Oxidase-Mimicking Properties Of Low-Valence State Manganese-Based Nanostructures And Their Application In Biomolecule Determination

Natural enzyme is a kind of biological macromolecules with high catalytic activity and substrate specificity.The inherent drawbacks of natural enzymes including poor stability as well as difficulty in extraction and high cost greatly hampered their wide applications.As a new generation of artificial enzyme,nanozyme is defined as nanomaterials with the characteristics of natural enzyme.Currently,it has been developed to simulate the structure and function of enzyme.Oxidase mimics are a kind of nanomaterials that can simulate the catalytic activity of natural oxidases.The reported oxidases have been applied in analytical chemistry,cancer therapy,environmental protection and anti-bacteria.Due to the adjustable catalytic activity,simple preparation and low cost,manganese-based nanomaterials with oxidase-like activity have gradually emerged in the fields of sensing,biomedicine and pollutant removal in recent years.However,most of the related reports focused on the oxidase-like properties of MnO₂ and Mn₃O₄ nanostructures.Therefore,the types and applications of manganese-based oxidase mimics are limited.In addition,the studies on the affinity between low-valence manganese-based oxidase mimics and catalytic substrate/activity of nanostructures are lacking.Therefore,this paper systematically investigated the oxidase-like properties of low-valence manganese-based nanostructures and their applications in biomolecule detection.Manganese carbonate nanoparticles（MnCO₃NPs,+2）with intrinsic oxidase activity were synthesized by hydrothermal method.The composition,morphology and structure of the samples were characterized by various methods.The oxidase-like activity and catalytic mechanism using 3,3’,5,5’-tetramethylbenzidine（TMB）as the substrate were explored.The relationship between MnCO₃NPs and substrate TMB affinity and catalytic activity was explored by combining reaction kinetics and Michaelis-Menten equation.In addition,a freshly sensitive and reliable glutathione in serum colorimetric method was developed based on glutathione regulating MnCO₃ oxidase-like activity.The linear range and detection limit are 0.5～18μM and 0.3μM（3σ/slope）,respectively.The experimental results show that MnCO₃ has excellent affinity for TMB,which may stem from its rich Mn²⁺,K_m=7.30μg/ml.However,the low V_max(1.29×10-8 M·s^-1)of MnCO₃ triggers our interest in studying the effects of Mn³⁺on the catalytic activity and substrate affinity of nanostructures.Therefore,we have prepared lamellar hydroxyl manganese oxides（MnOOH HNFs,+3）by hydrothermal method,and discussed the role of Mn³⁺in the catalytic process of MnOOH using TMB as substrate.However,the introduction of reactive cysteine into the system will inhibit the catalytic activity of Mn³⁺.Therefore,the colorimetric analysis of cysteine in plasma can be realized based on this principle.The linear range and detection limit are 0.05～18μM and 0.1μM（3σ/slope）,respectively.It was found that compared with MnCO₃,MnOOH exhibited oxidase-like activity in a wide p H range（2.6～7.6）and could catalyze the oxidation of TMB in neutral environment.The experimental results show that the catalytic activity of MnOOH was stronger than that of MnCO₃,while the substrate affinity was weaker than that of MnCO₃.Based on the above studies,we speculate that Mn²⁺may enhance the affinity of the substrate,and Mn³⁺may improve the catalytic activity.In order to verify the above-mentioned hypothesis,we synthesized Mn₃O₄ hexagonal nanosheets（Mn₃O₄ HNSs）with mixed valence of Mn²⁺/Mn³⁺via hydrothermal method.TMB was used as the substrate to study its oxidase like properties and its potential catalytic mechanism.The experimental results demonstrate that the Mn₃O₄ oxidase mimic with mixed valence of Mn²⁺/Mn³⁺has K_msimilar to MnOOH,but catalytic activity was weaker than that of MnOOH.It was found that dopamine could regulate the activity of Mn₃O₄ HNSs oxidase-like mimics.Based on this principle,a newly reliable dopamine in serum colorimetric detection approach was constructed,and its linear range was 3～60μM,and the detection limit is 0.3μM（3σ/slope）.The difference of catalytic activity of three kinds of oxidases could be attributed to their ability to produce hydroxyl radicals.Three manganese-based oxidase-like mimics were compared,Oxidase-like activity:a(Mn³⁺)>a(Mn²⁺/Mn³⁺)>a(Mn²⁺),substrate affinity:K_m(Mn³⁺)≈K_m(Mn²⁺/Mn³⁺)≈2K_m(Mn²⁺).