The present study reports the design of the Mn3O4-Cn electrode and its use for simultaneous detection of selenium and nickel in water. The designed electrode can be used as a convenient electrochemical device for on-site testing of Se (IV) and Ni (II) levels in affected regions. The best responses are obtained with 0.1 M phosphate buffer saline (PBS) and 5 mM Fe (CN)6 as supporting electrolyte. The scan rate and the number of cyclic repetitions have a great effect on peak shape and intensity. It is seen from our study that peak intensity is directly proportional to Se (IV) and Ni (II) concentrations in the range of 5 to 250 µg/L (correlation coefficients 0.952 and 0.984) when the optimized parameters are used. The detection limit of 0.533 µg/L Se (IV) and 0.718 µg/L for Ni (VI) with a response time of 18 s for 5–250 µg/L concentration is obtained, respectively. Enhanced analytical results for different water samples establish that the proposed method is appropriate for Se (IV) and Ni (II) detection. FESEM images confirm the Mn3O4-Cn nanocomposite formation on the electrode.

1. Introduction

Heavy metals in the environment pose a great hazard to organisms owing to their nonbiodegradability [1]. Numerous research laboratories are using systematic procedures to analyze metal ions in various mediums. This consists of atomic absorption spectrometry, atomic fluorescence spectrometry, high-pressure liquid chromatography, gas chromatography, and flow injection analysis [2]. But, these techniques have their own drawbacks. Electrochemical detection of metal ions has several advantages such as low cost, on-site availability, stability, repeatability, accuracy, and detection of multiple ions. Cyclic voltammetry (CV) is a method to recognize and differentiate redox behavior, stability, and surface area of the working electrode used for electrochemical sensing [3, 4].

This technique using modified electrodes is to provide selectivity toward few metal ions. However, the selection of working electrodes is vital for the achievement of CV analysis. There are many reports based on different metal oxide nanoparticles [513]. Manganese oxide nanoparticles are among the most suitable contenders as electrode material owing to their catalyst nature, low cost, stability, nontoxic nature, large surface area, low detection limit, natural abundance, and high energy density [1420]. John reported the excellent gas sensing properties of Mn3O4 nanorods [20]. Nowadays, due to the progress of nanoscience and nanotechnology, there are numerous ways of fabricating chemically modified electrodes using nanocomposites. A clever candidate to form nanocomposite with Mn3O4 is Chitosan. Chitosan (Cn) is a polysaccharide polymer with attractive properties like thin film-forming capability, good adhesion, and outstanding sensitivity [13]. Chitosan consists of volatile amino and hydroxyl functional groups which enable strong enzyme affinity and chemical modifications [18]. There are only a few works reported for metal ion detection based on these materials [24, 13]. Mn3O4 is known for its high electrocatalytic nature which makes it an excellent electrode for gas sensors and biosensors. However, a mixture of Cn and Mn3O4 is yet to be tried for trace metal ion detection. In the present work, an electrochemical sensor is developed for selenium and nickel detection in various water sources by modifying a platinum (Pt) electrode with manganese oxide (Mn3O4) and chitosan. Selenium and nickel are toxic to the environment if their levels are above the required limit. Freshwaters are being contaminated by the dumping of toxic metal ions as a result of industrialization. So, it is very vital to determine these metal ion traces in freshwaters before usage. To the best of our knowledge, this is the first report of electrochemical selenium and nickel sensor based on Mn3O4-Cn nanocomposite, that too for selenium and nickel level detection in lake water.

2. Experimental

2.1. Materials and Synthesis

All the chemicals are of analytical grade and used without further purification. Stock solutions of Se (IV), Ni (II) of 1000 µg/L, and ions like Cd2+, Mg2+, Fe2+, As2+, PO43−, I, Cu2+, Br, Pb2+, Li2+, Ba2+, SO42−, and Fe(CN)6 are brought from Merck and stored in refrigeration. Water for selenium and nickel detection is collected from a lake in South India, which is known for its toxicity due to the immense dumping of the waste from industrial and fertilizer factories. In commercial fertilizers, selenium is added in trace amounts. But, an ample amount is added up in the waste which polluted the surrounding water resources. Selenium and nickel act as a pollutant due to factory waste dumped into the water resources and lakes especially. Samples are collected from different sites of lakes and rivers during monsoon and summer. Anodic aluminum oxide (AAO) template with a length of 60 µm and having a pore diameter of 350 nm (Smart membranes, Germany) are purchased for the synthesis. We took 1 M manganese acetate (5.13 g) is dissolved in 100 ml of deionized (DI) water and stirred it for 20 minutes [20]. While stirring, we added 1 M ammonium hydroxide (100 ml) slowly. This solution is magnetically stirred for around one hour, resulting in a brown solution. Later, it is subjected to hydrothermal reaction, setting the temperature to 170°C for 7 hr. The resultant mixture is then washed with distilled water and dried at 60°C for 9 hr. The dried powder is annealed at 300°C for 2 hr, which produced pearl-shaped nanostructures, which are sonicated for 60 s in ethanol. This mixture is then filled into the pores of the AAO template by vacuum infiltration technique [20]. The AAO template is then air-dried at room temperature. This process leads to the development of aligned nanorods. These nanorods from AAO are separated to fabricate the biosensor, Mn3O4 nanorods are sonicated in ethanol for one hour, and the residue is washed again in ethanol. The residue obtained is then dried at 60°C for 5 hours, which resulted in an ensemble of Mn3O4 nanorods without any support.

2.2. Fabrication of Pt/Mn3O4/Cn Working Electrode

The working electrode is prepared where 20 mg of Mn3O4 nanorods separated from AAO is added to 5 ml of chitosan solution prepared when 1 gm chitosan is dispersed in 100 ml of 0.1 m acetic acid [13]. When the solution is stirred, Mn3O4-Cn suspension is formed. The Mn3O4-Cn electrode is formed when 2 µL of this aliquot is uniformly coated on the Pt working electrode by drop cast method. Performance of Pt/Mn3O4-Cn electrode for selenium and nickel detection in lake water is investigated as a real-time application. At first, the electrode is exposed to different trials to gain recovery when spiking different concentrations of selenium and nickel in supporting electrolytes [10, 11].

2.3. Characterization

Morphology of Mn3O4 nanorods separated from AAO and Mn3O4-Cn is characterized by Field Emission Spectroscopic Electron Microscopy (FESEM-JOEL). FESEM is also used to confirm nanocomposite formation. The Fourier Transform Infrared (FT-IR, Spectrum 400, Perkin Elmer) spectra of Mn3O4-Cn and Mn3O4-Cn/Se and Ni are used to justify the absorbed peaks related to each component of the material. CV is a three-electrode arrangement that consisted of a working electrode, a reference electrode, and a counter electrode. All voltammetry experiments are conducted at room temperature in a CV system (Biologic SP-200, Germany) which involved a Pt/Mn3O4-chitosan electrode (working electrode), platinum wire as a counter electrode, and Ag/AgCl as reference electrodes.

3. Results and Discussion

3.1. Field Emission Spectroscopy and Fourier Transform Analyses

The morphology of Mn3O4 nanorods separated from AAO is characterized using FESEM which is illustrated in Figure 1(a). The surface morphology of Mn3O4-Cn is observed using FESEM confirming nanocomposite formation as shown in Figure 1(b). It is seen that chitosan has closely anchored on Mn3O4 nanorods forming Mn3O4-Cn nanocomposite. The nanorods exhibit a well-aligned and active surface area for selenium mobilization. From FESEM, the average diameters of Mn3O4 and Mn3O4-Cn are 300 ± 2.3 nm and 335 ± 4.7 nm, respectively. The electrodes are thoroughly washed and rinsed with Millipore water.

3.2. Electron Impedance Spectroscopy

Electrochemical impedance spectroscopy (EIS) is an efficient method to understand interfacial electron transfer due to changes at the electrode surface while being modified [10]. Fe (CN)6 is used as the redox probe in impedance measurements conducted in PBS media at Mn3O4 and Mn3O4-Cn electrodes. EIS measurements are carried out using 1 Hz to 100 kHz with an alternating voltage of 5 mV and a bias potential of 0.5 V (Figure 2). The semicircle at higher frequencies denotes the electron transfer resistance (Rct), and the linear part at lower frequencies connotes the diffusion process. A definite semicircle is exhibited by Mn3O4 at a higher frequency. It is noted that the semicircle is reduced, which implies the charge relocation resistance once Mn3O4 is modified with Cn. This shows that Mn3O4-Cn is an excellent conducting material and it can accelerate electron transfer.

3.3. Electrochemical Characterization

When a potential is applied to the working electrode, it sweeps back and forth for a definite number of cycles according to the given range of voltage and speed of voltage sweep, and subsequent current at the working potential is measured [5]. This current is then plotted against the potential to draw a CV graph that provides a deep perception about the material regarding the anodic peak current (Ipa) from the oxidation process and about cathodic peak current (Ipc) from the reduction process that occurred on working potential. Potentials, where peak currents happen, are called peak potentials (Ep). We can examine the reversibility of reactions happening at the electrode surface by increasing the scan rates [6].

Electrochemical reversible reactions arise from fast electron transfer between ions and electrodes, which will be slower in irreversible reaction [610]. The applicability of modified Mn3O4-Cn as a potential electrochemical sensing platform for a metal solution such as Se (IV) and Ni (II) is investigated using cyclic voltammetry at a deposition potential of 400 mV. All the experiments are conducted at room temperature. The electrochemical analyses are carried out in 10 mL electrochemical cell with a known amount of Se (IV) and Ni (II) in 0.1 M phosphate buffer solution (pH 2) and 5 mM Fe (CN)6 as supporting electrolyte, respectively.

In CV, the electrolyte has a vital role in the dispersal of analyte types onto the electrode [1820]. Phosphate buffer solution (PBS) gives rise to a sharp and intense oxidation signal and is used as a supporting electrolyte in order to avoid all interferences of H+ and complex compound formation that happens while using HCl, HNO3, and H2SO4 [21]. PBS (0.1 M) with 5 mM Fe (CN)6 acting as a redox couple is used as the supporting electrolyte and provides a good response to selenium and nickel detection [21]. PBS is examined for pH from 1–6 in order to detect Se (IV) and Ni (II) using Mn3O4-Cn sensor [21]. We observe the optimized condition for metal ion detection takes place at pH = 2 for 100 µg/L Se (IV) and Ni (II). Voltammetric response of Pt/Mn3O4 and Pt/Mn3O4-chitosan electrodes in PBS (0.1 M) and 5 mM Fe (CN)6 is carried out at a scan rate of 100 mVs−1 (Figure 3). We tested for different scan rates too, but without any vast variation obtained for 100 mVs−1.

We noticed that the bare Pt electrode in PBS and Fe (CN)6 is not showing any peak current. As we used the Pt/Mn3O4 and Pt/Mn3O4-Cn electrodes, there is a current response. Peak current with Pt/Mn3O4-Cn electrode is more intense than that with other electrodes. This confirms that peak intensity is due to Pt/Mn3O4-Cn electrode only. Based on the profile of the voltammogram, peak current and peak potential of Se (IV) and Ni (II) in 0.1 M PBS and 5 mM Fe (CN)6 are obtained. Cyclic voltammograms of Mn3O4 and modified Mn3O4 are recorded with 0.1 M PBS with 5 mM Fe (CN)6 supporting electrolyte, at different scan rates (10 to 100 mV/s). Figure 4 illustrates anodic peak current plot against the square root of scan rate for Pt/Mn3O4 with R2 = 0.9768 and Pt/Mn3O4-Cn with R2 = 0.9978, respectively. Their respective equation curves obtained arewhere R is the correlation coefficient and ʋ is the scan rate [21, 22]. Evidently, peak currents of Mn3O4-Cn electrode are higher than Mn3O4, due to more active surface area and, hence, increased conductivity after adding Cn [21]. The linear dependence between scan rate and oxidation current indicates that selenium and nickel are adsorbed on the surface of the Pt/Mn3O4-Cn electrode which is due to a surface-confined electron transfer. Moreover, the current passing through the electrode is restricted by the diffusion of selenium and nickel to the electrode surface. This diffusion change is caused by the concentration gradient near the electrode [19, 20, 22, 23]. This gradient is affected by the concentration of selenium and nickel at the electrode and the rate at which it can diffuse through the solution [8, 21]. So, the diffusion coefficient can be obtained using Randles–Sevcik formula:

Therefore, the obtained diffusion coefficient is 8.2 × 10−6 cm2 s−1, where Ipa is anodic peak current, A is the active electrode surface area, n is the number of mobile electrons in PBS, ʋ is the scan rate, and C is the concentration of PBS [21]. Electroactive surface area is 0.055 cm2 and 0.043 cm2 for Mn3O4-Cn and Mn3O4, respectively. Afterward, cyclic voltammograms of metal ion solution of Se (IV) and Ni (II) concentrations between 5 µg/L and 250 µg/L are observed using the optimized conditions (Figure 4). The enhanced current response is due to the large specific surface area of the Mn3O4-Cn. The improved electrochemical response of Mn3O4-Cn arose due to the enhanced electroactive surface area and higher catalyst activity of Mn3O4 [4, 16]. This enhancement of electrochemical current response due to the uniform and well-aligned grid of Pt/Mn3O4–Cn electrode delivers a good pathway for the passage of electrons created during oxidation of selenium and nickel which are transported to the electrode surface through Mn ion [4, 16]. Also, the existence of nanorod-shaped morphology of Mn3O4–Cn electrode gives forth better electronic and ionic transport because of uniformly distributed Mn3O4–Cn rather than bulk matrix [4, 16]. Usually, the redox kinds get absorbed on the electrode surface, but due to the electrochemical property of Mn3O4, it would decrease the absorption of redox species which will enable the electron to be electrochemically reversible. This suggests that this sensor can increase the electron transfer rate at the electrodes which enhance its performance. More conductive pathways are created for electron transfer on the surface of Mn3O4-Cn in PBS [4, 16, 21]. The peak current of Se (IV) and Ni (II) increased from 400 mV to 1000 mV because of strong absorption by the Mn3O4-Cn sensor. Se (IV) and Ni (II) are adsorbed due to electrostatic interaction between the electrode and metal ions resulting in a high peak signal due to the intermetallic bond between Mn and ions [16, 19, 20, 23]. The high peak signal can also arise due to higher electron transfer capability, increase in electroactive surfaces, and conductivity resulting in higher sensitivity toward Se (IV) and Ni (II) ions. The potential is set at 400 mV and reversed at 1000 mV with a scan rate of 100 mV/s. During the analysis, oxidation peak and reduction peak are detected at 730 mV and 850 mV for Se (IV) with 500 mV and 600 mV for Ni (II), respectively (Figure 4) [1120, 23].

Those peaks are apparently associated with the redox reaction of Se (IV) and Ni (II) at the surface of Mn3O4-Cn. As the reduction of metal ions can occur by different mechanisms, the overall process that occurs on the surface of the electrode is considered to include reduction of Se (IV) to Se (0) and Ni (II) to Ni (0). This fact is confirmed by the alteration of the peak intensity during variation of Se (IV) and Ni (II) concentrations [11]. The analytes concentration played a vital role in the shifting of both cathodic and anodic peaks toward lower potential, but the potential difference of reduction and oxidation peaks is relatively consistent as shown in Figure 4. Based on the profile of the voltammogram, the peak current and the peak potential of Se (IV) and Ni (II) in 0.1 M PBS and 5 mM Fe (CN)6 are determined.

3.4. Variation of the Scan Rate

The effects of scan rate from 25 mV/s to 200 mV/s on the electrochemical response of Mn3O4/chitosan electrode in PBS (0.1 M) and 5 mM Fe (CN)6 toward 250 μg/L Se (IV) and Ni (II) are investigated. From Figure 5, one can observe that some anodic peaks appeared at potential around 900 mV to 1000 mV, while the cathodic peak is observed at a potential of around 350 mV to 400 mV. Anodic peak (1000 mV and 1100 mV) and cathodic peak (360 mV and 370 mV) are observed for Se (IV), respectively, while, for Ni (II), the anodic peak is from 410 mV to 650 mV with a cathodic peak between 800 mV and 1000 mV.

Majorly, electron transfer on the surface of Mn3O4-Cn is controlled by diffusion. We conclude that the working curves of Se (IV) and Ni (II) investigations are least limited by concentration. We observed that the reduction of Se (0) and Ni (0) is obtained toward the lower potential. The experiment is accomplished using Se (IV) and Ni (II) of 20 µg/L in 0.1 M PBS and 5 mM Fe(CN)6 to 7 times of repetition at a scan rate of 25 mV/s, which resulted in only a slight variation in current.

3.5. Calibration Curves and Detection Limit

The fabricated Mn3O4-Cn electrodes exhibit excellent reusability, repeatability, and stability. The electrodes are recyclable up to 35 successive runs with relative standard deviation <3%, while a substantial reduction is observed after it, which may be due to consecutive deposition of Se (0) and Ni (0) during reduction [22, 23]. Reproducibility of Mn3O4-Cn electrode is confirmed using five different sets, which shows outstanding reproducibility with a standard deviation of ∼0.8%. To estimate the stability of this electrode, CV response is recorded for 14 days. We noticed that the current response is diminished by ∼4.2%, which still could be enhanced by storage situations. Thus, the Mn3O4-Cn electrode provides improved stability, reusability, and reproducibility characteristics toward selenium (IV) and Ni (II). It is observed that a linear calibration equation exists from 5 to 250 μg/L. Linear regression model for Se (IV) and Ni (II) for 5 to 250 μg/L is fitted using the following expression:

The calibration curve is plotted using peak current versus the concentration of Se (IV) and Ni (II) under the recommended conditions from Figures 6 and 7.

There is a linear relationship held over the concentration ranges of 5–250 μg/L (correlation coefficient R2 = 0.952) for Se (IV) and Ni (II) (R2 = 0.984) as depicted in Figure 7.

The limit of detection (LOD) is calculated aswhere CL, SD, and m are the limit of detection, the standard deviation of the blank, and slope of the calibration graph, respectively [22]. LOD obtained is 0.533 μgL−1 Se (IV), and for Ni (II), it is 0.718 μgL−1, respectively, with a detection time of 18 s. The fabricated sensor displays a satisfactory detection limit and a varied detection range essential for practical utility. Table 1 reveals the superior characteristics of Mn3O4-Cn electrode for detection of selenium and nickel comparable to reported values.

3.6. Interference

Detection of Se (IV) and Ni (II) in the presence of additional commonly found ions in water is of high importance and significance [521, 23, 24]. Therefore, interference of various ions like Cd2+, Mg2+, Fe2+, As2+, PO43−, I, Cu2+, Br, Pb2+, Li2+, Ba2+, and SO42− is examined at a 5-fold concentration to that of 50 µg/L of selenium and nickel. The obtained results after reacting with each ion confirm the negligible interference on Se (IV) and Ni (II) for the current at 50 µg/L, 100 µg/L, and 100 µg/L concentrations of interfering ions (Figure 8). The species evaluated could not generate any current signal within the range of potentials used in voltammograms. However, a decrease in current is observed in the presence of 1000 µg/L of Li (II). But, the probabilities of existence of this much concentration of interfering ions along with Se (IV) and Ni (II) in real water are negligible. So, the developed sensor can be used for the detection of Se (IV) and Ni (II) in real water. It is important to access inference from organic compounds too since natural water contains them [24, 25]. We did the extraction of each organic compound from the sample and dried it. After the drying process, the weight of each compound is taken [1120, 23, 24]. The tolerance levels of organic compounds like L-cysteine, sodium dodecylbenzene sulfonate, polyethylene glycol mono-p-nonyl phenyl ether, and EDTA-2Na are 1.2 mg L−1, 0.9 mg L−1, 1.0 mg L−1, and 0.09 mg L−1, respectively [6].

3.7. Real-Time Analysis

The analysis is done for the purpose of the practical application of Pt/Mn3O4-Cn electrochemical sensor in environmental detection of Se (IV) and Ni (II) in the real sample, tap water (St. Berchmans College Lab) and lake water (South Kerala, India). The pH of the samples is at the optimum condition as mentioned above. The standard addition method is used as a validation method to evaluate the analytical applicability in the determination of selenium and nickel using the proposed technique. The acquired water samples are first filtered through a 0.45-micron filter to get rid of suspended particles and then added with 10 µg/L of selenium and nickel. With the standard addition method, 10 mL electrochemical cells comprising 2 mL of both tap water and lake water sample alternatively, 1 mL 0.1 M PBS, and known Se (IV) and Ni (II) concentrations are analyzed consecutively. Even without spiking, the Mn3O4-Cn electrode is sensitive enough to sense Se (IV) and Ni (II) in tap water ensuring its applicability and sensitivity. It is necessary to validate the accuracy of this technique; therefore, the electrodes are tested via a certified reference material (TM-15) with a concentration of 15 µg/L Se (IV) and Ni (II). However, another approach using ICP-MS for water samples is done to specifically rule out any interference from foreign ions. We observed reasonable recovery, which showed the capability of Mn3O4-Cn for detecting Se (IV) and Ni (II) in real water samples. The analysis is carried out in quadruple where a relative error of 5.98% is obtained for Pt/Mn3O4-Cn. However, no major differences between the theoretical and experimental values are observed. Table 2 summarizes the analytical results of Se (IV) and Ni (II) concentrations in tap water and lake water on four replicate analyses.

The lake water sample has higher concentrations of selenium with reference to the World Health Organization (WHO) guideline of 10 μg/L for Se (IV) and 20 μg/L for Ni (II). But, a higher concentration is noted during the summers while, in the monsoon season, lower concentrations are recorded. This might have occurred due to dilution from higher rainwater runoff and flows. The concentration of Se (IV) and Ni (II) in tap water are within the limit of WHO guidelines. Our projected technique could be used for the speciation investigation of natural water samples with adequate results. We also collected the sample from the lake during summer when the factory is closed due to a strike for above one month. The results are noteworthy as there is a decrease in the traces of Se (IV) and Ni (II) in the collected samples and detailed out in Table 3.

4. Conclusions

The detection of Se (IV) and Ni (II) using Mn3O4-Cn is successfully demonstrated. In low concentrations, CV is a more appropriate method as Se (IV) and Ni (II) exhibit characteristic voltammetric waves of peak heights relevant to selenium concentration. The working electrode of Mn3O4-Cn is fabricated and used to evaluate the voltammetric behavior of selenium and nickel using the cyclic voltammetry method. CV is a relevant technique to understand electron transfer at the electrode-solution interface, especially for low concentrations. Supporting electrolyte concentration, scan rate, and the number of cyclic repetition is observed for their influence on selenium and nickel peak shape and intensity The results from CV indicate that the presence of Mn3O4 helps to enable the electrochemically reversible electron transfer. Optimization of the parameters of this technique has been carried out for consistent and reproducible results by parallel assemblies of Mn3O4, which amplifies the current response. Mn3O4-Cn electrode offers advantages of low cost, low detection limit, low response time, and high sensitivity. The Se and Ni redox reaction appears to be kinetically faster and more reversible. Selenium and nickel concentrations are detected in tap water and lake water. Pt/Mn3O4-Cn is successfully working on the analysis of real water samples, and the results allied with the ICP-MS analysis with recoveries greater than 96%. Due to the practical protection of fabricated electrodes toward frequently found interfering ions during quantification, it is effectively verified for real water sample analysis. We had limitations in obtaining results for concentrations below 5 µg/L and for higher concentrations of the detected species. Further work is underway to demonstrate the use of these sensors for testing sediment samples providing rapid on-site analysis for selenium and nickel using differential pulse voltammetry.

Data Availability

Data obtained have been plotted in graphs.

Conflicts of Interest

The authors declare that there are no conflicts of interest.


The authors thank the School of Chemical Sciences, MG University, and CSIR-CECRI for providing facilities to conduct electrochemical analysis. The authors also acknowledge the Optoelectronics Department, Kerala University for FESEM. The authors thank the Central Instrumentation Facility, St. Berchmans College, Changanassery, for FTIR analysis using Spectrum 2.