Materials Chemistry and Physics xxx (2012) 1e9
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Materials Chemistry and Physics
journal homepage: www.elsevier.com/locate/matchemphys
Stable water-soluble iron oxide nanoparticles using Tiron
Katalin V. Korpany a, Fatemah Habib b, Muralee Murugesu b, Amy Szuchmacher Blum a, *
a
b
Department of Chemistry, McGill University, 801 Sherbrooke Street West, Montreal, QC, H3A 0B8 Canada
Department of Chemistry, University of Ottawa, Ottawa, ON, K1N 6N5 Canada
h i g h l i g h t s
< Ligand exchange for Tiron and dopamine resulted in water-soluble iron oxide NPs.
< Particles retained their superparamagnetic properties, crystal structure, and size.
< Analysis of exchange methods highlighted the importance of solvent selection.
< Synthesized nanoparticles can be fully redispersed after being dried and frozen.
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 6 July 2012
Received in revised form
2 October 2012
Accepted 7 October 2012
Success in biological and nanomaterial applications that rely on magnetic iron oxide nanoparticles
(IONPs) often depends on monodispersity, size, and aqueous stability of the synthesized particles. Here
we report a simple and efficient strategy to prepare monodisperse, ultrasmall, water dispersible
superparamagnetic IONPs. Monodisperse IONPs are initially synthesized in organic solvents using oleic
acid as a dispersant. The subsequent ligand exchange of oleic acid for dopamine and Tiron (4,5dihydroxy-1,3-benzenedisulfonic acid disodium salt) allows for superior colloidal stability in aqueous
media. Zeta potential measurements confirm the stability of the nanoparticles upon redispersal in water
or biologically relevant buffers. The synthesized particles also preserve their general shape, size, and
crystallinity after ligand exchange as evidenced by TEM and SAED measurements. Magnetic properties
are also maintained after the ligand exchange as verified by magnetometry and magnetic force
microscopy (MFM). An analysis of potential issues regarding this and other prior ligand exchanges is also
highlighted, which may aid others in future investigations.
Ó 2012 Elsevier B.V. All rights reserved.
Keywords:
Magnetic materials
Nanostructures
Electron microscopy
Magnetic properties
1. Introduction
Aqueous stable superparamagnetic iron oxide nanoparticles
(IONPs) are key components in many current technologies such as
magnetic resonance imaging contrast agents [1,2], magnetic separations [3], drug targeting [1,4], and hybrid inorganiceorganic
nanomaterials [5]. However, as nanoparticle properties depend
on nanoparticle size, stability, and dispersant identity, these
parameters must be strictly controlled for the successful use of
nanoparticles in functional materials.
Current syntheses of magnetic IONPs in aqueous media often
rely on co-precipitation methods that can be run on large scales but
suffer from polydispersity [6]. In some cases, ultrasmall (<15 nm)
nanoparticles are required to facilitate downstream nanomaterials
applications (that require templation on a small size regime) or to
* Corresponding author. Tel.: þ1 514 398 6237; fax: þ1 514 398 3797.
E-mail address: amy.blum@mcgill.ca (A.S. Blum).
maintain desired superparamagnetic properties [7]. As MRI
contrast agents, smaller particles possess a longer half life in the
bloodstream [8]. Furthermore, if particles are less than 10 nm in
diameter they can penetrate the endothelium, thereby increasing
their bioavailability [8]. To this end, the synthesis of ultrasmall
monodisperse IONPs that are stable in aqueous solution is critical to
the development of biological applications.
Highly monodisperse small IONPs are easily synthesized in large
quantities in organic solvents by either thermal decomposition [9]
or modified co-precipitation methods [10], utilizing oleic acid as
a capping agent for stabilization. However, these monodisperse
nanoparticles are only stable in organic solvents and are therefore
unsuitable for biological applications. To render these particles
aqueous stable, chemical modification of the oleic acid capping
group can be performed using oxidizing agents (KMnO4) [11] or
ozonolysis [12]. Unfortunately, the potential for excess oxidation of
the magnetite iron oxide core to maghemite makes these processes
undesirable. Aqueous stable IONPs are typically stabilized using
high molecular weight dispersants such as dextran, polyethylene
0254-0584/$ e see front matter Ó 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.matchemphys.2012.10.015
Please cite this article in press as: K.V. Korpany, et al., Stable water-soluble iron oxide nanoparticles using Tiron, Materials Chemistry and Physics
(2012), http://dx.doi.org/10.1016/j.matchemphys.2012.10.015
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K.V. Korpany et al. / Materials Chemistry and Physics xxx (2012) 1e9
glycol (PEG) and polyvinylalcohol (PVA) [13]. However, these
polymer dispersants are generally bulky and add considerably to
the hydrodynamic size of the nanoparticle, leaving them unsuitable
for applications requiring small nanoparticle diameters. A variety of
small carboxylates (citric acid, citrate, dimercaptosuccinic acid) [13]
can be used to electrostatically stabilize IONPs. Nevertheless, use of
citrate and citric acid, can lead to changes in surface geometry and
decreases in crystallinity of the iron oxide [14]. Small catechol
derivatives such as dopamine (Scheme 1) and catechol anchor
ligands have been comprehensively studied for the stabilization of
iron oxide particles, but many of these catechol anchor ligands are
PEGylated [15] or possess other bulky chemical modifications. The
synthesis of such groups can be taxing and generally results in
a large increase in hydrodynamic size of the grafted nanoparticle. In
spite of these drawbacks, chemical addition to the catechol anchor
has afforded a diverse collection of ligands with useful functionalization such as Ni(II)-NTA [3], streptavidin (for biotin complexation), or antibodies for specific cell targeting [16].
Ligand exchange remains an attractive method to maintain
monodispersity while rendering nanoparticles stable in aqueous
medium. The ligand exchange of oleic acid for dopamine (Scheme
1) has been previously investigated [2,17]. One catechol derivative, 4,5-dihydroxy-1,3-benzenedisulfonic acid (Tiron), has not
been previously studied for the stabilization of magnetic IONPs.
Tiron is a commercially available, cheap, non-bulky, highly charged
capping group, making it very attractive for the electrostatic
stabilization of nanoparticles. Yoe and Jones first presented Tiron as
an analytical reagent for the colorimetric determination of Fe3þ in
solution [18]. Its use was further extended to the analysis of other
metal ions such as and Ti4þ [19,20], Zn2þ, Os4þ [21], Cu2þ [22], UO2þ
2
4þ
3þ
3þ
3þ
3þ
[23], Co2þ [24], as well as Ce3þ, MoO24 ,Nb , Sc , Nd , Ho , Er
[25]. Due to its affinity for metal ions, Tiron (Scheme 1) has been
successfully used in the stabilization of bare metal oxides such as
SnO2 [26,27], ZrO2 [28,29], Al2O3 [28,30], TiO2 [28] and CuO [31].
Tiron has also been shown to adsorb to a-Fe2O3 (hematite) [32], and
allowed for shape control during particle growth. To our knowledge, no ligand exchange for Tiron has been performed using oleic
acid capped magnetic iron oxide nanoparticles. In most cases, Tiron
is negatively charged, because the two sulfonic acid groups have
very low pKa values (unreported) [33]. This property allows the
ligand it to act as an effective electrostatic dispersant when surface
bound to nanoparticles. We investigated the ligand exchange of
dopamine and dopamine/Tiron for oleic acid on ultrasmall IONPs to
ascertain if aqueous stable nanoparticles could be obtained.
Exchange methods developed were compared with respect to
the size, stability, and magnetic properties of nanoparticles
synthesized.
2. Experimental
2.1. Materials
All chemicals were used as received without further purification. Ferric chloride (FeCl3∙6H20) (reagent grade, 98%), ferrous
chloride (FeCl2∙6H20) (puriss. p.a. 99.0%), toluene (anhydrous,
99.8%), hexane (spectrophotometric grade, 95þ%), chloroform
Scheme 1. Structural formulas for (1) dopamine (DA) and (2) Tiron at neutral pH.
(CHCl3) (ACS reagent, 99.8%, contains amylenes as stabilizer, 350e
400 ppm), chloroform (CHCl3) (purum, 99.5% (GC)), dopamine
hydrochloride
(DA∙HCl)
(98%),
4,5-dihydroxy-1,3benzenedisulfonic acid disodium salt monohydrate (Tiron) (97%),
sodium chloride (99.5%), potassium chloride (KCl) (molecular
biology tested, 99%) and ACS reagent grade tris(hydroxymethyl)
aminomethane (Tris-base), sodium phosphate monobasic monohydrate, sodium phosphate dibasic heptahydrate, and potassium
phosphate monobasic were purchased from SigmaeAldrich (St.
Louis, MO). Tris(hydroxymethyl)aminomethane hydrochloride
(Tris-acid) was purchased from Fisher Scientific (Fair Lawn, NJ).
Sodium oleate (C18H33ONa) (97%) was purchased from TCI America
(Portland, OR). ACS reagent grade methanol (MeOH), hydrochloric
acid (HCl), and potassium hydroxide (KOH) were obtained from
ACP Chemicals (Montreal, QC). Ethanol (anhydrous) was obtained
from Commercial Alcohols Inc. (Brampton, ON).
All buffers and solutions were prepared using deionized (DI)
water (>15 MU) obtained using Diamond TII water purification
system (Barnstead, Dubuque, IA). If necessary, pH of DI water used
in the resuspension of synthesized nanoparticles was adjusted with
KOH. Where required, water was deoxygenated by N2(g) bubbling
for 30 min. All centrifugations were performed with a Sorvall
Legend RT centrifuge (Thermo Fisher Scientific, Waltham, MA)
using a F15-8x50c fixed angle rotor. Sonication steps were carried
out using a BransonicÒ model 2510 ultrasonicator (Branson Ultrasonic Corp., Danbury, CT).
2.2. Synthesis of magnetic iron oxide nanoparticles stabilized with
oleic acid (IONP-OA)
Oleic acid capped Fe3O4 iron oxide nanoparticles were prepared
according to the method reported by Jiang et al., [10,34], with slight
modification. Briefly, 4 mmol of FeCl3∙6H20, 2 mmol FeCl2∙6H20,
and 16 mmol of C18H33ONa were added to a 100 mL round bottom
flask charged with 12 mL ethanol, 9 mL of deoxygenated water, and
21 mL toluene. This mixture was refluxed at 74 C for 4 h under
N2(g). The resulting black mixture was cooled to room temperature
and 100 mL ethanol was added to precipitate the particles.
Precipitated particles were evenly distributed to five 50 mL polypropylene centrifuge tubes and centrifuged at 4000 g for 10 min
at 4 C to pellet. A clear, light orange supernatant was decanted off
and the residual black pellets redispersed in 5 3 mL of hexane by
vortexing. Redispersed particles were precipitated with 5 30 mL
of ethanol then centrifuged at 4000 g for 10 min at 4 C to pellet.
Redispersal, precipitation, and centrifugation was repeated once
more. The pelleted particles were redispersed in 5 30 mL hexane
and undispersed residues removed by centrifugation at 4000 g
for 5 min at 4 C. The resulting black supernatant was decanted, and
hexane removed to obtain nanoparticles in solid form. Nanoparticles (IONP-OA) were stored under N2(g) at 20 C until further
use.
2.3. Synthesis of dopamine-stabilized nanoparticles (IONP-DA)
As synthesized solid IONP-OA were defrosted and redispersed in
sufficient hexane to obtain a 1.25 mg mL1 stock solution. Four
milliliters of stock solution (5 mg) was aliquoted to a glass vial and
hexane removed by evaporation under N2(g) flow. The nanoparticles were redispersed in 14 mL chloroform by mild vortexing.
DA∙HCl (25 mg) was dissolved in 1 mL of methanol and added to
the redispersed nanoparticles in chloroform in 0.5 mL aliquots. This
mixture was shaken for 4 h at room temperature.
After 4 h, the mixture was pelleted by centrifugation at 4000 g
for 10 min at 4 C and the light tan supernatant decanted. DA∙HCl
(25 mg) dissolved in 10 mL methanol was added to the black
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K.V. Korpany et al. / Materials Chemistry and Physics xxx (2012) 1e9
nanoparticle pellet, and redispersed by mild vortexing. This
mixture was shaken for 1 h at room temperature.
The particles were purified by repeated centrifugation and
washing with methanol as follows. The ligand exchange mixture
was pelleted by centrifugation at 4000 g for 10 min at 4 C, the
supernatant decanted, and 10 mL of fresh methanol added to wash
the nanoparticles. This purification step was repeated once again,
and the purified nanoparticles pelleted by centrifugation at
4000 g for 10 min at 4 C. The supernatant was decanted and
residual methanol in the nanoparticle pellet evaporated under
nitrogen flow. The dried nanoparticles (IONP-DA) were subsequently redispersed in water by brief sonication.
2.4. Synthesis of dopamine/Tiron-stabilized nanoparticles (IONPDA/Tiron)
Detailed exchange methods for IONP-DA/Tiron, IONP-DA/Tiron
(with sonication) and IONP/Tiron (with amylene stabilized chloroform) are presented in Supporting Information. A summary of the
exchange methods is as follows: 4 mL of IONP-OA (5 mg)
(1.25 mg mL1 stock solution in hexane) was aliquoted to a glass
vial and hexane removed by evaporation under N2(g) flow. The
nanoparticles were redispersed in 14 mL chloroform by mild vortexing. DA∙HCl (25 mg) was dissolved in 1 mL of methanol. If
amylene-stabilized chloroform was used, then 10 mL 1 M HCl(aq)
was added to the DA in solution before proceeding. DA in methanol
was added to the redispersed nanoparticles in chloroform in 0.5 mL
aliquots and this mixture was shaken (4 h, 2.5 h with amylene
stabilized chloroform) or sonicated (30 min) at room temperature.
The mixture was then pelleted by centrifugation at 4000 g at 4 C
and the light tan supernatant decanted. 6.3 mg of DA∙HCl and
32.8 mg Tiron was dissolved in 10 mL of 95% methanol. The catechol
mixture was added to the pellet and the pellet redispersed by mild
vortexing. This mixture was shaken (1 h, 2.5 h with amylene
stabilized chloroform) or sonicated (30 min) at room temperature.
The particles were purified by repeated centrifugation and
washing with 95% methanol as follows. The ligand exchange
mixture was pelleted by centrifugation at 4000 g at 4 C, and the
supernatant decanted. Fresh 95% methanol (10 mL) was added to
the isolated pellet, and the mixture briefly vortexed to wash and
resuspend the particles. Centrifugation at 4000 g (4 C) repelleted the particles. To further purify the nanoparticles, the wash
and subsequent centrifugation step was repeated once more.
Finally, the supernatant was decanted and residual 95% methanol
in the nanoparticle pellet evaporated under nitrogen flow. The
dried nanoparticles were subsequently redispersed in desired
buffer or pH adjusted water by brief sonication.
2.5. Microemulsion formation via a two phase ligand exchange
with dopamine
supplemented to 10% (v/v) ethanol to yield a clear black-brown
nanoparticle product suspension.
2.6. Transmission electron microscopy (TEM) imaging and selected
area electron diffraction (SAED)
The shape, size, and crystalline nature of prepared nanoparticles
was analyzed by TEM imaging with a Philips CM200 TEM operating
at 200 kV, and selected area electron diffraction (SAED) patterns
were obtained in diffraction mode. IONP-OA and IONP-DA samples
for observation were prepared by submerging and incubating for
30 s a carbon-coated 200-mesh copper grid (CanemcoeMarivac,
Lakefield, QC) in the as purified IONP-OA in hexane or IONP-DA
redispersed in water. Grids were removed from the respective
solutions, excess solution wicked off via Kimwipe, and left to dry in
air. Grids used for IONP-DA/Tiron samples were pre-treated by
illumination with shortwave UV-light (254 nm) provided by
a handheld UV lamp (6 Watts, UVP, Upland, CA) for 18 h. As
synthesized IONP-DA/Tiron were redispersed in 1 mL 20 mM
sodium phosphate buffer pH 6.6, and 20 mL deposited on a UV pretreated grid. The droplet was incubated for 5 min at room
temperature, then excess removed with filter paper and the grid
left to dry in air. Two-phase exchange product TEM samples were
prepared by submerging a carbon coated 200-mesh copper grid in
the nanoparticle product suspension for 30 s, removed, and left to
air dry at room temperature. Nanoparticle and droplet Feret
diameters (defined as the maximum caliper diameter) were obtained from the TEM images using the software ImageJ 1.43u
(Wayne Rasband, National Institutes of Health, USA). Radial
intensity profiles were extracted from the SAED images using
ImageJ and measured ring radius was converted to dhkl values by
applying eq (1) [35]
dhkl ¼
Ll
D
(1)
where Ll (mm∙
A) is the TEM camera constant and D (mm) is the
measured ring radius from the SAED pattern.
2.7. Electrophoretic mobility and zeta (z) potential measurements
Electrokinetic properties were determined with a ZetaPlus
analyzer (Brookhaven Instruments, Holtsville, NY). All electrokinetic measurements were obtained with unfiltered nanoparticle
dispersions at 25 C and stated solution composition in Table 2. For
each sample, mobility and zeta potential values are reported as an
average of ten measurements, each of which was obtained over 3
cycles. Apparent zeta potentials were calculated using the ZetaPlus
software utilizing the Smoluchowski equation (Eq. (2)), which
directly calculates z potential from electrophoretic mobility (m)
m ¼
Two phase ligand exchange with dopamine was performed
based on the method outlined in Xu et al. [3] In a 25 mL Erlenmeyer
flask, 1 mL of 6.28 mg mL1 IONP-OA in hexane was added to 10 mL
hexane and 22.4 mg of DA∙HCl dissolved in 9 mL of water. The two
phase mixture was sonicated for 30 min at room temperature. After
30 min, the phases were allowed to separate yielding nanoparticles
at the interface of the clear colorless hexane and water phases. The
aqueous phase and nanoparticles were washed with 2 10 mL
fresh hexane. Fresh hexane (10 mL) was added and the mixture
sonicated for an additional 15 min. Phases were allowed to separate, yielding a dark black-brown aqueous phase. The hexane layer
was removed and the aqueous phase washed with 10 mL hexane.
The slightly opaque aqueous layer was removed to a glass vial and
3
z3
h
(2)
where 3 is the dielectric constant and h the viscosity of the medium.
Where required ionic strength (I) is reported, it is calculated
using Eq. (3)
Table 1
Summary of nanoparticle sizes, remanent magnetization (MR), and blocking
temperature (TB) of IONPs under study.
Nanoparticle
Feret diameter (nm)
MR (emu g1)
TB (K)
IONP-OA
IONP-DA
IONP-DA/Tirona
4.61 1.13
5.02 1.39
4.98 1.77
10.87
N/A
10.78
25
N/A
28
a
Exchange performed with amylene stabilized chloroform, with sonication.
Please cite this article in press as: K.V. Korpany, et al., Stable water-soluble iron oxide nanoparticles using Tiron, Materials Chemistry and Physics
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K.V. Korpany et al. / Materials Chemistry and Physics xxx (2012) 1e9
Table 2
Electrophoretic mobility and zeta (z) potential measurements obtained by the Smoluchowski formula of the different nanoparticle suspensions under various solution
compositions.
Nanoparticle
Solution composition
m (108 m2 s1 V1)
z (mV) (Smoluchowski)
IONP-DA
IONP-DA/Tiron
IONP-DA/Tiron (with sonication)
DI water pH 8 (with KOH)
50 mM sodium phosphate pH 7.00
DI water pH 8 (with KOH)
6.5 mM sodium phosphate pH 7
50 mM sodium acetate pH 4.92
50 mM sodium phosphate pH 5.94
50 mM sodium phosphate pH 7.00
e
50 mM sodium phosphate pH 7.99
0.25X PBS pH 7.4
50 mM TRIS pH 9.38
20 mM sodium phosphate pH 7.00
50 mM sodium phosphate pH 6.95
þ1.54
2.37
2.93
3.15
Ppt.a
Ppt.
2.22
2.56
1.58
2.91
3.24
3.16
2.86
þ19.67
30.29
37.54
40.34
Ppt.
Ppt.
28.44
32.74
20.20
37.27
41.49
40.48
36.63
IONP-DA/Tiron (amylene stabilized chloroform,
with sonication)
a
0.14
0.17
0.26
0.13
0.09
0.26
0.15
0.07
0.10
0.21
0.18
1.80
2.11
1.07
1.62
1.16
3.31
1.91
0.89
1.32
2.73
2.26
Particle suspensions precipitated out before measurements could be obtained.
I ¼
n
1X
c z2
2 i¼1 i i
(3)
where ci is the molar concentration of ion and zi the charge of
the ion.
2.8. Magnetic measurements
Direct current (dc) magnetic susceptibility measurements were
carried out using a Quantum Design SQUID MPMS-XL7 magnetometer at temperatures of ranging from 3 K to 300 K in applied dc
fields of 20 kOe to þ20 kOe. Measurements were performed on
samples of 16.8 mg for IONP-OA and 13.9 mg for IONP-DA/Tiron.
10 mL droplet of each solution was then deposited on freshly cleaved
mica (muscovite mica, grade V-4, SPI) and incubated for 30 min at
room temperature. Excess nanoparticle solution was wicked off and
samples dried completely in air.
Tapping mode MFM experiments were carried out using the
Cypher AFM system (Asylum Research) under ambient conditions
using low moment (w0.3 1013 emu) Co/Cr coated silicon
cantilevers (MESP-LM, Veeco, Santa Barbara, USA and PPP-LMMFMR, Nanosensors, Neuchatel, CH). Both probes have similar
properties, and upon use, gave similar results. Before imaging,
probes were remagnetized using a small NdFeB magnet. Image
analysis was carried out using Igor Pro v.6.22A (Wavemetrics Inc.,
Lake Oswego, USA).
3. Results and discussion
2.9. Magnetic force microscopy (MFM) of IONP-DA/Tiron
3.1. Ligand exchange of OA for DA or DA/Tiron
Magnetic properties of synthesized IONP-DA/Tiron and nanoscale TiO2 were imaged by magnetic force microscopy (MFM).
IONP-DA/Tiron samples for imaging were prepared by diluting as
synthesized IONP-DA/Tiron to a final concentration of 1 mg mL1
with 20 mM sodium phosphate buffer pH 7.14. 12 nm (by dynamic
light scattering) TiO2 nanoparticles (kind gift of R. Godin, McGill)
were diluted with 0.1 M HCl to a final concentration of 4 mg mL1. A
The general strategy for IONP phase transfer and ligand
exchange is outlined in Scheme 2. The as-synthesized IONP-OA is
redispersed in chloroform then dopamine in methanol is added. As
methanol is completely miscible in chloroform, a one phase
mixture forms, ensuring that further processing of the mixture by
shaking or sonication results in minimal emulsification. This is
Scheme 2. Summary of ligand exchange of oleic acid (OA) for dopamine (DA) or DA and Tiron on the iron oxide nanoparticle surface. Either shaking or sonication was used to
facilitate ligand exchange at each step. IONP-DA was only stable in water, whereas IONP-DA/Tiron was stable in water and a variety of buffers as outlined in Table 2.
Please cite this article in press as: K.V. Korpany, et al., Stable water-soluble iron oxide nanoparticles using Tiron, Materials Chemistry and Physics
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K.V. Korpany et al. / Materials Chemistry and Physics xxx (2012) 1e9
important as emulsions were found to lead to decreased yield of
phase transferred nanoparticles using the prescribed method.
Furthermore, nano-emulsions can mimic the appearance of waterstable nanoparticles [36], impacting their availability for biological
and self assembly applications. In either ligand exchange procedure, the first step always consists of an exchange of some of the
oleic acid capping groups for dopamine. Subsequent centrifugation
removes any exchanged oleic acid ligands from the nanoparticles,
and the particle pellet is redispersed into MeOH or 95% MeOH. For
IONP-DA particles, the second step consists of the addition of more
dopamine to complete the exchange. Alternatively, a mixture of
dopamine and Tiron is added to produce IONP-DA/Tiron. In the case
of IONP-DA/Tiron the addition of dopamine in the second step was
found to be absolutely required for the resulting nanoparticles to be
stable in buffer. Among ratios tested, a molar ratio of 1:3 (DA/Tiron)
was found to produce the most stable nanoparticles.
The ligand exchange and subsequent phase transfer occurs in
two parts for a few reasons. Firstly, for both IONP-DA and IONP-DA/
Tiron particles, separating the ligand exchange procedure into parts
allows for the removal of exchanged oleic acid ligand residue from
the reaction mixture via the first purification step. This purification
is thought to encourage the continued ligand exchange. Secondly, it
was determined that the concentration of dopamine or Tiron used
in each step greatly affected the stability of the resulting IONP-DA
or IONP-DA/Tiron particles (unpublished data). If the dopamine or
Tiron concentration is too high in either step, the obtained IONP-DA
or IONP-DA/Tiron particles could not redisperse in water. A similar
effect has been reported in reference to the coating of CuO nanoparticles with Tiron [31]. It has been reasoned that excess dispersant molecules in solution cause decreased suspension stability,
due to an increase in ionic strength. That is, an optimal amount of
dispersant is required to ensure a stable solution. It is probable that
at high dopamine or Tiron concentrations, the particles will flocculate to some extent and ligand exchange processes can be
inhibited. Therefore, in order for sufficient ligand exchange to
5
occur, the addition of catechol ligand must occur in multiple steps.
Lastly, separating the exchange procedure into two steps allows for
incorporation of Tiron on the IONP surface. Tiron was found to be
only soluble in water and some water/alcohol mixtures. In order for
the incorporation of Tiron to occur, partial exchange of oleic acid for
dopamine in the first step is necessary to obtain particles that are
soluble in a solvent mixture that Tiron is likewise soluble.
This method has several desirable qualities. Notably, the
exchange method does not require long reaction times, especially if
sonication is employed, nor excessive concentrations of reagents or
elevated temperatures. All solvents and reagents used are readily
and cheaply available and the ligands used do not require further
chemical modification to act as good dispersants. Retention of DA in
the ligand sphere of the highly stable IONP-DA/Tiron particles is
also advantageous, since the DA primary amine can be used in
further coupling chemistries to attach other molecules of interest
[37]. In addition, the synthesized IONP-DA/Tiron particles can be
redispersed in water or buffer (as required), or can be stored in the
long-term as a solid under nitrogen at 20 C until use.
3.2. Nanoparticle characterization
Iron oxide nanoparticles were individually stabilized with DA or
DA and Tiron ligands and maintained their general size and
spherical shape throughout the ligand exchange, as shown in Fig. 1.
Size distributions of the IONPs were obtained from analysis of >600
particles visualized by TEM (Table 1 and Fig. S1). It is also clear from
the TEM images that the particles are roughly spherical and are
dispersed as separate nanoparticles with no aggregates observed.
Particles that result from the ligand exchange procedures were
slightly less monodisperse and larger than the parent oleic acid
capped nanoparticles. This observation can likely be attributed to
the exposure of the catechol-iron oxide units to acidic conditions
during the exchange. Acidic conditions are known to decompose
iron oxide nanoparticles. Shultz et al. [38] examined this
Fig. 1. TEM of (A) oleic acid-capped IONPs (B) dopamine-capped IONPs and, (C) dopamine and Tiron-capped IONPs. (D) Selected area electron diffraction (SAED) of oleic acidcapped IONPs confirming crystalline nature of the particles and characteristic reflections for Fe3O4. (E) SAED of dopamine-capped IONPs shows crystallinity is not significantly
altered by the ligand exchange. (F) SAED of dopamine and Tiron-capped IONPs. The reflection corresponding to the (422) diffraction plane is not clearly visible in the SAED of
dopamine and Tiron-capped IONPs. The overall low intensity of the diffraction pattern is likely due to the lower nanoparticle deposition on the grid surface for dopamine and Tironcapped IONPs.
Please cite this article in press as: K.V. Korpany, et al., Stable water-soluble iron oxide nanoparticles using Tiron, Materials Chemistry and Physics
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K.V. Korpany et al. / Materials Chemistry and Physics xxx (2012) 1e9
decomposition behavior where bond cleavage between surface
catechol-iron oxide units of Fe2O3-DA nanoparticles is thought to
occur, leading to release of free iron hydroxide into solution and
eventual destabilization of the nanoparticle suspension. Unfiltered
suspensions of IONP-DA/Tiron, to which acid has been added to
supplement the exchange, show some enhanced background
contrast during TEM imaging, indicating the presence of heavy ions
in solution (Fig. 1c). This effect is likely aggravated by use of sonication to facilitate the ligand exchange.
In a qualitative sense, ligand exchange success could be visualized by nanoparticle deposition on the TEM under normal plating
conditions. Preparing TEM samples of IONP-DA/Tiron particles
using the same methods for IONP-OA or IONP-DA resulted in poor
deposition on the carbon coated grid. This result confirms the
highly charged nature of the IONP-DA/Tiron, illustrating the
incompatibility of highly charged particles with a hydrophobic
(carbon) surface. Upon grid pretreatment with UV radiation
(known to increase the hydrophilic nature of the carbon coating
[39]), subsequent nanoparticle deposition increased such that
suitable statistics on particle size and distribution could be obtained. SAED patterns (insets, Fig. 1) reveal that the crystal structure
of the nanoparticles is not altered to a large extent upon the
exchange of oleic acid for dopamine ligands. Measurements performed on the rings of the ED patterns for all nanoparticles under
study (Fig. S2) result in values of interplanar spacing (dhkl) that
match well with that of Fe3O4 (JCPDS card 19-0629). Only the most
intense reflections (220), (311), and (511) are obvious in the
diffraction data for the DA/Tiron coated nanoparticles, likely owing
to the lessened nanoparticle deposition on the grid surface available for diffraction studies.
3.3. Nanoparticle stability
Stability was investigated by electrophoretic mobility and zeta
potential determination (Table 2). Table 2 shows the effect of
varying solution composition on the electrokinetic data obtained,
as electrophoretic mobility and zeta potential depend on electrolyte concentration and pH. However, as the iron oxide particles
used are quite small, the application of the Smoluchowski formula
for the calculation of zeta potential may not be entirely appropriate
[40]. As such, mobility data is reported alongside zeta potential
values to avoid misinterpretation.
Particles coated with only dopamine are expected to have an
overall positive charge at pH values below the pKa of the dopamine
amine (pKaNH2 w9.1 [41]). This appears to be the case, as IONP-DA
has positive zeta potential and mobility values in water at pH 8.
Likewise, IONP-DA/Tiron particles are expected to have negative
zeta potential and mobility values since the sulfonic groups of Tiron
possess a negative charge at all pH values. Regardless of ligand
exchange method, all IONP-DA/Tiron samples tested presented
negative mobility and zeta potential. Precipitation of particles in
solutions below pH 7 may be attributed to the pHpzc of the bare
nanoparticle surface (pHpzc w 6.7 [15]); in which pH dependant
behavior is attributed to FeeOH surface active groups (eq (4) and
(5)) [42]. Likely the surface is not completely saturated with
ligands, and at pH > pHpzc these uncomplexed surface active
groups are deprotonated to FeeO imparting a negative charge on
the surface:
Fe OH þ OH #Fe O þ H2 O
(4)
If pH <pHpzc the surface FeeOH groups instead protonate to
yield FeeOHþ
2 resulting in a positive surface charge contribution:
At pH values below pHpzc, it is likely that the accumulation of
positive charge due to the presence of FeeOHþ
2 and protonated DA
amines closely matches the amount of negative charge provided by
the Tiron ligands, resulting in destabilization of the suspension.
Likewise, the increased stability of IONP-DA/Tiron in 50 mM TRIS
pH 9.38 can be explained by the additive effects of increased
negative charge on the exposed nanoparticle surface and decreased
ligand positive charge due to deprotonation of the DA amine.
Conversely, in the sodium phosphate buffer systems, increasing the
alkalinity of the nanoparticle solution does not always yield more
stable nanoparticles. This observation can be explained by consideration of the effect of ionic strength on zeta potential (Table 3) for
the IONP-DA/Tiron particles in phosphate buffer. Increases in ionic
strength of the solution in which particles are suspended will
compress the electrical double layer associated with the particles,
thereby decreasing their zeta potential [43].
The ligand exchange method used appeared to influence the
overall stability of the nanoparticles, with the acid supplemented
exchange (with amylene stabilized chloroform) yielding the most
negative electrokinetic measurements. The greater stability of
particles resulting from the acid supplemented exchange may be
explained by excess acid present that aids ligand exchange. Prior
work on the functionalization of iron oxide coreeshell nanoparticles with dopamine anchor ligands has required that the pH
value be adjusted to 4e5 in order for the ligand attachment to occur
[3]. The dependence of ligand attachment on solution pH is highlighted by the observation that IONP-DA/Tiron fails to phase
transfer if amylene stabilized chloroform is not supplemented with
small amounts of HCl(aq). Many chlorinated solvents contain
amylene (2-methyl-2-butene) or amylenes (mixture of amylene
and 2-pentene) as a stabilizing agents, which are known proton
scavengers [44]. If amylene stabilized solvents are used, the addition of small amounts of HCl(aq) to the exchange reaction was
found to be required in order for the ligand exchange to occur.
3.4. Magnetic measurements
The magnetic properties of the two samples, IONP-OA and
IONP-DA/Tiron, were studied using zero-field-cooled (ZFC) and
field-cooled (FC) magnetization measurements as a function of
temperature as well as magnetization vs. applied dc field hysteresis
loop measurements at 3 and 300 K. In the ZFC-FC experiment, the
samples were cooled to 3 K in the absence of a dc field then the
magnetization was measured as a function of temperature under an
applied field of 100 Oe from 3 to 300 K (ZFC). The magnetization
was then measured as the samples were cooled slowly back down
to 3 K (FC). ZFC-FC and magnetization vs. applied field plots are
shown in Fig. 2. The maximum in the ZFC curves indicates the
blocking temperature (TB) of the nanoparticles. This is observed at
25 K for IONP-OA and at 28 K for IONP-DA/Tiron (Fig. 2a). Above the
aforementioned TB, the nanoparticles are behaving as superparamagnets; they are free to align with the field by reversing their
Table 3
Relationship between ionic strength (I) and zeta potential (z) for IONP-DA/Tiron
produced via sonication.
Solution composition
I (mM)
z (Smoluchowski) (mV)
6.5 mM NaePa, pH 7
50 mM NaeP, pH 7.00
e
50 mM NaeP, pH 7.99
20 mM NaeP, pH 7.00b
50 mM NaeP, pH 6.95b
14
112
e
144
45
110
40.34
28.44
32.74
20.20
40.48
36.63
a
Fe OH þ Hþ #Fe OH2þ
(5)
b
1.62
1.16
3.31
1.91
2.73
2.26
NaeP ¼ sodium phosphate.
Particles synthesized with amylene stabilized chloroform.
Please cite this article in press as: K.V. Korpany, et al., Stable water-soluble iron oxide nanoparticles using Tiron, Materials Chemistry and Physics
(2012), http://dx.doi.org/10.1016/j.matchemphys.2012.10.015
Fig. 2. a) ZFCeFC curves for IONP-OA and IONP-DA/Tiron. A zoomed-in view is shown to the right. Magnetization vs. applied field plots at temperatures of 3 and 300 K are shown for
IONP-OA (b) and IONP-DA/Tiron (c).
Fig. 3. MFM of IONP-DA/Tiron (A) topography and (B) phase at a lift height (d) of 50 nm showing a strong magnetic signal. For comparison, MFM of TiO2 nanoparticles shows (C)
topography and (D) phase at d ¼ 50 nm. As TiO2 is not magnetic, there is no signal associated with the nanoparticles in the phase image.
Please cite this article in press as: K.V. Korpany, et al., Stable water-soluble iron oxide nanoparticles using Tiron, Materials Chemistry and Physics
(2012), http://dx.doi.org/10.1016/j.matchemphys.2012.10.015
8
K.V. Korpany et al. / Materials Chemistry and Physics xxx (2012) 1e9
magnetization while below the TB their magnetization is blocked or
irreversible. The magnetization vs. applied field measurements
were performed above the blocking temperatures, at 300 K (Fig. 2b,
c) where no remanent magnetization or coercivity is observed for
all three samples. Below the blocking temperatures, however, the
samples exhibit coercivity and remanent magnetizations of 250 Oe
and 10.87 emu g1 for IONP-OA and 250 Oe and 10.78 emu g1 for
IONP-DA/Tiron.
While the observed TB of 25 K for IONP-OA corresponds well
with the literature value of 24.4 K [10], IONP-DA/Tiron possesses
a slightly higher blocking temperature. An increase in blocking
temperature of magnetite nanoparticles upon ligand exchange of
oleic acid with dopamine for has been previously observed by
Nagesha et al. [17]. It was suggested that the functionalization
with dopamine partially restores the nanoparticle’s magnetic
‘dead’ layer; a zero-moment magnetically disordered surface layer.
The surface spin disorder is thought to be a result of the decreased
coordination of magnetic cations with oxygen ions as well as
broken (super) exchange bonds between spins [45]. Daou et al.
[45] has shown that the nature of the interaction of the ligand
with the nanoparticle surface influences the degree of spin
disorder, and therefore, the magnetic properties of the nanoparticle. As the binding of Tiron to the iron oxide nanoparticle is
similar to that of dopamine, the two ligands both bind through
a catechol anchor, likely the rise in blocking temperature for IONPDA/Tiron is due to the reorganization of the nanoparticle surface
layer.
(Fig. 4). An emulsion formed regardless of whether shaking or
sonication was used to facilitate the ligand exchange. The IONPs do
not appear to be aggregated (Fig. 4b), but rather situated at the oil/
water interface. When toluene is used in the exchange, shaking or
sonication would yield a milky emulsion that could not be separated, whereas using hexane produced a clear blackebrown
aqueous layer. Typical emulsions, in which droplet size is greater
than 100 nm, appear milky due to the scattering of visible light. In
contrast, nanoemulsions (droplet size less than 100 nm) appear
clear [36]. This coincides with our analysis of droplet diameter
(Fig. S3), where most droplets are below 100 nm in size. As highlighted by the emulsion in Fig. 4, the observation of a clear blacke
brown aqueous layer is not sufficient to assume that monodisperse
nanoparticles have been transferred to the aqueous phase. This
3.5. Magnetic force microscopy (MFM)
Tapping mode MFM images of prepared IONP-DA/Tiron (Fig. 3)
reveal that the nanoparticles remain magnetic after ligand
exchange. Topographic images are obtained concurrently with
phase images at lift height (d) to allow direct comparisons of
topography and magnetic force. The particles appear well
dispersed, as shown in the topographic image (Fig. 3a). Contrast in
the phase image (Fig. 3b) is related to the interaction energy
between the oscillating cantilever and the sample, resulting in
a phase shift in the cantilever oscillation [46]. In this case, the
interaction is attractive and results in a negative phase shift visualized by darkened areas of the phase image. These areas of contrast
correspond well to the nanoparticles as seen in the topographic
image. If d is increased, the magnitude of phase shift decreased. This
observation agrees with our interpretation, because magnetic fields
decrease rapidly with increasing distance. As a control to avoid
misinterpretation of phase contrast, nanoscale TiO2 (nonmagnetic) was imaged in MFM mode. In some cases, phase contrast
has been seen in traditionally non-magnetic nanoparticles [47],
attributing the phase shift to repulsive electrostatic forces between
the tip and sample. However, electrostatic forces responsible for the
phase contrast have been shown to typically manifest as positive
phase shifts, rather than negative (as seen with IONP-DA/Tiron). At
the same lift height (d ¼ 50 nm), clearly there is little phase contrast
seen with the TiO2 sample in comparison to the IONP-DA/Tiron
sample. It is reasoned that any phase contrast seen in the TiO2
imaging is most likely attributed to noise, since the contrast cannot
be associated with specific features on the topographic image.
Given the above observations, we are confident that the IONP-DA/
Tiron are indeed magnetic.
3.6. Two phase methods of ligand exchange result in emulsions
Attempting the ligand exchange of oleic acid for dopamine in
a two phase system [3] with the organic soluble IONP-OA in either
hexane or toluene and dopamine in water resulted in emulsions
Fig. 4. Emulsions form under different solvent conditions. (A) Sonication at room
temperature of a biphasic system with IONPs-OA dissolved in hexane and DA in water
results in an emulsion. (B) A closer look confirms the nanoparticles are intact and
situated at the interface of the two phases.
Please cite this article in press as: K.V. Korpany, et al., Stable water-soluble iron oxide nanoparticles using Tiron, Materials Chemistry and Physics
(2012), http://dx.doi.org/10.1016/j.matchemphys.2012.10.015
K.V. Korpany et al. / Materials Chemistry and Physics xxx (2012) 1e9
observation may serve as a warning to other nanoparticle ligand
exchange methods utilizing two phase mixtures. That is, TEM or
other suitable imaging or size analysis techniques must be
employed to confirm that water-stable monodisperse nanoparticles have been produced.
4. Conclusions
We have demonstrated that the ligand exchange of oleic acid for
Tiron and dopamine successfully renders the IONPs aqueous stable.
The exchange method outlined is quick, consumes minimal reagents,
and is conducted at room temperature making it an efficient
synthetic procedure. Importantly, the ligands used can be commercially obtained at minimal cost and do not require chemical modification to act as good dispersants. However, if desired, the residual
dopamine in the ligand sphere of IONP-DA/Tiron can be used as
a platform for further conjugation chemistry. We have also demonstrated the commercial value of the synthesized nanoparticles as
they can be fully redispersed after being dried and frozen, allowing
for convenient use and limiting oxidation during storage.
Experimentally, it was found that extra care must be taken to
ensure that the ligand exchange proceeds according to the protocol
utilized. If using solvent systems containing stabilizers that have
the potential to alter pH (ex. amylenes), it was determined that
modifications (i.e. addition of HCl(aq)) must be made to ensure the
ligand exchange proceeds at acidic pH. For similar ligand exchange
methods using two phase solvent systems, the potential for
emulsions must be considered, and TEM or similar imaging or size
analysis modalities should be used to confirm that singly dispersed
nanoparticles have been obtained.
Stability of the IONP-DA/Tiron is pH dependent with large
negative zeta potential values at alkaline pH, rendering the particles useful for biological applications at physiological pH. Magnetic
measurements confirm that the ligand exchange procedure did not
significantly alter the remanent magnetization or blocking
temperature of the nanoparticles implying that the general crystal
structure and size of the nanoparticles was maintained. MFM
imaging verifies the nanoparticles are indeed magnetic and well
dispersed. Enhancements achieved here in IONP monodispersity,
aqueous stability, and ease of synthesis may result in increased
efficiency and performance in further applications that require
ultrasmall aqueous stable magnetic IONPs.
Acknowledgments
K.V.K. thanks McGill University for Alexander McFee Memorial
Fellowship and J.W. McConnell Memorial Fellowship. We thank R.
Godin (McGill University) for the generous gift of TiO2 nanoparticles. We gratefully acknowledge the financial support of the
Natural Sciences and Engineering Research Council of Canada
(NSERC), the Canada Foundation for Innovation (CFI) and the Centre
for Self-Assembled Chemical Structures (CSACS).
Appendix A. Supplementary material
Supplementary material related to this article can be found at
http://dx.doi.org/10.1016/j.matchemphys.2012.10.015.
9
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Please cite this article in press as: K.V. Korpany, et al., Stable water-soluble iron oxide nanoparticles using Tiron, Materials Chemistry and Physics
(2012), http://dx.doi.org/10.1016/j.matchemphys.2012.10.015