ZnO/poly(nigrosine)/modified carbon paste electrode for selective sensing of vanillin in the presence of amaranth: a voltammetric study

Abstract

In the current work, a ZnO/Poly Nigrosine (ZnO/PN MCPE) modifier was added to the unmodified carbon paste electrode to increase its sensitivity for analyzing vanillin (VAN) with the company of amaranth (AMR) using a cyclic voltammetric approach. In order to analyse ZnO nanoparticles, the X-ray diffraction (XRD), scanning electron microscopy (SEM) and energy disperssive X-ray spectroscopy (EDS) studies was accomplished. The ZnO/PN MCPE succeeded in the development of electrochemical sensors for the detection of vanillin (VAN) by the ZnO/PN MCPE by cyclic voltammetry (CV) technique. Many different parameters was studied such as electrochemical investigation, scan rate, concentration, pH effect was studied. The simultaneous analysis of VAN in the presence of amaranth was studied. The detection limit and quantification limit was calculated and also oxidation of VAN was adsorption controlled. This profound method was successfully implemented for the investigation of vanillin in food samples. This modified electrode shows a very promising electrochemical sensing of VAN.

Supplementary Information

The online version contains supplementary material available at 10.1007/s13197-024-06122-w.

Introduction

Vanillin is an essential flavouring agent which is extensively used worldwide. It’s an organic compound have molecular formula C₈H₈O₃ and it’s a vanilla bean extract which is component of natural vanilla. VAN is also known as 4-hydroxy-3-methoxy benzaldehyde (O Moradi.A review on nanomaterial-based electrochemical sensors for determination of vanillin in food samples 2022; Vinod Kumar Gupta 2018; Venkatesh 2022; Tânia Regina Silva 2015a, 2015b; Taouri et al. 2021; Yiğit et al. 2018; Pushpanjali et al. 2020). Even though the yield of vanillin for each year was more than 12000t, but natural vanillin yield from vanilla was below 1% of each year production. This remainds the synthesised VAN was considerably more cheaper, hence it was synthesised through the chemical or biochemical methods. Due to the high cost of natural vanilla, in this contemporary world the synthetic VAN was widely applicable in food, non edible food, in perfumes and in medicinal applications as binding and flavouring agent in preparation of medicines etc.

Because of its antioxidant property this claims an application in stroage of milk in diary. VAN was used to nurse sickle cell anemia and cardiovascular disease. Limited use of VAN made an attention because considering it’s stern after cause effects such as shortness of breath, nausea, headache, kidney and liver damage and tempting odour of VAN cause addiction to those flavoured foods. In recent days, the usage of VAN was restricted for infant foods. The higher concentration of VAN in food samples was very effective (Nie et al. 2020; Abbasghorbani 2017; Kumari et al. 2023; Shahidi and Chekin 2022). Thus, it was crucial to analyze VAN in food samples.

The examination of VAN made extensive use of electroanalytical techniques in contrast to other techniques like fluorimetry, chromatography, and spectroscopy. By virtue of its benefits, the electrochemical approach was widely adopted. Excellent sensitivity, simplicity of selectivity of bioactive compounds, rapid response, and low cost are all demonstrated by the electrochemical approach (Filik 2016; Arpitha et al. 2024; Radhakrishnan et al. 2022; Sevcan erden, Zehra Yazan 2018; Karakaya and Kaya 2021; Madhuchandra and Kumara Swamy 2019). The most advantageous aspect was VAN's careful and prompt handling of food samples. Cyclic voltammetric analysis was preferred over the entire electrochemical methods in the current work owing to the reason of inexpensive, easy to analyze the results, and helpful in determining the rate constant, kinetics of heterogeneous electron transfer, and the impact of analyte diffusion rate (Arpitha et al. 2023; Yang et al. 2024; Razavi 2024; Peng et al. 2012; Hui et al. 2023).

Because of the over-potential and electrode fouling nature noticed the weak electrochemical signals in the direct determination of analytes with unmodified electrodes. This method particularly needs electrochemical expertise to analyse the sample with electrochemical determination. In our present work we have carried out the modification of carbon pate electrode (CPE). CPE was prioritized over other working electrodes because of its advantages like the potential window, large surface area, modest cost. Even the modification of CPE was done by different modifiers it has the proficiency of detection and sensing of the analytes (Radhakrishnan et al. 2022; Sevcan erden, Zehra Yazan 2018; Karakaya and Kaya 2021; Madhuchandra and Kumara Swamy 2019; Arpitha et al. 2023; Yang et al. 2024; Razavi 2024).

According to the literature survey many different modifiers were used for the analysis of VAN such as graphene modified GCE exhibited efficiently electrocatalytic oxidation for vanillin (Peng et al. 2012), the sensor based on Pt-Ser-B-GQD exhibits a high sensitivity for electrochemical detection of vanillin (Hui et al. 2023), CuO@SiO2 was coated on an electrode surface where it demonstrates high catalytic activity for the electro-oxidation of vanillin in phosphate buffer solution (Venkadesh et al. 2021) etc. For the first time ZnO/PN modified carbon paste electrode was used for the electrochemical analysis of vanillin. As compared to literature survey out modification process was simple and shows a promising application for the analysis of VAN in food samples.

ZnO/Poly nigrosine modified carbon paste electrode (ZnO/PN MCPE) was prepared. The ZnO nanoparticle was prepared by employing co-precipitation method. Nigrosine dye used as negetive staining of bacteria. It is also called as acid black—2 and has molecular formula C₂₂H₁₄N₆Na₂O₉S₂. Nigrosine (Scheme 1A) polymeric film was layered on surface of ZnO modified CPE by electropolymerisation method so that it increases the active sites and adhesiveness to the electrode surface (Sevcan erden, Zehra Yazan 2018; Karakaya and Kaya 2021; Madhuchandra and Kumara Swamy 2019; Arpitha et al. 2023; Yang et al. 2024; Razavi 2024). Hence ZnO/PN MCPE was choosen for the analysis of VAN in food samples in biological pH. Amaranth (AMR) (Scheme 1B) was used to treat anemia and consumption of bread made from amaranth increases healthy level of red blood cell and it also has good antioxidant property. There is currently no information available regarding the cyclic voltammetric analysis of VAN utilizing Poly Nigrosine/ZnO modified CPE.

Scheme 1.

A Structure of poly nigrosine. B Structure of amaranth

Our current work has a good novelty i.e. Vanillin (VAN) is very essential flavouring agent and daily consumption can cause addiction and leads to some diseases. Hence PN/ZnO modified carbon paste electrode (MCPE) for electrochemical sensing of VAN was developed for this cause. VAN is a restricted food for infants because higher consumption was very effective. Hence the detection of VAN concentration was necessary. So we developed a sensor for VAN in food samples by PN/ZnO MCPE. The modified electrode exhibits a very effective electrocatalytic activity for the determination of VAN.

Experimentation

Chemicals and reagent.

Graphite powder, siliicon oil, vanillin, amaranth (sigma aldrich), Sodium dihydrogen phospate and disodium hydrogen phosphate, zinc acetate, sodium hydroxide (NICE) all are of analytical grade chemicals, furthermore purification was not needed. Chemical solutions were prepared from double distilled water. CV was operated in CHI-660c workstation.

Synthesis of ZnO nanoparticles.

Several methods were there for the synthesis of ZnO nanoparticles such as sol–gel, hydrothermal, chemical vapour deposition, biosynthesis, chemical reduction etc. The benefits of co-precipitation over other techniques were its low cost, high yield, and pure product. Thus, the co-precipitation method of synthesis was selected. Here the 100 ml solution of zinc acetate (0.2 M) and NaOH (0.4 M) was prepared through deionised water. The zinc acetate solution was stirred continuously around 25–30 min, after this NaOH solution was added to above solution drop wise with continuous stirring until precipitation occurs (2 h). White precipitation was formed. The precipitate was centrifuged at 4000 rpm for about 30 min. It was filtered using whatman filter paper no. 42. Filtered precipitate was washed with alcohol, calcined at 600⁰ C for 2 h. Finally occurred precipitate was characterized by XRD, SEM and EDS analysis.

Fabrication of electrode

The fabrication of BCPE was carried out and it was reported in our earlier work (Venkadesh et al. 2021; Fayemi et al. 2018). The ZnO modified CPE was prepared in earlier step. 6 mg of ZnO nanoparticle, 70% of graphite powder and 30% of silicon oil was grinded in a mortar around 30 min. The prepared paste was filled to the cavity of the teflon tube, it was rubbed on butter paper. It is known as ZnO MCPE. Later the prepared ZnO MCPE was polymerized by Nigrosine dye by electropolymerisation method. Then it was known to be ZnO/PN MCPE.

Preparation of real sample solution

Samples from commercial products, including biscuits, tablets, and vanilla essence, were collected. A known quantity of crushed powder was taken individually and it was first dissolved in 10 ml of ethanol while being continuously stirred. It was then diluted in deionized water and stirred for approximately 20 min. This procedure was done for the preparation of the solution of vanilla extract, biscuit and tablet (which was bound by vanilla flavor). These solutions retain roughly thirty minutes each in a separate sonicator. After that, it was removed, filtered, and put to use for the analysis in pH 7.4 of 0.2 M PBS.

Results and discussion

Investigation of morphology of ZnO nanoparticle.

SEM analysis was performed to verify the surface morphology of ZnO nanoparticles. The surface looked like nanoflakes (Fig. 1A) by using EDS analysis, the production of ZnO nanoparticles was verified. The EDS analysis report's (Fig. 1B) peaks for C, O and Zn were noted. The XRD pattern obtained, which is displayed in Fig. 1C, is used to determine the nanoparticle's size. By using Debye–Scherrer Eq. 1, the size of the nanoparticle was determined (Arpitha et al. 2023; Kumar et al. 2015).

$$D=k\lambda /\beta cos\theta$$ 1

where, wavelength is denoted by λ, half maximum intensity is denoted as β, diffraction angle of the diffration peak as θ and K is constant.

Fig. 1.

A. SEM images of ZnO nanoparticles. B EDS plot of ZnO nanoparticles. C XRD pattern of ZnO nanoparticles

The particle size of the synthesised ZnO nanoparticle was 19.17 nm.

Study of VAN at varied ZnO nanoparticle MCPE.

At sweep rate of 50 mV/s the cyclic voltammogram of VAN at various ZnO MCPE compositions was examined in pH 7.4 PBS. During the formulation of the carbon paste, ZnO was added in increments of 2, 4, 6 and 8 mg. A voltammogram was reported for each of the following amounts: 2 mg, 4 mg, 6 mg and 8 mg of ZnO MCPE. The 6 mg ZnO MCPE exhibits a good improvement in the peak current (depicted in Figure S1) among other modified electrodes. Electropolymerization of poly Nigrosine in this work, 6 mg ZnO MCPE was therefore selected.

Electropolymerisation of nigrosine to ZnO MCPE.

A supporting electrolyte of 0.1 M NaOH (24 ml) was electropolymerized with 1 mM nigrosine (1 ml) to properly deposit the poly(nigrosine) over the electrode surface. Figure S2A shows a cyclic voltammogram of the polymerization of nigrosine between the potential window of -0.6 V to 1.4 V at a scan rate of 100 mV/s. The electrooxidation of 10 μM VAN over 5–25 multicle cycles at pH 7.4 PBS with a scan rate of 50 mV/s was also recorded as part of the multiple cycle process (inset Fig S2B). At 15 cycles, the peak current reached its maximum. As a consequence, the full process of electropolymerizing nigrosine required 15 cycles.

Effective surface area of the electrode.

By the electrochemical approach the active surface area of the bare and modified carbon paste electrode was evaluated. The comparision of the peak current response of BCPE (coloured black) and ZnO/PN MCPE (coloured red) was examined in K₄[Fe(CN)₆] electrolyte system in 1 M KCl with a sweep rate of 50 mV/s (Fig S3). In comparision with the peak current of BCPE the ZnO/PN MCPE shows good enhancement in the redox peak current and by Randles-sevick’s equation the active surface area of BCPE and ZnO/PN MCPE was found to be 0.038 cm² and 0.062 cm² individually (Eq. 2) (Arpitha et al. 2024, 2023; Arpitha and Kumara Swamy 2023).

$$Ipa=2.69\times {10}^5{n}^{3/2}A{D}_0^{1/2}{C}_0{\vartheta }^{1/2}$$ 2

where, Ip stands for peak current, $\nu$ and A are the scan rate in mV/s and area of the electrode respectively, n indicates number of electron transferred, C₀ and D₀ stands for concentration and diffusion co-efficient.

Voltammetric behaviour of VAN at BCPE and ZnO/Poly nigrosine MCPE.

Figure 2 depicts the cyclic voltammogram outline of 10 μM VAN at BCPE (dotted line), ZnO MCPE (coloured blue) and ZnO/PN MCPE (coloured red) in 0.2 M PBS of pH 7.4 with a sweep rate of 50 mV/s. Compared to BCPE and ZnO MCPE the ZnO/PN MCPE revealed good increament in redox peak current and a modest shift in the potential but in the case of BCPE the VAN shows lower current response with substantial peak in comparision with ZnO/PN MCPE. The procured result reveals that the modified electrode shows astonishing electrocatalytic performance of the developed sensor of VAN compared to other electrodes.

Fig. 2.

Cyclic voltammogram outline of 10 μM VAN at BCPE (dotted line), ZnO MCPE (coloured blue) and ZnO/PN MCPE (coloured red) in 0.2 M PBS of pH 7.4 with scan rate 50 mV/s

pH study

The electrochemical oxidation of VAN was studied by using CV technique in the PBS of different pH from 5.8—7.8. The peak current of VAN depends on the value of pH and with the increase in pH the peak current also increases upto maximum of pH 7.4 later on it decreases with the high pH value. The Ipa versus pH was shown in Fig. S4A. The pH 7.4 shows highest current response. Hence it was taken for entire work. The pH effect also indicates a notable effect on peak potential of vanillin. Peak potential was linearly dependent on the value of pH. It was proved to be that the peak potential values shifted to lower value with increases pH value shown in Fig. S4B, the plot of Epa versus pH. Dependency of peak potential versus pH based on following formula Ep (V) = 0.062 pH + 0.206 (R² = 0.997). A very good linear plot of Epa versus pH was procured. The procured slope value from this plot was 62 mV/pH unit almost proximate to knernst theoritical value that is 59 mV/pH unit. This tells in the oxidation of VAN even number of protons and electrons are involved in this process (Scheme 2).

Scheme 2.

Oxidation mechanism of VAN

Consequence of scan rate

The influence of the potential sweep rate on the electrochemical performance of the VAN was researched in PBS of pH 7.4 at ZnO/PN MCPE. Cyclic voltammogram was recorded for varied potential sweep rate ranging from 10—100 mV/s. Thereby increament in the peak current with little positive shift in peak potential which was displayed in Fig. S5A. The plot of Ipa versus sweep rate also exhibits good linearity Fig. S5B with linear regression Eq. 3.

$$Ipa\left(\mu A\right)=Ipa\left(\mu A\right)=4.075\vartheta \left(mV/s\right)+3.108$$ 3

with correlation co-efficient R² = 0.997.

This finding indicates that, at lower VAN concentrations, the adsorption process regulates the.

electro-oxidation of VAN rather than diffusion. Equation 4 (Peng et al. 2012) was utilized to calculate the.

heterogeneous rate constant (k⁰) for VAN (Table 1) based on the observational peak potential difference data (ΔEp). This equation was used for the experimental data when values of ΔΕp was found to be greater than 10 mV. The rate of reaction increases with increase in heterogeneous rate constant.

$$\mathrm{\Delta }{E}_p=201.39log(\vartheta /k_0)-301.78$$ 4

Table 1.

Variation of the voltammetric parameters gathered from the plots shown in Fig. S5A as a function of the potential sweep rate

Scan ratemV/s	Epa(V)	Epc(V)	∆Ep(mV)	k 0(s−1)
50	0.5698	0.5052	64.6	0.76
100	0.5935	0.5069	86.6	1.18
150	0.6021	0.5086	93.5	1.63
200	0.6075	0.5095	98	2.06
250	0.6100	0.5025	107.5	2.32
300	0.6153	0.5069	108.4	2.76
350	0.6223	0.5113	111	3.12
400	0.6250	0.5121	112.9	3.49
450	0.6275	0.5142	113.3	3.90
500	0.6284	0.5146	113.8	4.31

Consequence of concentration

The cyclic voltammogram of VAN at ZnO/PN MCPE for varied concentration in PBS of pH 7.4 with sweep rate of 50 mV/s was depicted in Fig. 3A. With the increment in concentration, the VAN peak current increased. The plot of Ipa against VAN concentration displays a good linearity [Fig. 3B] with the regression equation are Ipa (μA) = 0.043 (μM) + 1.618, R² = 0.995. The lowest detection limit and quantification limit is aquired according to Eq. 5 and 6. The LOD and LOQ of VAN perceived was 0.29 μM and 0.98 μM. The comparitive value of LOD with other work was tabulated in table S1. According to this compared to other works our modified electrode exhibits less detection limit.

$$LOD=\frac{3S}{M}$$ 5

$$LOQ=\frac{10S}{M}$$ 6

Fig. 3.

A cyclic voltammogram of VAN at ZnO/PN MCPE for varied concentration in PBS of pH 7.4 with sweep rate of 50 mV/s. B The graph of Ipa versus VAN concentration

S and M indicates standard deviation and slope.

Simultaneous analysis of VAN in presence of Amaranth

Figure 4 depicts the ZnO/PN MCPE was evidently develops the voltammetric resoluteness of VAN with amaranth in the solution mixture containing 10 μM VAN and 10 μM amaranth in PBS of pH 7.4 with scan rate of 50 mV/s. The oxidation peak potential of both the analyte were very nearer hence the chance of overlapping of both the analytes occur at BCPE. The ZnO/PN MCPE shows well separated peak with increment in the peak current than the BCPE. The problem of separation was achieved by ZnO/PN MCPE. The cyclic voltammogram of well defined separated two peaks of VAN and amaranth was shown at different potentials, for VAN it was 15 mV and for amaranth peak potential was 16 mV respectively. The procured result was enough to analyse VAN with amaranth at ZnO/PN MCPE.

Fig. 4.

Cyclic voltammogram of 10 μM VAN and 10 μM AMR at ZnO/PN MCPE in PBS of pH 7.4 with scan rate of 50 mV/s

Analytical performance

For the analysis of VAN in three commercially available real samples such as custard powder, vanilla extract, tablet were purchased from the local market. The real sample solutions were prepared. The proficiency of the ZnO/PN MCPE was checked by using the above real solution samples by standard addition method. The procured result was tabulated in Table 2. The ZnO/PN MCPE shows best sensing for VAN in the food and pharmaceutical samples.

Table 2.

Determination of VAN in commercially available samples (n = 3)

Samples	Spiked VAN sample (μM)	Found	Recovery (%)
Vanillin extract	10	9.6	96.84
	20	19.4	96.94
	30	28.6	95.3
Tablet	10	3.3	92.9
	20	18.6	93.3
	30	28.6	95.20
Custard powder	10	9.6	95.64
	20	19.7	98.6
	30	29	98

Conclusion

Using the cyclic voltammetric technique, the ZnO/PN MCPE demonstrated outstanding electrocatalytic activity of VAN in food samples within the context of this investigation. SEM, EDS, and XRD analysis were applied to characterize the produced ZnO nanoparticles. Here, ZnO/MCPE was created using the electropolymerization process and poly nigrosine dye. In comparison to BCPE, a larger surface area was fetched with ZnO/PN MCPE. The scan rate investigation, the influence of concentration, the simultaneous study, and the application in a real sample all yield successful findings. ZnO/PN MCPE shown a significant increase in VAN peak current when compared to BCPE. When comparing the detection limit to other study data analysis, very little was obtained. In the existence of AMR, the VAN showed two distinct peak separations.

Overall, the ZnO/PN MCPE exhibits exceptional electrocatalytic activity for VAN together with good sensitivity. The formulated sensor was successfully applied to the VAN that included actual sample analysis.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

S.B. Arpitha: Electrochemical Sensors experiments, Formal analysis, Writing—original draft B. E. Kumara Swamy: Conceptualization, Supervision, Writing—review and editing.

Funding

The authors have not disclosed any funding.

Data availability

The data will be given upon the request and with terms and conditions of Journal policy.

Declaration

Conflicts of interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Consent for publication

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