Synthesis of MnO2 sub-microspheres with effective oxidase-mimicking nanozymes for the colorimetric assay of ascorbic acid in orange fruits and juice

Abstract

Nanozymes, as an 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 compared with natural enzymes. Improving the sensitivity of nanozyme-based assays is highly required and depends on the nanozyme size, chemical structure and morphology. In the present work, an oxidase-mimicking nanozyme of MnO₂ sub-microspheres was synthesized from KMnO₄ and MnCl₂·4H₂O via a one-step hydrothermal method followed by calcination for 2 h at 300 °C. The oxidase-like activity of the sub-microspheres was evaluated through the 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 a low constant (K_m = 0.1 mM) compared with the natural enzyme HRP (K_m = 0.434 mM). The catalytic mechanism was deduced, whereby the oxidase-mimicking behavior is dependent on the formation of O₂˙⁻ radicals and partially dependent 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, and the limit of detection was 0.33 μM. The proposed method showed high selectivity and sensitivity for the detection of ascorbic acid. Further applications in real samples were also successfully demonstrated.