J Nanopart Res (2014) 16:2596
DOI 10.1007/s11051-014-2596-7
RESEARCH PAPER
Catechol versus bisphosphonate ligand exchange
at the surface of iron oxide nanoparticles:
towards multi-functionalization
Erwann Guénin • Yoann Lalatonne •
Julie Bolley • Irena Milosevic •
Carlos Platas-Iglesias • Laurence Motte
Received: 1 July 2014 / Accepted: 30 July 2014 / Published online: 9 October 2014
Ó Springer Science+Business Media Dordrecht 2014
Abstract We report an investigation of the ligand
exchange at the surface of iron oxide nanoparticles in
water. For this purpose we compared two strong
chelating agents on the iron oxide surface containing
catechol and bisphosphonate moieties. Interactions
between the coating agents (catechol/bisphosphonate)
and the nanoparticle’s surface were studied by FTIR
and DFT calculations. Ligand exchange experiments
were performed using sonication and the exchange
yield was characterized by FTIR and EDX. This
methodology allowed introducing bisphosphonates
with various functionalities (alkyne or biotin) permitting multi-functionalization.
Guest Editors: Carlos Lodeiro Espiño, José Luis Capelo
Martinez
This article is part of the topical collection on Composite
Nanoparticles
Electronic supplementary material The online version of
this article (doi:10.1007/s11051-014-2596-7) contains supplementary material, which is available to authorized users.
E. Guénin (&) Y. Lalatonne J. Bolley I. Milosevic L. Motte
Université Paris 13, Sorbonne Paris Cité, Laboratoire
CSPBAT, UMR 7244 CNRS, Bobigny, France
e-mail: guenin@univ-paris13.fr
C. Platas-Iglesias
Departamento de Quı́mica Fundamental, Universidade da
Coruña, Campus da Zapateira, 15008 A Coruña, Spain
Keywords Nanoparticle functionalization Ligand
exchange DFT simulation Catechol Iron oxide
nanoparticle Bisphosphonate Composite
Introduction
Metal oxide nanoparticles and especially iron oxide
nanoparticles represent an outstanding material for
numerous applications in biomedicine (Ling and
Hyeon 2012; Reddy et al. 2012; Tran et al. 2012).
Owing to their magnetic properties and their unique
surface to volume ratio, they can be used for biomolecules and cell sorting, biosensing, imaging, and
therapy such as hyperthermia or drug delivery (Collins
et al. 2013; Colombo et al. 2012; Pan et al. 2012; Wu
et al. 2010; Yigit et al. 2012). For these applications
the nanoparticle needs to be biocompatible and
requires functionalization to get specific properties
(Erathodiyil and Ying 2011; Kunugi et al. 2012;
Mahon et al. 2012). Many approaches have been
employed to realize nanoparticle functionalization
(Perrier et al. 2010; Sperling and Parak 2010;
Turcheniuk et al. 2013), one of the most popular
methodologies being the coating of the surface with
small bifunctional molecules which enables to protect
the nanoparticle by chelation while allowing the
insertion of the molecules of interest via reactive
functions. Two popular families of chelatants are
currently used for nanoparticle coating: catechol and
bisphosphonates. Catechol-functionalized molecules
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J Nanopart Res (2014) 16:2596
Scheme 1 Schematic
ligand exchange process at
the c-Fe2O3@CA and cFe2O3@HMBP
nanoparticle’s surfaces
derived from mussel adhesive protein are being
intensively studied as coating agents of superparamagnetic iron-oxide particles, to attain biocompatibility and increased colloidal stability (Amstad et al.
2011, 2009; Bolley et al. 2013b; Goldmann et al. 2010;
Ling et al. 2011; Yuen et al. 2012). Nevertheless
Reimhult’s group (Amstad et al. 2011, 2009) showed
that the chemical structure of the catechol is of crucial
importance for nanoparticle stability. Another family
of small bifunctional molecules, that is also the subject
of increasing interest, is the phosphonate family
(Basly et al. 2010; Georgelin et al. 2008; Papst et al.
2013; Tudisco et al. 2013) and among these molecules
the bisphosphonate compounds (Lalatonne et al. 2008;
Portet et al. 2001; Sandiford et al. 2013). Indeed, we
and others have thoroughly described their use for
stabilizing nanoparticles in aqueous media. These
molecules owing to their structure are also used to
further functionalize the surface. As part of our
research program on iron oxide nanoparticle functionalization, we decided to study the differences in
chelating properties between catechol and bisphosphonate. For this purpose we have selected two related
molecules such as caffeic acid (CA in the text) and
5-hydroxy-5,5-bis(phosphono)pentanoic acid (called
HMBP in the text, Scheme 1). These molecules are
only differing by their chelating groups and are
bearing the same carboxylic functionality on their
side chain. CA was previously used to transfer
123
surfactant-coated nanoparticles from an organic solvent to water (de Montferrand et al. 2013; Hu et al.
2012) and we recently showed that both CA and
HMBP can be used to elaborate multifunctional
nanoplatforms (Bolley et al. 2013a, b).
Here we examined the design of the nanoplatforms
with each individual entity. The nanoparticle surface
functionalization was characterized using Fourier
transform infra-red (FTIR) and energy dispersive
X-ray (EDX) measurements. Density functional theory (DFT) calculations were performed to further
elucidate the interactions between catechol/bisphosphonate groups and the nanoparticle’s surface.
We then evaluated CA/HMBP ligand exchange in
water (Scheme 1). The interest of sonication in this
exchange process was emphasized. Moreover, using
two other bisphosphonate derivatives differing by
their lateral chain and end functionality (HMBP-biotin
and HMBP-C:CH (Demay-Drouhard et al. 2013)),
we showed that this methodology could be used for
multi-functionalization of the nanoparticle surface.
Experimental details
Materials
Caffeic acid (C98.0 % (HPLC)), glutaric anhydride
(95 %), biotin (C99 % (TLC)), lyophilized powder,
J Nanopart Res (2014) 16:2596
dimethylamine (40 wt% in H2O), and tris(trimethylsilyl) phosphite were purchased from Sigma-Aldrich,
and were used without further purification. Streptavidine was bought from AnaSpec. Solvents: methanol
(RS HPLC), dichloromethane (RE amylene stabilized), and diethylether (RE stabilized) were purchased from Carlo Erba SDS. All the reagents were
used without further purification.
Water was purified with a Millipore system (resistivity 18.2 MX cm).
1
H NMR spectra (400.0 MHz), proton-decoupled
13
C NMR spectra (100.6 MHz), and proton-decoupled
31
P spectra (162.0 MHz) were recorded on a Bruker
Avance III 400 spectrometer. Chemical shifts are
reported in parts per million (ppm) on the d scale. The
residual solvent peaks were used as internal references
(1H NMR: CHCl3 7.26 ppm, H2O 4.79 ppm; 13C
NMR: CDCl3 77.16 ppm). 31P NMR spectra were
recorded using phosphoric acid (85 %) as the external
reference. Data are reported as follows: s = singlet,
d = doublet, t = triplet, q = quartet, qt = quintuplet, m = multiplet, and coupling constant(s) are
given in Hz. FTIR spectra were recorded as KBr
pellets on a Thermo Scientific Nicolet 380 FTIR
spectrophotometer and are reported in wavenumbers
(cm-1). High resolution Mass Spectrometry experiments were realized on a LTQ Orbitrap Velos
(Thermo Scientific) in positive and negative modes
using an ESI source.
The size and the zeta potential of the nano complex
were determined by dynamic laser light scattering
(DLS) on a Nano-ZS (Red Badge) ZEN 3600 device
(Malvern Instruments, Malvern, UK).
TEM images were obtained using a FEI CM10
microscope (Philips), and samples were prepared by
depositing a drop of nanoparticles suspension on
carbon-coated copper grids placed on a filter paper.
The average particle diameters were deduced from
TEM data measurements, simulating the diameter
distribution with a log-normal function.
The magnetic behavior at room temperature of the
as-synthesized nanoparticles was characterized using
a MIAplexÒ reader (Magnisense SA). MIAplexÒ
reader is a miniaturized chip detector system, measuring a signal corresponding to the second derivative
of the magnetization around zero field (de Montferrand et al. 2012; Lenglet 2009).
Page 3 of 13
2596
The MIAtekÒ sensor was used for the detection of
superparamagnetic nanoparticles in the streptavidin–
biotin binding procedure on MIAstripÒ (Motte et al.
2010). The MIAtekÒ sensor (Magnisense SA) measures a signal proportional to the third derivative of the
magnetization at zero magnetic field (Lenglet et al.
2008) (q3M(H)/qH3). Hence, the MIAtekÒ signal is
very sensitive and is proportional to the amount of
magnetic particles allowing detection of nanograms of
superparamagnetic materials.
Quantification of HMBP and CA coating and
grafting per particle was evaluated by EDX, FTIR,
and thermogravimetric analysis (TGA), respectively.
EDX microanalyses were performed using a TM 3000
tabletop microscope equipped with a Swift EDX-ray
3,000 microanalysis system (Oxford Instruments).
Samples were deposited as powder on a copper
surface, and data were collected using a 15 kV
accelerating voltage, studying ratio of iron vs phosphorus and knowing the average number of iron
atoms/particles. The number of molecules of HMBP
was determined by FTIR spectroscopy using OMNIC
8.1. The different FTIR spectra are normalized with
the iron oxide vibration band (500–600 cm-1) and then
the HMBP ratio per particle is determined from area
measurement of the phosphonic function vibration
bands (between 950–1200 cm-1). The TGA curves
were recorded using a LabsSys evo TG–DTA-DSC
16,000
device
manufactured
by
Setaram
Instrumentation.
Ligand exchange reactions were performed with a
USC200T ultrasonic bath (VWR) at a 45 kHz
frequency.
Chemical synthesis and characterization
The synthesis of HMBP (5-Hydroxy-5,5-bis(phosphono)pentanoic acid) was adapted from the previously described protocol (Guenin et al. 2004).
Condensation of glutaric anhydride (4 mmol) with
tris(trimethylsilyl) phosphite (8.8 mmol) in a 25-mL
round-bottom flask under an inert atmosphere without
any solvent led to the fully trimethylsilylated intermediate, which was not isolated. The reaction was
stirred for 4 h and the volatile fractions were then
evaporated under vacuum (0.1 Torr) at 50 °C. Methanol was added at 0 °C to the residue and the solution
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was stirred for another 4 h. After solvent evaporation,
the pH of mixture was adjusted to pH 2.3 and the
product was precipitated with a MeOH/H2O (9/1)
mixture. The resulting white solid was washed with
diethylether (3 9 50 mL). Lyophilization yielded the
desired product as a white powder (80 %).
M.p. [ 260 °C. IR (KBr, pH 7) 1,694, 1,464, 1,308,
1,213, 1,174, 1,149, 1,059, 983, 938, 923 cm-1. 1H
NMR (D2O, 400 MHz, 298 K): d 2.40 (m, 2 H,
CH2COOH), 1.85–2.00 (m, 4 H, HOCCH2CH2). 13C
NMR{1H} (D2O, 100.6 MHz,298 K): d 185.2
(COOH), 76.3 (t, 1JC,P 132.0 Hz, COH), 37.5 (CH2COOH), 36.0 (CH2COH), 22.4 (CH2CH2CH2). 31P
NMR{1H} (D2O, 162.0 MHz, 298 K): d 19.2 (s).
HMBP-C:CH and HMBP-biotin were synthesized
according to the already described protocol (DemayDrouhard et al. 2013). Tris(trimethylsilyl)phosphite
was added dropwise at -20 °C to 6-heptynoic acyl
chloride or biotin acyl chloride (freshly prepared)
without solvent and under inert atmosphere. When the
addition was completed, the reaction mixture was
allowed to stand at room temperature for 4 h. The
evolution of the reaction was monitored by 31P NMR
{1H}. Then, volatile fractions were evaporated under
reduced pressure (0.1 Torr) before being hydrolyzed
with methanol. After methanol evaporation, the product
was dissolved in water at pH 2.3 and lyophilized. The
product was then precipitated twice in a water/methanol
mixture [1:9]. Both products were isolated as their
sodium salts as white powders (Yield = 70 % for
HMBP-C:CH and 60 % for HMBP-Biotin).
HMBP-C:CH (1-hydroxy-1-phosphonohept-6ynyl)phosphonic acid
1
H NMR (400.00 MHz, 25 °C, D2O): d 2.29 (t, J4
(H–H) 2.6 Hz, 1H, HC:); 2.20 (td, J3(H–H) 7.2 Hz,
J4(H–H) 2.6 Hz, 2H, CH2C:); 1.91 (m, 2H, CH2COH); 1.63 (m, 2H, CH2CH2COH); 1.51 (qt, 2H,
CH2CH2CH2COH).
13
C NMR (100.63 MHz, 25 °C, D2O) : d 86.5; 74.0
(t, J1(P–C) 134.5 Hz, COH); 68.9; 33.1; 28.7; 23.0;
17.5.
31
P NMR (161.98 MHz, 25 °C, D2O): d 18.4
(t, J3(P–H) 13.9 Hz).
IR (KBr, pH 7): 3,541, 3,261, 2,953, 2,866, 2,113,
1,725, 1,467, 1,448, 1,383, 1,329, 1,265, 1,227, 1,167,
1,058, 986, 947, 911, 755, 738, 673, 656, 597, 547,
487, 455.
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J Nanopart Res (2014) 16:2596
HR-MS (ESI--Q Tof) C5H9O7P2 : m/z (M-H)-:
271.01359; calc: 271.0137.
HMBP-biotin (5-((3aS,6aR)-hexahydro-2-oxo-1Hthieno[3,4-d]imidazol-6-yl)-1-hydroxy-1phosphonopentylphosphonic acid (Figs. S1, S2
and S3)
1
H NMR (D2O, 400.00 MHz, 298 K): d 4.5 4 (m, 1 H,
CH biotin), 4.38 (m, 1 H, CH biotin), 3.30 (m, 1 H,
CHS biotin), 2.96–2.70 (m, 2H, CH2S biotin), 1.93 (m,
2H, HOCCH2), 1.52–1.71 (m, 4H, HOCCH2CH2CH2CH2), 1.36–1.42 (m,2H, HOCCH2CH2CH2CH2). 13C
NMR{1H} (D2O, 100.6 MHz, 298 K): d 165.4 (CO
biotin), 73.4 (t, 1JC,P 140.7 Hz, COH), 62.0 and 60.3
(CH biotin), 55.3 (CHS biotin), 39.7 (CH2S biotin),
33.3 (HOCCH2CH2CH2CH2), 29.1 and 27.7 (HOCCH2CH2CH2CH2), 23.1 (HOCCH2CH2CH2CH2).
31
P NMR{1H} (D2O, 162.0 MHz, 298 K): d 24.0
(s). HR-MS (ESI--Q Tof) C10H19N2O8P2S: m/z (MH)-: 389.0351; calc: 389.0337.
Nanoparticles synthesis and coating
The bare maghemite nanoparticles, cFe2O3 nanocrystals (average diameter 11.2 nm and size distribution
r = 0.3,) were synthesized according to the following
procedure. (Bolley et al. 2013b; Lalatonne et al. 2008)
Dimethylamine ((CH3)2NH) was added to an aqueous
solution of ferrous dodecyl sulfate (Fe(DS)2). The final
concentrations after the reactants were mixed were
2.7.10-2 mol L-1 and 0.1 mol L-1 for Fe(DS)2 and
dimethylamine, respectively. The solution was then
stirred vigorously for 2 h at 28.5 °C. 6 mL of HCl
(1 M) was then added in order to reach the isoelectric
point (around pH 7), inducing nanoparticle precipitation. The precipitate was isolated from the supernatant
using magnetic separation. After 10 washings at
neutral pH, the nanoparticles were then dispersed at
pH 2 in distilled water. At this stage, bare c-Fe2O3
nanocrystals were produced. The coating of c-Fe2O3
nanocrystals with CA or HMBP was done following
the protocols already described. The average number
of CA per nanocrystal was measured by thermogravimetric analysis. The average number of HMBP per
nanocrystal was determined by EDX analysis and
FTIR spectroscopy.
J Nanopart Res (2014) 16:2596
DFT calculations
All calculations presented in this work were performed employing the Gaussian 09 package (Revision
B.01) (Frisch et al. 2009). Full geometry optimizations
were performed employing DFT within the hybrid
meta-GGA approximation with the TPSSh exchangecorrelation functional. In these calculations we used
the standard 6-311??G(d,p) basis set for all atoms.
(Tao et al. 2003). No symmetry constraints have been
imposed during the optimizations. The stationary
points found on the potential energy surfaces as a
result of geometry optimizations were tested to
represent energy minima rather than saddle points
via frequency analysis. The default values for the
integration grid (75 radial shells and 302 angular
points) and the SCF energy convergence criteria
(10-8) were used in all calculations. All systems
containing Fe(III) were modeled in their high-spin
configurations (S = 5/2) using an unrestricted model.
IR spectra were simulated from the calculated harmonic frequencies and IR intensities obtained through
second derivatives, with the aid of the GaussView
program (Dennington et al. 2009). A half width at half
height of 304 cm-1 was applied. The frequencies used
to obtain the simulated spectra were unscaled.
Ligand exchange experiments
HMBP exchange on c-Fe2O3@CA nanoparticle
surface
To 5 mL of a c-Fe2O3@CA ferrofluid ([Fe] & 0.1 M,
pH 7), 2 mL of a HMBP solution (1 or 10 equivalent
per CA, pH 7) was added. The mixture was stirred or
sonicated for 4 h. In order to eliminate the excess of
coating molecules, the nanoparticles were purified as
follows: the pH was adjusted to 2 by addition of HCl
0.1 M, and the nanoparticles were separated by
magnetic decantation and washed five times with
HCl 10-2 M. The nanoparticles were then re-dispersed
in water at pH 7. The same protocol was followed for
HMBP-biotin and HMBP-C:CH on c-Fe2O3@CA.
CA exchange on c-Fe2O3@HMBP nanoparticle
surface
A solution of CA (1 or 10 equivalent per HMBP) was
prepared in 2 mL at pH 11. The pH was then adjusted
Page 5 of 13
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at 7 by addition of NaOH and mixed to 5 mL of a cFe2O3@HMBP ferrofluid ([Fe] & 0.1 M, pH 7). The
mixture was sonicated for 4 h. Removal of excess of
coating agent was performed as previously described.
c-Fe2O3 coating with an equimolar mixture of CA
and HMBP
65 mg of CA was dissolved in 2.5 mL of water at pH
11. The pH was then adjusted at 7 by addition of
NaOH and mixed with 2.5 mL of a HMBP (125 mg)
solution at pH 7. The resulting mixture was added
under vigorous stirring to 5 mL of bare c-Fe2O3
ferrofluid (([Fe] & 0.1 M). The pH was then adjusted
to 7 and the solution was stirred for 2 h. The pH was
then adjusted to 2 by addition of HCl 0.1 M and the
nanoparticles were separated by magnetic decantation
and washed five times with HCl 10-2 M. The
nanoparticles were then re-dispersed in water at pH 7.
Biotin/streptavidin assay
A 250 lL solution of c-Fe2O3@CA ? HMBP-Biotin
nanoparticles (3 wt% Fe2O3) was prepared in sodium
tetraborate buffer (0.01 M). 50 lL of Streptavidin
(5 mg mL-1) was added and the mixture was stirred
for 30 min. Nanoparticles were then isolated by
magnetic separation, washed three times, and redispersed in the buffer. The solution was then
deposited on the strip at the starting point. The
particles are allowed to migrate by capillarity.
Results and discussion
Nanoplatforms characterization
c-Fe2O3 nanoparticles were obtained by ‘‘soft synthesis’’ (Lalatonne et al. 2008) based on a direct micelle
protocol. The TEM images of deposited nanocrystals
indicated an average diameter of 11.2 nm and a size
distribution of r = 0.3, Fig. 1.
Coatings by HMBP and CA were performed as
previously described (Bolley et al. 2013b; Lalatonne
et al. 2008). c-Fe2O3@CA and c-Fe2O3@HMBP
nanoparticles both formed highly stable ferrofluids
in a large pH range, from pH 4 to pH 11. At pH 7.4
the hydrodynamic diameter and the zeta potential
are 16.7 nm/-50 mV and 20.3 nm/-51 mV,
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J Nanopart Res (2014) 16:2596
Fig. 1 TEM image and size
distribution for cFe2O3@CA
respectively. The second derivatives of magnetization,
measured with a MIAplex detector are characteristic of
superparamagnetic behavior (Fig. S4).
For c-Fe2O3@CA nanoparticles, the number of CA
molecules per particles was determined by TGA and
found to be 1,600 molecules/particles. For c-Fe2O3@HMBP nanoparticles, the grafting yield was deduced
by EDX measurements and found to be 1,800 molecules/particles. These results correspond to surface
areas of 24 and 22 Å2 occupied, respectively, by the
CA and HMBP ligands. These surface areas were in
good accordance with previously obtained results
(Bolley et al. 2013a; de Montferrand et al. 2012;
Lalatonne et al. 2008). To further elucidate the
interactions between nanoparticle surface and both
CA and HMBP, we performed FTIR spectroscopic
measurements and DFT calculations at the TPSSh/6311 ??G(d,p) level of theory (Fig. 1). In accordance
with previous observations, the spectrum of c-Fe2O3@CA nanoparticles presents a strong characteristic
band between 1,650 and 1,600 cm-1, attributed to the
asymmetric m(C=O) stretching vibration of the carboxylate function coupled with the m(C=C) vibration
of the alkene function, which is related to the
formation of a coordinate bond between the nanoparticle surface and the catecholic aromatic ring (Bolley
et al. 2013b; Shultz et al. 2007). The vibration bands
corresponding to C=C stretching between 1,550 and
1,650 cm-1 as well as vibration bands corresponding
to C–O stretching and bending (between 950 and
1,300 cm-1), also appeared modified upon surface
modification. Our DFT calculations performed on the
123
(HO)2Fe@CA system (Fig. 2) provide a calculated IR
spectrum in fairly good agreement with the experimental spectrum of c-Fe2O3@CA. In particular, the
simulated spectrum showed a broad band envelope
maximum at 1,595 cm-1 due to the asymmetric
m(C=O) vibration (1,628 cm-1 in the experimental
spectrum). The m(C=C) vibration was calculated at
1,493 cm-1 (1,488 cm-1 in the spectrum of c-Fe2O3@CA), and mainly involved the displacement of
carbon atoms placed one and two bonds away from the
oxygen atoms of the catechol moiety. The bands
calculated at 1,330 and 1,274 cm-1 corresponded to
the symmetric carbonyl stretch and the m(C–O)
vibration coupled to in-plane C–C–H deformations
(experimental values of 1,389 and 1,264 cm-1,
respectively).
The main discrepancy between the experimental
and calculated spectra was due to the overestimation
of the intensity of the latter two bands. The spectral
region 450–750 cm-1 was characterized by a broad
absorption band in the experimental and calculated
spectra. This band was associated to Fe–O stretching
vibrations that signal particle functionalization (Fig.
S5). The good agreement between the experimental
and calculated spectra confirmed that CA binds to the
particle surface through the catechol unit, which acts
as a bidentate chelating unit forming a five-membered
chelate ring. Furthermore, different model systems
with the catechol unit of CA acting as a bidentate
bridging unit or with CA binding to Fe through the
carboxylate group provided a poor agreement with the
experimental spectrum. The spectrum of c-
J Nanopart Res (2014) 16:2596
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Fig. 2 FTIR comparison of free ligand with ligand at the surface of the c-Fe2O3 nanoparticles (CA, panel a; HMBP, panel b) and
spectra obtained with DFT simulations for CA and the (HO)2Fe@CA system (c) and (OH)Fe–O–Fe(OH)@HMBP (d)
Fe2O3@HMBP nanoparticles presented significant
changes with respect to that of HMBP in the region
of the phosphonic vibrations (900–1,200 cm-1),
indicating the linkage of the bisphosphonic moiety
to the surface (Benyettou et al. 2011, 2009; Guénin
et al. 2012; Lalatonne et al. 2008). The other
characteristic vibration bands corresponding to the
carboxylate region (1,700–1,400 cm-1) experienced
minor changes upon grafting the molecule to the
surface. The theoretical prediction of the spectrum of
c-Fe2O3@HMBP was proved to be rather difficult.
The best agreement was obtained using the (OH)Fe–
O–Fe(OH)@HMBP model system, in which each
phosphonate function coordinates to a Fe center in a
bidentate chelate fashion (Fig. 2). The main
difference between the experimental and calculated
spectra arose from the band due to the P–O stretches
calculated at ca. 860 cm-1 that was observed at ca.
1,000 cm-1 for c-Fe2O3@HMBP. The reasons for the
poorer agreement between experimental and calculated spectra for c-Fe2O3@HMBP were probably
related to the presence of three oxygen atoms in each
phosphonate function that can potentially give mono-,
bi-, and tridentate modes. In addition, non-coordinated oxygen atoms were likely to be involved in
hydrogen-bonding interaction with OH groups on the
surface of the metal oxide nanoparticle, which was
expected to have a significant influence in the P–O
stretching vibrations (Pujari et al. 2014). The two
spectra showed the typical strong vibration bands of
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Fig. 3 Illustration of the ligand exchange yield evaluation using FTIR (a) and EDX (b) analysis. c-Fe2O3@HMBP (blue curve), cFe2O3@CA (red curve) and one example of hybrid exchange experiment c-Fe2O3@ CA ? HMBP (black curve). (Color figure online)
Fe–O that indicate coordination of the phosphonate
functions to the surface of iron oxide (Fig. S6).
CA versus HMBP ligand exchange
at the nanoparticle surface
Aiming to evaluate the ligand exchange at the nanoparticle surface (Scheme 1), we firstly added 1 or 10
equivalent of HMBP to the c-Fe2O3@CA nanoparticle
in water at pH 7 under stirring. The solution was left for
4 h after stirring at room temperature. The input of
sonication on the exchange process was also evaluated,
keeping the number of HMBP equivalents and the
exchange time unchanged. To further study the competitive attachment of the two molecules we also
performed the reverse experiment by adding CA onto
c-Fe2O3@HMBP nanoparticles. Finally, a coating
reaction starting with bare nanoparticles was also
evaluated by mixing the two ligands in equimolar
conditions. The molecular exchange yield was calculated by two different methodologies: FTIR and EDX.
We focused on the evaluation of the number of HMBP
molecules added or removed from the surface. The
number of molecules of HMBP was determined by
FTIR spectroscopy (figs S7 to S10). The different FTIR
spectra are normalized with the iron oxide vibration
band (500–600 cm-1) and then the HMBP ratio per
123
particle is determined from area measurement of the
phosphonic function vibration bands (between
950–1200 cm-1). One must note that in the spectral
range corresponding to vibration bands of the phosphonic function are observed in a spectral region where
no significant vibration bands were detectable for the cFe2O3@CA nanoparticle, thus allowing a good reliability of the FTIR evaluation. On the contrary, when trying
to evaluate the CA exchange yield difficulty arose as all
vibration bands of the CA were in position where
vibrations of HMBP could be found (Fig. 3a). Consequently, determining the amount of CA molecules was
not possible using FTIR spectroscopy. Using EDX, the
number of HMBP molecules present at the nanoparticle
surface was determined by studying the phosphorous/
iron ratio (Fig. 3b).
For each experiment, the hydrodynamic diameter
and zeta potential were measured. The zeta potential at
pH 7.4 changed only slightly. This is consistent with
the fact that the two initial nanoplatforms (c-Fe2O3@CA and c-Fe2O3@HMBP) present same zeta potential
(-50 mV) and to the fact that carboxylic acid function
on the c-Fe2O3@CA nanoplatform is substituted to the
carboxylic acid function of the HMBP and vice versa.
The hydrodynamic diameter (DH) ranged from 17 to
30 nm indicating a relatively low aggregation state
considering the DH of the two initial nanoplatforms
50
37
890
310
35
17
19
340
350
20
625
–
100
55
0
% HMBP (from FTIR)
990
–
HMBP/particle (FTIR)
665
865
1,800
750
330
440
385
HMBP (1 eq.)
Sonication 4 h
HMBP (10 eq.)
Stirring 4 h
HMBP (1 eq.)
Stirring 4 h
1,189
–
HMBP/particle (EDX)
770
CA ? HMBP
Stirring 2 h
–
–
–
–
Added ligand
Conditions
HMBP (10 eq.)
Sonication 4 h
c-Fe2O3@HMBP
Nanoparticles
Table 1 Results of the coating and the ligand exchange at the surface of nanoparticles
Page 9 of 13
c-Fe2O3@CA
CA (1 eq.)
Sonication 4 h
CA (10 eq.)
Sonication 4 h
c-Fe2O3
J Nanopart Res (2014) 16:2596
2596
(16.7 and 20.3 nm). The magnetic behavior at room
temperature was evaluated for each experiment and
compared to that of the starting nanoplatform. The
ligand exchange process did not induce changes in
magnetic properties as attested by the similar MIAplex
signatures (Fig. S4). Moreover, the magnetic signature
stability is also indicative of the relative low aggregation state already mentioned (Benyettou et al. 2011).
Table 1 reports the average number of coating molecules per nanoparticle and ligand exchange yields as
well as the results obtained by coating bare nanoparticles with an equimolar mixture of CA and HMBP.
Results obtained from EDX and FTIR were generally in good accordance with the HMBP quantification, varying only slightly between the two evaluation
methods. Ligand exchange at the surface of cFe2O3@CA nanoparticle showed that mild conditions
(stirring 4 h) promoted exchange at the surface but the
amount of HMBP introduced was limited to 20 %.
Only when using sonication and a large HMBP excess
(10 eq.) the exchange yield was enhanced, allowing
the addition of more HMBP up to 35 % HMBP onto
the surface. Sonication was already described to the
enhanced ligand exchange processes with other types
of ligands, such as silanes (Bloemen et al. 2012).
When studying the reverse exchange (adding CA onto
c-Fe2O3@HMBP nanoparticles), the addition of CA
appeared slightly more efficient as when using 10 eq.
under sonication more than half of the HMBP were
replaced (37 % remaining). Finally, when trying to
coat bare nanoparticles using both molecules in
equimolar quantity, only half of the theoretical
number of HMBP can be added. With respect to the
similar surface area occupied by both molecules, we
can reasonably hypothesize that approximately as
much CA was added. Taking together these results
indicated that the two chelating molecules have close
affinity for the iron oxide surface; however, CA
seemed slightly more affine. This result is in accordance with what was observed by Wang et al. (2006).
When iron oxide nanoparticles were coated with a
catechol-HMBP difunctional molecule, catechol
bonding to the surface was preferred.
Toward multifunctional nanoparticle
To conduct our study, molecules bearing the same
carboxylic functionality on their side chain were
chosen. In order to obtain nanoparticles with multiple
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2596 Page 10 of 13
J Nanopart Res (2014) 16:2596
Fig. 4 Ligand exchange on c-Fe2O3@CA with HMBP-C:CH and HMBP-Biotin and corresponding EDX spectra
functionalities, the ligand exchange methodology was
further used to introduce two different HMBPs onto
the c-Fe2O3@CA nanoparticle surface. A first set of
experiments were performed with an HMBP bearing
an alkyne function on the side chain (HMBP-C:CH).
Such functionality can be further used to introduce
molecules by two different click methodologies (Bolley et al. 2013a; Demay-Drouhard et al. 2013; Guénin
et al. 2012). A second HMBP bearing an ureido ring
fused with a tetrahydrothiophene ring typical of the
biotin motif (HMBP-Biotin) was also investigated
(Fig. 4 and Fig. S11). Both molecules were added
using the conditions described previously: 10 eq. of
HMBP molecules added to c-Fe2O3@CA at pH 7
under sonication for 4 h. EDX was used to evaluate the
quantity of HMBP molecule exchange at the surface,
123
which yielded 725 and 600 molecules/particles for
HMBP-C:CH and HMBP-Biotin, respectively. It
can be noted that in the case of addition of HMBPBiotin, the presence of sulfur was also detected by
EDX in the expected half ratio toward phosphorus.
Comparing the previous results with HMBP, the average number of HMBP-C:CH added to c-Fe2O3@CA
nanoparticle’s surface was similar. The exchange with
HMBP biotin was slightly less efficient, which could
be related to steric hindrance induced by biotin
function.
In the case of the c-Fe2O3@CA ? HMBP-Biotin
nanoparticles we further studied the availability of the
biotin moiety at the nanoparticle surface by performing
a magnetic version of the common streptavidin–biotin
binding experiment. This procedure used MIAstripÒ
J Nanopart Res (2014) 16:2596
Page 11 of 13
2596
Scheme 2 Streptavidin–
biotin binding procedure on
MIAstrip with cFe2O3@CA ? HMBPBiotin
(Lenglet et al. 2008; Motte et al. 2010) on which BSAbiotin is sprayed onto the test line (Scheme 2). The cFe2O3@CA ? HMBP-Biotin nanoparticles were conjugated to streptavidin and deposited on the strip at the
starting point (Scheme 2). Then the particles were
allowed to migrate by capillarity, the particles presenting available streptavidin at their surface were retained
on the test line. The detection was performed over the
strip measuring the MIAtekÒ signal of magnetic
particles. Such a measurement is directly proportional
to the magnetic material quantity (Geinguenaud et al.
2012). When the strip-test was performed with the cFe2O3@CA ? HMBP-Biotin nanoparticles, a MIAtek
signal of 150 a.u. was obtained on the test line whereas
no signal was obtained when using c-Fe2O3@CA
nanoparticles. This result demonstrated the specific
targeting of multi-functionalized nanoplatforms. Thus,
this confirmed the efficient ligand exchange at the
surface of the nanoparticle and the availability of the
biotin moiety to bind streptavidin.
Conclusion
We studied nanoplatforms consisting of iron oxide
coated with two bifunctional coating agents of the
catechol and HMBP family. These two molecules
differed only by their chelating moiety. Both nanoplatforms were studied by FTIR and DFT calculations which were performed to further elucidate the
interactions between catechol/bisphosphonate groups
with nanoparticle surface. Theoretical spectra
obtained from DFT calculations were reasonably in
good accordance with the observed FTIR spectra,
indicating that the attachment of both ligands was, as
expected, realized through their chelating moiety.
We then evaluated the ligand exchange onto the
surface in water under several conditions. We have
shown that both molecules can be exchanged. An
increase in ligand exchange was obtained using a
large excess of HMBP (10 eq.) and sonication. In
these experiments, the catechol ligand appeared
slightly more affine for the iron oxide surface than
the HMBP ligand. Using two other bisphosphonates
differing by their lateral chain and end functionality
(HMBP-biotin and HMBP-C:CH), we finally
showed that this methodology can lead to control
multi-functionalization of the nanoparticle surface.
The availability of biotin at the surface of the
nanoparticle to bind streptavidin was further evaluated using a magnetic biotin–streptavidin assay
demonstrating the specific targeting of multi-functionalized nanoplatforms paving the way for their
use as a magnetic bioprobe.
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2596 Page 12 of 13
Acknowledgments The authors thank the Magnisense
Corporation for providing a MIAplexÒ and a MIAtechÒ
Reader and MIAstripÒ. The authors also thank N. Liévre
(UFR SMBH, Université Paris 13, Bobigny, France). C. P.-I.
thanks Centro de Supercomputación de Galicia (CESGA) for
providing the computer facilities.
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