Preparation Of Two-dimensional Metal-organic Frameworks And Their Application In Immobilization Of Oxidoreductases

Glucose is a key physiological indicator,which should be measured in food quality analysis and diabetes management.Glucose oxidase(GOx)and horseradish peroxidase(HRP)are multifunctional oxidoreductases,and the dual-enzyme immobilization strategy can not only improve the sensitivity of glucose detection,but also improve the catalytic stability and reusability of the free enzyme.However,the strategy often suffers from low enzyme loading,limited mass transfer rate and high enzyme cost.Therefore,it is crucial to prepare carrier materials with good biocompatibility,high specific surface area,high mass transfer rate,and easy chemical modification.Two-dimensional metal-organic frameworks materials(2D MOFs)have high specific surface area,short diffusion path,abundant exposed active sites,and easy activation modification,which make them potential for application as immobilized enzyme carriers.In this paper,a series of large-size 2D MOFs were prepared by adjusting the solvent ratio to control the morphology and size of 2D MOFs.On this basis,two Cu-based 2D MOFs with large specific surface area and good water stability were selected as the carrier materials and successfully immobilized two oxidoreductases,namely GOx and HRP.The properties of the immobilized enzymes and their applications in glucose detection were investigated.The details are as follows.(1)Preparation of 2D MOFs and their morphology modulation.A series of 2D MOFs nanosheets with large specific surface area and excellent morphology were prepared using Co,Ni,Zn and Cu as metal nodes and porphyrin(TCPP)as organic ligands.The morphology and size of 2D MOFs were tuned by adjusting the reaction solvent ratio.The results showed that Co-TCPP nanosheets increased from 500 nm to about 30 μm;Zn-TCPP nanosheets increased from 2 μm to about 15 μm;Ni-TCPP changed from a nanowire of about 2 μm to a nanosheet of about10 to 20 μm;Cu-TCPP was a ruffled nanosheet of about 7 to 8 μm.Among them,compared with other 2D MOFs,Cu-TCPP has higher biocompatibility,larger size,higher uniformity,and higher stability in water.It has obvious advantages in terms of being immobilized enzyme carrier.(2)Construction and application of strategy for immobilization of GOx/HRP using Cu-based 2D MOFsCu-TCPP nanosheets were used successfully as carriers to immobilize GOx and HRP by two immobilization methods,namely physical adsorption and covalent linkage,to realize the dual enzyme cascade catalytic reaction.The adsorbed enzyme loading was calculated to be up to 187.9 mg/g and the covalent enzyme loading was up to 249.1mg/g.The enzymatic properties of the two immobilized enzymes and the free enzyme were investigated.The results showed that the optimum temperature of the immobilized enzyme was 40°C and the optimum p H was 6.Compared with the free enzyme,the thermal stability of the immobilized enzyme was increased by 20% when incubated at 60°C for 120 min,the mechanical stability was increased by 10% when sonicated for 120 min,and the relative activity in organic solvents such as acetonitrile was increased by about 20%.Based on the results of enzymatic performance investigation,the enzymatic activity of the covalent enzymes was more advantageous than that of the adsorbed enzymes.Immobilized enzyme had better repeatability and storage stability.It could maintainedmore than 50%,and 85% of the initial enzyme activity after 9 cycles and 15 days of storage,respectively.When these immobilized enzymes were applied to glucose assay,the linear detection ranges of the adsorbent enzyme and covalent enzyme were 0.005～1.2 m M and 0.005～1.6 m M,respectively,and their detection limit were 0.001 m M and 0.0006 m M,respectively.(3)Construction and application of strategy for immobilization of GOx by Cubased 2D MOFs nanoenzymesCu and metalloporphyrin TCPP(Fe)were used as the metal node and organic ligand,respectively,to prepare the 2D MOFs nanoenzyme Cu-TCPP(Fe)with peroxidase-like properties.And then the factors affecting the catalytic performance of Cu-TCPP(Fe)-like enzymes were investigated.Cu-TCPP(Fe)was used as a carrier to covalently immobilize GOx,which successfully realized the cascade reaction between the carrier nanoenzyme and the natural enzyme.The strategy reduced the enzyme cost and improving the enzyme catalytic stability.The results showed that the enzyme loading could reach 145.0 mg/g,and the immobilized enzyme had higher temperature and p H tolerance,thermal stability,and mechanical stability compared with the free enzyme.In addition,immobilized enzyme had high repeatability and maintained more than 50% of the initial enzyme activity after 5 cycles.The linear detection range of immobilized enzyme glucose was 0.02～1.6 m M,and the detection limit was 0.0005 m M.