Nonenzymatic electrochemical assay for hydrogen peroxide detection based on green synthesized MnO2 nanosheets

In this study, manganese oxide (MnO2) nanosheets were successfully prepared with a green and cost-effective method by using Gum Arabic as they both reduce agent and template in the absence of other additives. The composition, crystalline structure and morphology of the synthesized MnO2 nanosheets were studied using different characterization methods. The TEM and FESEM images show that the prepared MnO2 exhibits wrinkle-like nanolayer morphology. Then, the performance of the synthesized MnO2 was investigated for electrochemical detection of hydrogen peroxide (H2O2). The electrochemical sensing of H2O2 based on MnO2 nanosheets modified carbon ionic liquid electrode (MnO2/CILE) was constructed by a pasting method and its electrocatalytic performance was studied using cyclic voltammetry and amperometry. Compared with the bare CILE, the MnO2/CILE electrode exhibited good electrocatalytic behavior for H2O2 reduction in alkaline solutions. The fabricated nonenzymatic H2O2 sensor also showed a linear relationship over an extensive concentration range of 5.0 μM to 10.0 mM (r2 = 0.998) with a LOD of 1.0 μM and a response time of fewer than 5 s. In addition, the developed electrochemical sensor displayed excellent anti-interfering ability, good recovery and also good reproducibility on the H2O2 detection in urine samples. The simple synthesis of MnO2 nanosheets and good electrocatalytic activity reveal the promising usefulness of the fabricated sensor for detection of H2O2.


Introduction
Manganese oxide (MnO 2 ) nanostructures have been regarded as one of the most favorable inorganic materials due to its abundance, low cost, and fascinating catalytic properties [1]. Manganese oxide is among the strongest oxidants and has numerous polymorphic shapes such as α, β and γ type which show distinctive physical and chemical properties [2]. According to literature, synthesis methods such as hydrothermal synthesis [3], sol gel process [4], electrodeposition method [5] and template method [6] have been developed to obtain MnO 2 with a desired morphology. So far, MnO 2 nanostructures with various morphologies like nanorods [7], nanoflowers [8], nanosheets [9], nanospheres [10] and so on have been successfully synthesized. MnO 2 nanoparticles have been employed in various domains including catalysis [11], energy storage [9,12], electrochemical study [13], and biological applications [14]. In recent years, MnO 2 nanostructures have been extensively explored for biological applications such as biosensing [15], cell imaging [16], and drug delivery [16,17]. Also they have been widely studied in photocatalysis [18] and the oxidative degradation of organic/inorganic contaminants [19]. Due to good electrocatalytic properties and wide potential window, MnO 2 nanostructures have been extremely applied in the field of electrochemical sensing of various analytes like dopamine [20], glucose [21], hydrazine [22] and hydrogen peroxide [23][24][25]. However, the restrictions such as low electrical conductivity and mechanical instability severely hinder their applications as electrode materials [26]. Hence, conductive materials such as graphene nanosheets [27], graphite nanofibers [7] and carbon nanotube [28] have been hybridized with MnO 2 in order to maximize their electrochemical performance. This helps form hybrid or composite nanostructures, reduce the electrical resistance, and improve sensing performance.
Hydrogen peroxide (H 2 O 2 ) is a prominent biomolecule that shows a crucial role in pharmaceutical, environmental, textile, and food industries. H 2 O 2 is most frequently used as a universal oxidizing factor in different areas such as the degradation and elimination of pollutants from waste water [29]. It has also been considered as an important reactive oxygen species (ROS) which is generated as a by-product from most oxidases in cell and plays a vital role in different cellular signaling transductions [30]. Moreover, excess of H 2 O 2 in cell leads to damage in the DNA, cytokines, and defective cell growth [31]. In spite of other applications, H 2 O 2 is utilized as disinfectant agent in food industry such as a milk treatment [32]. Therefore, the selective and sensitive determination of H 2 O 2 with a simple, green and rapid analytical method has aroused considerable concerns in numerous applications. H 2 O 2 is also produced in most enzymatic reactions; thus, its measurement is necessary for the evolution of biosensors. Among various techniques for the accurate determination of H 2 O 2 , electrochemical methods have been accepted as an efficient quantification system with high selectivity and sensitivity and fast response reliability. The electrochemical sensing of H 2 O 2 were developed based on enzymatic [33] and nonenzymatic [23][24][25] sensors. However, enzymatic-based sensors such as horseradish peroxidase and hemoglobin are unstable, high cost and require complex immobilization processes that limit their application [33,34]. In order to overcome those obstacles, the developments of nanostructure materials for nonenzymatic detection of H 2 O 2 have gained growing attention. As one of the most applied metal oxide nanostructures, MnO 2 has received significant attention for H 2 O 2 determination [35][36][37][38]. Other functionalized MnO 2 nanoparticles with Ag nanoparticles have been demonstrated to be appropriate for electrochemical sensing of H 2 O 2 [39,40].
Gum Arabic (GA) is known as a natural, high soluble, nontoxic and highly branched polysaccharides which are derived from exudates of Acacia Senegal tree. GA as a natural polymer has various applications in the food, cosmetic and pharmaceutical productions. GA is a highly heterogeneous material which is composed of carbohydrate moieties consisting of galactose, rhamose, arabinose, glucuronic acid, 4-O-methylglucuronic acid and small fraction of arabinogalactan protein (AGP, 10.4% of total), and glycoprotein (>1% of total) [41]. Due to the multiple functional groups such as amine and carboxylate in GA and its good steric stabilization effect, it has been extensively applied as a stabilizing and reducing factor in the synthesis of nanoparticles. Typically, GA is used in the synthesis of various nanoparticles such as gold [42], CuO/Cu 2 O [43], silver [44], and ZnS [45] nanoparticles. In addition, GA can be used as a magnetic nanoparticles coating to prevent destabilization and agglomeration and produce reactive functions on the surface of nanoparticles [46]. In this study, ultrathin MnO 2 nanosheets were successfully prepared via a simple and green process with Gum Arabic as a both reactive template and reducing agent. The synthesis strategy developed here provides a simple and facile method to prepare MnO 2 nanosheets in large scale.
In previous reports carbon ionic liquid electrode (CILE) was introduced as a high performance electrode with special properties such as high rate of electron transfer [47]. Furthermore, CILE modified with various nanoparticles such as gold [48], palladium [49], copper hydroxide [50] nanoparticles and so on showed superior electrochemical properties and has been mainly used in the detection of different analytes. However, CILE electrode shows a weak response towards H 2 O 2 reduction. Therefore, MnO 2 nanosheets-modified carbon ionic liquid electrode was constructed as an electrochemical sensor. The performance of MnO 2 nanosheets modified with carbon ionic liquid electrode (MnO 2 /CILE) was investigated for nonenzymatic H 2 O 2 detection in alkaline solution. The proposed sensor gave a fast current response, good sensitivity, wide linear range, and eligible selectivity.

Apparatus
The surface morphology characterization of the nanomaterials was studied using field emission electron microscopy ((Hitachi S-4160 FESEM) and high resolution transmission electron microscopy (JEOL, JEM-2100T, 200 KV). The semi-quantitative chemical analysis of as-prepared nanomaterials was conducted by energy-dispersive x-ray spectroscopy (EDX). The structure and crystalline phase of the nanoparticles was analyzed by x-ray diffraction pattern (XRD, D8, Advance, Bruker, AXS diffractometer) with Cu Kα irradiation (λ=1.5418°A) in the range 20-80. A particle size analyzer (Horiba LB-550) was used to investigate the size distribution of the nanoparticles. The study of electrochemical behavior was performed on Autolab PGSTAT204-Compact and modular potentiostat/galvanostat system equipped with NOVA software. Cyclic voltammetry and amperometric experiments were carried out using a conventional three electrode system in 0.1 M NaOH solution. CILE and MnO 2 /CILE (1.8 mm in diameter) were used as the working electrodes, a platinum disk as the counter electrode and Ag/AgCl as the reference electrode at ambient conditions.

Synthesis of MnO 2 nanosheets
MnO 2 nanosheets were synthesized through a simple and green method as follows: firstly, 100 mg of Gum Arabic was dispersed at 20 ml of deionized water and the solution was stirred for 10 min. Then, 30 mg of KMnO 4 was dissolved in 20 ml of water and was added to the above solution under constant stirring. The mixture was heated at 60°C for 5 h. After the completion of reaction, the color of solution changed from purple into dark brown. The as-prepared MnO 2 nanosheets were collected and washed for several times by centrifugation using deionized water and ethanol. Then, they were dried at 50°C.

Preparation of electrode
CILE electrode was made by hand-mixing of graphite powder with ionic liquid (OPyPF 6 ) with ratio of 50:50 [47]. The MnO 2 /CILE electrode was constructed by mixing MnO 2 nanosheets: ionic liquid: graphite powder with a ratio of 5%: 45%: 50% w/w ratio, respectively. The paste was packed into the Teflon holder with the diameter of 1.8 mm and copper wire was used to prepare the electrical contact. The electrode surface was renewed mechanically by polishing the electrode on the surface of smooth paper.

Characterization of nanosheets
In this study, MnO 2 nanosheets were produced with reduction of potassium permanganate by GA. The product was characterized by EDX, XRD, FESEM, TEM and DLS methods. To study the effect of GA on the generation of MnO 2 nanosheets, the synthesis process was performed in the presence and absence of GA (2.5 g l −1 ) at 60°C. In the presence of GA, the color of solution changed from purple into dark brown and the reaction was completed after 5 h but in the absence of GA, no color change was observed even after 24 h and the reduction of permanganate was not occurred. Thus, ultrathin MnO 2 nanosheets were efficiently synthesized in the presence of Gum Arabic as a both reactive template and reducing agent. The formation of MnO 2 nanosheets was found to be dependent on the appropriate concentration of the KMnO 4 . The effect of the different amounts of KMnO 4 (10, 20, 30, 50 and 100 mg) was investigated in the presence of 2.5 mgl −1 of GA. At low concentrations of KMnO 4 (0.25, 0.50 and 0.75 g l −1 ) a brown colloidal stable MnO 2 nanosheets were obtained but at higher concentrations of KMnO 4 (1.25 and 2.50 gl −1 ) the nanosheets were aggregated and finally a brown precipitate was formed. Therefore, the amount of 30 mg of KMnO 4 was selected for synthesis of MnO 2 nanosheets.
Also, the effect of temperature on the synthesis process of MnO 2 nanosheets was examined by changing the temperature from 25 to 60°C. The rate of the reaction are strongly dependent on the temperature. At 25°C, the process was completed after 72 h. By increasing the temperature, the reduction reaction rate increased sharply and at 60°C the color of the solution changed from purple to brown after 5 h. Therefore, synthesis of MnO 2 nanosheets was performed at 60°C. Higher temperatures were not tested due to higher evaporation rates.
TEM images (figures 1(A)-(C)) were employed at different magnification to identify the morphology of the synthesized MnO 2 nanostructures. The TEM images clearly displayed 2D MnO 2 layered nanosheets with uniform morphology. The obtained MnO 2 nanosheets exhibited extra thin wrinkle-like nanolayer with only a few nanometers in thickness. XRD technique was utilized to identify the crystalline phase of MnO 2 nanosheets under our synthesis condition. Figure 1(D) exhibits the broad and weak diffraction peaks at 12.2, 25.2, 37.2 and 65.7 of MnO 2 nanosheets represent the (001), (002), (−111) and (020) significant reflection planes, respectively. The diffraction peaks of nanosheets could be perfectly categorized to the birnessite type MnO 2 (JCPDS 80-1098). The XRD pattern of birnessite type MnO 2 with broad and weak diffraction peaks also were recorded in previous reports [23,51]. The XRD pattern with broad peaks and no other characteristic reflections confirms the formation of MnO 2 nanostructures with high purity.
Furthermore, DLS was exploited to analyze the size distribution of MnO 2 nanosheets ( figure 2(A)). DLS analysis indicate that the average particle size of MnO 2 nanosheets is about 70 nm and a size distribution of nanoparticles is lower than 100 nm. The composition of as-synthesized nanosheets was confirmed by EDX analysis and the obtained EDX pattern is shown in figure 2(B). The EDX spectrum revealed the existence of manganese, oxygen, and carbon element in the structure of nanosheets. The FESEM image and elemental

The study of electrocatalytic performance of MnO 2 nanosheets
The electrocatalytic performance of MnO 2 nanosheets modified with carbon ionic liquid electrode (MnO 2 /CILE) was examined by cyclic voltammetry at the scan rate of 50 mVs −1 in 0.1 M NaOH solution.  net current for reduction of H 2 O 2 was not changed dramatically. Therefore, the amount of 5 % of MnO 2 nanosheets was selected as the optimum ratio for determination of H 2 O 2 with good sensitivity.
According to the previous studies, it seems that the reduction of MnO 2 to Mn(OH) 2 compounds with H 2 O 2 and then oxidation of Mn(OH) 2 to MnO 2 is the possible mechanism of electrocatalytic reduction of H 2 O 2 at MnO 2 modified electrodes [39].
The influence of scan rate on the current of H 2 O 2 reduction peak at MnO 2 /CILE was studied by cyclic votammetry in the 5-500 mVs −1 interval. As can be observed from figure 4, with increasing the scan rate, the reduction current increased linearly with the square root of scan rate with the high correlation coefficient of r 2 =0.993 (figure 4 inset). The reduction of H 2 O 2 at MnO 2 /CILE involves a diffusion controlled process. In order to examine the sensitivity of the MnO 2 /CILE toward H 2 O 2 detection, the amperometric analysis was used to evaluate the linearity between the current responses of fabricated electrode and H 2 O 2 concentrations. Figure 5  Besides, for investigation of the the repeatability of the reduction current of fabricates sensor, the response current in seven successive determinations on one electrode was measured at a H 2 O 2 concentration of 1 mM for MnO 2 /CILE. The relative standard division (RSD) was calculated to be 2.7%. The RSD value of the response   current in six modified electrodes, which were independently fabricated, was found to be about 3.5%, representing the excellent reproducibility of modified electrode. Furthermore, the stability of MnO 2 /CILE was also investigated after one month. The results showed the current response to H 2 O 2 remained 93% of its initial value, indicating the adequate stability of the prepared electrode. The concentration of H 2 O 2 in the urine samples were determined in order to examine the feasibility of the fabricated sensor based on MnO 2 /CILE for practical analysis. 0.5 ml of the urine sample was diluted with 9.5 ml of 0.1 M NaOH solution and the known concentrations of H 2 O 2 were regularly added and the reduction currents were obtained at −0.4 V by amperometry. The measurement results are summarized in table 2. The developed sensor displayed a good recovery in the range of 97%-106%, suggesting that the fabricated H 2 O 2 sensor is reliable and has potential in the practical analysis of real samples.

Conclusion
In this study a green and reliable method was established for the synthesis of MnO 2 nanosheets and then the prepared nanosheets were applied to construct a nonenzymatic H 2 O 2 sensor. MnO 2 /CILE electrode has great ability in electrocatalyzing the reduction of H 2 O 2 and can be utilized for amperometric detection of H 2 O 2 . It displays high sensitivity, extensive linearity and excellent selectivity against common interfering substances in H 2 O 2 determination. Also, the fabrication method was simple and convenient. The suggested sensor was effectively used for the practical detection of H 2 O 2 in the urine.