A nanostructured label-free platform based on an ultrathin film for ultrasensitive detection of a secosteroid hormone

Article abstract of DOI:10.1039/c6ra04740h

We report the electrocatalytic activity of perovskite-type LaNiO₃-nanoxide (LN-NO) on secosteroid hormone oxidation in alkaline solution. LN-NO was synthesized by the Pechini method and calcined at 973 K for 2 h under air atmosphere. Subsequently, the LN-NO material was studied by high-resolution transmission electron microscopy (HR-TEM), energy-dispersive X-ray (EDX), X-ray photoelectron spectroscopy (XPS), X-ray diffractometer (XRD) and electrochemical techniques such as cyclic voltammetry (CV), square wave voltammetry (SWV) and electrochemical impedance spectroscopy (EIS). The Rietveld refinement by the XRD pattern indicated the presence only the LN-NO. Optimized electrocatalytic activity was achieved using the LN-NO architecture, on a label-free platform, and nanostructures with sizes ranging between 50 and 100 nm were well distributed throughout the nanoxide. The detection of the secosteroid was performed at a low potential (0.46 V vs. Ag/AgCl) in a range between 0 and 2.6 × 10^-5 mol L^-1, with a detection limit of 8.3 × 10^-7 mol L^-1, which is considerably competitive with similar devices. The application of the LN-NO nanostructured label-free platform as a voltammetric sensor showed a good sensitivity of 17.75 A M^-1. Finally, the use of LN-NO as a low-cost alternative to carbon nanomaterials (nanotubes and graphene) has the potential to be an excellent approach to sensor development.

Full text of DOI:10.1039/c6ra04740h

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A nanostructured label-free platform based on an ultrathin film for
ultrasensitive detection of a secosteroid hormone
Paulo A. RaymundoꢀPereiraa,*, Alan R. F Limab, Sergio A. S. Machadoa
a São Carlos Institute of Chemistry, University of São Paulo, PO Box 780, 13560ꢀ970
São Carlos, SP, Brazil
b Institute of Chemistry, University of São Paulo, PO Box 26077, 05508ꢀ000 São Paulo,
SP, Brazil
*Corresponding author:
São Carlos Institute of Chemistry, University of São Paulo, CP 380, CEP 13566ꢀ590,
São Carlos, São Paulo, Brazil.
Eꢀmail address: pauloaugustoraymundopereira@gmail.com (Paulo A. Raymundoꢀ
Pereira)
Tel.: +55 16 33518098; fax: +55 16 33518350.
1

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Abstract
We report the electrocatalytic activity of Perovskiteꢀtype LaNiO3ꢀnanoxide (LNꢀNO) on
secosteroid hormone oxidation in alkaline solution. LNꢀNO was synthesized by the
Pechini method and calcined at 973 K for 2 h under air atmosphere. Subsequently, the
LNꢀNO material was studied by highꢀresolution transmission electron microscopy (HRꢀ
TEM), energyꢀdispersive Xꢀray (EDX), Xꢀray photoelectron spectroscopy (XPS), Xꢀray
diffractometer (XRD) and electrochemical techniques such as cyclic voltammetry (CV),
square wave voltammetry (SWV) and electrochemical impedance spectroscopy (EIS).
The Rietveld refinement by the XRD pattern indicated the presence only the LNꢀNO.
Optimized electrocatalytic activity was achieved using the LNꢀNO architecture, on a
labelꢀfree platform, and nanostructures with sizes ranging between 50–100 nm were
well distributed throughout the nanoxide. The detection of the secosteroid was
performed at a low potential (0.46 V vs. Ag/AgCl) in a range between 0 and 2.6 × 10−5
mol L−1, with a detection limit of 8.3 × 10−7 mol L−1, which is considerably competitive
with similar devices. The application of the LNꢀNO nanostructured labelꢀfree platform
as a voltammetric sensor showed a good sensitivity of 17.75 A M−1. Finally, the use of
LNꢀNO as a lowꢀcost alternative to carbon nanomaterials (nanotubes and graphene) has
the potential to be an excellent approach to sensor development.
Keywords: Nanostructured Perovskiteꢀtype oxide, Endocrine disruptors, Sensing,
Vitamin D3, Secosteroid hormone.
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1. Introduction
Vitamin D3, or cholecalciferol (Figure 1), is a secosteroid (a steroid molecule
with one open ring)1 with an endocrine mechanism of action2. Despite its classification
as a vitamin (defined as biomolecules not synthesized in the human body) it is
sequentially synthesized by the body in the liver, skin and kidneys2 in a route catalyzed
by UVꢀsunlight. Vitamin D3 acts as a prehormone in cellular synthetic cycles in human
body.
Figure 1 – Threeꢀdimensional structure of Vitamin D3.
Vitamin D deficiency (VDD) has become a worldwide health issue, affecting
more than one billion individuals3 with significant consequences for the immunological
system, and infectious and cardiovascular diseases4. Furthermore, it can directly
influence the absorption of phosphorus and calcium in the body, and has been
associated with symptoms of others diseases such as cancer5. Due to this
hypovitaminosis D, health professionals have recommended the consumption of vitamin
D in dietary sources including fatty fishes, eggs, liver and fish liver oils1 and in some
cases also in vitamin supplements6. However, this can lead to another problem:
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probably due to its slow metabolization in the body, cholecalciferol can be excreted and
released into the environment in wastewater.
Cholecalciferol (Vitamin D3) is used in baits to control the house mouse, through
a hypercalcemia mechanism7. It is therefore important to develop analytical
methodologies for the quantification of cholecalciferol in water. Moreover,
cholecalciferol is considered an “Endocrine Disrupter Chemical” (EDC), as are steroids
in general, and can be found in superficial and underground waters. Published works
show that endocrine disrupters can increase the incidence of testicular, ovarian and
breast cancers, reduce fertility by diminishing the number of spermatozoids and
promote fish feminization8ꢀ10. Thus, the bioaccumulation of steroids in the environment
is a problem that must be addressed. In this context, researchers have been motivated to
develop analytical methodologies to identify and quantify these substances in the
environment. Currently, the development of methodologies for the remediation,
detection and quantification of endocrine disruptors in the environment is an objective
for researchers in several fields, especially in clinical and environmental analysis. Three
methods are currently used for the determination of vitamin D3: HPLCꢀMS,
11,
radioimmunoassay (RIA) and nonꢀisotopic automated determination5,
12. These
methods are precise, accurate, free from interference and reliable. However, they are
also expensive, time consuming, and require specialized analysts for their operation.
On the other hand, electrochemical methods have a number of advantages, such
as their low cost, high sensitivity, and easy operation5, 13. They also have the potential
for miniaturization and automation, and are suitable for inꢀfield/onꢀsite monitoring
using simple portable devices that generate fast qualitative and quantitative responses14.
They can therefore be proposed as an excellent alternative to classical methods15.
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Nowadays only three studies were found in literature: i) in the study by
Canevari15 et. al., cholecalciferol was determined with an electrode composed of a
hybrid silica/graphene oxide thin film modified with amorphous nickel (II) hydroxide
particles; ii) directly determined using an immunosensor with SPR transduction5 and iii)
the low solubility of cholecalciferol in water requires the use of mixed organic/water
solvents for analysis, as reported by Cincotto and coꢀworkers13. The limited number of
publications in this subject indicates the necessity of further work on cholecalciferol
electroanalytical determination in order to develop simple methodologies for its
quantification.
Specifically, nanoꢀscaled LaNiO3 perovskiteꢀtype oxide have received the
attention of many researchers due to their excellent physical and chemical properties
and their suitability for applications in a variety of fields, such as ferroelectric, highꢀTc
superconductivity, nonꢀvolatile memory effects, magnetic or sensor materials, in
catalysis and in optical applications 16ꢀ18. The main property of good electrically
conducting oxide lead to applicability as electrode material in electronic devices17.
The purpose of this study was to evaluate a new carbonꢀfree nanomaterial
composed of a LaNiO3 perovskiteꢀtype oxide, synthesized by the Pechini method19ꢀ21, as
an advanced electrode modifier for the electroanalytical determination of cholecalciferol
using simple, fast, and lowꢀcost voltammetric techniques. Thus, this work outlines the
development of an electrochemical sensor based on a LaNiO3 perovskyteꢀtype nanoxide
coated glassy carbon surface and its application in the identification of cholecalciferol in
creek water, demonstrating the accuracy and efficiency of the proposed sensor.
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2. Experimental Section
2.1 Materials
The reagents used were of analytical standard and used as received. Lanthanum
oxide (La2O3, ≥ 99.9%), nickel nitrate (Ni(NO3)2.6H2O, ≥ 98.5%), anhydrous citric acid
(CA, ≥99.5%), dimethylformamide (DMF, ≥99.9%), sodium hydroxide (NaOH, ≥98%)
and Vitamin D3 (≥98%) were purchased from Sigma–Aldrich (St. Louis, MO, USA).
Ethylene glycol (EG, ≥ 99.9%) was purchased from J.T. Baker®. Sodium hydroxide was
obtained from Merck (Darmstadt, Germany). Highꢀpurity deionized water (resistance,
18 Mꢁ cm) was obtained from a Millipore MilliꢀQ system. Sodium hydroxide solution
(pH 13) was prepared and employed as supporting electrolyte. All protocols were
performed at approximately 25 °C (room temperature).
2.2 Synthesis of LaNiO3 Nanoxide
The Pechini method21 is one of the most versatile synthesis techniques to obtain
highly pure oxides with a high degree of homogeneity and dispersion, and, for this
reason, chosen to prepare the LNꢀNO sample22, 23. The main characteristic of this
method is an intermediate viscous resin, where the cations are chemically
trapped, avoiding any segregation or formation of secondary phases20. Initially, CA and
EG were dissolved in deionized water at 333 K under constant stirring. Then,
stoichiometric quantities of lanthanum nitrate and nickel nitrate were added in this
solution to complete the desired stoichiometry. Small quantities of diluted nitric acid
(P.A. Synth) were used in order to favor the dissolution of La2O3. The molar ratio of
citric acid/metal cations was fixed at 1.8:1, while the mass ratio of citric acid/ethylene
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glycol was adjusted to 1:4. The volume of the resulting solutions was reduced until a
viscous green resin was obtained, which was then heat treated at 353 K for 5 h. The
polymeric resin obtained was heat treated at 623 K for 3 h. The obtained precursor
powder was calcined at 973 K for 2 h under air atmosphere. These procedures were
detailed and outlined and detailed in the flow chart (Scheme S1 – Supplementary
information).
2.3 Apparatus
Highꢀresolution transmission electron microscopy (HRꢀTEM) images were
captured with a FEI TECNAI G² F20 transmission electron microscope operating at 200
kV. The powders were ultrasonically suspended in ethanol for 30 min, and the
suspension was deposited on carbonꢀcoated copper grids. The Xꢀray photoelectron
spectrum (XPS) of the LNꢀNO was obtained using a VSW HA 100 hemispherical
electron analyzer using an Al anode as the Xꢀray source, operated at 12 KeV and 15
mA. The correction of the binding energies for charge was obtained using the reference
Si2p line from silica, which was set at 103.4 eV24.
Xꢀray powder diffraction (XRD) analysis was performed on a Shimadzu XRDꢀ
6000 diffractometer equipped with CuKα radiation (λ=1.54178 Å and graphite
monochromator) operating in continuous scan mode at 0.02° min−1. The measurements
were collected at room temperature with a scan range between 5° and 90°.
Electrochemical measurements were performed using a model PGSTAT 302
Autolab electrochemical system (Eco Chimie) and controlled by GPES 4.9.7 software.
All analyses were conducted in a 25 mL thermostated glass cell at 25 °C, with a threeꢀ
electrode configuration: a glassy carbon electrode (diameter 3.0 mm) coated with
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LaNiO3 nanoxide film as the working electrode, an Ag/AgCl (3 mol L–1 KCl) as a
reference and a platinum foil (1.0 cm²) as an auxiliary electrode. The solution within the
cell was neither stirred nor aerated during the measurements. Electrochemical
impedance spectroscopy (EIS) spectra were obtained with a PGSTAT 302 system,
controlled by FRA2 software, in the frequency range between 0.1 Hz and 100 kHz with
an amplitude of 10 mV and under open circuit potential (OCP) conditions in 1.0 mol L–1
NaOH solution containing 5.0 mmol L–1 of K3[Fe(CN)6]/K4[Fe(CN)6]25.
2.4 Sensor Preparation
The GC electrode surface with a geometric area of 0.07 cm2 was polished prior
to modification with 0.3 ꢀm alumina slurries, rinsed thoroughly with doubleꢀdistilled
water, sonicated for 3 min in ethanol and 3 min in water, and dried in air. On the
electrode surface, a thin film of the LaNiO3 nanomaterial was coated according to the
following procedure. Previously, a solution containing 2 mg of LaNiO3 and 1.0 mL of
DMF was obtained, which was subjected to 2 h of ultrasonication. A film coating layer
was prepared on the GC electrode surface by casting the suspension using a
micropipette (10 µL) as shown in Figure 2.
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Figure 2 – Schematic illustration of the steps involved in the preparation of the surface
for detection.
3. Results and Discussion
3.1 Morphological and structural performance
Highꢀresolution transmission electron microscopy was used to obtain
information about the structure of the LaNiO3 nanomaterial (LNꢀNO) (Figure 3). The
micrographs confirmed that LNꢀNO (size range 50–100 nm) presented a spherical shape
and was well distributed over the whole surface, showing no evidence of aggregation.
The EDX spectrum was used to confirm the presence of lanthanum, nickel and oxygen
in the material. The Cu and C signals were caused by the copper composition of the
grids used for HRꢀTEM analysis. The distribution of the LaNiO3ꢀnanoxide provides
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good electrocatalytic properties to the material, and the resulting material has potential
as a substrate for the development of electrochemical sensors.
Figure 3 – High Resolution TEM micrographs of the LNꢀNO and EDX spectrum of the
material.
The XRD pattern of LaNiO3 shown in Figure 4 fitted perfectly with the
corresponding perovskite structure (ICSD 33ꢀ0711), with no evidence of secondary
phases. The sample was refined by the Rietveld method through the GSAS program.
The residual pattern shows an excellent fit between the theoretical and observed Xꢀray
pattern (Table S1), it can be easily seen that there is little variation in the diffraction
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angle as shown in Figure 4. The data used for the theoretical model were those available
in the ICSD database (Code 173477)26.
Calculated
Observed
Obs - Calc
Bragg
15
30
45
60
75
90
2
θ
/ degree (CuK
α
radiation)
Figure 4 – Rietveld graphic of LaNiO3 nanoxide calcined at 973 K for 2h.
Surface chemical features in the LaNiO3 nanoxide material were evaluated by Xꢀ
ray photoelectron spectroscopy (XPS) measurements. The binding energies determined
from fitting the spinꢀorbit components La3d, Ni3p, O1s and C1s for the main peaks of
the XPS spectra are summarized in Table S2. The O1s profile is basically constituted by
three peaks at 528.7, 531.1 and 532.8 eV, which can be attributed to lattice oxygen O2−,
O− (including carbonates or nitrates, and hydroxyl groups (OH−)) and adsorbed H2O
compounds, respectively 27ꢀ32. Similarly, two peaks are detected in the C1s zone. The
first, at approximately 284.6 eV, can be attributed to surface contamination from
atmospheric hydrocarbons while the second at approximately 286.6 eV is common to
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carbonate species27, 29. Since Laꢀbased perovskites are basic materials, they can be
easily carbonated or hydrated upon exposure to ambient atmosphere 27, 29
.
The La 3d5/2 core level spectra, taken at normal emission and grazing emission
angle can be deconvoluted into two doublets, attributed to oxide and hydroxide groups.
The main oxide band is at 833.5 eV binding energy and the satellite band is at 837.7 eV.
The main line corresponding to the lanthanum bonding with hydroxyl species lies at
835.5 eV and the satellite band at 838.8 eV 27, 29, 30, 33. Our results are in according to
reported in the literature31. The Ni 3p profile is basically constituted by three peaks at
66.4, 68.5 and 71.6 eV that can be assigned to Ni2+ 3p3/2 and Ni3+ 3p3/2 consistent with
previously reported results30, 34, 35. The absence of binding energies for the spin orbit
components Ni 2p1/2 and Ni 2p3/2 between 890 and 850 eV shows that there was no
formation of metallic Ni, NiO or Ni(OH)2 species (or hydrated nickel oxide NiO.H2O
species)15, 36
.
3.2 Electrochemical performance
The effect of the number of cycles on the voltammetric response of the LNꢀNO
electrode was evaluated in 0.5 mol L–1 NaOH solution at pH 13 and is presented in
Figure 5. It is known that in alkaline conditions, metallic nickel begins to be
electrochemically oxidized to nickel hydroxide (Ni + 2OH– → Ni(OH)2 + 2e–) at
potentials more positive than ꢀ0.68 V vs. SCE. At potential values more positive than
0.40 V the Ni(OH)2 is further oxidized to NiOOH (Ni2+ → Ni3+) 37. For a boronꢀdoped
diamond (BDD) surface modified with NiNP, the nickel oxyhydroxide is
electrogenerated, with the peak potential located at 0.345 V, in 1.0 M NaOH 37. In the
case of the LNꢀNO modification proposed in this work, the oxidation peak stabilizes at
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0.465 V vs. Ag/AgCl, after 30 potential cycles. The potential shift in the oxidation peak,
considering the results presented in Reference 32, is evident in Figure 5A and can be
justified by the stabilization caused by the presence of lanthanum in the perovskite
structure of the oxide. A continuous increase in the anodic (Ipa) and cathodic (Ipc) peak
currents with successive scans is also observed, which can be associated with an
increase in the electroactive surface area of the electrode. Apparently, during the
potential scanning, there is some dissolution or cavitation in the electrode surface,
increasing the available area for the next cycle. Here, the selective dissolution of
lanthanum is an unwelcome possibility, which would leave only the conventional
NiOOH surface. In order to elucidate such modification of the surface characteristics,
another EDX spectrum was obtained after the electrode surface had been submitted to
the activation procedure (150 voltammetric cycles). The result was quite similar to that
in Figure 3, indicating the presence of the same lanthanum perovskite on the electrode
surface, so there was no evidence of any selective dissolution.
The available surface area of the LNꢀNO layer is proportional to the value of the
peak current at +465 mV. The anodic peak currents were measured from the cyclic
voltammograms showed in Figure 5A, plotted as a function of cycle number and
presented on Figure 5B. It can be easily seen the surface area increases linearly with the
number of voltammetric cycles until the 100th cycle. Between cycle numbers 100 and
150 there is a trend towards stabilization. The increased current is due to the greater
access of OH− species to the nanoxide due to increased roughness factors and,
consequently, more Ni2+ is oxidized to Ni3+ 37
.
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60
40
55
50
45
40
35
30
150th
20
1st
0
ꢀ20
ꢀ40
ꢀ60
25
B
A
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0
20 40 60 80 100 120 140 160
E vs. Ag/AgCl / V
Cycle number
Figure 5A – Cyclic voltammograms for LaNiO3ꢀNanoxide/GCE in 0.5 mol L–1 NaOH,
at a scan rate of 50 mVs−1. The figure shows the first and 5 – 150th scans. Figure 5B –
Relationship between cycle number and the values of the anodic current for the number
of cycles between 1 and 150 as determined from Figure 3A.
The influence of OH– concentration on the voltammetric profile was evaluated
and is shown in Fig. S1(A), where the cyclic voltammograms of the 150th cycle of the
nanoxide electrode in 0.1, 0.5 and 1.0 mol L–1 NaOH solutions are presented. The
anodic peak currents and the corresponding peak potentials are presented as a function
of the NaOH concentration in Fig. S1(B). It can be easily seen that an increase in OH−
concentration led to an increase of peak current and a shift to less positive potential
values. This behavior is indicative of the involvement of OH− anions in the electrode
reaction for NiOOH formation, consistent with reports in the literature14. Due to the less
positive potential of the anodic peak and the higher value for the oxidation peak current,
the concentration of 1.0 mol Lꢀ1 was chosen for the following experiments.
The influence of the inversion potential on the oxide layer formation is shown in
Figure S2. The voltammetric profiles were evaluated in 1.0 mol L–1 NaOH solution at
pH 13 with the 150th cycle registered. Figure S2(A) shows typical cyclic
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voltammograms of the nanoxide electrode recorded between 0 and: (a) 500, (b) 550 and
(c) 600 mV inversion potential. The anodic peak current and anodic peak potential are
presented as a function of the inversion potential in Figure S2(B). It is clearly seen that
an increase in the range of potential led to an increase in the peak current and a shift to
less positive potential values (see Fig. S2(B)). The linear dependence of the anodic peak
current with the inversion potential suggests that the amount of LaNiO2OH produced on
the electrode surface increases linearly because the time window where the potential is
suitable for the electrode reaction to occur is larger, with increased formation of the
mixed oxyꢀhydroxide 38. Due the higher amount of mixed oxyꢀhydroxide produced in
the electrode surface, the range potential between 0 and 600 mV was chosen for the
following experiments.
The final parameter that influences the modified electrode performance and,
therefore, had to be optimized is the amount of nanoxide suspension added to the glassy
carbon electrode surface. The optimization procedure was carried out by cyclic
voltammetry in 1.0 mol L–1 NaOH solution (pH=13), using a potential range of 0.20 to
0.60 V vs. Ag/AgCl, after the modification of the GC electrode surface with aliquots of
2.0 to 10 ꢂL of nanoxide suspension. It is observed in Figure S3 that the anodic peak
current goes reached a maximum value when 8 ꢂL of the nanocomposite solution was
dropped onto the electrode surface. This behavior can be ascribed to the crescent
thickness and roughness of the ultrathin film, with a corresponding increase in the
amount of active electrocatalytic sites on the surface of the electrode, as reported to
conducting polymers by Martin and Teixeira 39. Above the volume of 8 ꢂL the decrease
in the peak current observed is due to increasing electrical resistance on the surface of
the electrode. This result was consistent to reported in others modified surfaces39, 40 and
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can be attributed to a balance between better dispersion of the nanoxide on the electrode
surface for smaller coating volumes and a higher electrical resistance provided by large
volumes added. Considering the results presented, the volume of 8 ꢂL solution of
nanocomposite was utilized for further studies.
The standard voltammetric profile of the LaNiO3 electrode in 1.0 mol L−1 NaOH
solution (pH 13) is presented in Figure 6, using the optimized parameters discussed
above. Cyclic scans were performed in unstirred solution between 0.2 and 0.60 V vs.
Ag/AgCl at a potential scan rate of 50 mV s−1, in the positive potential direction first.
Such cyclic voltammograms presented two current peaks, an anodic one at +0.412 V
(EPA) and the corresponding cathodic one at +0.353 (EPC). The peaks can be attributed
to the reversible process (ꢃE = 59 mV) involving one single electron transfer to the
NiII/NiIII pair. The surface concentration of electroactive sites on the modified surface
(Γ/mol cm–2) was estimated from the backgroundꢀcorrected electric charge (Q), under
the anodic peaks, in accordance with the theoretical relationship41: Γ = Q/nFA, where Q
(C) is the electric charge, n is the number of electrons transferred; F is the Faraday
constant (96,485.34 C mol–1); and A is the electrode geometric area. In the experimental
conditions described above, Q was found to be about 2.93 x 10–5 C, and the estimated
surface concentration was found to be equal to 4.34 x 10–9 mol cmꢀ2.
The impedance spectrum under the same conditions as Figure 6A is displayed in
Figure 6(B) as complex plane plots (Nyquist diagrams) for the two electrodes (GC and
LNꢀNO). The diagrams consisting of semicircles and diffusion straight lines were
analyzed with a Randle’s modified equivalent circuit [Rs(CPE[RctZW])], where Rs is the
solution resistance, Rct is the charge transfer resistance, ZW is the Warburg impedance
and CPE is a constant phase element. The apparent heterogeneous electron rate constant
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was determined using Kapp = RT/F²RctCA, in which F is the Faraday constant (96,485.34
C molꢀ1), C is the probe redox concentration in solution (5.0 mmol Lꢀ1), R is the gas
constant (8.3145 J K mol), T is the temperature (298 K), A is the geometric area (0.07
cm²) and Rct is the chargeꢀtransfer resistance obtained by fitting the data. The Nyquist
diagrams were fitted with a modified Randles circuit. It is worth highlight the charge
transfer resistance decreasing from 3,590 Ω for bare GC to 30.24 Ω with LNꢀNO
modification surface. Kapp values were calculated for bare GC and modified electrodes
and the values obtained were 3.65 and 0.03, respectively. Rct is smaller for GC/LNꢀNO
because of the electrocatalytic capacity of the nanoxide, which promotes faster electron
transfer.
600
A
B
(a) GC Bare
(b) GC/LaNiO3
4000
3000
2000
1000
0
100
80
60
40
20
0
500
400
300
200
100
0
0.412
0
100 200 300 400 500 600
(b)
(a)
Z' /
ꢁ
(a)
(b)
ꢀ20
0.353
0.2
0.3
0.4
0.5
0.6
0
1000
2000
3000
4000
E vs. Ag/AgCl / V
Z' /
ꢁ
Figure 6 (A) – Cyclic voltammograms for bare GC (a) and LaNiO3ꢀnanoxide/GC (b)
electrodes in 1.0 mol L−1 NaOH solution (pH 13). (B) – Complex plane impedance
spectra (Nyquist diagrams) for bare GC (a) and GC modified with nanoxide LaNiO3 (b)
electrodes. The measurements were performed in 0.1 mol Lꢀ1 KCl solution containing
5.0 x 10ꢀ3 mol Lꢀ1 of the redox probe [Fe(CN)6]4ꢀ/3ꢀ
.
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In order to understand the electrochemical behavior of nickel in strong alkaline
medium, a scan rate study was carried out. Figure S4(A) shows cyclic voltammograms
of the LaNiO3/GC in 1.0 mol L–1 NaOH solution recorded at several scan rates between
5 and 100 mV s−1. Figure S4(A) shows that the peakꢀtoꢀpeak potential separation
increased from 55 mV at 5 mV s−1 to 88 mV at 600 mV s−1. In according to the report
by Sedenha and coꢀworkers37, this increase indicates a limitation in the chargeꢀtransfer
kinetics. In Figure S4(B), a linear relationship can be observed when Ipa and Ipc are
plotted against the scan rate, which suggests an electrochemical activity controlled by
surfaceꢀbound species. This performance is expected, as reported in the literature37, as
the redox species (Ni(OH)2 and NiOOH) are immobilized on the electrode surface.
The relationship between variations in the peak potential and the logarithm scan
rate may be used to calculate parameters such as anodic electron transfer coefficients
(αa) and the apparent electrochemical rate constant (ke) for immobilized redox centers
on the electrode surface, similar to the method described by Laviron42. Figure S4(C)
shows the relationship of EP (V) with the log scan rate (V.s−1) obtained with the
GC/LNꢀNO in 1.0 mol L−1 NaOH solution. There is a fair linear dependence on the
peak potential only in the scan rate range of 0.04–0.10 V.s−1 and the slope of the linear
segment is equal to 2.3003RT/αanF for the anodic peak. The value obtained for αa was
0.97. The apparent electrochemical rate constant can be obtained using the equation ke =
2.303 αanFva/RT, in which the value scan rate (va) is estimated by extrapolation of the
anodic linear branch at higher scan rates and evaluating its intersection with the constant
peak potential. The apparent electrochemical rate constant was calculated as 1.82 s−1 (to
va = 0.020 V.s−1). The ke is a measure of the kinetic facility of a redox couple. A system
with a high electrochemical rate constant will achieve equilibrium on a short time scale.
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3.3 Applicability
The electrocatalytic performance of the optimized LaNiO3ꢀnanoxide/GC
electrode was evaluated for the oxidation of cholecalciferol (Vitamin D3) by square
wave voltammetry (SWV). Figure 7 shows the respective wave voltammograms
obtained in 1.0 mol L–1 NaOH solution containing 1.21 x 10–5 mol Lꢀ1 of vitamin D3.
From curve (b) it is evident that in the presence of cholecalciferol a wellꢀdefined
irreversible oxidation peak appears at +0.46 V vs. Ag/AgCl. Such an electroꢀoxidation
peak has been previously reported in the literature and was attributed to the
electrochemical mechanism illustrated15, 43 in the inset of Figure 7.
1.8
1.6
1.4
(b)
1.2
(a)
1.0
0.8
0.6
0.4
0.2
(A)
0.0
0.1
0.2
0.3
0.4
0.5
0.6
E vs. Ag/AgCl / V
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Figure 7 (A) – Electroꢀoxidation process of cholecalciferol (Vitamin D3) on the
LaNiO3ꢀnanoxide/GC electrode in the absence (a) and presence (b) of 1.21 × 10–5 mol
Lꢀ1 of vitamin D3. Measurements were carried out in 1.0 mol L–1 NaOH solution (pH
13). (B) – Schematic representation of the electrocatalytic reaction of vitamin D3 on the
LaNiO3ꢀnanoxide/GC electrode.
The dependence of the voltammetric profiles on the cholecalciferol
concentration was obtained and is presented in Figure 8. It can be easily observed that
the anodic peak current at +0.46 V vs. Ag/AgCl increased proportionally with the
cholecalciferol concentration in the range from 0 to 2.6 × 10–5 mol L–1, suggesting that
the modified electrode presents an excellent electrocatalytic response and thus can be
used for the determination of cholecalciferol in real samples. This is a very important
conclusion since electrochemical determination of cholecalciferol is quite difficult, with
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only a few reports in the literature. In those studies the cholecalciferol was electroꢀ
oxidized in a much more positive range of potentials (1.30 V44, 1.18 V43, 1.05 V45, 0.66
V13 and 0.51 V15 vs. Ag/AgCl) than that presented in this work using the LaNiO3ꢀ
nanoxide/GC electrode (+0.46 V vs. Ag/AgCl). This achievement is important because
at low overpotentials the possibility of interfering compounds decreases significantly,
thus increasing the selectivity of the analytical methodology. The analytical curves of
the peak currents (Ip) versus vitamin D3 concentrations are linear in the range between 0
and 2.6 × 10–5 mol L–1 (Figure 8(B)) with a linear regression equation of Ipa (mA) =
9.35 × 10–9 + 17.75 Cvitamin D3 (mol L−1) with a correlation coefficient of 0.999 (n = 14).
The limit of detection was calculated in according to equation LOD = yB + 3SB, with the
values yB (= a) and SB (= Sy/x) previously calculated where a is the intercept and Sy/x is
the standard deviation of yꢀresiduals of leastꢀsquares line (linear regression). Whereas,
the sensitivity was calculated by the slope of leastꢀsquares line (linear regression), b =
∑[(xi – x)(yi – ӯ)]/∑(xi ꢀ x)² 46, 47. The limit of detection (LOD) of cholecalciferol was
calculated as 8.2×10−7 mol L−1. The excellent results in the electroanalytical
performance highlighted the electrocatalytic activity into LNꢀNO in the electroꢀ
oxidation of cholecalciferol indicating that this sensor can be excellent alternative to
Vitamin D3 detection.
Although there is a previous report with a lower detection limit, using a glassy
carbon electrode modified with a SiO2/GO/Ni(OH)2 material (3.26 nmol L−1) 15, the
proposed electrode has the advantage of simple preparation and much lower cost for the
nanoxide material, which is carbonꢀfree (not requiring graphene and carbon nanotubes).
The cost to obtain 1 g of LNꢀNO was about 64, 172 and 37 times lower than graphene,
carbon nanotubes: singleꢀwalled and multiꢀwalled, respectively.
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1.6
1.4
1.2
1.0
1.8
1.5
1.2
0.9
0.6
0.3
0.45
0.54
Evs. Ag/AgCl / V
(A)
0.0
0.1
0.2
0.3
0.4
0.5
0.6
E vs. Ag/AgCl / V
0.4
0.3
0.2
0.1
0.0
(B)
0
5
10
15
20
25
Ccholecalciferol (Vitamin D ) / ꢀ
mol L-1
3
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Figure 8 (A) – SWV for the LaNiO3ꢀnanoxide/GC electrode at a range of
cholecalciferol (vitamin D3) concentrations (0 to 2.6 × 10–5 mol L–1) in NaOH, pH 13.
(B) – Plot of current intensity versus cholecalciferol concentration.
The reproducibility and repeatability of the LaNiO3ꢀnanoxide/GC electrode were
tested using the square wave voltammetry technique. The measurements were
performed ten times in the presence of 15 µmol L–1 of vitamin D3 with the same
electrode and with five different sensors prepared at room temperature under the same
conditions. The LaNiO3ꢀnanoxide/GC electrode presented good repeatability and good
reproducibility without any significant loss of electrocatalytic activity with relative
standard deviations (RSD) of 1.9% and 2.7%, respectively.
Table 1 list the efficiency of the GC/LNꢀNO electrode for vitamin D3
determination compared with other electrodes modified. The limit of detection this
sensor can be considerably competitive with similar devices for the determination of
Vitamin D3 in comparison with most modified materials.
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Table 1: Selected electroanalytical approaches used for sensing Vitamin D
performances with four transducers different.
Sensing Layer
Transduction Sensitivity
LOD
Linear
range
Reference
5
SPR
DPV
DPV
2.8 mºmL
mg−1
2 ꢂg mL−1
5–50 ꢂg
mL−1
Au/SAM/Antibody
Au/SAM/Antibody
Glassy carbon
5
0.020 mA
mL ng−1
0.025 µA /
µmol Lꢀ1
10 ng
mL−1
20–200 ng
mL−1
5.0 × 10−6
5.0 × 10−5
mol L−1
13
0.118
µmol L−1
ꢀ
15
SWV
SWV
9.2 µA /
µmol Lꢀ1
3.26 ×
10−9 mol
L−1
8.3 × 10−7
mol L−1
0.5ꢀ2.5
ꢂmol L−1
GC/
SiO2/GO/Ni(OH)2
17.75 A /
mol Lꢀ1
0 ꢀ 2.6 ×
10−5
This work
GC/LN-NO
3.5 Real Sample Analysis and Interference of Coexisting Substances
The LaNiO3ꢀnanoxide/GC electrode was used to detect cholecalciferol (vitamin
D3) in untreated river water to evaluate the validity of the proposed analytical methods.
No electroanalytical signals were observed after the addition of river water to NaOH
solution, which indicates a level of cholecalciferol contamination below the limit of
detection of the proposed methodology, if any. After spiking the samples with vitamin
D3, the analytical methods were able to recover 98–100ꢄ% of the added analyte. These
data suggest that the proposed LaNiO3ꢀnanoxide/GC electrode could be used to
determine the concentration of vitamin D3 in environmental water samples. Potential
sources of interference in the identification and determination of cholecalciferol were
investigated.
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Various Vitamins structurally related with Vitamin D3 such as Vitamins A, E,
B2, B3, B6, C, P and K were added into the NaOH solution in the presence of 1.5ꢄ×ꢄ10–5
mol L–1 cholecalciferol were tested as potential interfering compounds. Firstly, SWV to
Vitamin D3 electrooxidation (fixed in 1.5 × 10−5 mol L−1) was performed. Subsequently,
3.2 and 6.4 × 10−6, 4.9 and 9.8 × 10−7, 0.92 and 1.8 × 10−6, 5.2 and 10.0 × 10−6, 1.3 and
2.6 × 10−6, 1.1 and 2.2 × 10−6, 2.3 and 4.7 × 10−6, 8.6 × 10−5 of Vitamins A, E, B2, B3,
B6, C, P and K, respectively, were added were added individually, and the SWV were
recorded (Figure S5). No additional analytical signals were detected for all interfering
compounds within the ranges studied suggesting that the sensor was specific for the
Vitamin D3 detection. The effect of the presence of each substance was performed from
the currents measured, at the optimized experimental conditions, employing GC/LNꢀNO
surface for Vitamin D3 and interfering compound. Figure 9 shows relative current
percentages for Vitamin D3 after each interfering added. However, interfering
compounds showed small variation of interference on the Vitamin D3 oxidation current.
Considering that the Vitamin D3 signal is interferenceꢀfree, we can estimate the
percentages of interference of the Vitamin A, E, B2, B3, B6, C, P and K, and the values
obtained were of 1.6 and 2.9, 3.9 and 5, 7.5 and 7.5, 8.7 and 8.6, 9.1 and 9.7, 11 and
11.4, 12 and 12.8, 13.5 and 12%, respectively. This indicate the coꢀadsorption processes
on the LNꢀNO surface15 but there are not impediment of the nanoxide knowing the
interference percentage of each compound48. The relative standard deviation was
calculated using RSD = 100S/Xm and the value obtained was of 4.1%. Therefore, we
can conclude that the nanoxide LaNiO3 was able to detect Vitamin D3 in the presence of
these interferences. Moreover, it is relevant to highlight the little shift in the sensitivity
of the GC/LNꢀNO surface does not restrain its use for the Vitamin D3 detection in the
presence of possible interferences substances and can be applied in real samples15.
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The developed sensor was specific for secosteroid due to synergetic effect
between LaNiO3 perovskiteꢀtype oxide and Vitamin D315, as well as, an excellent
electrically conducting oxide used to electrode material in electronic devices17. These
nanomaterials can be applied together in the development of electrochemical sensors for
direct Vitamin D3 quantification in biological15 and environmental matrices.
Figure 9 – Effect of the presence of Vitamins A, E, B2, B3, B6, P, C and K on the
voltammetric responses obtained for 1.5 x 10–5 mol L–1 Vitamin D with the LNꢀNO/GC
sensor.
Conclusions
In conclusion, this work reports the synthesis of a LaNiO3ꢀnanoxide with
perovskiteꢀtype structure using the Pechini method and its use as a nanostructured labelꢀ
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free platform for sensing. The optimized electrode was electrochemically characterized
in an alkaline medium and applied as a voltammetric sensor for a secosteroid hormone
(Vitamin D3). The nanostructured platform showed good analytical performance (low
detection limit, wide working range, high sensitivity and good reproducibility)
compared with carbon nanomaterials. In addition, the electrochemical properties of LNꢀ
NO confirm the high potential of this costꢀeffective nanomaterial, making it competitive
with respect to the more commonly used graphene and carbon nanotubes. Thus, LNꢀNO
can be an much cheaper alternative for sensor development. The low detection limit,
along with the ease of preparation and manipulation, make the LNꢀNO nanostructured
platform an ideal candidate for sensing in other applications.
Acknowledgments
The authors are thankful to grants from the São Paulo Research Foundation (FAPESP),
CAPES and CNPq for research support. The scholarship granted by the São Paulo
Research Foundation, FAPESP (2012/17689ꢀ9) to Paulo Augusto RaymundoꢀPereira is
gratefully acknowledged. Thiago C. Canevari is specially acknowledged for the XPS
measurements.
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