispersed amine functionalized nanocrystals in water.

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overlaid with the IR absorptions of Si–O–Si in the range of 1130–

1200 cm�1 and of Si–CH2–R in range 1250–1200 cm�1 producing

a wide peak at 1109 cm�1.45,46 Peaks at 1560 cm�1 and 1632 cm�1

are attributed to N–H bending and –NH2 scissoring band,

respectively, while the peak at 3416 cm�1 is due to N–H stretching

band for terminal amine groups. Silanized magnetic nano-

particles show decreased saturation magnetization of 22.11 emu

g�1 (Fig. 7). The coercivity was estimated to be 14.1 G. The mean

hydrodynamic size after silanization and the particle size distri-

bution is presented in Fig. 8c. There is a small increase in mean

particle size and a shift in particle size distribution. The mean size

has increased from about 12.7 nm to 12.9 nm after silanization.

The silanization process only yields a thin layer of silica coating.

TEM image of the silanized nanoparticles (Fig. 8d) shows that

the layer owing to silanization is too thin to be visible under

TEM. The quantum yield of the silanized Tb-doped g-Fe2O3 was

estimated to be 1.95%, which is attributed to less surface

passivation.

The silanized water-dispersible nanocrystals were further used

for in vitro cytotoxicity studies. Cytotoxicity studies were con-

ducted for three different types of nanocrystals for comparison:

cadmium selenide (CdSe) quantum dots, undoped and doped

g-Fe2O3 nanocrystals. The studies were based on conditions

described in American National Standard ISO 10993-5 for

testing of biomaterials. About 42% cell viability was determined

This journal is ª The Royal Society of Chemistry 2009

via direct contact of CdSe after 48 h, indicating toxicity, while no

toxicity was observed for the remaining nanocrystals (Fig. 9a).

For leachate obtained at 70 �C, no significant toxicity was

observed for all nanocrystals tested (Fig. 9b). However, about

71% cell viability was observed for leachates obtained at 121 �C

for CdSe, indicating a decrease in cell metabolism most possibly

due to toxicity. For g-Fe2O3 and Tb-doped g-Fe2O3, almost

100% of the cells were viable, indicating that the nanocrystals

were non-toxic (Fig. 9c). We further determined the concentra-

tions of metal ions in the 121 �C leachate using ICP-OES.

A concentration of 5.82 ppm of Cd ions was found in the leachate

and hence it was deduced that the toxicity was due to the toxic Cd

ions present. Fe and Tb ions were found in the undoped and Tb-

doped g-Fe2O3 leachate at concentrations of 3.32 ppm Fe, and

4.48 ppm Fe and 1.421 ppm Tb, respectively. However, cell

viability in the presence of these nanocrystals was almost 100%,

and hence it was concluded that Fe and Tb ions were not toxic

under the current method of toxicity evaluation. The cytotoxicity

test of the current nanocrystals is preliminary. More studies on

the toxicity of the nanocrystals by using different cell lines

and observing cell morphological change upon exposure to the

nanocrystals have been undertaken.47 In view of the leaching of

Tb ions from the Tb-doped g-Fe2O3, a kinetic study was

conducted by varying the leaching time from 1 to 5 h at 121 �C.

The higher temperature condition at 121 �C is an accelerated test

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Fig. 9 HepG2 cell viability using (a) direct contact method; (b) leachate

obtained at 70 �C; (c) leachate obtained at 121 �C, 24 h, 48 h.

Fig. 10 Variation of PL intensity of Tb-doped g-Fe2O3 with leaching

time. Leaching performed at 121 �C. Inset: variation of PL intensity at

545 nm emission with time.

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condition used to determine what might be expected at longer

periods at a body temperature of 37 �C. The PL intensity at

545 nm transition (5D4 / 7F5) was monitored and the result

shows that PL intensity decreased with longer leaching time since

more ions were leached out (Fig. 10). The PL intensity decreased

to approximately 60% after 2 h and almost diminished to zero

after 5 h.

3702 | J. Mater. Chem., 2009, 19, 3696–3703

4. Conclusions

In summary, a facile method has been developed to synthesize

magnetic-fluorescent bifunctional Tb-doped iron oxide nano-

crystals. These iron oxide nanocrystals are highly monodispersed

and around 13 nm in diameter. The as-obtained nanocrystals are

of superparamagnetism with the saturation magnetization

strength of 30 emu g�1. Besides, these nanocrystals exhibit room

temperature green photoluminescence under 235 nm excitation

wavelength. We have also shown that the nanocrystals can be

amine-functionalized and are non-toxic. The reported bifunc-

tional nanocrystals are envisioned to have potential in integrated

imaging technology, of which MRI and florescence microscopy

can be combined to provide better resolution in tissue and

cellular imaging. We also believe that these water-dispersible

bifunctional nanocrystals could be further engineered to provide

up-conversion emission, which is more effective for deep tissue

imaging. This work is currently in progress.

Acknowledgements

We thank Dr Jeyagowry T. Sampanthar (Institute of Chemical

and Engineering Sciences) for assistance in XPS measurements,

Mr Patrick Lai for assistance in VSM measurements and

discussion, and Miss Teo Ailing for conducting cell viability

experiments. Gautom K. Das gratefully acknowledges a research

scholarship from Nanyang Technological University. This

work is supported by Center of Advanced Bionanosystems

(M61120003), Nanyang Technological University.

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