Abstract
A transition metal oxide, MnO2, doped reduced graphene oxide (rGO) was prepared to develop a 2D-carbon-metal oxide composite modified glassy carbon electrode (GC) for sensitive electrochemical determination of nitrite ion (NO2−). GO was first synthesized and reduced to rGO followed by a MnO2 doping process. The obtained MnO2-rGO composite was then characterized with Raman spectroscopy, infrared spectroscopy, and scanning electron microscopy techniques. The MnO2-rGO coated on GC electrodes were electrochemically characterized using cyclic voltammetry, electrochemical impedance spectroscopy and optimized for the determination of NO2− using differential pulse voltammetry. A linear calibration curve was obtained between 0.1 and 5.5 μM with LOD and LOQ values of 0.02 μM and 0.06 μM, respectively. The selectivity of the proposed sensor was tested in different substances and no significant interference was found. The sensor validation test showed that the precision (% RSD) and accuracy of the system were around 1.95–2.73% and (− 3.5)–2.5%, respectively. Finally, real sample tests with commercially available juice samples and tap water confirmed that the method could detect the spiked NO2− in real samples. As a result, the sensitive and easy determination of NO2− has been achieved using MnO2-rGO composite materials in real samples with good recovery values and minimum interference.
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Introduction
Nitrite ion (NO2−) is a widely used additive in chemical and pharmaceutical industries and can be a serious threat above certain exposure levels in food and water (Iammarino et al. 2013). Although it is mainly utilized for food preservation, it can also be used as a dyeing agent (El‐Apasery 2008), corrosion inhibitor (Reou and Ann 2008), and bleach (Ikeda et al. 1999). It is a very well-known pollutant for the environment and human health with fatal ingestion dose levels between 8.7 µM and 28.3 µM (da Silva and Mazo 1998). These values could even go down to 2.17 µM for drinking water (da Silva and Mazo 1998). NO2− accumulation in the human body can lead to very serious health problems such as methemoglobinemia due to the irreversible oxidation of hemoglobin. Infant methemoglobinemia which is known as ‘the blue baby syndrome’ is developed congenital or early in life due to this reaction leading to oxygen deficiency (Chan 2011). Therefore, rapid, sensitive, and easy determination of the dangerous levels of NO2− in food samples and drinking water is very important for human health.
The determination of NO2− can be problematic due to its easy oxidation but the water solubility facilitates the analysis in aqueous solutions. The current methods in the literature for the determination of NO2− include spectroscopic (Yang et al. 2018, Singh et al. 2019), chromatographic (Croitoru 2012, Antczak-Chrobot et al. 2018), and electrochemical methods (Terbouche et al. 2016, Sudarvizhi et al. 2018, Yan et al. 2018). The spectroscopic and chromatographic methods have several limitations such as difficulty in sample preparation, longer test durations, interferences, and selection of column filler (Moorcroft et al. 2001). Electrochemical detection, on the other hand, is more advantageous because it can eliminate these problems for the determination of NO2−. NO2− can be determined using several different approaches such as voltammetric (Esaifan and Hourani 2009, Manea et al. 2010) and amperometric (Baciu et al. 2015). However, the direct NO2− determination from aqueous samples are not widely studied especially using electrochemical methods.
Graphene is one of the most widely used one-atom-thick planar sheets comprising a sp2-bonded carbon structure as two-dimensional materials (Chen et al. 2012). Graphene oxide (GO) and its derivatives such as reduced graphene oxide (rGO) and graphene nanoribbons (GNR) have been more popular than graphene because of their functionality in many applications (Zor et al. 2015, Dideikin and Vul 2019). The derivatives are used as electrode materials and catalysts for electrochemical sensors (You et al. 2015, Çelik et al. 2016). The incorporation of transition metal oxides into 2D-based carbon materials such as MnO2 and rGO to benefit the enhanced performance of the composite material has been studied in the literature for their good electrocatalytic effect in supercapacitor applications (Jadhav et al. 2019) and the detection of trace metals in water samples (Mnyipika et al. 2021).
Herein, we have developed a detection strategy for the analysis of nitrite in food samples and water using manganese oxide decorated-rGO coated glassy carbon electrodes. (MnO2-rGO/GC). MnO2 doped rGO was first prepared and characterized with Raman spectroscopy, infrared spectroscopy (IR) and scanning electron microscopy (SEM) techniques. Then, MnO2-rGO/GC electrodes were prepared and electrochemically characterized using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) and optimized for the determination of NO2− using differential pulse voltammetry (DPV). The sensitive and easy determination of NO2− has been achieved in commercially available juice samples and tap water with good recovery values and minimum interference.
Experimental
Materials
All chemicals used in this study were at analytical grade and purchased from Merck, Fluka, Sigma-Aldrich, and Riedel. Argon (HPLC grade, 99.999%) was used for oxygen removal. Electrochemical experiments were performed at room temperature (24 ± 1 °C) using a three-electrode system where GC electrode (BASi, model: MF-2012), Pt wire and Ag/AgCl (Sat.) electrode as working electrode, auxiliary electrode, and reference electrode, respectively. All aqueous solution and washing steps were performed using ultra-pure water (UPW, 18.2 MΩ cm) which was obtained from Human Power I (Human Corp., Korea).
Preparation of MnO2 doped reduced graphene oxide electrodes
The schematic for the preparation of the MnO2 doped reduced graphene oxide electrodes is shown in Fig. 1. Briefly, GO was synthesized according to modified Hummer’s method reported elsewhere (Işık et al. 2021). GO is then reduced using hydrazine (Chua and Pumera 2016). First, 3 g of GO was dispersed in 1 L UPW in an ultrasonic bath (Bandelin, Sonorex, 35 kHz) and mixed with 1 mL of hydrazine under reflux at 80 °C for 12 h. The black suspension was then centrifuged and washed with acetone and UPW multiple times. The final crude product (rGO) was dried in a vacuum oven at 55 °C overnight. To prepare MnO2 doped rGO electrodes, 26.4 mg rGO and 1.08 g MnCl2 + H2O was first mixed in isopropyl alcohol and left in an ultrasonic bath for 1 h. 0.6 g KMnO4 was then dissolved in 20 mL of UPW and added to the previous mixture (Erkal et al. 2015, Güleşen et al. 2019, Erkal-Aytemur et al. 2019). The final mixture is stirred at 85 °C for 30 min and centrifuged for 10 min at 5000 rpm. The solid precipitate was washed with UPW three times and left to dry in a vacuum oven at 55 °C overnight. MnO2 doped rGO electrodes were finally obtained by drop coating 5, 10, 15, 20 and 25 µL suspensions of 10 mg of MnO2-rGO in 1 mL acetonitrile on GC electrode surface and denoted as MnO2-rGO/GC. 15 µL suspensions of GO and rGO in acetonitrile were also coated on GC electrodes for comparison purposes and denoted as GO/GC and rGO/GC, respectively.
Characterization of electrodes and sensor calibration
Before any optimization experiments, GC, GO/GC, rGO/GC, and MnO2-rGO/GC electrodes were characterized using CV and EIS in the presence of 2 mM K3Fe(CN)6/K4Fe(CN)6 redox couple in 0.1 M KCl. Infrared spectroscopy (Bruker, Tensor-27, Germany), SEM–EDX (Nova, NanoSEM-650, Belgium), and Raman spectroscopy (Horiba, Jobin-Ivon, France) analysis were also used for the characterization of GO, rGO, and MnO2-rGO. The effect of the coating volume and the calibration of the prepared sensor was performed using the DPV in the presence of 5 µM NO2− and (0.1–5.5 µM) NO2− concentrations, respectively. The stability of the prepared sensor was calculated using intraday and inter-day (7 consecutive days) experiments. Interference studies were also conducted using 100 μM Na+, Ca2+, Zn2+, Cu2+, NO3−, Cl−, SO32−, SO42−; citric acid, ascorbic acid, catechol, and hydroquinone to demonstrate the selectivity of the sensor. Finally, real sample tests were performed using different beverages and tap water. The beverages were first filtered and diluted before electrochemical tests.
Results and discussion
The successful modification of different electrode configurations was first confirmed using CV and EIS using a redox couple as shown in Fig. S1a and 1b. The voltammograms show that MnO2-rGO/GC electrode had the highest anodic peak current ca. 29.6 ± 0.08 µA. There is a significant current increase response of 80% from rGO/GC indicating the high electrocatalytic performance of the MnO2-rGO/GC electrode. Furthermore, EIS results (Fig. S1b) show that a low charge transfer resistance (Rct) of the MnO2-rGO/GC electrode compared to other modified electrodes and the bare electrode was obtained. This is because of the highly conductive nature of the MnO2-rGO/GC electrode allowing fast electron transfer between the redox couple and the electrode surface. The experimental data were fitted using the Randles equivalent circuit (Garyfallou et al. 2017). The equivalent circuit designs and calculated Rct values of the redox couple on the electrode are given in Table S1. The electrochemical characterization results revealed that CV and EIS results were in parallel with each other confirming the electron transfer rates of the redox couple were increased with MnO2-rGO/GC electrode.
Figure 2a shows the D and G bands of graphite, GO, rGO, and MnO2-rGO obtained from Raman spectroscopy. The ID/IG Raman intensity ratios of graphite, GO, rGO, and MnO2-rGO were 0.06, 0.94, 1.10, 1.02, respectively. This shows that the D band intensity of GO is increased compared to graphite because of the change in sp2 hybridization due to the chemical oxidation of graphite. The ID/IG Raman intensity ratio of rGO is higher than GO indicating a further change in sp2 hybridization due to reduction GO. The ratio of MnO2-rGO was decreased compared to rGO but still was slightly over 1. Furthermore, the characteristic peak of Mn–O lattice vibrations can also be seen at around 625 cm−1. These Raman spectroscopy results are similar to previously reported literature (Han et al. 2014).
IR spectra of GO, rGO, and MnO2-rGO are also given in Fig. 2b. The IR spectrum of GO shows a broad peak between 3000 and 3700 cm−1 corresponding to O–H stretching of –COOH (Yeter et al. 2021). The C=O (carbonyl) and C–O–C peaks appeared at 1700 cm−1 and 1280 cm−1, respectively (Erkal-Aytemur et al. 2019). In the IR spectrum of rGO, the intensity of the peaks that appeared in the spectrum of GO is dramatically decreased but C=C stretching was seen at 1550 cm−1 (Uluok et al. 2015). The IR spectrum of MnO2-rGO revealed that the C–N peak also appeared at 1280 cm−1 in addition to the peaks that appeared in the spectrum rGO (Çelik et al. 2016).
SEM micrographs of GO, rGO, and MnO2-rGO show typical structural patterns of corresponding graphene structures (Fig. 2). The two-dimensional sheet-like structure with visible edges of individual sheets is evident of the multiple lamellar layer structure of GO (https://doi.org/10.4236/graphene.2017.61001). On the other hand, rGO micrograph shows a similar structure to its predecessor, but more wrinkled and possibly smaller sheets were obtained after the reduction (https://doi.org/10.3390/nano9081180). The micrograph of MnO2-rGO shows typical but non-uniform clusters of oxide crystals (https://doi.org/10.1007/s00604-018-3064-3). MnO2 existence on rGO sheets was also confirmed by EDX analysis (Fig S2). The mean percentages of the elements (by weight) were as follows: 68.48% C, 27.41% O, 3.30% Mn, and ~ 0.81% of other impurities.
Although MnO2-rGO/GC electrode showed better electroanalytical performance than other modified and/or unmodified electrodes in the presence of a redox couple, further voltammetric analysis has been performed to confirm the electrocatalytical effect of different electrode configurations for NO2− oxidation. Figure 3a shows the CV results of coated GC electrodes tested in 0.1 mM NO2− and the anodic peak potentials are given in Table S2. All modified GC electrodes showed catalytic activity for NO2− oxidation in which MnO2-rGO/GC electrode showed the highest peak current of 48.2 ± 0.8 µA. These results of MnO2-rGO/GC also confirmed that the electrooxidation of NO2− was a diffusion-controlled process (Fig. S3); therefore, the quantitative determination of NO2− can be performed using voltammetric techniques.
Upon confirming the suitability of the MnO2-rGO/GC electrode and the electrochemical technique for quantitative NO2− analysis, optimization studies have been conducted. First, the amount of MnO2-rGO was optimized by coating different volumes of 10 mg/ml MnO2-rGO (in acetonitrile) suspension on GC electrode and dried under IR lamb. The resulting electrodes were tested using DPV between 0.4 V and 1.2 V (vs Ag/Cl) in the presence of 5 µM NO2− (in PBS, pH 7). Figure 3b, c shows that the best peak current was obtained for 15 µL coating (~ 2 mg/cm2 surface coverage); therefore, it was chosen as an optimized coating volume.
Next, the calibration curve was obtained using DPV for the optimized suspension coating volume for 0.1 µM–5.5 µM NO2− concentration range in PBS, pH 7 (Fig. 3d). The anodic peak currents were then plotted against respective concentrations (Fig. 3e) and the analytical performance parameters are calculated as shown in Table 1. The calibration curve was linear between 0.1 µM and 5.5 µM NO2− concentration range with a sensitivity value of ca. 5.18 µA/µM. The LOD and LOQ of the proposed analysis method were calculated as 0.02 µM (S/N ratio of 3) and 0.06 µM, respectively. A comparison of the electroanalytical sensors proposed in this study with other reported electrodes is listed in Table 2. The developed sensor performance is comparable with the recently published sensors for NO2− determination. The performance of the developed nitrite sensor shows high stability, repeatability, and selectivity.
For the validation of the developed analysis strategy, precision and accuracy tests were also performed using two different NO2− concentrations (2 and 4 µM). The inter-day and intra-day experiments were revealed that the RSD % and RE % of the system were around 1.95–2.73% and (− 3.5)–2.5%, respectively. The validation experiments were conducted with 6 individually prepared electrodes, and the results are presented in Table 3. The results indicate that the method is working within a negligible margin of error in terms of its precision and accuracy for both inter and intra-day conditions.
Several different potential interfering anion and molecules were also investigated to test the sensitivity of the developed method. 100 µM of each interfering substance was tested with 10 µM NO2− and the obtained results are presented in Table 4. The results showed that the change in the peak current was not significant; therefore, the method was considered as sensitive to NO2− in the presence of the selected interferences.
The real sample tests were performed using commercially available juices and water. Different juices were first filtered and diluted for the recovery experiments. Table 5 shows the obtained results for three different juices and tap water for six individually prepared electrodes per sample. The results showed that the method could detect the spiked NO2− within a confidence level of 95%.
Conclusion
A sensitive and rapid electrochemical NO2− sensor has been developed using MnO2 doped rGO coated on GC electrodes. Spectroscopic and microscopic characterization techniques confirmed the MnO2 existence on rGO sheets and electrochemical characterization showed electroanalytical activity of MnO2. Furthermore, MnO2-rGO/GC electrodes showed electrocatalytic activity toward NO2− oxidation in aqueous media. DPV was utilized to obtain the linear calibration curve which has LOD and LOQ values of 0.02 µM and 0.06 µM, respectively. The linear detection range was found between 0.1 μM and 5.5 μM, and the sensor showed very good selectivity in the presence of thirteen different interfering substances. The validation of the sensor was further confirmed with inter and intra-day experiments resulting in RSD % and RE % values around 1.95–2.73% and ( − 3.5)–2.5%, respectively. These precision and accuracy values suggest that the proposed sensor may be very advantageous for the use of the sensor during same-day or consecutive days for food/water quality testing on site. The detection of the spiked NO2− in real samples showed that the sensitive and easy determination of NO2− could be achieved using MnO2-rGO composite materials with good recovery values and minimum interference. The proposed sensor may be useful for food safety and water-quality monitoring based on its comparable performance with the most recent studies in the literature.
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Yılmaz-Alhan, B., Çelik, G., Oguzhan Caglayan, M. et al. Determination of nitrite on manganese dioxide doped reduced graphene oxide modified glassy carbon by differential pulse voltammetry. Chem. Pap. 76, 4919–4925 (2022). https://doi.org/10.1007/s11696-022-02218-9
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DOI: https://doi.org/10.1007/s11696-022-02218-9