MnO 2 nanorods modified screen-printed electrode for the electrochemical determination of Sudan dye in food sample

A novel MnO 2 nanorods modified screen-printed electrode was fabricated and used as a voltammetric sensor for Sudan determination. MnO 2 nanorods were characterized using Field emission-scanning electron microscopy (FE-SEM). Electrochemical measurements were performed using cyclic voltammetry (CV), linear sweep voltammetry (LSV), differential pulse voltammetry (DPV), and chronoammperometry (CA). The MnO 2 nanorods on the electrode surface act as an excellent catalyst for the Sudan oxidation reaction. Our modified electrode presents good electrocatalytic activity toward Sudan, a short response time of <10 s, a low detection limit of around 0.08 µM, and linear detection range from 0.25 to 300.0 µM


Introduction
All food dyes can be divided into two basic, general categories: natural dyes and artificial dyes.The natural food dyes are derived from grapes, saffron, paprika, carrots, beets, and algae and are used to color a variety of foods.The artificial food dyes (AFCs), mostly derived from petroleum, contain a single or more azo functional groups (-N=N-), most frequently connecting the two aromatic parts.People associate specific colors with specific flavors, therefore, the colors of food can affect their perception of taste, especially their perception of sweets and beverages.Artificial dyes may improve on natural variations in color, may enhance colors that occur naturally, or may provide color to colorless and "fun" food, thereby making it appear more attractive and appetizing, e.g., adding a red, yellow or green color to gummy sweets, which would naturally be colorless [1,2].
Sudan red I (1-phenylazo-2-naphthol), a synthetic azo dye, is considered to be a genotoxic carcinogen and classified as a category 3 carcinogen by the International Agency for Research on Cancer (IARC) (1975).Its presence is prohibited in foodstuffs for any purpose at any level worldwide.Unfortunately, a variety of foodstuffs contaminated with Sudan dyes and Para red, particularly Sudan red I, have been detected throughout Europe and Asia [3][4][5].High-performance liquid chromatography-mass spectrometry (HPLC-MS) has been proven to be an excellent method for the direct determination of Sudan reds (I, II, III, and IV).Although sensitive and specific, this method can be time-consuming and expensive.Therefore, it is necessary to develop a rapid and economical method for detecting Sudan red I [6].
In this context, electrochemical sensors based on screen-printed electrodes (SPEs) have gained increasing interest as analytical tools for food analysis since SPEs provide great advantages that make these kinds of sensors have the important characteristics of ideal sensors: ease of use, low cost, and portability [40][41][42].So, the screen-printed technology has significantly contributed to the transition from the traditional unwieldy electrochemical cells to miniaturized and portable electrodes that meet the needs for on-site analysis.Although a screen-printed electrode (SPE) is not as robust as a conventional electrode, such as glassy carbon or gold disk, and the surface of its working electrode is not as perfect as the one of a mirror-like polished solid electrode, the advantages of SPEs regarding cost and size led to their increasing use in the last years as transducers in sensing.The use of SPE-based sensors in the control of food spoilage as complementary analytical tools to the conventional methods allows a rapid screening at any point of the food production chain, preventing the occurrence of foodborne illnesses and the reduction of food waste [43].
In this research work, an electrochemical sensor modified with MnO 2 nanorods (MnO 2 -NRs) was designed using a screen-printed electrode for the determination of Sudan.

Apparatus and chemicals
All the electrochemical measurements were carried out on a PGSTAT302N potentiostat/galvanostat Autolab.The measurement cell consisted of SPE (DropSens; DRP-110: Spain) containing a graphite counter electrode, a graphite working electrode, and a silver pseudo-reference electrode.Solution pH values were determined using a 713 pH meter combined with a glass electrode (Metrohm, Switzerland).Sudan and other chemicals used were analytical grade and were purchased from Merck.Orthophosphoric acid and its salts (NaH 2 PO 4 , Na 2 HPO 4 and Na 3 PO 4 ) were utilized to prepare the phosphate buffer solutions (PBSs), and sodium hydroxide was used for adjusting the desired pH values (pH range between 2.0 and 9.0).MnO 2 nanorods (MnO 2 -NRs) were synthesized in our laboratory.So that first, 0.316 g of KMnO 4 was dissolved in deionized water (30 ml) under stirring.Then, 1.The achieved solution was transferred into a Teflon Lined autoclave at 160 °C for 6 h.Next, it was cooled down at room temperature to gather the products via centrifugation, followed by washing with ethanol and deionized water several times.The product was finally dried in an oven at 60 °C for 12 h.Figure 1 shows the FE-SEM image of MnO 2 -NRs.The surface areas of the MnO 2 -NRs/SPE and the unmodified SPE were obtained by CV using 1 mM K 3 Fe(CN) 6 at various scan rates.Using the Randles-Ševčik equation for MnO 2 -NRs/SPE, the electrode surface was found to be 0.122 cm 2 which was about 3.9 times greater than un-modified SPE.

Electrochemical behavior of Sudan at the surface of various electrodes
The effect of the electrolyte pH on the oxidation of 70.0 μM Sudan was investigated at MnO 2 -NRs/SPE using differential pulse voltammetry (DPV) measurements in the PBS in the pH range from 2.0 to 9.0.According to the results, the oxidation peak current of Sudan depends on the pH value and increases with increasing pH until it reaches the maximum at pH 7.0 and then decreases at higher pH values.The optimized pH corresponding to the higher peak current was 7.0, indicating that protons are involved in the reaction of Sudan oxidation.
The electrochemical behavior of Sudan was also investigated by CV.The cyclic voltammetry obtained using the bare SPE (trace b) and MnO 2 -NR/SPE (trace a) in 0.1 M PBS (pH 7.0) in the presence of 100.0 μM Sudan is shown in Figure 2. On a bare SPE, a signal with a low oxidation current of 3.2 μA was obtained with a peak potential of 680 mV.In contrast, MnO 2 -NRs/SPE exhibited an enhanced sharp anodic peak current (I pa =11.0 μA) at a much lower overpotential E p = 610 mV.These results confirmed that the MnO 2 -NRs/SPE improved the sensitivity of the modified electrode by enhancing peak current and decreasing the overpotential of the oxidation of Sudan.

Effect of scan rate on the determination of Sudan at MnO 2 -NRs/SPE
The influence of the scan rate () on the peak currents (Ipa) of Sudan at MnO 2 -NRs/SPE was investigated by LSV (Figure 3). Figure 4 shows the voltammetric response of 100.0 μM Sudan at MnO 2 -NRs/SPE at different scan rates in the range of 5 to 400 mV/s.The oxidation peak current of Sudan increases linearly with increasing scan rate.A linear regression equation was obtained from the plot I pa vs.  1/2 (square root of scan rate) as follows; I pa = 1.4523  1/2 + 0.8751 (R 2 = 0.9990) for the oxidation process, which indicates that the reaction of Sudan at MnO 2 -NRs/SPE is diffusion controlled.

Chronoamperometric analysis
The analysis of chronoamperometry for Sudan samples was performed using MnO 2 -NRs/SPE at 0.66 V.The chronoamperometric results of different concentrations of Sudan in PBS (pH 7.0) are demonstrated in Figure 5.The Cottrell equation for the chronoamperometric analysis of electroactive moieties under mass transfer limited conditions is as in equation ( 1): where D represents the diffusion coefficient (cm 2 s -1 ), and C b is the applied bulk concentration (mol cm -3 ).Experimental results of I vs. t -1/2 were plotted in Figure 6A, with the best fits for different concentrations of Sudan.The resulting slopes corresponding to straight lines in Figure 6A were then plotted against the concentration of Sudan (Figure 6B).The mean value of D was determined to be 4.5×10 -5 cm 2 /s according to the resulting slope and Cottrell equation.

Calibration curve
Because DPV commonly has a higher sensitivity than cyclic voltammetry, the DPV technique was applied for the quantitative detection of Sudan. Figure 7 shows the differential pulse voltammograms of Sudan at various concentrations using MnO 2 -NRs/SPE (step potential of 0.01 V and pulse amplitude of 0.025 V).As seen, the oxidation peak currents of Sudan enhance gradually by increasing its concentration.The oxidation peak currents (I pa ) show a good linear relationship with the concentrations of Sudan ranging from 0.25 to 300.0 μM.The linear equation is I pa = 0.0952C Sudan + 1.296 (R 2 = 0.9995) (Figure 8).Also, the limit of detection, Cm, of Sudan was calculated using the equation ( 2 where, m is the slope of the calibration plot (0.0952 μA/ μM) and S b is the standard deviation of the blank response obtained from 15 replicate measurements of the blank solution.The detection limit of 0.08 μM was obtained for the determination of Sudan using this method.

Analysis of real samples
The real samples for the analysis were prepared and quantified by the DPV method.The developed sensor was applied to detect Sudan in tomato paste and ketchup sauce samples.The results are summarized in Table 1.Each measurement was repeated 3 times.The recovery and relative standard deviation (RSD) values confirmed that the MnO 2 -NRs/SPE sensor has a great potential for analytical application.

Conclusion
Modifications of the screen-printed electrode with MnO 2 nanorods for sensing Sudan in food samples were investigated.MnO 2 -NRs/SPE electrodes were used as Sudan sensors by using CV, LSV, CA and DPV techniques.The results showed that the electrodes gave linearity of 0.25 to 300.0 µM, and a detection limit of 0.08 µM.The diffusion coefficient for Sudan using MnO 2 -NRs/SPE, 4.5×10 -5 cm 2 /s was obtained.The analysis of real food samples spiked with sudan gave satisfactory results with recovery values between 97.5 and 105.0 %.

Figure 4 .
Figure 4. Plot of anodic peak current vs. ν 1/2 at different scan rates in the range of 5 to 400 mV/s

Table 1 .
The application of MnO 2 -NR/SPE for determination of Sudan in real samples (n=3)