Construction Of Biosensing System Based On Enzyme Cascade Reaction

Natural enzymes are the molecular recognition elements in biosensing,and have unique advantages over other catalysts such as mild conditions,high catalytic efficiency,and strong specificity.At the same time,it is easily inactivated and expensive,which makes the use of natural enzymes greatly limited.Enzyme mimicking have the same catalytic properties as natural enzymes,and they are favored by many researchers because of their low cost and easy availability.However,the synthetic process of mimic enzyme materials is cumbersome and the mimic enzyme cascade reactions needs to be further strengthened.The traditional enzyme cascade reaction is limited to the product of the previous enzymatic reaction is the substrate of the subsequent enzymatic reaction,which makes the enzyme cascade reaction is very limited in the practical application process.In this paper,a novel enzyme cascade reaction system based on biological enzymatic reactions to produce mimic enzymes was studied,which broke through the limitation of product/substrate and provided a broad space for the application of cascade reactions.The specific research contents are as follows:?1?Prussian blue nanoparticles?PBNPs?synthesized by natural enzymes acid phosphatase?ACP?trigger reactions has mimic peroxidase activity,and it can catalyze the oxidation of classical chromogenic substrate TMB in the presence of H₂O₂.PPi can strongly bind Fe³⁺and hinder the formation of PBNPs,which further affect the oxidation of substrates.Under acidic conditions,PPi can be hydrolyzed by acid phosphatase?ACP?,and the product after hydrolysis has no significant effect on the production of mimic enzymes.Based on this principle,we have established a novel enzyme cascade reaction sensing system and apply it to ultra sensitive detection of ACP.?2?Mercury ferricyanide?Hg₂[Fe?CN?₆]?synthesized by natural enzymes ACP trigger reactions has high mimic peroxidase activity,it can catalyze the oxidation of substrate TMB at lower H₂O₂ concentrations.At the same time,experiments have found that ACP hydrolysis of PPi reaction can trigger the generation of the enzyme mimicking Hg₂[Fe?CN?₆].Based on the coupling of bio-enzymes and nano-materials enzyme mimicking Hg₂[Fe?CN?₆],we have established a multifunctional enzyme cascade reaction sensing system.This multifunctional enzyme cascade reaction sensing system has dual signal amplification and can be used for highly sensitive detection of glucose and ACP.The absorption intensity of reaction solution increased with the increase of the ACP concentration,and the absorbance variation was linearly related to the ACP concentration in the range of 0.03-50 U/L.The detection limit was 0.007U/L.?3?Under visible light irradiation,the common small molecule material potassium ferricyanide K₃[Fe?CN?₆]has the activity of simulating horseradish peroxidase?HRP?and can oxidize the chromogenic substrate TMB.PPi can strongly inhibit the mimic enzyme activity of K₃[Fe?CN?₆],which leads to the decrease of the oxidation degree of the substrate and the absorbance of the solution.Under alkaline conditions,alkaline phosphatase?ALP?can regulate the activity of K₃[Fe?CN?₆]mimetic enzyme by hydrolyzing PPi reaction.We have established a new type of enzyme cascade reaction sensing system and used it for immunoassay to detect carcinoembryonic antigen?CEA?.