Determining Kinetics Parameters of Oxidase-Like Ce/Fe-Bimetallic Nanozymes Toward 3,3′,5,5′-Tetramethylbenzidine Oxidation

Introduction

Native enzymes suffer some disadvantages such as instability in harsh reaction conditions, difficult recovery, etc [1-10]. In contrast, nanomaterials with enzyme-like activity reveal high stability against pH and temperature changes along with excellent kinetic performances [11-19]. In fact, with the development of nanoscience, several types of nanomaterials were introduced with unique spectral, optical, catalytic, and stability as a result of the fast development of nanoscience in recent years, some of them reveal significant enzyme-like activity especially peroxidase-like properties with significant advantages over native enzymes for example, high pH and thermal stability, excellent reusability, and high storage stability [20-43]. Up to now, nanozymes have been utilized in organic dye biodegradation, battery development, sensor, and biosensor design, especially after the first report of COVID-19, they applied for its clinical sensing [44-64]. In this field proving the catalytic mechanism of the reaction is an attractive research topic. Hence, herein, kinetics parameters, Km and Vmax, of oxidase-like Ce/Fe-bimetallic nanozymes toward 3,3′,5,5′-tetramethylbenzidine oxidation were determined. The Michaelis–Menten kinetic model was used for the evaluation of the kinetic of the nanozyme-mediated oxidation of 3,3′,5,5′-tetramethylbenzidine. To provide the quantitative and accurate values of kinetics parameters, Km and Vmax, the linear plot of Lineweaver–Burk was constructed.

Experimental

Synthesis of Nanozymes

To synthesize the oxidase-like Ce/Fe-bimetallic nanozymes, 0.4 g fumaric acid was added to 25.0 mL water (solution#1). Besides, 0.3 g (NH₄)₂[Ce(NO₃)₆] and 0.3 g Fe(NO₃)₃.9H₂O were introduced to 25 mL water (solution#2) . Afterward, solution#1 was added drop by drop to solution#2, totaling 50 mL, and stirred for 1 hour. The reaction mixture was then heated at 120 ºC for 2 hours to complete the synthesis process.

Kinetic Studies

The Michaelis–Menten kinetic model was used for the evaluation of the kinetic of the nanozyme-mediated oxidation of 3,3′,5,5′-tetramethylbenzidine. To provide the quantitative and accurate values of kinetics parameters, Km and Vmax, the linear plot of Lineweaver–Burk was constructed. It is notable that 3,3′,5,5′-tetramethylbenzidine was used as the standard substrate [65-67].

Results and Discussion

The kinetics of the oxidation process involving TMB chromogenic agents on oxidase-like Ce/Fe-bimetallic nanozymes were investigated. This examination involved analyzing the kinetic components of the Michaelis-Menten equation using the following formula;

V0=(Vmax[S])/(Km+[S]

where V0 represents the initial velocity, Vmax stands for the maximum reaction velocity, [S] denotes the concentration of chromogenic agents, TMB, and Km signifies the Michaelis constant as previously reported in the literature [68-75]. To do this, a series of experiments were carried out for the oxidation of different concentrations of TMB in different concentrations of 0.015-0.2 mM over the developed nanozymes. The oxidation reaction rate was then calculated in nM min-1. Afterward, the Michaelis-Menten curve was plotted by plotting the rate as a function of TMB concentration. The results are shown in Figure 1. As can be seen in this figure, the rate of the oxidation process was increased by increasing the TMB concentration and then leveling off. Based on the Michaelis-Menten curve, a Vmax as high as 55. 5 nM min-¹ was provided for the oxidase-like Ce/Fe-bimetallic nanozymes, revealing their high catalytic activity. Besides, the Km value was found to be about 0.05 mM. Moreover, Vmax/Km was calculated as a crucial indicator of enzyme catalytic efficiency, the results revealed a ratio of 1.10 ×10-3 min-1 from the non-linear Michaelis- Menten curve.

Figure 1: The non-linear Michaelis-Menten curve of the oxidase-like Ce/Fe-bimetallic nanozymes

Besides, the linear Burk diagram was also constructed by plotting the 1/V against 1/[TMB]. The results are shown in Figure 2. As can be seen in this figure, based on the linear-Burk diagram, a Vmax as high as 67.56 nM min-1 was provided for the oxidase-like Ce/ Fe-bimetallic nanozymes, revealing their high catalytic activity.Besides, the Km value was found to be about 0.067 mM, revealing the high affinity of the oxidase-like Ce/Fe-bimetallic nanozymes to TMB. It is notable that the results of the non-linear Michaelis-Menten curve and linear Burk diagram are in good agreement with each other. Moreover, Vmax/Km was calculated as a crucial indicator of enzyme catalytic efficiency, the results revealed a ratio of 1.0×10-3 min-1 from the linear-Burk diagram which is close to that of the results of the non-linear Michaelis-Menten curve. It is notable, that the summary of the kinetic parameters of the oxidase-like Ce/Fe-bimetallic nanozymes provided from linear-Burk diagram and non-linear Michaelis-Menten curve are represented in Table 1.

Figure 2: The linear-Burk diagram of the oxidase-like Ce/Fe-bimetallic nanozymes.

Table 1: Kinetic parameters of the oxidase-like Ce/Fe-bimetallic nanozymes provided from the linear-Burk diagram and non-linear Michaelis-Menten curve

Conclusions

Herein, kinetics parameters, K_m and V_max, of oxidase-like Ce/ Fe-bimetallic nanozymes toward 3,3′,5,5′-tetramethylbenzidine oxidation were determined. The Michaelis–Menten kinetic model was used for the evaluation of the kinetic of the nanozyme-mediated oxidation of 3,3′,5,5′-tetramethylbenzidine. To provide the quantitative and accurate values of kinetics parameters, K_m and V_max, the linear plot of Lineweaver–Burk was constructed. The results exhibited a V _max as high as 67.56 nM min-1 for the oxidase-like Ce/Fe-bimetallic nanozymes. Besides, K_m was found to be as low as 0.06 mM for the as-prepared nanozymes, revealing the high affinity of the nanozymes toward 3,3′,5,5′-tetramethylbenzidine. Moreover, the ratio V_max/K_m was estimated as a reliable index of catalytic efficiency of the nanozymes, revealing a high value of 1.0×10^-3 min-1.

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