Unlabeled Fluoresence Detection Of Hydrogen Peroxide And Glucose Based On Bifunctional MOF Nanozymes

Natural enzymes are widely used in the fields of medicine,agriculture and chemical industry due to their high catalytic activity,high substrate specificity and sensitive reaction.However,the preparation and purification of natural enzymes cost more,most of the natural enzymes are unstable and easy to inactivate,so people began to pay attention to the research of nanomaterial mimic enzymes.In recent years,the simulated peroxidase activity of MOFs material has been discovered and applied to colorimetric sensing system.However,it is necessary to add chromogenic reagent as the substrate in most colorimetric sensor systems.Most chromogenic agents are harmful to human body due to the presence of aromatic amines.Fluorescent MOF sensors reported in recent years also require additional fluorescent labeling,which makes the sensing system more complex.Therefore,unlabeled nanoscale enzymes with the dual functions of catalysis and emission fluorescence was developed in this paper.The research on the simulation enzyme of dual-function nanoscale materials developed a sensitive,economical and simple new idea for the multi-enzyme cascade analysis.In this paper,two nanomaterials Ni-MOF and MIL-68（Fe）were prepared,and their simulated enzyme activities and applications in the detection of H₂O₂ and glucose were investigated.（1）Two-dimensional（2D）Ni based metal-organic framework（MOF）nanosheets were synthesized by solvent-thermal method.The morphology,composition and structure of functional groups were characterized by scanning electron microscopy（SEM）,powder X-ray diffraction（XRD）and FT-IR spectrum.In the presence of H₂O₂,Ni-MOF can catalyze 3,3’,5,5’-tetramethylbenzidine（TMB）to produce color reaction,which proves that it has the properties of simulated peroxidase.The steady-state kinetic studies showed that the affinity of Ni-MOF nanosheets to 3,3’,5,5’-tetramethylbenzidine（TMB）and H₂O₂ substrates was higher than that of natural horseradish peroxidase（HRP）.The apparent Km of Ni-MOF to H₂O₂ is approximately an order of magnitude lower than that of HRP.Then,Ni-MOF nanosheet was applied to establish an H₂O₂ fluorescence sensor which deserves a wide linear range of 0.1～20 m M（R2=0.9993）and a low detection limit of 7.4×10-5 M.Glucose was transformed into H₂O₂ by glucose oxidase（GOx）catalytic oxidation,which further developed this nano-sensor for the detection of glucose in human serum.Its linear range and detection limit were 8～30 μM（R2=0.9919）and 4×10-6 M,respectively.This method provides a simple,economical and sensitive method for the determination of H₂O₂ and glucose.It has been successfully applied to the actual sample analysis with good reproducibility and high accuracy.（2）MIL-68（Fe）nanometer material was synthesized by solvent-heat method.The morphology,composition and structure of the functional groups were characterized by means of scanning electron microscopy（SEM）,powder X-ray diffraction（XRD）and FT-IR.MIL-68（Fe）was found to have simulated peroxidase activity,and the effects of temperature and p H on catalytic activity were investigated.The study of steady state kinetics shows that the catalytic kinetics of MIL-68（Fe）nanoparticles conforms to the typical Michaelis-Menten equation.The results show that the michaelis constant Km with TMB as the substrate is 15.2,and the michaelis constant Km with H₂O₂ as the substrate is 0.126.The affinity of MIL-68（Fe）nanoparticles to the substrate H₂O₂ is much greater than HRP and the affinity of Ni-MOF nanoparticles to H₂O₂,which theoretically indicates that MIL-68（Fe）nanoparticles can be used for sensitive detection of H₂O₂.