Peroxidase Like Activity of Quinic Acid Stabilized Copper Oxide Nanosheets

Abstract

Water-soluble copper oxide nanosheets (CuO nanosheets) were synthesized via a chemical reduction method and used as peroxidase enzyme mimetics. The synthesized CuO nanosheets were characterized by using XRD and TEM. The obtained CuO nanosheets in fact possessed an intrinsic enzyme like catalytic activity identical to that found in natural peroxidases, which have applications as detection tools and in water treatment.

Keywords: Water soluble CuO nanosheets; Peroxidase enzyme activity; Quinic acid

Introduction

Enzymes, a kind of proficient biocatalyst play key role in almost all in vivo reactions. Reactions catalyzed by enzymes are of extensive significance due to their high specificity, efficiency and need of mild reaction conditions. Additionally, enzyme based analysis has a broadscope of applications in various fields such as biochemistry, chemical technologies, clinical diagnosis and in environmental science. On the other hand, the availability of limited natural sources, instability at different temperatures, PH and high cost of purification has made enzymes restricted to limited applications. Hence there is a need for the construction of new stable, temperature and PH resistant, low cost materials which can replace the natural enzymes in their function and properties [1]. However, various enzyme mimetics have been synthesized for different enzymes such as serine protease [2], hydrolase [3], superoxide dismutase [4], dioxygenase [5,6], lipase [7], nitrile hydratases [8], phosphodiesterase [9-12], aldolase [13], cytochrome P450 [14], ligase [15] and acylase [16].

Now-a-days, much attention has been focused on the development of peroxidase mimetics due to its high importance in enzymatic analysis and in waste water treatment. Several peroxidase mimetics such as metal porphyrin [17], hemin [18], metal hexacyanoferrate [19,20], Schiff base complex [21], metal phthalocyanine [22], hemeatin [23] and carboxyl groups containing mesoporous polymers [24] have been used for enzymatic analysis.

Recently, scientists have proved that the iron oxide magnetic nanoparticles are found to possess intrinsic peroxidase mimetic activity [25]. Later, several nanoscale peroxidase mimetics have been developed to their potential applications [26–39]. The present study describes the intrinsic peroxidase activity of quinic acid stabilized copper oxide nanosheets sheets. Here, we have synthesized the water soluble copper oxide nanosheets and used as peroxidase enzyme mimics.

Chemicals and Materials

Experimental procedure

Copper (II) sulfate, Sodium hydroxide, Sodium borohydride, Quinic acid, Hydrogen peroxide, O-Dianisidine and all other solvents were obtained from Sigma-Aldrich Chemicals, Bangalore.

Preparation of CuO nanosheets

The copper oxide nanosheets were synthesized as per our reported procedure elsewhere [40]. Briefly, equal volume of aqueous NaOH (0.01 M) and NaBH₄ (0.1 M) were added into a beaker containing the same amount of CuSO₄ (0.01 M) and Quinic acid (0.02 M) mixture and stirred vigorously at room temperature (30°C). Reaction was continued for about a half an hour with stirring to obtain copper oxide nanosheets. The product was then purified by repeatedly washing with double distilled water and ethanol which was finally dried at 60°C in hot air oven.

Investigation of peroxidase like catalytic activity of CuO nanosheets

To examine the peroxidase like catalytic activity of the synthesized CuO nanosheets, the catalytic oxidation of o-dianisidine (as colourless peroxidase substrate, reduced form) was tested in the presence of H₂O₂ as oxidizing agent. The oxidation of o-dianisidine was carried out using phosphate buffer (p^H 7.0) in the presence of CuO nanosheets (100 μl) for 200 seconds at 25°C. Later, hydrogen peroxide (10mM) was added to start the reaction. The catalytic activity of CuO nanosheets was carefully measured by the development of colour due to oxidation of o-dianisidine using UV-Visible spectrophotometer at 430 nm.

Kinetic parameters analysis

Steady-state kinetic experiments were done by changing the H₂O₂ concentration from 10 to 50mM at a fixed concentration of o-dianisidine (0.7mM) prepared in phosphate buffer (pH 7.0). The change in the absorbance was measured using a UV-Visiblespectrophotometer in the time scan mode at 430 nm. The catalytic parameters were then obtained by fitting the absorbance data to the Michaelis–Menten equation as,

V = V_max[C]/K_m + [C]

Where V_max is the maximal velocity of reaction, V is the initial velocity and C represents the substrate concentration, and K_m is the Michaelis-Menten constant.

Results and Discussion

Characterization of the copper oxide nanosheets

The copper oxide nanosheets were synthesized via a chemical reduction method. Figure 1 shows the XRD pattern of the produced CuO nanosheets which represents the formation of CuO nanosheets with a monoclinic structure (JCPDS No. 96-410-5686). Peaks in standard X-ray diffractogram of CuO obtained from JCPDS database had been matched with that of the obtained CuO nanosheets to find out any impurity present or not. Figure 1 shows the absence of characteristic impurity peaks in the XRD diffractogram of CuO nanosheets after comparison with standard diffractogram of CuO nanosheets which confirm the formation of pure CuO nanophase. The average crystalline size of the CuO nanosheets was calculated by using Debye-Scherrer formula as about 192 nm.