**E**ffi**cient Multifunctional Catalytic and Sensing Properties of Synthesized Ruthenium Oxide Nanoparticles**

#### **Ruby Phul <sup>1</sup> , Mohammad Perwez <sup>2</sup> , Jahangeer Ahmed <sup>3</sup> , Meryam Sardar <sup>2</sup> , Saad M. Alshehri <sup>3</sup> , Norah Alhokbany <sup>3</sup> , Mohd A. Majeed Khan <sup>4</sup> and Tokeer Ahmad 1,\***


Received: 23 June 2020; Accepted: 7 July 2020; Published: 13 July 2020

**Abstract:** Ruthenium oxide is one of the most active electrocatalyst for oxygen evolution (OER) and oxygen reduction reaction (ORR). Herein, we report simple wet chemical route to synthesize RuO<sup>2</sup> nanoparticles at controlled temperature. The structural, morphological and surface area studies of the synthesized nanoparticles were conducted with X-ray diffraction, electron microscopy and BETsurface area studies. The bifunctional electrocatalytic performance of RuO<sup>2</sup> nanoparticles was studied under different atmospheric conditions for OER and ORR, respectively, versus reversible hydrogen electrode (RHE) in alkaline medium. Low Tafel slopes of RuO<sup>2</sup> nanoparticles were found to be ~47 and ~49 mV/dec for OER and ORR, respectively, in oxygen saturated 0.5 M KOH system. Moreover, the catalytic activity of RuO<sup>2</sup> nanoparticles was examined against the Horseradish peroxidase enzyme (HRP) at high temperature, and the nanoparticles were applied as a sensor for the detection of H2O<sup>2</sup> in the solution.

**Keywords:** ruthenium oxide; nanoparticles; electrocatalysts; sensing

#### **1. Introduction**

The present generation is largely dependent on fossil fuelsto meet the present energy requirements—for instance, oil, coal, or natural gases. However, these energy demands fulfilled by these products directly affect the environment. The burning of fossil fuels leads up to the emissions of carbon dioxide gas (a greenhouse gas), which is affecting the world significantly through global warming, change in weather patterns and several other noteworthy geographical changes [1]. In addition, we know fossil fuels are nonrenewable resources, hence they will eventually deplete, so alternatives must be found. Therefore, the development of efficient, inexpensive and eco-friendly sources of energy has become a significant and crucial task for the researchers [2]. Scientists have investigated the use of renewable resources such as solar, wind, tidal, biomass, geothermal energy etc. The major research trend of today's era is the water-splitting phenomena for energy generation. Water splitting via electrocatalysis or photocatalysis is a clean, environmentally friendly and renewable source of energy for fuel cells, batteries and hydrogen generation [3–5]. In the presence of electro/photocatalyst, the water molecule splits into hydrogen and oxygen gas (i.e., H2O → H<sup>2</sup> + <sup>1</sup>/<sup>2</sup> O2). The evolved

hydrogen gas is used in fuel cells as a fuel which further reacts with O<sup>2</sup> to produce an electric current. The evolved oxygen gas participates in the combustion reaction of fuel cells to generate power.

Forthe past few decades, researchers made headway in creating robust and efficient catalysts for the oxidation of water. Several noble metals viz. Pt, Ir, Ru and their oxide-based catalysts have been developed for oxidation–reduction reactions [6–8]. Various other earth-abundant metal-based catalysts such as Mn, Fe, Co, Cu and W were also reported for their role in water splitting reactions [9–13]. In a previous research era, stupendous research has been done in the fabrication of active electrode material for oxygen evolution reactions (OERs). The main limitation with this method is that the OER at the anode gives rise to a high energy loss. Therefore, the emphasis has been given on attaining a high oxygen evolution rate at a low overpotential by optimizing the overall water splitting reaction. Electrode corrosion and low current densities are also the major disadvantages of conventional anode materials for OER [14]. Currently, ruthenium oxide has been used in the fabrication of dimensionally stable anodes (DSA), which havebeen employed to yield chlorine [15]. In addition, it has been used as a heterogeneous catalyst for the low temperature dehydrogenation of NH<sup>3</sup> [16], HCl [17], and methanol [18], respectively. Further, ruthenium oxide was reported to work as anexcellent electrode material for OERs [19,20] and hydrogen evolution reactions (HERs) [21,22]. The metallic conductivity of RuO2, along with IrO2,is of the order of 10<sup>4</sup> ohm−<sup>1</sup> cm−<sup>1</sup> [23]. IrO<sup>2</sup> shows high corrosion-resistance, whereas RuO<sup>2</sup> shows better OER activity [24]. Among all the other transition metal oxides, RuO<sup>2</sup> and IrO<sup>2</sup> are considered as the best electrocatalytic materials for electrolysis of water in acidic as well as an alkaline mediums [25,26].

In the past few years, nanocrystalline RuO<sup>2</sup> particles were synthesized by thermal evaporation [27], nanocasting [28], cryogenic decomposition of RuO<sup>4</sup> [29] and electro-spinning [30]. Recently, M. P. Browne et al. [31] synthesized a series of MnxOy/RuO2/Ti mixed oxide anode materials via a thermal decomposition method for OERs in alkaline medium. They have shown that electrocatalysts were containing different concentrations of Mn viz. 10%, 25% and 90% show almost similar or improved OER activity as compared to pure RuO2. Gustafson et al. [32] have synthesized RuO<sup>2</sup> nanocatalyst for chemical and photochemical oxidation of water, which showed better catalytic performance as reported in the literature.

Presently, the artificial enzymes, i.e., nanoenzymes, are receiving significant attention of researchers due to their low cost, high catalytic property and thermal stability as compared to the natural enzymes. Prototypically, Gao et al. discovered that magnetite nanoparticles exhibit intrinsic peroxidase-like activity similar to that of a natural peroxidase enzyme [33]. H2O<sup>2</sup> plays a vital role as an intermediate in food, pharmaceutical, clinical, and environmental analysis [34–36]. So, the detection of H2O<sup>2</sup> has been done by using different nanoparticles. Besides that, H2O<sup>2</sup> possesses a strong oxidizing property which may lead to different types of disorders in the body [37–39]. Hence, the detection of hydrogen peroxide is of a great practical feature.

Herein, we report the redox reaction of water (OER/ORR) happening through as-synthesized ruthenium oxide nanoparticles in 0.5M KOH electrolytic solution at room temperature under different atmospheres (air, N<sup>2</sup> and O2). Further, the as-synthesized ruthenium oxide nanoparticles were also used as a sensor for the detection of H2O<sup>2</sup> in solution. Moreover, the synthesis of RuO<sup>2</sup> nanoparticles was carried out through a simple, environmentally friendly and cost-effective wet chemical method at 80 ◦C followed by annealing at 300 ◦C for 6h.

#### **2. Results and Discussion**

The powder X-ray diffractometry was used for the structural analysis of the as-synthesized nanoparticles. Figure 1a shows the X-ray diffraction pattern of the nanocrystalline RuO2. The obtained diffraction peaks are as follows at Bragg's angles of 27.8◦ , 34.9◦ , 39.8◦ , 54.0◦ , 57.6◦ , 59.2◦ , 65.2◦ , 66.7◦ , 69.2◦ , 73.8◦ , 82.9◦ and 87.3◦ corresponds to the planes (110), (101), (200), (211), (220), (002), (310), (112), (301), (202), (321) and (222), respectively, which were correlated to a tetragonal unit cell of ruthenium oxide. The reflection pattern could be indexed to a pure tetragonal phase of RuO2(JCPDS No. 065-2824).

No peaks from any impurity or other phase or metallic Ru were detected, which affirms the formation of monophasic RuO<sup>2</sup> nanoparticles. RuO2(JCPDS No. 065-2824). No peaks from any impurity or other phase or metallic Ru were detected, which affirms the formation of monophasic RuO<sup>2</sup> nanoparticles.

The microstructure and surface texture of as-synthesized ruthenium oxide nanoparticles wereinvestigated through SEM studies. Figure 1b shows the SEM micrograph of the as-synthesized nanoparticles. Further analysis of the SEM micrograph depicted the highlydense and agglomerated RuO<sup>2</sup> nanoparticles. The nanoparticles aggregate randomly to form almost spherical shape with an average diameter of 28 nm, which is as per the TEM analysis. The microstructure and surface texture of as-synthesized ruthenium oxide nanoparticles wereinvestigated through SEM studies. Figure 1b shows the SEM micrograph of the as-synthesized nanoparticles. Further analysis of the SEM micrograph depicted the highlydense and agglomerated RuO<sup>2</sup> nanoparticles. The nanoparticles aggregate randomly to form almost spherical shape with an average diameter of 28 nm, which is as per the TEM analysis.

**Figure 1.** (**a**) X-ray diffraction pattern and (**b**) scanning electron microscope (SEM) micrograph of Ruthenium oxide nanoparticles. **Figure 1.** (**a**) X-ray diffraction pattern and (**b**) scanning electron microscope (SEM) micrograph of Ruthenium oxide nanoparticles.

The detailed structural analysis, shape and size distribution of RuO<sup>2</sup> nanoparticles was done with the help of TEM studies. The low magnification TEM micrograph is shown in Figure 2a, which indicates the formation of tiny sized tetragonal RuO2 nanoparticles with slight agglomeration. Figure 2a also reveals that the small-sized nanoparticles tend to form large tetragonal structures, which are in accordance with X-ray diffraction studies. Furthermore, High Resolution Transmission Electron Microscope (HRTEM) analysis revealed the crystal structure, phase and growth direction of as-synthesized nanoparticles. Figure 2b shows the typical HRTEM image of ruthenium oxide nanoparticles, which depicts the well-resolved lattice fringes with an average lattice distance of 3.210±0.05 Å and 2.560±0.05 Å corresponding to (110) and (101) planes, respectively, of ruthenium oxide nanoparticles. The TEM average size distribution histogram of ruthenium oxide nanoparticles are shown in Figure 2c, which indicates that the particle size ranges from 5 nm to 35 nm, as the various small particles have combined to form a single massive particle. The average grain size was The detailed structural analysis, shape and size distribution of RuO<sup>2</sup> nanoparticles was done with the help of TEM studies. The low magnification TEM micrograph is shown in Figure 2a, which indicates the formation of tiny sized tetragonal RuO<sup>2</sup> nanoparticles with slight agglomeration. Figure 2a also reveals that the small-sized nanoparticles tend to form large tetragonal structures, which are in accordance with X-ray diffraction studies. Furthermore, High Resolution Transmission Electron Microscope (HRTEM) analysis revealed the crystal structure, phase and growth direction of as-synthesized nanoparticles. Figure 2b shows the typical HRTEM image of ruthenium oxide nanoparticles, which depicts the well-resolved lattice fringes with an average lattice distance of 3.210 ± 0.05 Å and 2.560 ± 0.05 Å corresponding to (110) and (101) planes, respectively, of ruthenium oxide nanoparticles. The TEM average size distribution histogram of ruthenium oxide nanoparticles are shown in Figure 2c, which indicates that the particle size ranges from 5 nm to 35 nm, as the various small particles have combined to form a single massive particle. The average grain size was found to be ~20nm by using TEM micrograph as well as a size distribution plot.

found to be ~20nm by using TEM micrograph as well as a size distribution plot.

**Figure 2.** (**a**) TEM micrograph, (**b**) HR-TEM image and (**c**) size distribution histogram of Ruthenium **Figure 2.** (**a**) TEM micrograph, (**b**) HR-TEM image and (**c**) size distribution histogram of Ruthenium oxide nanoparticles.

oxide nanoparticles. The specific surface area of as-prepared RuO<sup>2</sup> nanoparticles was estimated by using a multipoint BET equation that showed the linear relationship in the P/P<sup>0</sup> range of 0.05–0.35. Figure 3a shows the BET plot for RuO<sup>2</sup> nanoparticles. The specific surface area was found to be 64.5 m2g−1 which agrees with the earlier reported value [19]. The BJH (Barrett-Joyner-Halenda) model was used to determine the pore size. The pore size distribution plot of ruthenium oxide nanoparticles (Figure 3b) gives the pore radius value of 16 Å , which lies in the range of mesoporous materials. The pore The specific surface area of as-prepared RuO<sup>2</sup> nanoparticles was estimated by using a multipoint BET equation that showed the linear relationship in the P/P<sup>0</sup> range of 0.05–0.35. Figure 3a shows the BET plot for RuO<sup>2</sup> nanoparticles. The specific surface area was found to be 64.5 m2g −1 , which agrees with the earlier reported value [19]. The BJH (Barrett-Joyner-Halenda) model was used to determine the pore size. The pore size distribution plot of ruthenium oxide nanoparticles (Figure 3b) gives the pore radius value of 16 Å, which lies in the range of mesoporous materials. The pore radius was also determined by the DA (Dubinin-Astakhov) plot, as shown in Figure 3c, and it was found to be 13.5 Å, which is a bit smaller than the BJH results.

radius was also determined by the DA (Dubinin-Astakhov) plot, as shown in Figure 3c, and it was

found to be 13.5 Å , which is a bit smaller than the BJH results.

,

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**Figure 3.** (**a**) BET, (**b**) BJH and (**c**) DA pore radius plots of ruthenium oxide nanoparticles. **Figure 3.** (**a**) BET, (**b**) BJH and (**c**) DA pore radius plots of ruthenium oxide nanoparticles.

The electrocatalytic activity of as-prepared nanoparticles for oxygen evolution reaction (OER) and oxygen reduction reaction (ORR) was evaluated by cyclic voltammetry (CV), linear sweep voltammetry (LSV) and Tafel polarization curves in 0.5M KOH electrolyte solution. The CV plots for OER (anodic sweep) and ORR (cathodic sweep) by ruthenium oxide electrode at the scan rate of 25 mVs−1 in the air (black), N2 (red) and saturated O2 (blue) atmosphere are shown in Figure 4a. The CV curves show that the OER starts from the low potential value of ~1.5 V vs. RHE (reversible hydrogen electrode). Figure 4a shows that the as-synthesized RuO<sup>2</sup> nanoparticles generate more current in O<sup>2</sup> saturated (17.5 mAcm−2) as compared to air (11.5 mAcm−2) and N2 (10.5 mAcm−2) atmosphere at 1.55 V vs. RHE at 25 mVs−1 for the oxygen evolution reaction. The ORR activity of ruthenium oxide nanoparticles in alkaline medium is also shown in Figure 4a. It was observed that as-synthesized RuO2 nanoparticles show an almost comparable ORR reaction in all the atmospheric conditions. The LSV measurements optimized the electrocatalytic activity of ruthenium oxide nanoparticles in 0.5M KOH electrolyte at the scan rate of 25 mVs−1. Figure 4b shows the LSV curves under air, nitrogen and O<sup>2</sup> saturated atmospheres. It was observed that the onset potential for OER was found to be ~1.5 (O2) and ~1.61 V (air and N2) vs. RHE. Notably, the resulting current density (current/area of the electrode) of RuO2 electrode is directly related to the amount of oxygen evolved from the electrolysis of water. The geometric electro-active surface area of the working electrode could be estimated from the Randles–Sevik equation [40]. The current density of RuO<sup>2</sup> electrode at ~1.7 V versus RHE at 25 mVs−1 was found to be ~9.6 mAcm−2 (in N2), ~11.9 mAcm−2 (in air) and ~15.5 mAcm−2 (in O2). The significance of LSV measurements is to find the onset potential, reaction kinetics and mechanism of the reaction, i.e., to identify the number of electrons taking part in the electrochemical reaction. Figure 4c shows the LSV curves of RuO<sup>2</sup> nanoparticles for the oxygen reduction reaction in air, N2, and O<sup>2</sup> saturated 0.5M KOH at the scan rate of 25 mVs−1. From this study, we clearly observed that The electrocatalytic activity of as-prepared nanoparticles for oxygen evolution reaction (OER) and oxygen reduction reaction (ORR) was evaluated by cyclic voltammetry (CV), linear sweep voltammetry (LSV) and Tafel polarization curves in 0.5M KOH electrolyte solution. The CV plots for OER (anodic sweep) and ORR (cathodic sweep) by ruthenium oxide electrode at the scan rate of 25 mVs−<sup>1</sup> in the air (black), N<sup>2</sup> (red) and saturated O<sup>2</sup> (blue) atmosphere are shown in Figure 4a. The CV curves show that the OER starts from the low potential value of ~1.5 V vs. RHE (reversible hydrogen electrode). Figure 4a shows that the as-synthesized RuO<sup>2</sup> nanoparticles generate more current in O<sup>2</sup> saturated (17.5 mAcm−<sup>2</sup> ) as compared to air (11.5 mAcm−<sup>2</sup> ) and N<sup>2</sup> (10.5 mAcm−<sup>2</sup> ) atmosphere at 1.55 V vs. RHE at 25 mVs−<sup>1</sup> for the oxygen evolution reaction. The ORR activity of ruthenium oxide nanoparticles in alkaline medium is also shown in Figure 4a. It was observed that as-synthesized RuO<sup>2</sup> nanoparticles show an almost comparable ORR reaction in all the atmospheric conditions. The LSV measurements optimized the electrocatalytic activity of ruthenium oxide nanoparticles in 0.5M KOH electrolyte at the scan rate of 25 mVs−<sup>1</sup> . Figure 4b shows the LSV curves under air, nitrogen and O<sup>2</sup> saturated atmospheres. It was observed that the onset potential for OER was found to be ~1.5 (O2) and ~1.61 V (air and N2) vs. RHE. Notably, the resulting current density (current/area of the electrode) of RuO<sup>2</sup> electrode is directly related to the amount of oxygen evolved from the electrolysis of water. The geometric electro-active surface area of the working electrode could be estimated from the Randles–Sevik equation [40]. The current density of RuO<sup>2</sup> electrode at ~1.7 V versus RHE at 25 mVs−<sup>1</sup> was found to be ~9.6 mAcm−<sup>2</sup> (in N2), ~11.9 mAcm−<sup>2</sup> (in air) and ~15.5 mAcm−<sup>2</sup> (in O2). The significance of LSV measurements is to find the onset potential, reaction kinetics and mechanism of the reaction, i.e., to identify the number of electrons taking part in the electrochemical reaction. Figure 4c shows the LSV curves of RuO<sup>2</sup> nanoparticles for the oxygen reduction reaction in air, N2, and O<sup>2</sup> saturated 0.5M KOH at the scan rate of 25 mVs−<sup>1</sup> . From this study, we clearly observed that the ORR activity performed significantly better in an O2-saturated system compared to other systems

the ORR activity performed significantly better in an O2-saturated system compared to other systems as expected. The ORR activity in air could be due to the dissolved oxygen in system. Note that very weak ORR activity in N<sup>2</sup> was also observed, which could be due to the presence of oxygen as expected. The ORR activity in air could be due to the dissolved oxygen in system. Note that very weak ORR activity in N<sup>2</sup> was also observed, which could be due to the presence of oxygen content (5%) in N2. The onset potential for ORR was found to be 0.7 V vs. RHE. Choronoamperometric (CA) measurements demonstrated the stability and the electrocatalyticactivity of the ruthenium oxide electrode at a fixed potential (1.5V vs. RHE) in O2-saturated 0.5M KOH for 200 s. Figure 4d shows the CA curves, which demonstrate that the material is stable, and the constant current is generated with time. It was observed that the resulting current densities were consistent with time. The CA experiments also showed that on turning off the potential, the water redox reaction was stopped instantly, and therefore the current density dropped to zero. The current density obtained was directly proportional to the amount of gas evolved during the OER. The surface area of the electrode material used, and the Faradaic and Non-Faradaic processes were responsible for the resulting current density. *Catalysts* **2020**, *10*, x FOR PEER REVIEW 6 of 13 content (5%) in N2. The onset potential for ORR was found to be 0.7 V vs. RHE. Choronoamperometric (CA) measurements demonstrated the stability and the electrocatalyticactivity of the ruthenium oxide electrode at a fixed potential (1.5V vs. RHE) in O2-saturated 0.5M KOH for 200 s. Figure 4d shows the CA curves, which demonstrate that the material is stable, and the constant current is generated with time. It was observed that the resulting current densities were consistent with time. The CA experiments also showed that on turning off the potential, the water redox reaction was stopped instantly, and therefore the current density dropped to zero. The current density obtained was directly proportional to the amount of gas evolved during the OER. The surface area of the electrode material used, and the Faradaic and Non-Faradaic processes were responsible for the resulting current density.

**Figure 4.** (**a**) CV, (**b**) LSV (OER), (**c**) LSV (ORR) curves of Ruthenium oxide photoanode in air, N<sup>2</sup> and O2 saturated 0.5M KOH electrolyte vs. RHE at the scan rate of 25 mVs−1 and (**d**) CA curves of Ruthenium oxide nanoparticles for OER activity at 1.5 V vs. RHE in O2-saturated 0.5M KOH. **Figure 4.** (**a**) CV, (**b**) LSV (OER), (**c**) LSV (ORR) curves of Ruthenium oxide photoanode in air, N<sup>2</sup> and O<sup>2</sup> saturated 0.5M KOH electrolyte vs. RHE at the scan rate of 25 mVs−<sup>1</sup> and (**d**) CA curves of Ruthenium oxide nanoparticles for OER activity at 1.5 V vs. RHE in O<sup>2</sup> -saturated 0.5M KOH.

The Tafel polarization studies determined the kinetics of the reaction, i.e., electrolysis of water. It was observed that reaction kinetics strongly depends upon the size, surface area, morphology and orientation of electrocatalysts [14,41]. Figure 5a,b shows the Tafel polarization plot of ruthenium oxide nanoparticles for the water redox reactions (OER and ORR) in air, and O2, respectively. The linear curve fitting calculated the value of Tafel slopes of ruthenium oxide nanoparticles, and it comes out to be 76, and 47 mVdec−1 in air, and O2, respectively, for OER while for ORR, these values were found to be 48 (air), and 49 mVdec−1 (O2) with the experimental error of ±5. The effective electro-active catalysts for water splitting could lower the Tafel slope values to sustain the high activity, stability and to enhance the efficiency by reducing the loss of energy during the The Tafel polarization studies determined the kinetics of the reaction, i.e., electrolysis of water. It was observed that reaction kinetics strongly depends upon the size, surface area, morphology and orientation of electrocatalysts [14,41]. Figure 5a,b shows the Tafel polarization plot of ruthenium oxide nanoparticles for the water redox reactions (OER and ORR) in air, and O2, respectively. The linear curve fitting calculated the value of Tafel slopes of ruthenium oxide nanoparticles, and it comes out to be 76, and 47 mVdec−<sup>1</sup> in air, and O2, respectively, for OER while for ORR, these values were found to be 48 (air), and 49 mVdec−<sup>1</sup> (O2) with the experimental error of ±5. The effective electro-active catalysts for water splitting could lower the Tafel slope values to sustain the high activity, stability and to enhance the efficiency by reducing the loss of energy during the electrochemical reactions [42].

electrochemical reactions [42]. The comparison in the current electrocatalytic activities viz.

OER/ORR of RuO2 nanoparticles with the literature has been tabulated in Table 1.

RuO<sup>2</sup>

The comparison in the current electrocatalytic activities viz. OER/ORR of RuO<sup>2</sup> nanoparticles with the literature has been tabulated in Table 1. *Catalysts* **2020**, *10*, x FOR PEER REVIEW 7 of 13

**Figure 5.** Tafel polarization curves of Ruthenium oxide nanoparticles for OER and ORR in (**a**) air, and (**b**) O2 saturated atmosphere at 10 mVs−1 . **Figure 5.** Tafel polarization curves of Ruthenium oxide nanoparticles for OER and ORR in (**a**) air, and (**b**) O<sup>2</sup> saturated atmosphere at 10 mVs−<sup>1</sup> .


**Table 1.** Comparison of OER/ORR activity of RuO<sup>2</sup> nanoparticles with other reported literature.

nanoparticles 0.5M KOH <sup>25</sup> 1.5 <sup>47</sup> <sup>49</sup> Present work The catalytic activity of as-synthesized nanoparticles depends on the concentration of H2O2, and the reaction temperature just like that of HRP [33]. The catalytic oxidation of TMB substrate by RuO<sup>2</sup> nanoparticles at a range of H2O<sup>2</sup> concentration and temperature is shown in Figure 6. It was observed that to attain maximum activity; the RuO<sup>2</sup> nanoparticles required a very high concentration of H2O<sup>2</sup> (1M) as compared to HRP and on further increasing the concentration of H2O<sup>2</sup> the catalytic activity was suppressed (Figure 6a). The effect of temperature on the catalytic activity of the RuO<sup>2</sup> nanoparticles was checked in the temperature range of 20–90 °C. Figure 6b indicates that the catalytic activity of RuO<sup>2</sup> nanoparticles increases with the increase in temperature till 60 °C and with further increase in temperature up to 90 °C the activity was quenched. Whereas, HRP shows maximum activity at 30 °C and it shows no activity at higher temperature, i.e., above 60 °C. The as-synthesized nanoparticles showed better activity at a wide range of temperature compared to HRP catalyst. The thermal stability of the RuO<sup>2</sup> nanoparticles and HRP was determined by incubating the reaction mixture with TMB substrate at 80 °C for 90 min, and the aliquots were taken at regular time intervals. It was found that the catalytic activity of RuO<sup>2</sup> nanoparticles was almost preserved until 90 min at 80 °C while the HRP showed no activity in similar reaction conditions (Figure 6c). We have also analyzed the sensitivity of RuO2 nanoparticles for the detection of H2O<sup>2</sup> in solution. The aliquots containing different concentration of H2O2 and The catalytic activity of as-synthesized nanoparticles depends on the concentration of H2O2, and the reaction temperature just like that of HRP [33]. The catalytic oxidation of TMB substrate by RuO<sup>2</sup> nanoparticles at a range of H2O<sup>2</sup> concentration and temperature is shown in Figure 6. It was observed that to attain maximum activity; the RuO<sup>2</sup> nanoparticles required a very high concentration of H2O<sup>2</sup> (1M) as compared to HRP and on further increasing the concentration of H2O<sup>2</sup> the catalytic activity was suppressed (Figure 6a). The effect of temperature on the catalytic activity of the RuO<sup>2</sup> nanoparticles was checked in the temperature range of 20–90 ◦C. Figure 6b indicates that the catalytic activity of RuO<sup>2</sup> nanoparticles increases with the increase in temperature till 60 ◦C and with further increase in temperature up to 90 ◦C the activity was quenched. Whereas, HRP shows maximum activity at 30 ◦C and it shows no activity at higher temperature, i.e., above 60 ◦C. The as-synthesized nanoparticles showed better activity at a wide range of temperature compared to HRP catalyst. The thermal stability of the RuO<sup>2</sup> nanoparticles and HRP was determined by incubating the reaction mixture with TMB substrate at 80 ◦C for 90 min, and the aliquots were taken at regular time intervals. It was found that the catalytic activity of RuO<sup>2</sup> nanoparticles was almost preserved until 90 min at 80 ◦C while the HRP showed no activity in similar reaction conditions (Figure 6c). We have also analyzed the sensitivity of RuO<sup>2</sup> nanoparticles for the detection of H2O<sup>2</sup> in solution. The aliquots containing different concentration of H2O<sup>2</sup> and colourimetric reagent were taken and analyzed via spectrophotometry. Figure 6d shows the absorbance at 505 nm, which increases nearly linearly as the concentration of H2O<sup>2</sup> increased.

colourimetric reagent were taken and analyzed via spectrophotometry. Figure 6d shows the

absorbance at 505 nm, which increases nearly linearly as the concentration of H2O<sup>2</sup> increased.

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**Figure 6.** (**a**) H2O<sup>2</sup> concentration optima and (**b**) temperature optima plots, (**c**) thermal stability (at 80 °C for 90 min under the standard reaction conditions) and (**d**) sensitivity (at 505 nm for the different concentrations of H2O2) of ruthenium oxide nanoparticles. **Figure 6.** (**a**) H2O<sup>2</sup> concentration optima and (**b**) temperature optima plots, (**c**) thermal stability (at 80 ◦C for 90 min under the standard reaction conditions) and (**d**) sensitivity (at 505 nm for the different concentrations of H2O<sup>2</sup> ) of ruthenium oxide nanoparticles.

#### **3. Experimental**

**3. Experimental** Ruthenium (III) chloride (RuCl3.xH2O, Alfa Aesar, Haverhill, MA, USA, 99.9%), NaOH(Merck India Ltd., Mumbai, India), hydrogen peroxide (50% *w*/*v*, Merck India Ltd.), HRP and Tetramethylbenzidine (SRL, Mumbai, India) were used without further purification. 1.3 mmol of RuCl3.xH2O was dissolved in 50 mL of water in a two-neck flask, followed by the dropwise addition of 1.5M aqueous NaOH to a pH ~8. The solution was continuously stirred and refluxed at 80 °C untilthe black precipitate was observed. The black precipitate was collected through centrifugation process and repeatedly washed with double-distilled water followed by ethanol to remove the Clions. The Clions present in the supernatant liquid were checked with the aqueous solution of silver nitrate. The black precipitate was dried at 100 °C in the hot-air oven and grounded to form a fine powder. Further, the black colour powder was annealed at 300 °C for 6 h in a high temperature furnace to obtained black colour ruthenium oxide nanoparticles. The as-synthesized nanoparticles were further employed to study the catalytic activity. Ruthenium (III) chloride (RuCl3.xH2O, Alfa Aesar, Haverhill, MA, USA, 99.9%), NaOH(Merck India Ltd., Mumbai, India), hydrogen peroxide (50% *w*/*v*, Merck India Ltd.), HRP and Tetramethylbenzidine (SRL, Mumbai, India) were used without further purification. 1.3 mmol of RuCl3.xH2O was dissolved in 50 mL of water in a two-neck flask, followed by the dropwise addition of 1.5M aqueous NaOH to a pH ~8. The solution was continuously stirred and refluxed at 80 ◦C untilthe black precipitate was observed. The black precipitate was collected through centrifugation process and repeatedly washed with double-distilled water followed by ethanol to remove the Clions. The Clions present in the supernatant liquid were checked with the aqueous solution of silver nitrate. The black precipitate was dried at 100 ◦C in the hot-air oven and grounded to form a fine powder. Further, the black colour powder was annealed at 300 ◦C for 6 h in a high temperature furnace to obtained black colour ruthenium oxide nanoparticles. The as-synthesized nanoparticles were further employed to study the catalytic activity.

#### *3.1. Physical Characterization 3.1. Physical Characterization*

Powder X-ray diffraction (XRD) was performed using a RigakuUltima IV X-ray diffractometer with Ni-filtered Cu-Kα radiation (λ = 1.5416 Å). The morphological features of synthesized ruthenium oxide nanoparticles were determined by Zeiss EVO40 Scanning Electron Microscope (SEM) at an accelerating voltage of 20 kV. Transmission electron microscopic (TEM) analysis was carried out on FEI Technai G<sup>2</sup> 20 HRTEM (High Resolution Transmission Electron Microscope) with an accelerating voltage of 200 kV. The surface area and the pore size of as-synthesized nanoparticles Powder X-ray diffraction (XRD) was performed using a RigakuUltima IV X-ray diffractometer with Ni-filtered Cu-Kα radiation (λ = 1.5416 Å). The morphological features of synthesized ruthenium oxide nanoparticles were determined by Zeiss EVO40 Scanning Electron Microscope (SEM) at an accelerating voltage of 20 kV. Transmission electron microscopic (TEM) analysis was carried out on FEI Technai G<sup>2</sup> 20 HRTEM (High Resolution Transmission Electron Microscope) with an accelerating voltage of 200 kV. The surface area and the pore size of as-synthesized nanoparticles were determined using

were determined using Brunauer-Emmett-Teller (BET) surface area analyzer (Model: Nova 2000e, Quantachrome Instruments Limited, Boynton Beach, FL, USA) at liquid nitrogen temperature (77K).

Brunauer-Emmett-Teller (BET) surface area analyzer (Model: Nova 2000e, Quantachrome Instruments Limited, Boynton Beach, FL, USA) at liquid nitrogen temperature (77K).

#### *3.2. Electrochemical Measurements*

The electrolysis of water for OER and ORR was carried out with a three-electrode electrochemical work station (potentiostat/galvanostat, CHI 660E, Shenzhen, China) at room temperature in alkaline medium (0.5 M KOH) to investigate the redox behaviour of the synthesized RuO<sup>2</sup> nanoparticles. Pt wire, Ag/AgCl and glassy carbon electrodes were used as the counter, reference, and working electrodes in the electrochemical analyzer. The reference electrode (Ag/AgCl) was converted to the reversible hydrogen electrode (RHE) as per the conversion equation, i.e., Nernst Equation at 25 ◦C.

$$\rm{E\_{RHE}} = \rm{E\_{Ag/AgCl}} + 0.059 \,\rm{pH} + 0.197 \,\rm{V}$$

The slurry was prepared by sonicating 2.5 mg of catalysts in 0.5 mL of isopropanol with 0.1 mL of Nafion solution for 10 min. Then a drop of the slurry was cast on the surface of glassy carbon and dried at 60 ◦C in vacuum oven [46,47]. The loaded amount of the nanoparticles was of ~0.30 mg/cm<sup>2</sup> on the GC electrode, and the area of the working electrode was 0.07 cm<sup>2</sup> . Freshly prepared electrodes were used for the electrochemical measurements. Cyclic Voltammetry (CV), linear sweep voltammetry (LSV) and Tafel measurements were done by applying redox potential versus Ag/AgCl electrode for OER and ORR at the scan rate of 25 mV s−<sup>1</sup> in 0.5 M KOH electrolyte at room temperature in air, nitrogen and saturated oxygen atmosphere.

### *3.3. Catalytic Activity of RuO<sup>2</sup> Nanoparticles and HRP*

The catalytic activity of as-synthesized nanoparticles and HRP was checked by peroxidase substrate Tetramethylbenzidine (TMB) [48]. In a typical procedure, 200 µL of RuO<sup>2</sup> nanoparticles (2 mg/mL), 1.5 mL TMB, and 5 µL H2O<sup>2</sup> were added in 1mL of 0.02M acetate buffer (Ph~4.5) and incubated in a water-bath at 30 ◦C for 10 min and the progress of the reaction was monitored by A Shimadzu UV-2450 spectrophotometer. The leaching of the ions from the reaction mixture was checked by incubating a suspension of RuO<sup>2</sup> nanoparticles in acetate buffer (5 mg/mL, Ph~3.5) (marked as control) for 10 min at 45 ◦C; the nanoparticles were removed from the reaction mixture, and the catalytic activity of the reaction mixture was analyzed. No activity was found in control. In order to investigate the effect of H2O<sup>2</sup> concentration on the activity of as-synthesized nanoparticles, it was examined by varying the concentrations of H2O<sup>2</sup> (0.002–2M). The effect of change of temperature on the activity of RuO<sup>2</sup> nanoparticles was determined by incubating the reaction mixture from 20 to 90 ◦C. The obtained results were compared with the activity of HRP enzyme over the same range of parameters.
