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A new chemically modified electrode
based on titanium dioxide nanoparticles (TiO2-NPs) has been developed.
Aluminium was incorporated into the TiO2-NPs to prepare aluminium
doped TiO2 nanoparticles (Al-TiO2-NPs). Aluminium doped
TiO2 nanoparticles-modified screen printed carbon electrode (Al-TiO2-NPs/SPCE)
was employed as easy, efficient and rapid sensor for electrochemical detection
of vanillin in various types of food samples. Al-TiO2-NPs were
characterized by energy-dispersive X-ray (EDX), transmission electron
microscopy (TEM), and X-ray diffraction (XRD) and analyses showing that the
average particle sizes varied for the Al-NPs (7.63 nm) and Al-TiO2-NPs
(7.47 nm) with spherical crystal. Cyclic voltammetry (CV) and linear sweep
voltammetry (LSV) and were used to optimize the analytical procedure. A
detection limit of vanillin was 0.02 µM, and the relative standard deviation (RSD)
was 3.50 %, obtained for a 5.0 µM concentration of vanillin. The
electrochemical behaviour of several compounds, such as vanillic acid, vanillic
alcohol, p-hydroxybenzaldehyde and p-hydroxybenzoic, etc., generally present in
natural vanilla samples, were also studied, to check the interferences with
respect to vanillin voltammetric signal. The applicability was demonstrated by
analysing food samples. The obtained results were compared with those provided
by a previous method based on liquid chromatography for determination of

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Keywords: Aluminium doped TiO2
nanoparticles; screen-printed carbon electrode; electrochemical detection;
vanillin; food samples.


* Corresponding author: A. Ríos, [email protected]


1.       Introduction


(4-hydroxy-3-methoxybenzaldehyde, C8H8O3) is
one of the world’s flavour extracts obtained primarily from Vanillia, a specie
of tropical climbing. Although the production of vanillin every year more than
12,000 tons, the natural vanillin from Vanilla is less than 1%; the remainder
is synthesized much more cheaply via biochemical and/or chemical processes 1. For the time being, several
analytical methods have been reported for determination and detection of vanillin
in various types of food samples or vanilla extracts, including fluorescence 2, capillary electrophoresis (CE) 3, liquid chromatography 4, and GC-MS 5. These have high cost and involve time-consuming sample
pretreatment processes. Because vanillin is an electro-active compound and it
is possible to measure the quantity of vanillin in vanilla and in the final
products by electrochemical detection (ECD) through the study of its oxidation.
ECD is important method for quantitative determination of vanillin due to their
easy to use, fast response, high sensitivity, and cheap instrumentation 6–11. Various electrochemical methods, such as amperometry, square-wave
voltammetry (SWV) or differential pulse voltammetry (DPV) for the detection and
determination of vanillin in various types of food samples have been reported
and discussed  in literature 12,13.

In recent years, many
reports on screen-printed carbon electrodes (SPCEs) technology have been used
to develop various electrochemical sensors that detect target molecules in
various sectors, such as biomedical environmental and agri-food 14,15. SPCE is planar shape, thus can be used as droplet on sensor, using
typically few microliters (µL) of the
sample in miniaturized system. Moreover, it is low-cost and can be used as a
disposable sensor with possibility used in large-scale implementation. The
oxidation of vanillin at SPCE and others most unmodi?ed electrode surfaces
represents a serious problem which arises from the poor reproducibility with high
over-potential resulted from a fouling effect, which results in rather poor sensitivity
and selectivity 16,17. To avoid these problems, the modification of electrochemical
working electrode is an excellent alternative. In this sense, many researchers
have attempted to diminish the over-potentials by using various modi?ed
electrodes such as, graphene 6,18, silver nanoparticles (Ag-NPs) 8, gold nanoparticles (Au-NPs) 9,10, and multi-walled carbon nanotubes (MWCNTs)
19. However, the performance of some
electrodes was still ineffective enough. Thus it is necessary
to develop new types of electrode devices by the preparation synthesis and
preparation of new materials (modi?ers). That regard, as modifiers for
electrode surface, titanium dioxide nanoparticles (TiO2-NPs) had highly
interest of many researchers and a many amount of research was applied in the
previous decades 12,13,19. The increase in the TiO2-NPs efficiency such as to ensnare
the charge carrier is accomplished by doping with transition metals and
transition metal oxides such as, copper (Cu) 20, cadmium oxide nanoparticles (CdO-NPs) 21, silver nanoparticles (Ag-NPs) 22, gold nanoparticles (Au-NPs) 23 and ruthenium (Ru) 24.

Here, the
synthesis of aluminium doped TiO2 nanoparticles (Al-TiO2-NPs),
which were characterized by TEM and XRD. The sensitivity of the developed
electrode is compared with SPCE, TiO2-NPs/SPCE, and Al-TiO2-NPs/SPCE
for vanillin detection. The results show that a composite film of Al-TiO2-NPs/SPCE
is more sensitive compared to SPCE and TiO2-NPs/SPCE. Furthermore,
the electrochemical behaviour of vanillin at modified SPCE with Al-TiO2
(Al-TiO2-NPs/SPCE) was investigated. The performance of the modified
electrode (Al-TiO2-NPs/SPCE) is also demonstrated for the
determination and detection of vanillin in various types of food samples
obtaining good selectivity, stability and high sensitivity.


Experimental section


Reagents, standards and samples


All the starting
materials were purchased with very highly purity. Aluminium acetylacetonate (Al(acac)3,
99%), lithium aluminium hydride (LiAlH4, 95%), mesitylene (97%),
titanium (IV) oxide (anatase, powder, 235 mesh), 4-hydroxybenzaldehyde (98%) and
4-hydroxy-3-methoxybenzyl alcohol (98%) were purchased from Sigma-Aldrich (St.
Louis, MO, USA). Nafion 117 solution (5% in a mixture of lower aliphatic
alcohols and water), vanillin, vanillic acid, 4-hydroxybenzoic acid were
purchased from Fluka Chemie (Buchs, UK). Ethanol and phosphoric acid were
purchased from Panreac Quimica S.L.U. (Barcelona, Spain). Vanillin solution was
prepared in ethanol and stored while tightly covered in the dark until use. Vanillin
stock solutions were frequently diluted to working standard solutions with
phosphate buffer at pH 6.3. The pure water was obtained by a Milli-Q system
(Millipore). Ethanol and phosphoric acid were obtained from Panreac Quimica
S.L.U. (Barcelona, Spain).

Vanillin extract
samples (sample A and sample B) were purchased from different local markets
(Ciudad Real, Spain). These extracts were filtered through a sintered filter,
and diluted directly in phosphate solution.


Apparatus, instruments and chromatographic conditions


detection was carried out on a CH Instruments Model 800D Series (Austin, Texas,
USA) all the experiments were carried out using a screen-printed carbon
electrodes (SPCEs) system (Dropsens DRP-C110) housed in the home made
electrochemical flow cell. Transmission electron microscopy (TEM) micrographs
were measured on a Jeol JEM 2011 operating at 200 kV and equipped with an Orius
Digital Camera (2 × 2 MPi). The digital analysis of the HRTEM micrographs was
done using Digital Micrograph TM 1.80.70 for GMS 1.8.0 Gatan. The samples were
prepared by deposition of a drop of the synthesized material suspension onto a
lacey carbon/format-coated copper grid. XRD patterns were measured on Philips
model X´Pert MPD diffractometer using a CuKa source (? =1.5418 Å), programmable
divergence slit, graphite mono-chromator and proportional sealed xenon gas
detector. The samples were made with a voltage of 40 KV, intensity 40 mA and an
angular range of 20 to 70 degrees (2?), a step of
0.02 degrees 2? and a time per step
of 1.50 sec.

Agilent 1200
liquid chromatography system was used as a chromatographic system. It was consisted
from a LC pump, a vacuum degasser, a micro well-plate autosampler (5 ?L
injection loop), a thermostatted column compartment and a Diode-Array Detector
(DAD). The data was processed using the PC computer with ChemStation Software. An
appropriate reversed-phase C18 analytical column Luna 5µm PFP
(2) 100A (150 x 4.6 mm) was used for the separation of the analytes present in
vanillin extract samples. Elution was done quite under isocratic conditions, by
using a mixture of acetonitrile/phosphate (20:80 v/v) as a mobile phase, 40 µL
the injection volume was injected and flow-rate was set at 1.0 mL min?1.
The detection wavelength was at 265 nm.


Preparation of aluminium doped TiO2
nanoparticles (Al-TiO2-NPs)


At first, Al-NPs were prepared
according the previously described procedures 25, with inserting some changes. The aluminium acetylacetonate
(Al(acac)3, 10 mmol) was added to a three-neck round bottom which
was already contained mesitylene solution with magnetic bar. Then lithium
aluminium hydride (LiAlH4, 30 mmol) was added to the mixture. The
reaction was purged with N2 gas during reflux with stirring for 72
hours at 165 °C. After cooling to 25
°C, a gray-colored precipitate was was
formed which was crushed and kept to dry under low pressure for 5 hours. Wash the
isolated solid product well with 25 ml portions of cold methanol three times,
to avoid any high temperatures (exothermic reaction) between solvent and
Al-NPs. The unreacted materials were washed three times with methanol. The
resulting product was filtered and dried at 25 °C under low pressure. The preparation of Al-TiO2-NPs
started by mixing of TiO2 (0.5 g) previously digested in nitric acid
(0.1 M, 25 mL) during 3 hours and Al-NPs (0.5 g) previously prepared. The final
product was filtered and dried at 25 °C under low
pressure, obtaining an Al-TiO2-NPs as light-gray powder.


Preparation of modified electrodes


were ultrasonically dispersed in pure water (0.5% Nafion,
v:v), The concentrations of 1 mg mL-1 were obtained individually. On
the SPCE forming a layer of thin films from nafion-solubilized nanoparticles
are more uniform distribution than those casted by organic solvents 26. TiO2-NPs and Al-TiO2-NPs were used. Dropsens
SPCEs (DRP-110), with carbon as a working electrode with a disk-shaped of 4 mm
of diameter, were used to fabricate the electrode. So, 2 µL of the dispersed
NPs was casted onto the surface of the SPCE. After drying the modified SPCE by
using infrared light lamp for 15 min, rinsing with pure water. The electrode is
ready for use.


Results and discussion


Characterization of aluminium doped TiO2
nanoparticles (Al-TiO2-NPs)


X-ray (EDX) elemental mapping and transmission electron microscopy (TEM)
micrograph for the aluminium and aluminium doped titanium nanoparticles (Al-TiO2-NPs)
are shown in Figure 1. The Figure shows that different materials have different
surface morphologies. Figure 1 (A and C), shows the TEM and EDX micrographs of
the aluminium particles without doping (this image was obtained before
aluminium was mixed with TiO2-NPs.), and a higher magnification is
also presented. A large number of precipitates were distributed homogeneously in
the aluminium grains. The distribution of the precipitates for aluminium, which
leads to increase in hardness and tensile properties. Figure 1 (B and D), shows
the TEM and EDX micrographs of the fully mixed precursor. It shows two large
particles in the lower region, which are Al-NPs and TiO2-NPs, were
distributed homogeneously, indicating that a large amount of Al-NPs has been
doped in the TiO2-NPs. Figure 2, shows XRD patterns measured for
Al-NPs (Figure 2 (A)) and Al-TiO2-NPs (Figure 2 (B)). The XRD
patterns show that the nanoparticles involved aluminium as a major component.
The XRD patterns and all the positions of the peaks are attributed to face-centered
cubic (fcc) crystal structure of aluminium 25, and anatase TiO2 27, as shown in Figure 2. A closer look at the figure shows that the
Al-TiO2-NPs involved aluminium as a major component mixed with TiO2-NPs
as minor components, as shown in Figure 2 (b) 28. Calculations based on the Scherrer equation (D=K?/?cos?) 29, show that the average particle sizes varied for the Al-NPs (7.63
nm) and Al-TiO2-NPs (7.47 nm). The XRD results confirm the TEM
micrograph results discussed above.





 Voltammetric behaviour
of vanillin at the Al-TiO2-NPs/SPCE


The modified screen
printed carbon electrodes (SPCEs) by aluminium doped TiO2
nanoparticles (Al-TiO2-NPs/SPCE) were ?rst characterized by cyclic
voltammetry (CV) to test their behaviour for the oxidation of vanillin, as
shown in Figure 3 (C). In order to find the role of Al-NPs, the cyclic
voltammograms of (A) SPCE, (B) TiO2-NPs/SPCE, and (C) Al-TiO2-NPs/SPCE
in the presence of 250 µM vanillin were recorded, as shown in Figure 3. The
electrochemical behaviours of the TiO2-NPs/SPCE and Al-TiO2-NPs/SPCE
were studied by using the CV technique, as shown in Figure 3 (B and C). All cyclic
voltammograms reported here were obtained in the presence of H3PO4
electrolyte (0.1M and pH=6.3) with 250 µM vanillin and scan rate at 50 mV s-1.
The cyclic voltammograms observed for the bare SPCE electrode (without TiO2-NPs
and Al-TiO2-NPs, Figure 3 (A)). In contrast, the cyclic voltammograms
observed for the TiO2-NPs/SPCE and Al-TiO2-NPs/SPCE
modified electrode exhibited only an oxidation peak in the presence of
vanillin, within the potential window between 0.00 and 1.20 V. The data suggest
that the oxidation reaction of vanillin on Al-TiO2-NPs/SPCE is
totally irreversible. The oxidation peak current at the SPCE electrode for
vanillin (Figure 3 (A)), we therefore have a rather low and broad peak current.
The oxidation current of vanillin (0.58 V) on Al-TiO2-NPs/SPCE
(Figure 3 (C)) was better than that on the bare SPCE. Compared with the SPCE,
TiO2-NPs/SPCE and Al-TiO2-NPs/SPCE, a significant
enhancement in the anodic current (0.58 V) was achieved at the Al-TiO2-NPs/SPCE
(Figure 3 (C)), indicating that the high conductivity and high surface area of
the TiO2-NPs/SPCE improve the catalytic activity and increase the
effective electrode area toward the vanillin oxidation obviously indicating
that the Al-TiO2-NPs/SPCE can be used to determine vanillin.


 Optimization of
experimental parameters


electrochemical response of the modified Al-TiO2-NPs/SPCE toward the
determination of vanillin were optimized by analyzing a standard solution (10
µM) of vanillin using linear sweep voltammetry (LSV) technique. The parameters
a?ecting the determination of vanillin, such as electrolyte, pH,
Al-TiO2-NPs amount, scan rates, adsorption time, accumulation
conditions and stability of the electrodes, were investigated. In order to find
the optimal parameters, one parameter was varied and the other parameters were held
?xed at their reference values.

The influence of
various supporting electrolytes were tested, such as NH4Ac, HCl, H3PO4,
HNO3, H2SO4 and NaOH (each 0.1 M). The results
indicated that when H3PO4 solution (0.1 M) was used, the
oxidation peak current was a higher sensitivity than other. For this study, 0.1
M H3PO4 solution was chosen to act as supporting
electrolyte. On the other hand, the effect of 0.1 M H3PO4
supporting electrolyte pH on the electrochemical behaviour of the modified Al-TiO2-NPs/SPCE
for the vanillin determination was studied. The variations of the oxidation
peak potential as well as the peak current with respect to changes in the pH of
the electrolyte in range (1.40 – 6.30), with an increase
the pH, manifesting that protons have taken part in their
the electrode reaction processes. Figure S1 shows, the relation between the
oxidation peak potential and supporting electrolyte pH. The effect of
modification amount of Al-TiO2-NPs on the SPCE surface for the
determination of vanillin was also studied by using LSV, different modification
volumes were tested: 2.0, 4.0 and 6.0 ?g. The 2.0 ?g gave the best result of
oxidation peak current sensitivity than other counterparts. The effect of scan
rate on the oxidation peak current of vanillin (10 µM) by using LSV was
studied. Different scan rates were tested 20, 30, 50 and 70 mV s-1.
The 50 mV s-1 scan rate gave the best result of oxidation peak
current sensitivity than other counterparts. In this study, 50 mV s-1
scan rate was used. The accumulation step of 10 µM vanillin after 120 second
was performed under 0.0 V, at a fixed accumulation potential 0.0 V, the peak
current increased progressively with accumulation time up to 120. Thereafter,
the peak current increased much slightly as further increasing the accumulation
time, also shown in Figure S2. This phenomenon could be explained to the
saturated adsorption of vanillin on the electrode surface. Then, the stability
of the modified electrodes was examined under the best conditions. Five
electrodes were made by the same procedure. Moreover, the stability of the modified
electrode was also investigated. When the modified electrode (Al-TiO2-NPs/SPCE)
was studied with eight segments, the peak current kept 99.0 % of the original
peak is also shown in Figure S3.


Analytical applications


According the
previous optimum conditions, the analytical purposes parameters of the system
were recorded by linear sweep voltammetry (LSV) technique, with a scan rate of
50 mV s-1. The calibration graph showed a linear range for standard vanillin
solutions of 0.07 to 20 µM. The LSV voltammograms obtained for different
concentrations of vanillin. Table 1, shows the results of linear range, slope, intercept
and the regression coefficient (R2) of the calibration curve for
vanillin. The precision of the method for standard solutions (investigated
after analyzing 10 series of 10 replicates) and the relative standard deviation
(RSD) was calculated to be 3.5 % at the 5 µM concentration of vanillin. The
theoretical limit of detection (LOD) was founded to be 0.02 µM are expressed as
the analyte concentration giving a signal equivalent to the blank signal plus
three times its standard deviation (3?). The developed method provides
clear and good advantages in terms of sensitivity with method previously-published
reports for the electrochemical determination of vanillin 16,17 that involve the use of others vanillin sensors and electrochemical
detection (see Table 2).


The influence of some
components in the determination of vanillin was studied for various common
interfering substances in the samples to be analyzed, including vanillic acid,
vanillic alcohol, p-hydroxybenzaldehyde and p-hydroxybenzoic acid. These substances
were chosen because they are known to be present in natural vanilla. A compound
was considered as interference if it caused an analytical variation of more
than 5% when compared to the analytical signal obtained in the absence of the
interfering compound. The results were indicated that no interferences were
observed for the interferent/analyte ratios investigated (Table 3).

The applicability
of the proposed methodology for the determination of vanillin in two vanillin
extract samples (Sample A and Sample B) was investigated. These vanillin
extract samples were purchased from local markets and prepared according the
section 2.1. Sample A and Sample B were found to contain vanillin. The results
obtained are shown in Table 4. This was compared with a blind analysis of the vanillin
extract samples using the modified HPLC-DAD method 30 described in the section 2.2, which provided a chromatogram
represented in Figure 4. The results showed, both methods are in close
agreement for supporting the validity of this method.




In summary, the development of an
aluminium doped TiO2 nanoparticles have been described, and the prepared
hybrid nanoparticles have been checked in SPCE, as new working electrode for detection
of vanillin in extract vanilla samples with good analytical performance. The use
of the developed working electrode (Al-TiO2-NPs/SPCE) allows the
simple, effective and rapid method for electrochemical detection of vanillin in
extract vanilla samples. The Al-TiO2-NPs/SPCE electrode was found to
increase the sensitivity higher than the commercial electrodes tested in
vanillin determination, therefore evaluating the effectiveness of the proposed
approach to improve the sensitivity of the working electrodes in screen printed
carbon electrodes. 

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