Synthesis of MnO2 Sub-microspheres with Effective Oxidase Mimic Nanozymes for Colorimetric Assay for Ascorbic Acid in Orange Fruit and Juice

Abstract

Nanozymes as alternative to natural enzymes have the advantages of good catalytic efficiency, high stability, easy storage, low-cost, and the ability to be used under extreme conditions when compared to natural enzymes. Improving the sensitivity of nano-enzymatic assays is highly significant and depends on the nanozyme size, chemical structure and morphology. In the present work, an oxidase-mimicking nanozyme of MnO2 sub-microspheres were synthesized from KMnO4 and MnCl2.4H2O by a one-step hydrothermal method followed by calcination for 2 h at 300 °C. The oxidase-like activity of the sub-microspheres was evaluated by conversion of 3, 3′, 5, 5′-tetramethylbenzidine (TMB, colorless) to ox-TMB (blue color). The kinetic results showed that the catalytic behavior of the MnO2 sub-microspheres follows Michaelis-Menten kinetics with low constant (Km = 0.1 mM) compared to the natural enzyme HRP (Km = 0.434 mM). The catalytic mechanism was deduced that oxidase mimicking is dependent on the formation of O2•− radicals and partially on OH˙ radicals. An efficient colorimetric platform capable of sensitive determination of ascorbic acid was developed based on fading of the blue color of ox-TMB by ascorbic acid. The linear detection range was from 1.0 to 100 µM with a limit of detection of 0.33 µM. The proposed method showed high selectivity and sensitivity for the detection of ascorbic acid. Further applications in real samples have also been successfully demonstrated.