Ionic liquid coated zerovalent manganese nanoparticles with stabilized and enhanced peroxidase-like catalytic activity for colorimetric detection of hydrogen peroxide

Nanomaterials based colorimetric detection is an area of vital importance in the field of sensing applications. The nanoparticles are the main component of colorimetric sensor in replacing the natural enzyme based sensor. In this context, zero valent nanoparticles have revolutionized the field of optical sensing especially due to easily shift of electron, facile and low cost of preparation, and ease of surface modification. In this work, zero valent manganese nanoparticles (ZV-Mn NPs) are prepared through a simple and very quick method and modulated with new type of ionic liquid (IL). As-prepared materials were characterized through FE-SEM, HR-TEM, BET, FTIR, and XRD. Subsequently, the peroxidase like catalytic activity of pure and modified ZV-Mn NPs to catalyze oxidation of N,N′,N,N′-tetramethylbenzidine (TMB) in the presence of hydrogen peroxide (H2O2) investigated. Moreover, the absorbance peak is observed at wavelength 652 nm. The enhanced catalytic activity of ZV-Mn NPs was attributed to the fast transfer of electron mechanism in between substrate and H2O2. The coating of IL on ZV-Mn NPs permitted a low limit of detection 0.2 μM with a linear range of 10–280 μM. This work can find wide spread interest in the colorimetric sensing applications. In order to verify the successful demonstration of H2O2 sensor, we have applied it in the dairy milk products with satisfactory results.


Introduction
Hydrogen peroxide (H 2 O 2 ) is one of the important constituents of the human body, which is formed as a byproduct of oxidative metabolism in the organism. A large number of oxidases through catalytic reaction produces H 2 O 2 . There is a key role of naturally existing peroxidase in the biological system for the catalytic degradation of H 2 O 2 [1]. The concentration of hydrogen peroxide is regulated by peroxidase, which is normally produced by the kidneys and red blood cell of the human body [2]. In this sequence, the concentration of hydrogen peroxide is controlled by the peroxidase, but the favorable working conditions are required (e.g., temperature, pH, and time, etc) [3]. Hydrogen peroxide is considered to be a significant mediator in food, clinical, industrial, pharmaceutical, and environmental analyses [4]. Many analytical techniques were widely utilized for the detection of hydrogen peroxide such as electrochemical [5][6][7][8][9][10][11][12][13][14], titrimetry [15], fluorescence [16,17], and colorimetric [18][19][20][21][22][23][24][25][26]. In comparison with other reported methods, there are considerable advantages of colorimetric detection of various analytes over other conventional sensing techniques e.g., costeffectiveness, facile and reliable method, and practicability and quick response. The visualization of the color variations to the unaided eye during the course of the reaction is the most attractive characteristic of colorimetric sensing. Subsequently, there exist two different routes of colorimetric detection of hydrogen peroxide based on enzyme and nanomaterials [20]. Detection based on nanomaterials has been considered to be more suitable than natural enzymes. Natural enzymes have high selectivity and specificity to the substrates; however, some drawbacks are associated with enzymes, such as high cost, instability, denaturing phenomenon, gentle handling, and required controlled environment [27].
Since in the past two decades, natural enzymes were replaced with artificial nano-enzymes having the remarkable advantages like, easy preparation, cost-effectiveness, large surface area, high catalytic activity, stability, and comfort in manipulation as compared to natural enzymes [28]. This is all possible just because of the integration of nanotechnology to substitute natural enzymes containing many bottlenecks [29]. In this context, large number of nanoparticles (NPs), including magnetic NPs, noble metals NPs, metal oxide NPs, polyoxometalates, conjugated polymers, and carbon materials as well as their derivatives have been employed as a nanozymes to displace the natural enzymes [30][31][32][33]. However, artificially designed biomimetic NPs mainly suffer from poor dispersion, low catalytic activity, less surface area, and precipitation under harsh atmospheric conditions. Therefore, it is inevitably needed to search for new approaches to prepare the low-cost biomimetic NPs to overcome the above limitations of nanozymes. The combination of ionic liquids (ILs) and nanoparticles can be employed to obtain high dispersibility, simple procedure of preparation, large surface area, and high as well stable catalytic activity under various pH and different temperatures [29,34].
Ionic Liquids (ILs) prepared by the combination of fused salts and maintained the low melting point below 100°C are the fascinating characteristics of organic solvents in the field of chemistry. ILs have been recognized as an organic solvent having negligible vapor potential, wide temperature range, high stability, and tune-able properties with suitable functionalization of cations and anions, as well the capability of dispersing numbers of materials [35]. These versatile characteristics of ILs have been used in a variety of applications [36,37]. Furthermore, in order to enhance the catalytic performance of catalyst various literature have been reported that ILs have the properties to enhance the catalytic performance and stabilize the reactive catalytic species or reaction intermediates [38]. More significantly, ILs have been used as a modulator in artificial enzymes as well as stabilizing mediator to thermally stabilize the enzymatic product, consequently make it possible to occur the catalytic reactions at high temperature [39].
In this work, all of these unique features are integrated to design a novel construct of nanoparticles modified with an ionic liquid to synergistically enhance the stability and detection limits of colorimetric sensors. Zerovalent Manganese (Mn) nanoparticles (ZV-Mn) NPs were used as promising peroxidase mimics for colorimetric detection of H 2 O 2 . Mn NPs were prepared by a quick, low cost, and facile method and characterized with various characterization tools, such as FE-SEM, HR-TEM, BET, FTIR, and XRD. The asprepared ZV-Mn NPs were modified with a new type of IL i.e. 1-H-3-Methylimidazolium formate (HMIM OF) to enhance the catalytic activity, high thermal stability, and high dispersibility as compared to other peroxidase mimetics for colorimetric detection of H 2 O 2 .

Characterization
Double beam Perkin Elmer UV-vis spectrophotometer Lambda-25 (UV-25, Perkin Singapore) was utilized to record the UV-vis absorption spectra in a 10 mm disposable cuvettes capacity of containing solution 2 ml and 1 nm bandwidth setting in the range of 400-800 nm.
Field Emission Surface morphology of ZV-Mn NPs was studied using the Field emission scanning electron microscope (FESEM, JEOL JSM7100F Japan). Accelerated voltage of 10 kV applied to take images at alter magnification. The internal structure, the shape of the material and the elemental distribution of as prepared Mn NPs were studied through a Tecnai G2 F30 S-TWIN 300 kV / FEG High resolution transmission Electron Microscopy (HR-TEM) of the as-prepared materials equipped with the BRUKER energy dispersive spectrometer (EDS). The surface area, pore volume, pore size and adsorption and desorption of Nitrogen gas of ZV-Mn NPs were measured through surface area and porosity analyzer Brunauer-Emmett-Teller (BET; Mike Tristar II 3020 Version 3.02, USA) of Micrometrics Instrument Corporation. The functional groups attached to ZV-Mn NPs were studies through Fourier-transform Infrared (FTIR) spectra using Thermo Fisher Scientific (Nicolet iS 50) spectrometer over the wave range of 400-4000 cm −1 at a resolution of 8 cm −1 . The crystal structures of ZV-Mn NPs were investigated using as-prepared powder by Bruker D8 advanced X-ray diffractometer (Germany, Bruker Corporation with Cu K α radiation, λ = 1.5418 Å) over the 2θ range of 20°-80°w ith a step of 0.02 degree. All refinement of the XRD was done by MJAD 6.5 software.

Synthesis of Manganese nanoparticles
Sodium borohydride (NaBH 4 , 140 mM) solution was prepared separately in the deionized deoxygenated water used to avoid any oxidation. At the same time, the manganese Chloride (MnCl 2 .4H 2 O, 20 mM) was prepared in a solution of 3 mL ethanol absolute and 22 mL deionized water. The solution of sodium borohydride was added dropwise into manganese chloride solution through titration followed by the simultaneous formation of particles of Mn NPs having black color. During titration, the resulting black materials were stirred for the formation of homogeneity. The proposed reaction for this process is given as follows; ( ) ( ) An excess amount of borohydride solution was used for the better formation of ZV-Mn NPs. However, the nanoscale zerovalent manganese (ZV-Mn) was allowed to be aged overnight. After aging, the prepared ZV-Mn was separated by filtration apparatus and micron filter paper (0.45 μM) was used to separate solution. To remove the surface impurities attached with particles, the current particles were washed through deionized (DI) water three times and then same with anhydrous ethanol and dried in Oven at 100°C for overnight. After drying, the dried particles were calcined in the furnace at 500°C for 5 h. at the ramp of 5°C/min followed by ground to get homogenous powder of ZV-Mn NPs. Repeated the same calcination in order to eradicate any possibility of impurity in powder. It was observed that the prepared materials oxidized quickly in the air. Beside other characterizations, the prepared materials were utilized for the sensing applications.

Preparation of ionic liquid
The 1 H-3-Methylimidazolium formate was prepared using the modified protocol reported by Petrović et al [40]. 1-methylimidazole (0.01 mol) was neutralized with formic acid (0.01 mol) in two neck flasks under the cooling condition and subsequently, the resulting mixture was stirred at room temperature for 12 hr to obtain the desired IL medium. Furthermore, to avoid the presence of the unreacted reactants in the solution, the solution was led to the rotary evaporator for 8 hr at 90°C under the moist environment at low pressure. The prepared ionic liquid (after subjecting to rotary evaporator) was characterized using NMR Bruker Avance (500 MHz).

Coating of nanoparticles
IL coated Mn NPs were prepared in such a way: initially, ZV-Mn NPs (5 mg) was added into the protic IL 1-H-3-Methylimidazolium formate (HMIM OF) (1 mL) and mechanically ground for 30 min in the pestle-mortal until a well-dispersed ionic liquid coated ZV-Mn NPs was achieved. Then prepared dispersed solution was further used for characterizations and colorimetric applications.

Peroxidase like-catalytic activity of ionic liquid coated nanoparticles
The peroxidase-like activity was evaluated through colorimetric detection of H 2 O 2 using N,N,N,Ntetramethylbenzidine (TMB) as a chromogenic substrate. The procedure was carried out as follow; 35 μL of stock solution of dispersed IL coated NPs, 100 μl of TMB (10 mM), 100 μl of Ionic Liquid and 575 μL of phosphate buffered saline were mixed. Subsequently, to investigate the optical change, 100 μL of H 2 O 2 (2.8 mM) was added into the reaction solution and was incubated for 5 min as a result a dark blue color was observed.
Furthermore, UV-vis spectroscopy measurement was used to understand and measure the quantitative relation among IL coated NPs, TMB and varying concentration of H 2 O 2 of the resultant solution.

Detection of Hydrogen peroxide
The detection of hydrogen peroxide was carried out in a similar to the above description but only difference was adding different concentrations of H 2 O 2 were mixed in ascending order in total volume of prepared solution very properly. The total solution was incubated for 5 mint at different temperature in the physiological environment, and then the resulting solution was diluted with water for the absorption spectroscopy measurements.

Characterizations analysis
The inset in figure S1(a) is available online at stacks.iop.org/MRX/7/035018/mmedia presents surface morphology of the ZV-Mn NPs powders. In the image of scanning electron microscopy (SEM) all the particles were distributed homogeneously, which is helpful in generating high area for catalytic activity. High resolution transmission electron microscopy (HR-TEM) images indicated that the particle size of ZV-Mn NPs was less than 50 nm and are well dispersed figure S1(b-c). The small size of ZV-Mn NPs provide a greater surface to volume ratio of the material and thus is responsible for high reactivity due to the greater number of active sites of the reaction. This can be associated with an intense homogeneous nucleation kinetics, where a large number of nucleation sites are formed, mainly due to the molar excess of borohydride added to the manganese salt solution that helped in providing the rapid reduction of the metallic ions; thus, without having time to form nanoparticle clusters, as a result the particle size is nanometric size. The planes provided by x-ray diffraction were verified by the TEM image in figure S1(d). The energy dispersive x-rays analyses were also carried out for ZV-Mn NPs to confirm the presence of the targeted elements at the nano-level with the BRUKER supported energy dispersive x-rays analyses associated with TEM. The elemental mapping is shown in figure S1(e-h). It can be clearly identified that all elements of ZV-Mn NPs are presented in the results confirming the proper synthesis of the proposed ZV-Mn NPs. Moreover, in-line with SEM results, the crystallinity, and homogeneity in the size distribution of particles are confirmed by the XRD analysis where sharp peaks corresponding to reflection from planes are observed. For pores size distribution and surface area analysis of fabricated zero-valent manganese catalyst, nitrogen adsorption-desorption measurements were used, which is shown in figure 1(A-B). From figure 1(A), it's quite obvious that it belongs to type IV isotherm, which confirms an excellent interaction between adsorbate and adsorbent [41]. The pores sizes were found to be bigger than the molecular diameter of N 2 (3.5 Å), which can be detected by nitrogen adsorption-desorption experiment [42]. In our case, the pore volume was in the range of 10-100 nm, which confirms the mesoporosity of the material [43] see figure 1(B). The specific surface area, pore volume, and pore size were found to be 6.0568 m 2 g −1 , 0.018187 cm 3 g −1 , and 12.011 nm respectively. The H 2 O 2 can be easily absorbed on the fabricated catalyst due to its large surface area that increases the effectiveness of the reaction. Moreover, it was revealed by the BET results that fabricated catalyst can be used easily for H 2 O 2 and other organic pollutants sensing due to its large surface area.
Fourier transform Infrared (FTIR) was performed in order to get information and identify the attached functional groups with ZV-Mn NPs in the range of 500-4000 cm −1 , as shown in figure S2. Many peaks were observed in the spectra of manganese nanoparticles such as, at 632.54 cm -1 ascribed to O-Br stretching because the preparation involved the borohydride and partially oxygen got attached. Additionally, a peak observed at 1010 cm −1 that can be assigned to the stretching vibration of O-H hydroxyl group bending belongs to physisorbed water molecules present on the surface of the adsorbent. Likewise, two more strong peaks at 1351 cm −1 and 1604 cm −1 were observed, which can be attributed to the bending vibrations of a hydroxyl group to the metal oxides such M-OH (M=Mn) [44,45].
In order to get the structural information of ZV-Mn NPs at room temperature, X-ray diffraction analysis was performed as depicted in figure S3. There amorphous peaks of MnO 2 were observed at 23.18°, 53.48°, 67.98°, and 72.32°, which are perfectly matched by JADE 6.5 software. Furthermore, strong peaks were observed for These results showed that the as-synthesized materials have enough presence of nZV-Mn; as presented in figure  S3. It has been reported that the oxidation of Mn to MnO 2 and Mn 2 O 3 nanocrystal can take time but it also depends upon environmental conditions and size of particles [46]. From this study, considering the peak at degrees, average particle size has been estimated by using Debye-Scherrer formula; Where k and λ are the shape factor (∼0.90) and the wavelength of the x-ray (1.5405 Å), respectively. Whereas, ω represents the full width at half-maximum (FWHM) of specified peak and θ is the diffraction angle and D is particle diameter size. The calculated mean crystallite size of the ZV-Mn NPs was approximately 25 nm.

Peroxidase like activity of Ionic Liquid coated nanoparticles
The peroxidase-like activity of nanoparticles has been investigated by the catalytic oxidation of TMB substrate in the presence of H 2 O 2 , along with IL as a stabilizing and modulating to enhance the biomimetic activity of a single assay [17,22,27]. IL has been widely utilized in the field of biomedical applications. Many methods have been reported to stabilize the nanoparticles and enhance the catalytic activity but coated moieties block the diffusion of other molecules through the surface. Subsequently, the idea was followed to determine the stability of IL, asprepared nano-zerovalent manganese nanoparticles were dissolved in phosphate buffered saline in the presence and absence of an appropriate proportion of IL. Figure 2(C) shows that IL has displayed a highly dispersive media and enhanced the dispersibility of ZV-Mn NPs for a longer time while on the other side agglomerates/ precipitates were formed. IL can stabilize the nanoparticles and can be the possible reason to the formation of ionic layer on the surface of the metal nanoparticles as well as to the enhancement of the catalytic activity. Furthermore, the peroxidase-like catalytic activity of IL coated ZV-Mn NPs was investigated. The catalytic activity of IL coated ZV-Mn NPs was found to be many folds higher than pure ZV-Mn NPs and the intense blue color product was observed in the IL coated ZV-Mn NPs system than that of pure ZV-Mn NPs system under the same experimental conditions, as depicted in the inset of figure 2(A-B). The catalytic activity can be attributed to the metal centers. The peroxidase substrate adsorbed on the surface of IL coated ZV-Mn NPs as a result formation of π-π interaction occurred. However, the interaction of H 2 O 2 with metal ions in the presence of molecular oxygen and their conversion to double ·OH helped in the catalytic activity as well as in the oxidation of TMB. The ZV-Mn NPs easily oxidize in air, get redox state Mn +1 , and yield the product of H 2 O 2 in the aqueous solution following the Fenton type of oxidation mechanism, the same Fenton type reaction was employed on zero-valent iron nanoparticles as well in iron-cupper nanoparticles [47,48].
The redox reaction can be expressed as following equation; ( ) Hence, it is observed from the above chemical reactions that higher oxidation states i.e. Mn +1 / Mn +2 play a key role in the peroxidase catalytic activity of manganese nanoparticles. Furthermore, H 2 O 2 absorbs on the surface of manganese nanoparticles and produces the Mn +1 to generate the hydroxyl ·OH, the ·OH further help in the oxidation of TMB substrate to generate an intense blue color. In addition, control experiment was performed where the absorbance of pure ZV-Mn NPs was found to be negligible as compared to IL coated ZV-Mn NPs, as shown in figure 2(A-B). The coating of IL over the manganese nanoparticles played a vital role in the enhancement of catalytic activity. The presence of pi-electron density in both of the imidazolium cations and carboxylate anions in IL were employed to enhance the medium conductivity. The high salvation power of IL is considered to play a positive role in the enhanced catalysis process [30] as well the presence of large number of cations and anions and their weak interactions with oxidized products were further expected to improve the activity. The chemical structure of IL (1-H-3methylimidazolium formate) is presented in figure S4. Likewise, the decomposition of H 2 O 2 into OH radical that is used for further catalyze the oxidation of chromogenic substrate TMB can be associated with the presence of nascent acidic hydrogen of the cationic part of IL.
Interestingly, the color of a catalyzed product was stable for more than 25 h.; afterwards, a slight color loss was observed as compared to the oxidized product in pure buffer medium without IL (maximum degradation occurred approximately after 1 h.) and the relative degradation of catalytic activity of oxidized product shown in figure S5. The oxidized product turns into a colorless product with the passage of time while the IL due to its attractive properties not only stabilized the nanomaterial but also enhanced the long-term stability of TMB. It has been reported in the literature that instability is one of the alarming constituents of the catalyzed product whereas our work has demonstrated the long term stability. Up to the best of our knowledge, very rare data of long-time stability of the catalyzed product is available in literature and stability of catalyzed product can pursue a vital role in biomedical research. It has been verified that IL is not only a powerful stabilizing agent but also modulate and increase the catalytic properties of manganese nanoparticles without disrupting the surface properties. Although various types of ILs have been reported in the literature but for the catalytic activity, specific kind of IL (1-H-3-methylimidazolium formate) must be used having the aromaticity in its structure.

Optimization of experimental conditions
Optimization of experimental conditions play a crucial rule in order to obtain the high catalytic activity of artificial enzymes depending upon various factors, amount of biomimetic materials, volume of IL in reaction mixture, concentration of TMB and H 2 O 2 . The maximum peroxidase-like catalytic activity was obtained under following conditions; pH 7.4, 25°C, 5 min incubation time, 35 μl IL coated NPs, 10 mM TMB and 2.8 mM H 2 O 2 as shown in figure 3 (A-B). Moreover, our system worked at various pH value of buffer medium and high temperature due to the good stability of IL, indicating the stable catalytic activity of ZV-Mn NPs and the obtained catalyzed products.
The kinetic parameters were measured for the catalytic oxidation of TMB in the presence of H 2 O 2 to determine the peroxidase-like catalytic activity of nanomaterials by using Line weaver-Burk plots including Michaelis -Menton (Km) and maximum initial velocity (Vm) by changing the concentration of TMB and H 2 O 2 . The kinetic parameters measured for IL coated ZV-Mn NPs and pure ZV-Mn NPs data are presented in table 1. To perform the kinetic analysis, the concentration of one substrate was varied while the other was kept constant. The comparative study was performed to measure the kinetic parameters of both IL coated ZV-Mn NPs as well as the pure ZV-Mn NPs. The obtained kinetic parameters for IL coated ZV-Mn NPs was many folds better than the pure ZV-Mn NPs. The lower Km value suggests that the lower concentration of oxidizing agent (H 2 O 2 ) is required to achieve maximum catalytic response with IL based ZV-Mn NPs.

Detection of hydrogen peroxide
A simple calorimetry assay was used to detect hydrogen peroxide based on the optimized experimental condition and biomimetic IL coated ZV-Mn NPs. There was a color change in a calorimetric method and this method was used to determine the direct relationship between the concentrations of H 2 O 2 and obtained absorbance at 652 nm for IL coated ZV-Mn NPs as shown in figure 4(A). The method enabled us to detect the H 2 O 2 with a linear range of 10-280 μM, the obtained limit of detection (LOD) was 0.20 μM; calibration curve presented in figure 4(B), and the standard deviation was 5 to 6% as shown in table S1. There are various methods  reported to detect H 2 O 2 but the calorimetric method offers a very simple procedure and can be visualized with naked eyes. There is no need for any complex instruments or technical persons. In addition, the generation of OH radical as a result of adsorption of H 2 O 2 on the nano-surface which are subsequently involved in the oxidation of TMB to give a blue-green color product.

Interference study
The selectivity of the proposed calorimetric detection based on the prepared IL coated ZV-Mn NPs as a peroxidase mimetic was further explored by studying the potential interfering compounds. The relative activity extracted from the investigating system of various interfering compounds such as dopamine, uric acid and ascorbic acid is shown in figure 5. From figure 5, it can be seen that the other interfering compounds having elevated concentration as compared to glucose showed very less response confirming the selectivity of the purposed peroxidase-like catalytic activity of IL coated ZV-Mn NPs induced by H 2 O 2 .

Real sample analysis
It is pretty known to everyone that H 2 O 2 is used as stabilizer in commercial milk products but their residual must be controlled due to its harmful effects on the skin of human beings. The accredited maximum H 2 O 2 quantity allowed by US FDA is 0.05 wt% (ca. 15 μM) [49]. Therefore, the sensitive and selective detection of H 2 O 2 is mandatory for the long time storage of pasteurized milk used in practical and healthy life. In order to confirm the

Conclusion
The present work demonstrated the peroxidase like catalytic activity of nano zerovalent manganese. In addition, the new type of IL namely 1 H-3-Methylimidazolium formate was utilized to modify the surface of ZV-Mn NPs to synergistically enhances its catalytic performance in oxidation of TMB by H 2 O 2 . The impact of work is associated with demonstration of ZV-Mn NPs as a peroxidase mimetic and application of IL coated ZV-Mn NPs for fast, highly sensitive and selective colorimetric detection of H 2 O 2 . The easy to prepare peroxidase mimetic, low degradation oxidized product, stable, and environmentally friendly procedure were investigated. This concept can be further extended to modulate the properties of nanozymes towards construction of colorimetric assays.