A new chemically modified electrodebased on titanium dioxide nanoparticles (TiO2-NPs) has been developed.Aluminium was incorporated into the TiO2-NPs to prepare aluminiumdoped TiO2 nanoparticles (Al-TiO2-NPs). Aluminium dopedTiO2 nanoparticles-modified screen printed carbon electrode (Al-TiO2-NPs/SPCE)was employed as easy, efficient and rapid sensor for electrochemical detectionof vanillin in various types of food samples. Al-TiO2-NPs werecharacterized by energy-dispersive X-ray (EDX), transmission electronmicroscopy (TEM), and X-ray diffraction (XRD) and analyses showing that theaverage 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 sweepvoltammetry (LSV) and were used to optimize the analytical procedure. Adetection 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.
Theelectrochemical behaviour of several compounds, such as vanillic acid, vanillicalcohol, p-hydroxybenzaldehyde and p-hydroxybenzoic, etc., generally present innatural vanilla samples, were also studied, to check the interferences withrespect to vanillin voltammetric signal. The applicability was demonstrated byanalysing food samples. The obtained results were compared with those providedby a previous method based on liquid chromatography for determination ofvanillin.Vanillin(4-hydroxy-3-methoxybenzaldehyde, C8H8O3) isone of the world’s flavour extracts obtained primarily from Vanillia, a specieof tropical climbing. Although more than 12,000 tons of vanillin are produced everyyear, the natural vanillin from Vanilla is less than 1%; the remainder issynthesized much more cheaply via biochemical and/or chemical processes 1. For the time being, manyanalytical methods have been proposed for vanillin determination in varioustypes 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 samplepretreatment processes.
Because vanillin is an electro-active compound and itis possible to quantify the vanillin amount in vanilla and in the finalproducts by electrochemical detection (ECD) through the study of its oxidation.ECD is important method for quantitative determination of vanillin due to theireasy to use, fast response, high sensitivity, and cheap instrumentation 6–11. Various electrochemical methods, such as amperometry, differentialpulse voltammetry (DPV) or square-wave voltammetry (SWV) for the determinationand detection of vanillin in various food samples have been reported inliterature 12,13.In recent years, manyreports on screen-printed carbon electrodes (SPCEs) technology have been usedto develop various electrochemical sensors that detect molecules in varioussectors, such as biomedical environmental and agri-food 14,15. SPCE is miniaturized and planar shape, thus can be used as drop onsensor, using few microliters of the sample.
Moreover, it is low-cost and canbe used as a disposable electrode with large-scale production capability. Theoxidation of vanillin at SPCE and others most unmodi?ed electrode surfacesrepresents a serious problem which arises from the high over-potential togetherwith poor reproducibility resulted from a fouling effect, which cause ratherpoor selectivity and sensitivity 16,17. In order to avoid these disadvantages, the modification ofelectrochemical working electrode is an excellent alternative. In this sense,many researchers have attempted to diminish the over-potentials by using variousmodi?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 someelectrodes was still not enough good. Thus it isimportant to develop new types of electrode devices by the synthesis andpreparation of new materials (modi?ers).
In this regard, as electrode surfacemodi?ers, titanium dioxide nanoparticles (TiO2-NPs) captured great interestof researchers and a considerable amount of research was executed in theprevious decades 12,13,19. The augment in the ef?ciency of TiO2-NPs such as to ensnarethe charge carrier is achieved by doping with transition metals and transitionmetal 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.Herein we reportthe synthesis of aluminium doped TiO2 nanoparticles (Al-TiO2-NPs),which were characterized by TEM and XRD. The sensitivity of the developedelectrode is compared with SPCE, TiO2-NPs/SPCE, and Al-TiO2-NPs/SPCEfor vanillin detection. The results show that a composite film of Al-TiO2-NPs/SPCEis more sensitive compared to SPCE and TiO2-NPs/SPCE. Further, weinvestigated the electrochemical behaviour of vanillin at modified SPCE withAl-TiO2 (Al-TiO2-NPs/SPCE).
The performance of themodified sensor is demonstrated by the determination of vanillin in foodsamples obtaining good selectivity, stability and high sensitivity. 2.All the startingmaterials 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%) werepurchased from Sigma-Aldrich (St. Louis, MO, USA). Nafion 117 solution (5% in amixture of lower aliphatic alcohols and water), vanillin, vanillic acid,4-hydroxybenzoic acid were purchased from Fluka Chemie (Buchs, UK). Ethanol andphosphoric acid were purchased from Panreac Quimica S.L.
U. (Barcelona, Spain).Vanillin solution were prepared in ethanol and stored in the dark until use.Working standard solutions were made by appropriate dilution of the vanillinstock standard solution with phosphate buffer at pH 6.3. Water was puri?ed witha Milli-Q system (Millipore).
Ethanol and phosphoric acid were obtained fromPanreac Quimica S.L.U. (Barcelona, Spain). Vanillin extractsamples (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.
Electrochemicaldetection was carried out on a CH Instruments Model 800D Series (Austin, Texas,USA) all measurements were carried out using a screen printed carbon electrodes(SPCEs) system (Dropsens DRP-C110) housed in the home made electrochemical flowcell. Transmission electron microscopy (TEM) micrographs were measured on aJeol JEM 2011 operating at 200 kV and equipped with an Orius Digital Camera (2× 2 MPi). The samples were prepared by deposition of a drop of the synthesizedmaterial suspension onto a lacey carbon/format-coated copper grid. The digitalanalysis of the HRTEM micrographs was done using Digital Micrograph TM 1.
80.70 forGMS 1.8.0 Gatan. XRD patterns were measured on Philips model X´Pert MPDdiffractometer using a CuKa source (? =1.5418 Å), programmable divergence slit,graphite mono-chromator and proportional sealed xenon gas detector. The sampleswere made with a voltage of 40 KV, intensity 40 mA and an angular range of 20to 70 degrees (2?), a step of 0.
02degrees 2 Theta and a time per step of 1.50 sec.Agilent 1200liquid chromatography system was used as a chromatographic system. It wasequipped with a LC pump, a vacuum degasser, a micro well-plate auto sampler (5?L injection loop), a thermostatted column compartment and a Diode-ArrayDetector (DAD). The data was processed using the Agilent ChemStation software.
Reversed-phase C18 analytical column Luna 5µm PFP (2) 100A (150x 4.6 mm) was used for the separation of the analytes present in vanillinextract samples. Elution was performed under isocratic conditions, by using amixture of acetonitrile/phosphate 20/80 (v/v) as a mobile phase, at a flow-rateof 1.0 mL min?1. The injection volume was 40 µL. Detection wavelengthwas performed at 265 nm. At first, Al-NPswere prepared according the previously described procedures 25, incorporating some modifications.
This modified synthesis involvesthe addition of aluminium acetylacetonate Al(acac)3 (10 mmol) tomesitylene which was already placed in a three-neck round bottom flask withmagnetic bar. Then lithium aluminium hydride LiAlH4 (30 mmol) wasadded to the mixture. The reaction was purged with N2 during refluxwith stirring for 72 hours at 165 °C. Thereaction mixture was cooled down to 25 °C. A gray-colored precipitate was crushed and dried under lowpressure for 5 hours. The crude product was washed with 25 ml of cold methanolat least three times, to avoid any exothermic reaction between solvent andAl-NPs. The unreacted starting materials were washed with methanol three times.The final product was filtered and dried at 25 °C under low pressure.
The preparation of Al-TiO2-NPsstarted 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 finalproduct was filtered and dried at 25 °C under lowpressure, obtaining an Al-TiO2-NPs as light-gray powder.Nanoparticleswere dispersed in water (0.5% Nafion, v:v) by ultrasound sonicator, obtainingindividual concentrations of 1 mg mL-1.
Films formed fromnafion-solubilized nanoparticles are more uniform than those casted by organicsolvents 26. TiO2-NPs and Al-TiO2-NPs were used. DRP-110SPCEs (Dropsens, Spain), with carbon as a working electrode, were used toprepare the modi?ed-SPCE. Each modi?ed SPCE was prepared by casting 2 µL of thedispersed NPs onto the surface of the electrode. After drying under infraredlight (IR) for 15 min, rinsing with pure water and ready for using. Energy-dispersiveX-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 differentsurface morphologies.
Figure 1 (A and C), shows the TEM and EDX micrographs ofthe aluminium particles without doping (this image was obtained beforealuminium was mixed with TiO2-NPs.), and a higher magnification isalso presented. A large number of precipitates were distributed homogeneously throughthe aluminium grains in the peak aged conditions. It is clear that the increasein hardness is brought about by the distribution of the precipitates foraluminium. Figure 1 (B and D), shows the TEM and EDX micrographs of the fullymixed precursor. It shows two large particles in the lower region, which areAl-NPs and TiO2-NPs, were distributed homogeneously, indicating thata 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 aluminiumas a major component. The XRD patterns and all the positions of the peaks are foundto be in good agreement with the face-centered cubic (fcc) form of aluminium 25, and anatase TiO2 27, as shown in Figure 2. A closer look at the figure shows that theAl-TiO2-NPs involved aluminium as a major component mixed with TiO2-NPsas 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 (7.63nm) and Al-TiO2-NPs (7.47 nm). The XRD results confirm the TEMmicrograph results discussed above. The modified screenprinted carbon electrodes (SPCEs) by aluminium doped TiO2nanoparticles (Al-TiO2-NPs/SPCE) were ?rst characterized by cyclicvoltammetry (CV) to test their behaviour for the oxidation of vanillin, asshown in Figure 3 (C).
In order to find the role of Al-NPs, the cyclicvoltammograms of (A) SPCE, (B) TiO2-NPs/SPCE, and (C) Al-TiO2-NPs/SPCEin the presence of 250 µM vanillin were recorded, as shown in Figure 3. Theelectrochemical behaviours of the TiO2-NPs/SPCE and Al-TiO2-NPs/SPCEwere investigated by using the CV technique, as shown in Figure 3 (B and C).All cyclic voltammograms were obtained in the presence of 0.
1 M H3PO4electrolyte (pH=6.3) with 250 µM vanillin at 50 mV s-1 scan rate.The cyclic voltammograms observed for the SPCE electrode (with the absence ofTiO2-NPs and Al-TiO2-NPs, Figure 3 (A)). In contrast, thecyclic voltammograms observed for the TiO2-NPs/SPCE and Al-TiO2-NPs/SPCEmodified electrode exhibited only an oxidation peak in the presence ofvanillin, within the potential window in the range from 0.00 to 1.20 V. Itsuggests that the oxidation reaction of vanillin on Al-TiO2-NPs/SPCEis totally irreversible.
The oxidation peak of vanillin is observed at the SPCE(Figure 3 (A)) with a low and broad peak current. The oxidation current ofvanillin (0.58 V) on Al-TiO2-NPs/SPCE (Figure 3 (C)) was higher thanthat on the 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 theAl-TiO2-NPs/SPCE (Figure 3 (C)), indicating that the highconductivity and high surface area of the TiO2-NPs/SPCE improve thecatalytic activity and increase the effective electrode area toward thevanillin oxidation obviously indicating that the Al-TiO2-NPs/SPCEcan be used to determine vanillin.
Theelectrochemical response of the modified Al-TiO2-NPs/SPCE toward thedetermination of vanillin were optimized by analyzing a standard solution (10µM) of vanillin using linear sweep voltammetry (LSV) technique. The parametersa?ecting the determination of vanillin, such as electrolyte, pH,Al-TiO2-NPs amount, scan rates, adsorption time, accumulationconditions and stability of the electrodes, were investigated. In order to findthe optimal parameters, one parameter was changed and the other parameters were?xed at their optimal values.The influence ofdifferent supporting electrolytes were tested, including NH4Ac, HCl,H3PO4, HNO3, H2SO4 andNaOH (each 0.1 M). The results indicated that when 0.1 M H3PO4 solutionwas used, the oxidation peak current was a higher sensitivity than other.
Forfurther study, 0.1 M H3PO4 solution was chosen as thesupporting electrolyte. On the other hand, the effect of 0.1 M H3PO4supporting electrolyte pH on the electrochemical response of the modified Al-TiO2-NPs/SPCEtoward the determination of vanillin was studied. The variations of the oxidationpeak potential as well as the peak current with respect to the change of theelectrolyte in the pH range (1.40 – 6.30), with anincrease the pH, manifesting that protons have taken partin the electrode reaction processes.
The relationship between the oxidationpeak potential and pH is also shown in Figure S1. The effect of modificationamount of Al-TiO2-NPs on the SPCE surface for the determination ofvanillin was also studied by using LSV, different modification volumes weretested: 2.0, 4.0 and 6.0 ?g. The 2.0 ?g gave the best result of oxidation peakcurrent sensitivity than other counterparts. The effect of scan rate value onthe oxidation peak current of 10 µM of vanillin by using LSV was studied.
Different scan rates were tested 20, 30, 50 and 70 mV s-1. The 50 mVs-1 gave the best result of oxidation peak current sensitivity thanother counterparts. In this study, 50 mV s-1 scan rate was used. Theaccumulation step of 10 µM vanillin after 120 second was performed under 0.0 V,the peak current increased progressively with accumulation time up to 120second at a fixed accumulation potential 0.
0 V. Thereafter, the peak currentincreased much slightly as further increasing the accumulation time, also shownin Figure S2. This phenomenon could be attributed to the saturated adsorptionof vanillin on the surface of the electrode.
Then, the stability of themodified electrodes was examined under the best conditions. Five electrodeswere made by the same procedure. Moreover, the stability of modified electrodewas also studied. When the modified electrode (Al-TiO2-NPs/SPCE) wasstudied with eight segments, the peak current kept 99.0 % of the original peakis also shown in Figure S3.
Using thepreviously optimized conditions, the analytical parameters of the system wereexamined by linear sweep voltammetry (LSV), using a scan rate of 50 mV s-1.A linear calibration graph was obtained using standard vanillin solutions inthe range of 0.07–20 µM. The linear sweep voltammograms obtained for differentconcentrations of vanillin under the optimum experimental conditions. Thelinear range, slope and intercept of the calibration curve are given in Table 1,along with the regression coefficient (R2) for vanillin. Theprecision of the method for aqueous standard solutions (evaluated as therelative standard deviation (RSD) obtained after analyzing 10 series of 10replicates) was 3.5 % at the 5 µM concentration of vanillin.
The theoreticallimit of detection (LOD), is expressed as the analyte concentration giving asignal equivalent to the blank signal plus three times its standard deviation(3?), was 0.02 µM. This method provides clear and good advantagesin terms of sensitivity with respect to other existing alternatives methods 16,17 that involve the use of others vanillin sensors and electrochemicaldetection (see Table 2).The influence ofpotential interferences in the analytical signal of vanillin was studied forsome common compounds in the samples to be analyzed, such as vanillic acid,vanillic alcohol, p-hydroxybenzaldehyde and p-hydroxybenzoic acid. Thesecompounds were chosen because they are known to be present in natural vanilla. Acompound was considered as interference if it caused an analytical variation ofmore than 5% when compared to the analytical signal obtained in the absence ofthe interfering compound. All the results are shown in Table 3.
Nointerferences were observed for the interferent/analyte ratios investigated.The applicabilityof the proposed methodology was evaluated by determining vanillin in twovanillin extract samples (Sample A and Sample B). These vanillin extractsamples 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 areshown in Table 4. This was compared with a blind analysis of the vanillinextract samples using the modified HPLC-DAD method 30 described in the section 2.
2, which provided a chromatogramrepresented in Figure 4. The results showed, both methods are in closeagreement for supporting the validity of this method. In summary, the development of analuminium doped TiO2 nanoparticles have been carried out, and theobtained hybrid nanoparticles have been tested in SPCE, as novel workingelectrode for detection of vanillin in extract vanilla samples with good analyticalperformance. The use of the developed working electrode (Al-TiO2-NPs/SPCE)allows the simple, effective and rapid method for electrochemical detection ofvanillin in extract vanilla samples.
The Al-TiO2-NPs/SPCE electrode wasfound to increase the sensitivity higher than the commercial electrodes tested,therefore evaluating the effectiveness of the proposed approach to improve thesensitivity of the working electrodes in screen printed carbon electrodes.