Precise determination of the nanoparticle concentration and ligand density of engineered water-soluble HgSe fluorescent nanoparticles

Article abstract of DOI:10.1039/c5ra26268b

HgSe nanoparticles (NPs) have been recognized as the ultimate product of mercury detoxification in biological systems. However, the exact mechanism of their formation and distribution in animals is not understood, and thus well-characterized engineered NPs would be invaluable to investigate such metabolic products. In this work, HgSe NPs were sonochemically synthesized and transferred for the first time to aqueous media using dihydrolipoic acid (DHLA) as a capping ligand. A thorough characterization of the engineered HgSe NPs was carried out. Different complementary analytical techniques, including high resolution-transmission electron microscopy (HR-TEM) and X-ray powder diffraction (XRD), provided information about the morphology and crystal structure of the NPs. X-ray photoelectron spectroscopy (XPS) and energy dispersive X-ray spectrometer (EDX) analysis were used in order to assess the chemical composition and purity of the same HgSe NPs. Moreover, Fourier transform infrared spectroscopy (FT-IR), nuclear magnetic resonance (NMR), dynamic light scattering (DLS), zeta potential determinations and asymmetric flow field-flow fractionation (AF4) coupled on-line to fluorescence and inductively coupled plasma-mass spectrometry (ICP-MS) detectors confirmed that the DHLA ligands were bound onto the surface of the NPs making them stable in aqueous solutions. Fluorescence measurements evidenced that the engineered NPs exhibit a photoluminescence emission centred at 575 nm. Finally, the Se/Hg molar ratio of the NPs was accurately and precisely determined by ICP-MS to be 0.96 ± 0.03. Such valuable information was used to precisely determine the nanoparticle concentration in aqueous solution (in terms of number of nanoparticles per unit volume) and the ligand density, in terms of number of DHLA molecules per nanoparticle (2500 ± 125).

Full text of DOI:10.1039/c5ra26268b

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Precise determination of the nanoparticle concentration and
ligand density of engineered water-soluble HgSe fluorescent
nanoparticles
Received 00th January 20xx,
Accepted 00th January 20xx
Diego Bouzas-Ramos, Mario Menéndez-Miranda, José M. Costa-Fernández*, Jorge Ruiz Encinar*
and Alfredo Sanz-Medel
DOI: 10.1039/x0xx00000x
www.rsc.org/
HgSe nanoparticles (NPs) have been recognized as the ultimate product of mercury detoxification in biological systems.
However, the exact mechanism of their formation and distribution in animals is not understood and thus well-
characterized engineered NPs would be invaluable to investigate such metabolic products. In this work, HgSe NPs were
sonochemically synthesized and transferred for the first time to aqueous media using dihydrolipoic acid (DHLA) as capping
ligand. A thorough characterization of the engineered HgSe NPs was carried out. Different complementary analytical
techniques, including high resolution-transmission electron microscopy (HR-TEM) and X-ray powder diffraction (XRD),
provided information about the morphology and crystal structure of the NPs. X-ray photoelectron spectroscopy (XPS) and
energy dispersive X-ray spectrometer (EDX) analysis were used in order to assess the chemical composition and purity of
the same HgSe NPs. Moreover, Fourier transform infrared spectroscopy (FT-IR), nuclear magnetic resonance (NMR),
dynamic light scattering (DLS), zeta potential determinations and asymmetric flow field-flow fractionation (AF4) coupled
on-line to fluorescence and inductively coupled plasma-mass spectrometry (ICP-MS) detectors confirmed that the DHLA
ligands were bound onto the surface of the NPs making them stable in aqueous solutions. Fluorescence measurements
evidenced that the engineered NPs exhibit a photoluminescence emission centred at 575 nm. Finally, the Se/Hg molar
ratio of the NPs was accurately and precisely determined by ICP-MS to be 0.96 ± 0.03. Such valuable information was used
to precisely determine the nanoparticle concentration in aqueous solution (in terms of number of nanoparticles per unit
volume) and the ligand density, in terms of number of DHLA molecules per nanoparticle (2500 ± 125).
these NPs are thought to be the ultimate metabolic product of
the Hg detoxification mechanism in biological systems.7,9 In
fact, several studies reported the presence of HgSe NPs in
Introduction
Since the discovery of the protective effect of selenium (Se) on
the intoxication by mercury (Hg) in laboratory rats,1 extensive
studies have demonstrated the toxicological antagonism
between both elements in animals.2,3 Nowadays it is well-
known that the accumulation of Hg often accompanies Se
accumulation, typically at a 1:1 molar ratio, in tissues of
marine mammals4 and in organs of Hg mine workers.5 This
equimolar Hg:Se ratio might be assigned to the presence of
mercury selenide (HgSe) nanoparticles (NPs) in the organs.
Actually, HgSe granules were found in the liver of cetaceans
and were assumed as the final metabolic product.6 Further
studies proposed that Se-containing biomolecules provide
effective and efficient pathways for converting some toxic Hg
compounds to chemically and biologically inert HgSe NPs.7,8
Although the mechanism is still far to be fully understood,
tissues of various marine mammals,10,11 sea birds12 and most
recently in human brain.13
In order to shed new light towards a better understanding
of the formation, distribution, metabolism and final fate of
HgSe NPs in biological systems, the use of well-characterized
water-soluble engineered NPs is needed to initiate basic in-
vitro and in-vivo studies. In this context, a detailed and
comprehensive physicochemical characterization of the
engineered NPs is required to ensure comparative and reliable
data.14 When synthesizing novel NPs their full characterization
in dry state is still indispensable (e.g. size, shape and chemical
composition of the core), but their properties in solution (e.g.
surface potential and hydrodynamic diameter) must also be
carefully characterized in order to understand their behaviour
in biological systems.15,16 Furthermore, it is imperative to
determine the nanoparticle concentration in terms of number
of NPs in a given sample, since NP toxicity issues are
commonly linked to NP concentration-dependent effects.15
During the past decades, only
a small number of
publications are dealing with the synthesis of HgSe NPs.
Conventional methods reported for the preparation of
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mercury chalcogenides include solid state reactions at
The precursors used for the synthesis of the NPs were
elevated temperature17 and reactions of metal cations with selenium powder (99.99%) from Sigma-Aldrich (Milwaukee,
hydrogen chalcogenides in aqueous solution.18 However, the WI, USA), mercury(II) acetate (≥98%) from Panreac (Barcelona,
solid-state reactions are not easily controllable, while the use Spain) and ethylenediaminetetraacetic acid disodium salt
of the gaseous and highly toxic hydrogen chalcogenides has dihydrate (EDTA, ≥99%) from Fluka (Basel, Switzerland).
some important practical limitations. Additionally, the Sodium hydroxide and absolute ethanol were obtained from
obtained products are not nanosized. More recently, Merck (Darmstadt, Germany) and Sigma-Aldrich, respectively.
preparation of metal chalcogenides has been described by
Water-solubilization of HgSe NPs was achieved by coating
means of a solvothermal synthesis19 and nanocrystalline them with DHLA ligands synthesized in our laboratory.28 For
mercury chalcogenides using a room-temperature conversion such purpose, DL-Thioctic acid (98%), sodium bicarbonate and
route,20 for both methods long reaction times are required. sodium borohydride (≥96%) were purchased from Acros
Alternatively, the sonochemical method has been recently Organics (Geel, Belgium), Merck and Fluka, respectively.
shown to be a quick and versatile technique to prepare HgSe Potassium tert-butoxide (≥98%), anhydrous methanol and
nanostructures.21 Because of the unique reaction effect of the anhydrous toluene, all of them acquired from Sigma-Aldrich,
ultrasound irradiation, this method has a rapid reaction rate, were also used during the solubilization process. L-Cysteine
tuneable reaction conditions and the capacity to form HgSe hydrochloride monohydrate (≥99%) was purchased from
NPs with uniform shapes, narrow size distributions and high Merck. Nitric acid (65%, v/v) from Merck was additionally
purity.22,23 The effects of ultrasound result from the process purified by subboiling distillation to operate with the minimum
called acoustic cavitation: the formation, growth and implosive background levels of metallic impurities. External FIA (Flow
collapse of bubbles in a liquid. During the ultrasonic radiation, Injection Analysis) calibrations using Merck certified 1000 mg
intense heating of the bubbles occurs.24 These unique reaction L-1 standards of Se, Hg and S were used in the ICP-MS analysis.
conditions, temperatures of roughly 5000 K and pressures of
Ammonium acetate and sodium dodecyl sulphate (SDS),
about 500 bar, provide an ideal atmosphere for the from Sigma-Aldrich, were used for the carrier in the AF4
preparation of nanomaterials. While the sonochemical instrument.
synthesis of HgSe NPs in aqueous solutions has been recently
described,25,26 the final product of the synthesis is essentially Instrumentation
insoluble and, furthermore, an appropriate characterization of
The synthesis of the HgSe NPs was carried out using a Vibra-
the obtained NPs is still lacking today.
Cell VC250 high-intensity ultrasonic probe (Sonics & Materials,
Herein, we present a simple and comprehensive protocol
USA). An ultrasonic bath (JP Selecta, Spain) was used to
to produce and thoroughly characterize HgSe NPs in water
improve the acid digestion of the DHLA-capped NPs before
media ready to be used in environmental and biological
ICP-MS analysis.
studies. A sonochemical synthesis has been employed and the
HR-TEM images were obtained on a JEM-2100F (JEOL,
resulting HgSe NPs were then transferred for the first time to
Japan) transmission electron microscope with a copper grid,
aqueous media by capping them with dihydrolipoic acid
using an accelerating voltage of 200 kV. The JEM-2100F was
(DHLA) ligands. Combination of the quantitative data produced
equipped with an energy dispersive X-ray spectrometer (EDX).
by inductively coupled plasma-mass spectrometry (ICP-MS),
XRD measurements were performed using a X´Pert Pro
directly or coupled to a powerful separation technique for NPs
diffractometer (PANalytical, Netherlands). X-ray photoelectron
(asymmetric flow field-flow fractionation, AF4), with
spectroscopy (XPS) analysis was performed using a Phoibos
complementary techniques such as Fourier transform infrared
100 hemispherical analyzer with a MCD-5 detector (SPECS,
spectroscopy (FT-IR), nuclear magnetic resonance (NMR), X-ray
Germany).
powder diffraction (XRD), high resolution-transmission
Infrared spectra were taken in a Varian 670 FT-IR
electron microscopy (HR-TEM) and dynamic light scattering
spectrometer (Agilent, Australia) equipped with a Golden Gate
(DLS) provided an in-depth characterization of the engineered
attenuated total reflectance (ATR) device consisted of a
HgSe NPs produced. In this context, elemental stoichiometry
diamond crystal with one internal reflection. Room
1
temperature H-NMR spectra were recorded on a Bruker AV-
of the core, ligand density on the surface and nanoparticle
concentration, in terms of the number of nanoparticles per
600 spectrometer (600.15 MHz).
unit volume, could be precisely determined.
Elemental measurements were carried out on a 8800 Triple
Quadrupole ICP-MS (Agilent, Japan) using a MicroMist
nebulizer. A two-position and six-port Rheodyne injection
Experimental
valve (IDEX Health & Science, Germany) was used to allow an
Reagents and materials
on-line flow injection analysis (FIA) of the samples.
Zeta potential and DLS measurements were carried out
with a Zetasizer Nano ZS (Malvern Instruments, UK). All these
measurements were performed using DTS1070 disposable
capillary cells and ZEN0040 disposable cuvettes, respectively.
UV/vis spectrophotometric measurements were performed in
All the chemical reagents used in the experiments were of
analytical grade and used as received without further
purification. Deionized ultrapure water (18.2 MΩ cm) was
obtained with a Milli-Q system (Millipore, Bedford, MA, USA)
and used throughout the work.
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Genesys 10S Spectrophotometer (Thermo Scientific,
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a
XPS measurements of the HgSe NPs powder were
Germany). Fluorescence spectra were recorded on a LS-45 performed under ultra-high vacuum conditions (10-9–10-10
Luminescence Spectrophotometer (Perkin Elmer, USA) using a mbar) using Mg X-ray radiation (1253.6 eV) at 200 W. Low
fixed excitation wavelength of 400 nm with both excitation resolution spectrum was taken with pass energy of 90 eV,
and emission slit width of 10 nm. The measurements of whereas high resolution spectra were recorded at pass energy
absorbance and fluorescence were carried out using of 30 eV. Analysis of the spectra was performed using the
conventional Quartz SUPRASIL cuvettes (Hellma Analytics, Computer Aided Surface Analysis for X-ray Photoelectron
Germany). The AF4 system used was an AF2000 (Postnova Spectroscopy software (CasaXPS).
Analytics, Germany). The fluorescence detector on-line
coupled to the AF4 was a 1200 Series FLD (Agilent, Japan).
ICP-MS analysis. Simultaneous detection of Se, Hg and S
was carried out with a ICP-MS equipped with a triple
quadrupole,29 due to its capabilities to detect, free of
interference and with high sensitivity, typically highly
interfered elements such as Se and especially S.30 For such
purpose, oxygen was introduced in the collision/reaction cell
at a flow rate of 0.35 mL min-1. Se and S were detected in mass
Experimental procedures
Sonochemical synthesis of HgSe NPs. HgSe NPs were
synthesized using Se powder and Hg(OAc)2 as precursors via
the sonochemical procedure described by Kristl et al.25 with
slight modifications. Briefly, 5 mmol of Se powder was
dissolved in 50 mL of 5 M NaOH solution. The mixture was
irradiated with ultrasounds via a direct-immersion high-
intensity probe (80 W cm-2) for 30 min, and simultaneously an
equimolar amount of Hg2+ in a 0.1 M EDTA solution was slowly
added. After the EDTA addition, the solution was left irradiated
during 3 h at room temperature. After ultrasounds irradiation,
the resulting suspension was centrifuged and the black
precipitate was washed in sequence with volumes of 0.1 M
EDTA, water and absolute ethanol. Finally, the washed powder
was left to dry in an open vessel to air.
→ →
shift MS/MS mode (80Se+ 96SeO+ and 32S+ 48SO+,
respectively) after their reaction with oxygen in the cell. Hg,
which does not react with oxygen, was measured in on-mass
MS/MS mode (202Hg+
→
202Hg+). The integration time for the
targeted isotopes 76Se, 78Se, 80Se, 200Hg, 202Hg, 32S and 34S was
0.1 s. Each operation conditions were daily optimized using a
tuning solution. Integration of the FIA peaks was performed
using the MassHunter software (Agilent). ICP-MS operating
conditions are summarized in Table S1 (see ESI).
Elemental ratios (Se/Hg and Se/S) in the DHLA-HgSe NPs
were determined by performing first an external FIA
calibration using Se, Hg and S standards (see Fig. S1 and S2 in
the ESI). A 0.1% (w/v) solution of cysteine in 1% nitric acid was
used as carrier for the determination of the Se/Hg ratio. The
addition of cysteine to the carrier during the measurement of
the Se/Hg ratio was essential to eliminate any adsorption of
mercury or memory effects in the ICP-MS.31 Conversely, a 1%
nitric acid solution was used as carrier for the determination of
the Se/S ratio inasmuch as sulphur detection would have not
been possible if cysteine was present in the carrier. For the
determination of these elemental molar ratios, the
synthesized DHLA-HgSe NPs were injected directly to the ICP-
MS. The experimentally obtained Se/Hg and Se/S area ratios
could be then translated into the Se/Hg and Se/S molar ratios
using the corresponding elemental calibration curves
previously obtained.
Preparation of DHLA and transfer of HgSe NPs to aqueous
media. Pure DHLA was prepared following the procedure
described by Clapp et al..28 Colloidal suspension of water-
soluble NPs was prepared by dispersing 20-50 mg of insoluble
sonochemically synthesized HgSe in 0.4-1.0 mL of freshly
prepared DHLA. The mixture was heated to 80 °C for 2 h while
stirring and incubated overnight with agitation. The resulting
solution was diluted in 4-5 mL anhydrous methanol and an
excess of potassium tert-butoxide was slowly added in order to
allow the deprotonation of the terminal carboxyl groups on
the DHLA. The formed precipitate of DHLA-capped HgSe was
sedimented by centrifugation and these new surface-modified
NPs with DHLA were dispersed in water (pH~12). An additional
purification step, consisting in an ultrafiltration using a 100
kDa cutoff ultracentrifugal filter and the subsequent
resuspension of capped NPs in alkaline water (pH>7) was
required to remove excess of reagents. Experimental
conditions used for ultrafiltration were 5000 rpm and 5 min
(x5 cycles).
In order to measure the total mass of Hg, Se and S in the
given sample, a complete digestion of the DHLA-capped HgSe
NPs was needed and the ICP-MS measurements were
performed as described above. The total mass of Hg and S and
the determined elemental molar ratios were used for the
determination of the nanoparticle concentration and the
ligand density of the NPs. Aqua regia was used to perform the
digestion of the NPs solution. A purification step before
digestion was required to remove the possible excess of DHLA
in solution by ultrafiltration using a 100 kDa cutoff membrane
filter, at 5000 rpm and 5 min (x3 cycles).
XRD and XPS measurements. For indexing and
microstructural analysis of the synthesized HgSe NPs, X-ray
powder diffraction measurements were performed employing
a X´Pert Pro diffractometer with Bragg-Brentano configuration.
This powder diffractometer was equipped with a Johansson
Ge(111) monochromator producing a monochromatic Cu Kα1
radiation (λ = 1.5405982 Å) and a PIXcel detector. The powder
sample was placed on a zero-background silicon sample
holder. XRD pattern from 20° to 100° (2θ) were recorded at
room temperature with a step size of 0.053° (2θ) and a
counting time of 500 s per step.
AF4 – Fluorescence – ICP-MS analysis. A 2 g L-1 ammonium
acetate, with 0.01% (w/v) of SDS, buffered solution (pH 10)
was employed as carrier in the AF4 system.
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Fig. 1 HR-TEM images of the (a) HgSe NPs, and (b) DHLA-capped HgSe NPs.
Fig. 2 Powder XRD pattern of the engineered HgSe NPs. The vertical lines
indicate the diffraction peak characteristics of the pure cubic tiemannite
structure (JCPDS no. 03-065-9275).
After fractionation, the effluent was first directed to the
fluorescence detector (being operated at an excitation
wavelength of 400 nm and at emission wavelength of 575 nm)
and then on-line to the ICP-MS detector equipped with a triple
quadrupole. Experimental conditions for the AF4 separation of
the DHLA-HgSe NPs are summarized in Table S2 (see ESI). ICP-
MS operating conditions are the same as in the ICP-MS
analysis section, but in this experiment only Se and Hg were
monitored because S detection was hampered by the use of
the required SDS. Se/Hg molar ratio of the on-line separated
engineered NPs was determined by external FIA calibration
using inorganic standards of Hg and Se.
The actual formation of the HgSe NPs was confirmed by
HR-TEM. A nanoparticle population in the purified product,
after the synthesis reaction, with a size of 23 ± 2 nm (1 SD,
n=50) was observed (Fig. S3 in the ESI). An HR-TEM image of
the corresponding HgSe NPs can be seen in Fig. 1a, which
shows the presence of NPs with spherical shape and low
polydispersity.
HgSe NPs crystal structure and purity estimation
The phase purity and crystal structure of the resulting products
were characterized by powder XRD analysis. The XRD pattern
of the HgSe is shown in Fig. 2. The intense and sharp
diffraction peaks indicates that the obtained nanosized
particles were well crystallized. All of the reflections of the
pattern can be indexed to the standard peaks of the pure cubic
phase of HgSe (tiemannite) with lattice constants of a = b = c =
0.6085 nm (space group F-43m), in good agreement with the
reported pattern JCPDS no. 03-065-9275. No peaks of any
other phases were detected, indicating the high purity of the
product. The nanocrystal size, τ, was also calculated from the
three major diffraction peaks of the XRD pattern using the
Scherrer equation given in 5:
Results and discussion
Sonochemical synthesis of HgSe NPs
The synthesis of the HgSe NPs was attempted here employing
a sonochemical approach25 and using Se and Hg(OAc)2 as metal
precursors as described in the Experimental section. The
mechanism of the sonochemical formation of HgSe NPs is
probably related to the radical species generated from water
molecules ultrasound energy absorption, Equation 1 below.
The extreme conditions occurring during the ultrasound
irradiation in aqueous solutions lead to the formation of H· and
OH· radicals.32 While the exact mechanism is still a subject of
speculation, a likely reaction mechanism can be as follows:
τ = (K λ) / (β cosθ)
(5)
H2O → H· + OH· (1)
where K is the so-called shape factor, which usually takes a
value of ca. 0.9; λ is the X-ray wavelength used (1.5405982 Å);
β is the width of the observed diffraction line at its half
intensity maximum (FWHM), in rad; θ is the Bragg angle, in
rad.21 The calculated nanoparticle size was 23 ± 1 nm (1 SD,
n=3), a value in good agreement with those observed
previously by HR-TEM. We could also estimate that the total
number of atoms in a HgSe nanoparticle of 23 nm is about
226000 atoms using the volume of the unit cell.
Se0 + 2OH- + 2H· → Se2- + 2H2O (2)
Hg(OAc)2 + EDTA → [Hg(EDTA)]2+ + 2OAc- (3)
[Hg(EDTA)]2+ + Se2- → HgSe + EDTA (4)
That is, the H· radicals generated from the water would be
able to reduce Se0 into Se2- (Equation 2) that quickly combine
with [Hg(EDTA)]2+ ions present in solution giving rise to HgSe
NPs, as shown in Equation 4. The complexing agents, e.g.
EDTA, plays a crucial role in the formation of HgSe NPs, since
they form a stable complex with Hg2+ that could affect the
nucleation rate and growth of the NPs.23 In this sense, it is the
EDTA complex that releases Hg2+ for the reaction with Se2- and
bringing about HgSe nanoparticles, while preventing the
formation of HgO.25
XPS measurements may provide further information about
the composition and purity of the obtained HgSe NPs. The low
resolution XPS spectrum of the sample is shown in Fig. 3a. No
peaks from eventual impurities present in the sample were
observed. The high resolution XPS spectra taken for Hg4f and
Se3d regions are shown in Fig. 3b and 3c, respectively. The two
strong peaks detected at 100.6 and 104.6 eV were assigned to
the Hg4f binding energy and the peak at 54.6 eV corresponded
to the Se3d transition.
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change or modification in the HgSe nanoparticle cores was
observed.
Both the native HgSe and the DHLA-capped NPs were then
characterized by DLS in order to investigate their
corresponding size distribution and hydrodynamic diameter.
The hydrodynamic diameter increased from 71 ± 1 nm (1 SD,
n=5) for native HgSe to 78 ± 2 nm (1 SD, n=5) for DHLA-capped
NPs (see Fig. S4 in the ESI). This increase can be assigned to the
contribution of the DHLA capping to the surface coating. It
should be considered that DLS provides information about the
hydrodynamic diameter of dispersed NPs. Therefore, it takes
into account not only the NP core but also the ligand and the
solvatation layers. This explains the significant differences
observed with data provided by HR-TEM and XRD (only
diameter of the inorganic HgSe core is given here). The
polydispersity index (PdI), which indicates the variability in the
particle size in a given sample, was also assessed using DLS.
Results showed that the performed sonochemical synthesis of
HgSe NPs provided a single distribution with an adequate PdI
of 0.15 ± 0.02 (1 SD, n=5). The PdI of the DHLA-capped NPs
was 0.10 ± 0.02 (1 SD, n=5), indicating a significant lower
polydispersity of the colloidal suspension of capped NPs. Such
decrease of the PdI value likely points to an electrostatic
stabilization of the DHLA-capped HgSe NPs in aqueous solution
via the negatively charged carboxylate groups of the DHLA
ligands attached on the surface. This results in significant
nanoparticle repulsion and therefore less aggregation.
In this context, the zeta potential is an important tool for
understanding the state of the nanoparticle surface and so
predicting the colloidal stability of NPs (which should be small
enough to remain as a stable colloidal suspension). Zeta
potential measurements of the DHLA-capped HgSe NPs were
carried out at different pH values ranging from 7 to 12, as
DHLA-capped NPs are stable in alkaline aqueous media
(pH>7).28 All the zeta potential values of the DHLA-HgSe NPs in
the selected pH range were lower than -35 mV, being more
negative values when increasing the pH (see Fig. S5 in the ESI).
Therefore, DHLA-capped HgSe NPs exhibited high colloidal
stability in the assayed aqueous solutions.34 The obtained
negative zeta potential values also agreed well with the
negative surface charge provided by carboxylate-terminated
DHLA ligands. It is worth noting here that DHLA-capped HgSe
NPs were stable at such basic pH aqueous medium for ca.
several weeks, before any visual precipitation was observed.
Additionally, FT-IR spectroscopy and NMR were used to
confirm that the DHLA ligands were attached to the surface of
the DHLA-capped HgSe NPs. FT-IR spectra of both native and
capped HgSe NPs were collected in the spectral range between
600 and 4000 cm-1 (see Fig. S6 in the ESI). Several spectral
bands characteristic of the DHLA, such as the symmetric and
antisymmetric stretching vibration of –CH2- at 2920 and 2845
cm-1, the deformation vibration of –CH2- group at 1415 cm-1
and the stretching vibration of C-S at 660 cm-1, are only
present in the FT-IR spectrum of the DHLA-capped HgSe NPs.
All of these bands are attributed to the DHLA capping groups.35
Fig. 3 (a) Low resolution XPS spectrum of the HgSe NPs. High resolution XPS
spectra of the (b) Hg4f, and (c) Se3d.
Peak areas of Hg4f and Se3d were measured and the
quantification of the peaks showed that the Hg:Se atomic ratio
was approximately 56:44. This result seems to point out that
the engineered NPs have a slight excess of Hg, a feature that
have already been observed for these type of NPs.23,25
Unfortunately, accuracy associated was very high (10% as
provided by the instrument supplier).
Solubilization and characterization of DHLA-capped HgSe NPs
As previously described, the capping of the HgSe NPs, to turn
them stable in aqueous media and compatible with
bioanalytical applications, was carried out with DHLA as
surface ligands anchored onto the surface of the HgSe NPs
through the two sulphur atoms of their thiol (mercapto)
groups.33 Binding of DHLA to the HgSe NPs was driven by
favourable interactions between thiol groups of the bidentate
ligands and the inorganic surface of the NPs. This modification
provides the NP surface with hydrophilic end groups,
rendering them stable in aqueous environments. Such
mechanism is similar to what has been already reported for
CdSe/ZnS Quantum Dots.28 The HR-TEM image of the DHLA-
capped HgSe is shown in Fig. 1b, which shows that the
synthesized DHLA-capped NPs were spherical and well
dispersed. Also, compared to the original NPs, no significant
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capped HgSe NPs by triple quadrupole ICP-MS. It is worth
mentioning that the standard deviation (SD) associated to each
result provided all along the following sections corresponds to
the propagated uncertainty of different replicates, considering
uncertainties associated to each value used in the
corresponding computation.
The inherent quantitative character of the ICP-MS
response for precise multielemental characterization of
nanostructures37 enabled us to determine accurately and more
precisely the elemental stoichiometry of the DHLA-capped
HgSe NPs. The Se/Hg molar ratio was directly determined in a
colloidal suspension of DHLA-HgSe NPs, in order to avoid any
potential losses or contaminations during acidic digestion. The
measured 80Se/202Hg area ratio was then translated into a
Se/Hg molar ratio (0.96 ± 0.02 for 80Se/202Hg; 1 SD, n=4) using a
calibration curve obtained with Se and Hg standard solutions
(Fig. 4a). It must be noted that possible differences between
the nebulization efficiency of the NPs and the ICP standards
should not affect the accuracy of the measured values because
ratios were used instead of absolute intensities.29 Multi-
isotope capabilities of ICP-MS allowed measuring an
alternative isotope ratio 78Se/200Hg in order to internally
validate the result and check for possible interferences. The
Se/Hg molar ratio obtained from 78Se/200Hg measurement
(0.96 ± 0.01; 1 SD, n=4) was identical to the value obtained by
measuring the most abundant 80Se/202Hg and closely matched
with the predicted value for a tiemannite crystal structure
(space group F-43m) with a 1:1 (Se:Hg) stoichiometry.
Fig. 4 (a) Plot of the Se/Hg area ratio experimentally obtained by ICP-MS versus
the theoretical Se/Hg molar ratio for the six elemental standards mixtures
prepared. Two independent area elemental ratios were measured in the DHLA-
HgSe NPs: (●) ∆)
80Se/202Hg and ( 78Se/200Hg. (b) Plot of the Se/S area ratio
experimentally obtained by ICP-MS versus the theoretical Se/S molar ratio for
the seven elemental standards mixtures prepared. Two independent area
elemental ratios were measured in the DHLA-capped HgSe NPs: (
78Se/32S. RSD values were below 3% in each point of the calibration curves.
◊)
76Se/34S and
(■)
Interestingly, the use of the ICP-MS equipped with a triple
quadrupole analyser allowed the measurement of the low
abundant S present after DHLA derivatization, typically highly
interfered by ICP-MS.29,30 In a similar way, a Se/S molar ratio of
16.2 ± 0.6 was obtained for the DHLA-HgSe NPs using a
calibration curve built from the adequate S and Se ICP
standards (see Fig. 4b). Similarly to the Se/Hg molar ratio
determination, two independent Se/S isotope ratios were
measured: 16.5 ± 0.3 for 78Se/32S (1 SD, n=4) and 16.0 ± 0.5 for
76Se/34S (1 SD, n=4). Besides, the elemental composition of the
engineered NPs was calculated using the Se/Hg (0.96 ± 0.03)
and Se/S (16.2 ± 0.6) molar ratios determined above. The
obtained composition for the DHLA-HgSe NPs was 49.5 ± 2.4%
of Hg, 47.5 ± 2.3% of Se and 2.93 ± 0.14% of S, values in
accordance to the previous observed EDX results. It is worth
highlighting that expanded uncertainties for both elemental
1
Furthermore, H-NMR spectra of the free DHLA and DHLA-
HgSe NPs (Fig. S7 in the ESI) verified the binding of the thiol
groups of DHLA to the surface of the NPs. As can be seen (Fig.
S7b), the two thiol resonance peaks at 1.2-1.3 ppm in the
spectrum of free DHLA, which are characteristic of the reduced
dithiol moiety,36 are not present in the DHLA-HgSe NPs
spectrum (Fig. S7a). The other peaks between 1.3 and 2.7 ppm
(characteristic of the rest of the DHLA ligand) remained intact
after the surface modification of the NPs, except for a small
shift which is attributed to a change in its environment.36
Finally, EDX analysis was employed to determine the
composition of both native and capped HgSe NPs. The EDX
spectrum obtained for the native HgSe NPs (Fig. S8a in the ESI)
only exhibited peaks corresponding to Se (47 ± 1%; 1 SD, n=3)
and Hg (53 ± 1%; 1 SD, n=3) in an equimolar mixture. In
contrast, the EDX pattern of the DHLA-capped HgSe (Fig. S8b in
the ESI) showed that these NPs were composed not only of Se
(47.0 ± 1.0%; 1 SD, n=3) and Hg (50.5 ± 2.1%; 1 SD, n=3) but
also of S (2.5 ± 1.2%; 1 SD, n=3). These results confirmed that,
as expected, the DHLA molecules were successfully
incorporated to the surface of the HgSe NPs and the products
are rather pure as shown by the absence of impurity peaks in
Fig. S8 (Cu signal comes from the grid used as sample holder).
ratios and composition were below
respectively.
4 and 5% RSD,
Determination of the nanoparticle concentration
As said before, the described nanoparticle synthesis produced
spherical NPs with a diameter of 23 ± 1 nm. Thus, the volume
of each individual spherical nanoparticle was theoretically
calculated. Alternatively, the total volume of HgSe NPs was
calculated as well from the total mass of Hg measured by ICP-
MS and, using the molar ratio Se/Hg previously determined,
the mass of Se.
Elemental analysis of DHLA-HgSe NPs
The determination of elemental stoichiometry of the NPs,
ligand density and nanoparticle concentration relies on the
simultaneous quantification of Hg, Se and S of the DHLA-
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Fig. 5 Fluorescence emission spectra in water media (λex = 400 nm) of DHLA-
capped HgSe NPs (line in red) and free DHLA (line in blue). The absorbance
spectrum of the DHLA-capped HgSe NPs is also included (line in black).
Fig. 6 Fractogram of the DHLA-capped HgSe NPs with fluorescence detection at
575 nm (line in black) and ICP-MS detection: 202Hg (line in blue) and 80Se (line in
green).
Once the total mass of NPs has been computed and using
the HgSe density (i.e. 8.23 g cm-3) it is possible to calculate the
total volume of the HgSe NPs in given sample. Nanoparticle
concentration in the measured sample could then be
estimated dividing such total NP volume by the individual NP
volume.
Again, the expanded uncertainty (5% RSD) was estimated
taking into account the corresponding uncertainties associated
to the moles of NPs and S present in the solution.
Optical characterization of the DHLA-capped HgSe NPs
solution
Nanoparticle concentration is a critical and very important
parameter to be measured for downstream applications.15 It is
evident that concentrations calculated exclusively in terms of
mass of solid in a given volume of solution provides a very
limited information. Toxicological effects it would depend on
the number of NPs and their size polydispersity more than NP
mass. Using ICP-MS results and assuming that the water-
solubilized HgSe NPs in the measured sample were spherical
with an average diameter of 23 nm (HR-TEM and XRD results),
a nanoparticle concentration of 9.5 ± 0.5 nM (1 SD, n=8), tha
corresponds to 57 x 1011 NPs mL-1, was determined in the
solution of DHLA-capped HgSe NPs measured.
The optical properties of the engineered NPs in aqueous
solution were first evaluated by their UV-vis absorbance and
fluorescence spectra. Absorption spectrum of the solubilized
HgSe NPs was recorded in a water solution of pH 12 and it
showed an excitonic shoulder at ca. 390 nm (see Fig. 5).
Moreover, the synthesized NPs displayed an intense sharp
fluorescence emission at 575 nm. Fig. 5 shows the observed
fluorescence spectrum of the DHLA-capped HgSe NPs for an
excitation wavelength of 400 nm.
The determination of the molar extinction coefficient of
nanocrystals enables to perform fast and simple concentration
determinations of NPs by just UV-vis absorption
measurements. Thus, the molar extinction coefficient of the
synthesized NPs was determined from the absorbance values
at the excitonic absorption shoulder (390 nm) plotted as a
function of the different molar concentrations of the NPs
assayed (Fig. S9 in the ESI). Samples containing NPs at different
concentrations were prepared from a stock solution of DHLA-
capped HgSe NPs (9.5 nM), characterized by ICP-MS, HR-TEM
and XRD. The slope of the regression line (extinction
coefficient of the DHLA-capped HgSe NPs sample) turned out
to be 2521 ± 6 (x105) M-1 cm-1 (1 SD, n=3). In this way, for
future bioanalytical applications of such water-solubilized HgSe
NPs, as the molar extinction coefficient is known, a simply
measurement of the absorbance at 390 nm would directly give
the number nanoparticle concentration of the working
solution.
Of course, the number of atoms per HgSe nanoparticle
could be easily determined as well. In this way the number of
atoms per HgSe nanoparticle could be precisely computed,
222000 ± 8400 atoms (1 SD, n=8). This value was in accordance
with the theoretical number of atoms per nanoparticle
estimated previously by XRD.
Determination of the ligand density
The same digested solution of the capped NPs has been used
for the quantification of the nanoparticle ligand density which
is of great importance, as well as technically challenging,27
since it will determine NP stability and reactivity in different
media. In this context, the number of DHLA molecules per
HgSe nanoparticle was also computed using the total number
of NPs, calculated as described above, and the total mass of S
measured by the ICP-MS. A purification step before the
digestion of the NPs and the subsequent ICP-MS measurement
was performed by ultrafiltration to remove the possible excess
of DHLA in solution. Once the total number of S atoms per NP
was determined and taking into account that each DHLA
molecule has two atoms of S, the ligand density calculated for
the DHLA-capped HgSe NPs sample was 2500 ± 125 molecules
of DHLA per nanoparticle (1 SD, n=8).
DHLA-HgSe NPs analysis by AF4 on-line coupled to
fluorescence and ICP-MS
AF4 is a separation technique that provides a size-dependent
separation and characterization of nanoscale particles
dispersed in aqueous media.38 It has demonstrated its great
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Journal Name
potential to separate different types of NPs with a low risk of studies of HgSe NPs, shedding light into their formation in
sample degradation or aggregation due to the minimal animal tissues. Interestingly, the newly produced aqueous-
interaction with the separation membrane.29,38 Furthermore, stable DHLA-HgSe NPs are fluorescent, which make them ideal
coupling the AF4 to fluorescence and sensitive elemental to investigate their fate and interactions in biological systems.
specific detectors, such as ICP-MS, could provide not only a
nanoparticle size separation but also molecular and elemental
information29 of the DHLA-HgSe NPs in the aqueous phase.
The obtained fractogram of the DHLA-capped HgSe NPs is
shown in Fig. 6. It is apparent that there is only one peak
Acknowledgements
This work was supported by the Spanish Ministry of Education
(MINECO-13-CTQ2013-49032-C2-1-R) and Principado de
eluting at
~34 min, which could correspond with a unique
Asturias
(FC-15-GRUPIN14-092).
Agilent
Technologies
population of DHLA-HgSe NPs. As expected, this peak
contained the two elements monitored, Hg and Se.
Interestingly, the elemental molar ratio of the nanoparticle
population could be determined using external FIA calibration
and inorganic standards. In this sense, the obtained Se/Hg
molar ratio was 0.98 ± 0.01 (1 SD, n=3), which matches well
with the values obtained previously by the other techniques
used in this study. Unfortunately, S information could not be
obtained as we resorted to a small amount of an anionic
surfactant such as SDS in the carrier (0.01%, w/v) in order to
prevent nanoparticle aggregation and minimize the
interactions between HgSe NPs and the permeation
membrane. Additionally, a significant fluorescence emission at
575 nm was observed in such population of NPs. Undesired
populations were not observed neither in the fluorescence nor
ICP-MS fractograms. Therefore, the coupling between AF4
with fluorescence and ICP-MS allowed to corroborate not only
that the derivatized DHLA-HgSe NPs were stable and still
fluorescent in a salt-containing and dynamic water medium
but also the slight molar excess of Hg in the NP and the
presence of a single nanoparticle population.
Foundation is also acknowledged. Diego Bouzas-Ramos and
Mario Menéndez-Miranda acknowledge the Ph.D. grants
(BP14-137 and BP12-046, respectively) from Principado de
Asturias (Spain).
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Fluorescent HgSe NPs were synthesized and water-stabilized. Full characterization,
including ligand density and nanoparticle concentration, makes them ideal candidates
as standards to investigate HgSe NPs metabolism and toxicity.