Simultaneous electrochemical sensing of dihydroxy benzene isomers at cost-effective allura red polymeric film modified glassy carbon electrode

A simple and simultaneous electrochemical sensing platform was fabricated by electropolymerization of allura red on glassy carbon electrode (GCE) for the interference-free detection of dihydroxy benzene isomers. The modified working electrode was characterized by electrochemical and field emission scanning electron microscopy methods. The modified electrode showed excellent electrocatalytic activity for the electrooxidation of catechol (CC) and hydroquinone (HQ) at physiological pH of 7.4 by cyclic voltammetric (CV) and differential pulse voltammetric (DPV) techniques. The effective split in the overlapped oxidation signal of CC and HQ was achieved in a binary mixture with peak to peak separation of 0.102 V and 0.103 V by CV and DPV techniques. The electrode kinetics was found to be adsorption-controlled. The oxidation potential directly depends on the pH of the buffer solution, and it witnessed the transfer of equal number of protons and electrons in the redox phenomenon. The limit of detection (LOD) for CC and HQ was calculated to be 0.126 μM and 0.132 μM in the linear range of 0 to 80.0 μM and 0 to 110.0 μM, respectively, by ultra-sensitive DPV technique. The practical applicability of the proposed sensor was evaluated for tap water sample analysis, and good recovery rates were observed. Electrocatalytic interaction of ALR/GCE with dihydroxy benzene isomers.


Background
Over the past few decades, the rapid progress of science and technology to fulfil the desire of human beings led to many advancement as well as became a threat to environment (Xia et al. 2021;Ali & Jain, 2004;Gupta et al. 2009;Asfaram et al. 2015;Ali et al. 2013). As the proverb says "the science of today is the technology of tomorrow," many industries were established to make the best use of pure science in the production of applied commercial products (Steven, 1998;Goyal et al. 2009aGoyal et al. , 2009bDehghani et al. 2016;Gupta et al. 2014aGupta et al. , 2014bGupta et al. , 2014cGoyal et al. 2009aGoyal et al. , 2009bGhaedi et al. 2015;Gupta et al. 2015aGupta et al. , 2015b. These chemical industries have a fundamental association with the national economy and people's lives (Han et al. 2020;Gupta et al. 2015aGupta et al. , 2015bGupta et al. , 2015cGupta et al. 2014aGupta et al. , 2014bGupta et al. , 2014c. The industry refers to the collective name of enterprises and units which are engaged in the manufacture, research and development of the chemical engineering, lubricant refining, metallurgy, energy storage, light industry, petroleum products, the environment, drug, environmental safety and the military division (Gavrilescu & Chisti, 2005;Schmid et al. 2002;Goyal et al. 2005;Gupta et al. 2015c;Karthikeyan et al. 2012;Robati et al. 2016;Saravanan et al. 2015b). However, the waste effluent coming out as a side product in all the manufacturing industries may cause big impact on environmental pollution and can become toxic to human beings and aquatic creatures (Ahammad et al. 2011). Therefore, the quantification of the toxic molecules in the waste sample is of prime concern (Buleandra et al. 2014;Gupta et al. 2015aGupta et al. , 2015bGupta et al. , 2015cGupta et al. 2014aGupta et al. , 2014bGupta et al. , 2014cGupta et al. 2012;Saravanan et al. 2013aSaravanan et al. , 2013bSaravanan et al. , 2013c. Most phenolic moieties are toxic, and they are the basic feed stocks of the manufacturing industries (Wang & Hasebe, 2011;Saravanan et al. 2013aSaravanan et al. , 2013bSaravanan et al. , 2013cSaravanan et al. 2015b). Catechol (CC) and hydroquinone (HQ) are the isomers of dihydroxy benzene, which are widely used in paint, leather, pharmaceutical, pesticide, cosmetic and plastic industries, and obviously, the effluent from these manufacturing industries contain traces of CC and HQ which in turn become toxic to human and animals (Wang et al. 2007a(Wang et al. , 2007b(Wang et al. , 2007cKumar et al. 2017). Due to this these, molecules were recognized as an ecological hazard (He et al. 2014). The drawback of using traditional spectroscopic methods for their determination is tedious and not consistent (Chao & Suatoni, 1982;Nagaraja et al. 2001;Sun et al. 2000;Mesa & Mateos, 2007).
Allura red AC (ALR) (see Scheme S1, supplementary file) is one of the azo dyes extensively used in the colouring confectionery, soft and alcoholic drinks, ice cream, candy and bakery products (Pliuta et al. 2020). This simple azo dye can be efficiently used to modify the bare GCE by electropolymerization technique. We followed a protocol proposed by Mansour et al. for the electropolymerization of allura red AC on GCE, which they employed for the flow injection analysis determination of HQ and CC using a two-line flow injection manifold with a single-sensor/double-pulse amperometric detection (Mansour et al. 2019). In this study, we reported the modification procedure for the GCE by simple electropolymerization of ALR by cyclic voltammetric (CV) technique in a basic supporting electrolyte solution. The fabricated ALR glassy carbon electrode (ALR/GCE) showed electrocatalytic activity in the discrimination of overlapped signals of CC and HQ in a binary mixture which is practically not possible at bare GCE. The ALR/GCE was electrochemically characterized; the effect of scan rate showed adsorption kinetics at ALR/GCE. Mechanism of analytes with varying pH was proposed, and the fabricated modified electrode was applied for the tap water sample analysis and good recovery results were obtained.

Main reagents
The allura red AC (ALR), hydroquinone and catechol were obtained from Himedia, and double-distilled water was used to prepare standard stock solutions of concentrations 25.0 mM, 2.5 × 10 −3 M and 2.5 × 10 −3 M, respectively. The 0.2 M buffer solution of unique ionic strength and preferred pH was prepared from a mixture of NaH 2 PO 4 •H 2 O and Na 2 HPO 4 . Before any electrochemical measurements, the working electrode was cleaned with 1 μm, 0.3 μm and 0.05 μm of α-alumina slurry on the polishing pad and ultrasonicated in an equimolar mixture of ethanol and water for 15 min, later rinsed with double-distilled water. All the chemicals were of analytical grade and used as received without any additional treatment.

Instrumentation
AUTOLAB potentiostat with PGSTAT 302 was used for all the electrochemical experiments. The surface morphology of the working electrodes was characterized by using ultra-high-resolution field emission scanning electron microscope (FESEM, FEI, & Nova NanoSEM450) instrument operating at 25 kV. The working electrode used for this study was glassy carbon electrode with 3.0 mm in diameter. A platinum wire was used as a counter electrode and Ag/AgCl, saturated KCl, as a reference electrode. All the electrochemical experiments were carried out at an ambient temperature of 25 ± 0.1°C; the corresponding redox potentials were recorded with respect to Ag/AgCl electrode.

Fabrication of ALR/GCE and its electrochemical characterization
In order to boost the performance of the bare GCE, electropolymerization of allura red was carried out on the glassy carbon working electrode by preparing 1.0 mM aqueous solution of ALR monomer along with 0.1 M NaOH solution as a supporting electrolyte. The potential window was maintained from − 0.8 to 1.2 V with a fixed scan rate of 0.1 Vs −1 for ten consecutive cycles as shown in Figure S1. During the progression of electropolymerization, the voltammogram has gradually descended with increased number of cycles, indicating the growth of the ALR polymer chain on the surface of GCE. After few consecutive cycles, the voltammogram tends to be constant with stable current signal reflecting the achievement of saturation level in the electropolymerization phenomenon (He et al. 2014). As the number of cyclic sweeps increases, a decreased electrocatalytic property was observed at the fabricated electrode due to the thickness of polymeric film (Ganesh et al. 2015;Wang et al. 2007aWang et al. , 2007bWang et al. , 2007cWang et al. 2006). Therefore, it was concluded that ten sweep cycles of CV are best suitable to get a strong electrocatalytic response. Figure S2A represents the CVs for the [Fe(CN) 6 ] 4-/3in the electrochemical cell with 1 M KCl as the supporting electrolyte at bare GCE and ALR/GCE electrodes with 0.05 Vs −1 scan rate. From the graph, it was observed that the CV response obtained at fabricated ALR/GCE was with sharp and static enhancement, and peak potential separation was observed to be 0.064 V, which was a characteristic voltammogram of [Fe(CN) 6 ] 4-/3redox couple (Kumar et al. 2017). The Randles-Sevcik equation was used to calculate the active surface area of the working electrodes (Ganesh et al. 2015;Kumar et al. 2019), which was found to be 0.0289 cm 2 and 0.0319 cm 2 for bare GCE and ALR/GCE, respectively. The approximate surface coverage of ALR deposit on GCE was calculated by Eq. 1 (Wang, 1994;Ganesh et al. 2018).
Here, Γ, A, Ip, n and υ represents the surface coverage concentration, surface area of the working electrode, peak current, number of electrons involved and scan rate, respectively. F, R and T have their usual significance. The Γ was calculated to be 0.1006 × 10 −10 M/cm 2 for ALR/GCE electrode. Figure S2B showed the surface morphology of the bare GCE (a) and ALR/GCE (b), which showed that the surface imperfections were covered by a thin layer of ALR, which proficiently diminished the trapping of analytes at ALR/GCE surface (Mansour et al. 2019).

Electrocatalytic response of CC and HQ in a binary mixture at ALR/GCE
The electrocatalytic oxidation of CC and HQ was carried out by using CV technique. Figures 1 and 2 represent the CVs obtained for the oxidation of 40.0 μM CC and 40.0 μM HQ in 0.2 M buffer of pH 7.4 at bare GCE (curve a) and ALR/GCE (curve b), respectively, with a scan rate of 0.05 Vs −1 . It can be observed from both figures that the voltammetric response of CC and HQ at bare GCE (curve a) was wide, with poor in sensitivity, and the oxidation signals were located at 0.225 V and 0.182 V, respectively. However, the ALR/GCE (curve b) favoured the oxidation process of both analytes, and the signals were observed at 0.181 V and 0.108 V, respectively, for CC and HQ. The refinement of shift in the oxidation signal was due to the electrocatalytic capability of the ALR/GCE towards the electrooxidation of dihydroxy benzene isomers. The chief task of the ALR/GCE is to resolve the overlapped oxidation signals of CC and HQ isomers, which is practically impossible at bare GCE. Due to the similarity in chemical structure and fouling of the electrode surface, bare GCE fails in the judgment of oxidation signals of these isomers. Figure 3 evidently shows the cyclic voltammograms for equimolar binary mixture (40.0 μM) of CC and HQ with 0.2 M buffer of pH 7.4 at 0.05 Vs −1 scan rate. At bare GCE, overlain wide voltammogram was observed at an oxidation potential of 0.208 V which is of no technical significance. However, at ALR/GCE, a strong separation was obtained for CC and HQ at potentials which were as similar to their individual determination. The difference in oxidation peak separation was calculated to be 0.102 V, which was more adequate for the interference-free determination of these isomers in a dual mixture. At fabricated ALR/GCE, the oxidation of HQ becomes easier than CC; furthermore, the oxidation potential of HQ shifts to negative side and oxidized well before reaching the oxidation potential of CC which leads a successful separation of these targeted analytes (Wang et al. 2007a(Wang et al. , 2007b(Wang et al. , 2007c. The results obtained at CV are again confirmed by ultra-sensitive differential pulse voltammetry (DPV) method due to the absence of background current as shown in Fig. 4. The distinguishable signals were not obtained at bare GCE (curve a), and a broad overlapped signal was observed at 0.179 V. However, the ALR/GCE (curve b) showed selectively resolved oxidation peaks at 0.147 V and 0.044 V for CC and HQ, respectively. The peak potential separation was determined to be 0.103 V. This peak potential difference was more sufficient for simultaneous determination of dihydroxy benzene isomers.

Impact of scan rate and pH
The impact of varying scan rates on the determination of equimolar (40.0 μM) mixture of CC and HQ in 0.2 M buffer of pH 7.4 was examined. It can be seen from Fig. 5 that there was an increase in peak current with the increase in scan rate, and a slight shift in the redox potentials were observed, which was in accordance with the Randles-Sevcik equation (Kumar et al. 2019). It can be observed from Figure S3A and S3B that a good linearity was observed for the plots of anodic peak current (Ipa) versus scan rate (υ) with correlation coefficient (r 2 ) of 0.9945 and 0.9949, respectively, for CC and HQ. On the other hand, the relationship between the Ipa and the square root of scan rate (υ 1/2 ) of CC and HQ gives a r 2 value of 0.9909 and 0.9902, respectively, as shown in Figure S3C and S3D. According to the previous reported literature, the linear establishment of Ipa versus υ suggests an electrode phenomenon dominated by the adsorption-controlled process at ALR/GCE (Ganesh et al. 2015;Kumar et al. 2019). To calculate the values of rate constant (k 0 ), Eq. 2 was used (Bard & Faulkner, 2001;Avendano et al. 2007).
And the obtained values of k 0 for electrooxidation of CC and HQ were tabulated in Table 1.
The electrochemical experiments performed in aqueous solutions directly depend on the pH of the supporting electrolyte. Figure 6a showed the effect of variation of buffer pH on the oxidation of equimolar (40.0 μM) binary  mixture of CC and HQ by CV technique. As the pH of the supporting electrolyte increases, the redox potential shifts towards the least potential. A linear graph was observed between oxidation peak potential (Epa) and pH of the buffer solution, and the slope of the linearity was found to be 0.0615 and 0.0596 for CC and HQ respectively, as in inset Fig. 6b.

Concentration study
The concentration studies of both the targeted analytes were carried out at ALR/GCE by ultra-sensitive DPV technique. Figures 7a and 8a showed an increment in the current signal due to the increase in concentration of CC and HQ. The linearity graphs of Ipa versus concentration of CC and HQ were shown in insets Fig. 7b and 8b, respectively. The linear regression equations obtained are as follows: Ipa μA ð Þ ¼ 0:0386 C 0 μM=L ð Þ þ 0:7751; r 2 ¼ 0:9931 À Á and Ipa μA ð Þ ¼ 0:0393 C 0 μM=L ð Þ þ 1:1810; r 2 ¼ 0:9898 À Á for CC and HQ, respectively. The limit of detection (LOD) and limit of quantification (LOQ) were calculated by using Eqs. 3 and 4, where S is the standard deviation of the six blank measurements and M is the slope of the calibration graph (Wang et al. 1994;Ganesh et al. 2018).
The LOD values of CC and HQ were calculated to be 0.126 μM and 0.132 μM, respectively, which are relatively lower than our own previous reports and available recent literature reports as tabulated in Table 2 (Kumar et al. 2017;He et al. 2014;Shen et al. 2017;Zheng et al. 2013;Wang et al. 2006;Zhang et al. 2015;Xu et al. 2015;Lai et al. 2014;Ganesh & Swamy, 2016;Ganesh et al. 2017;Da-Silva et al. 2013;Qi & Zhang, 2005;Li et al. 2009;Yang et al. 2009;Ding et al. 2005;Zhao et al. 2009;Peng & Gao, 2006;Wang et al. 2007a, 2007b, 2007c, Vilian et al. 2014Li et al. 2012;Si et al. 2012;Yin et al. 2011;Alshahrani et al. 2014;Dong et al. 2008;Umasankar et al. 2011;Hu et al. 2012;Zhang & Zheng, 2007;Sukanya et al. 2020;Harisha et al. 2018;Chetankumar et al. 2020;. Because of the similar oxidation potential, an individual determination of either CC or HQ in a binary mixture is a task with high difficulty and these isomers interfere each other in their simultaneous determination. The interference study was performed by keeping the concentration of one analyte constant and varying the concentration of the other one by ultra-sensitive DPV technique. We recorded the DPV in the absence of analyte (blank reading) to show there is no appearance of oxidative current signal at ALR/GCE at zero concentration of the analyte. Figure 9a shows that as the concentration of CC was varied in a linear range of 0.0 to 100.0 μM by keeping the HQ concentration constant (20.0 μM), we can observe an increase in peak current due to an increase in the concentration of CC only. In case of HQ too, the concentration was increased in a linear range of 0.0 to 100.0 μM and that of CC was kept constant (20.0 μM) as shown in Fig. 10a. From both Figs. 9a and 10a, it can be clearly observed that the increase in the current signal was due to the increase in concentration of any one of the analytes and the other one showed constant signal which indicated that interference-free simultaneous detection is possible at ALR/GCE electrode. Moreover, in both insets Fig. 9b and 10b, the peak current relationship with increasing concentration was linear. Therefore, the results reflect a successful interference-free and simultaneous determination of dihydroxy benzene isomers at ALR/GCE.

Sample analysis
As in the previous literature (Zhang et al. 2019;Alshik-Edris et al. 2019), to assess the performance of the proposed method for anti-interference determination of the dihydroxy benzene isomers, the CC and HQ determination in tap water was tested and the obtained results were tabulated in Tables 3 and 4, respectively. When a known amount of CC was added to the tap water sample containing HQ (10.0 μM), a recovery of 98.0 to 101.0% was obtained. Similarly, when a known quantity of HQ was added to the tap water sample containing CC (10.0 μM), a good recovery of 99.2 to 102.0 % was obtained. Therefore, these results are evident for the analytical applicability of the proposed ALR/GCE.

Conclusion
In the present study, we demonstrated a simple and convenient way of modifying the glassy carbon electrode by electropolymerization of allura red by cyclic voltammetric method. The fabricated working electrode showed minimization of over potential and excellent electrocatalytic activity towards the discrimination of dihydroxy benzene isomers' oxidative signals, which is practically impossible in bare working electrode. The impact of scan rate and pH study reveals the adsorptioncontrolled kinetics with equal number of protons and electrons transfer. The anti-interference study reflects  that the current signal of catechol and hydroquinone were independent and depend on their individual concentration in a binary mixture. The analytical application of the proposed electrochemical sensor was investigated by employing it to the water sample analysis by adding a known quantity of one analyte by keeping the other one constant, which yielded a satisfactory recovery results. Overall, a sensitive, selective, cost-effective, analytically applicable and reproducible electrochemical sensor was fabricated for the electroanalysis of dihydroxy benzene isomers.
Additional file 1: Scheme S1. Chemical structure of allura red. Figure  S1. CVs observed during the electropolymerization of ALR on GCE. 1.0 mM solution of ALR with 0.1M NaOH at 10 cycles with 0.1 Vs −1 scan rate. Figure S2.