Construction Of Biomolecular Self-assembled Oxidase Mimicking Enzyme And Its Detection Research On Application

Natural enzymes are characterized by high catalytic efficiency,strong substrate selectivity and mild catalytic conditions,and are involved in almost all metabolic processes in cells.The excellent properties of natural enzymes have led researchers to develop artificial analogues based on their structure and catalytic function.Through the aggregation of fluorene methoxycarbonyl groups（Fmoc）,Fmoc-modified amino acids have excellent self-assembly ability.In this paper,a highly active supramolecular oxidase catalyst was constructed by Fmoc-amino acid and Cu²⁺coordination assembly,and the mimic enzyme structure design,performance mechanism research and detection application exploration work were carried out.The main research contents are as follows:（1）In this paper,copper-dependent molecular oxidase mimics were used as probes to achieve selective and sensitive detection of amino acids by colorimetry.The oxidase mimic is self-assembled from commercially available guanosine monophosphate（GMP）,Fmoc-lysine,and Cu²⁺to catalyze the oxidation of 2,4-dichlorophenol（DCP）to a red product with maximum absorbance at 510 nm.The concentration and type of amino acids will reduce the catalytic activity of the mimic enzyme to varying degrees,thereby reducing the change of color and absorbance in the reaction,most significantly in the presence of histidine,and the limit of detection（LOD）is 6.4 n M.Theoretical simulations and experimental results（UV-Vis spectroscopy,fluorescence emission spectroscopy,and electron paramagnetic resonance spectroscopy）indicate that amino acids may participate in the coordination of copper centers and change the structure of the mimic enzyme activity centers.The sensing platform is characterized by fast response,simple fabrication,high sensitivity/selectivity,and is also suitable for the detection of other biomolecules such as nucleotides.（2）NAD⁺/Fmoc-K/Cu²⁺complexes with oxidase activity were prepared by using NAD⁺and Fmoc-lysine as ligands and assembling with Cu²⁺coordination.In the presence of oxygen,Cu²⁺/Fmoc-lysine catalyzes the oxidation of NADH to form NAD⁺,which binds to Cu²⁺/Fmoc-lysine to enhance the catalytic activity of Cu²⁺.By introducing glucose dehydrogenase and glucose,the cycle of NAD⁺/NADH is realized,and gluconic acid with industrial value is generated.The binding mode of ligands to metal ions and the regulatory mechanism of catalytic ability were explored,which provided research ideas for the efficient construction of mimic enzyme activity centers.