Preparation Of The NU-1000 Films Functionalized Electrode For Regeneration Of NADH Cofactor

Enzymatic reaction is a kind of reaction using oxidoreductases as the biocatalyst,it has the advantages of high selectivity and can be carried out under mild reaction conditions.Enzymatic reaction shows extensive application potential in fine chemicals production,drug synthesis,etc.However,most oxidoreductases are niacinamide adenine dinucleotide（NAD（H））-dependent dehydrogenases,which needs to consume NADH continuously in the process of enzymatic reaction.Due to the high price of cofactor NADH,it very meaningful to achieve the regeneration of cofactor NADH.Among the methods for cofactor NADH regeneration,electrochemical method has been widely studied due to the advantages of low cost and controllable potential.Electrochemical regeneration of cofactor NADH can be divided into direct and indirect electrochemical approaches.Direct electrochemical requires a high overpotential to achieve the direct reduction of NAD⁺on the electrode,which will not only produce 1,4-NADH with enzymatic activity,but also produce byproducts without enzymatic activity.Indirect electrochemical regeneration of cofactor NADH refers to the introduction of mediator in the direct electrochemical system to reduce the applied overpotential,and only 1,4-NADH is produced to participate in the enzymatic reaction.Currently,（2,2’-bipyridine）（pentamethylcyclopentadienyl）-rhodium complex is the most efficient non-enzyme catalyst for NADH regeneration.Fixing the Rh-complex on the electrode is a key strategy to achieve efficient electrochemical regeneration of cofactor NADH,it can not only realize the recycling of the Rh-complex,but also avoid the deactivation of mediator caused by direct contact with the redox enzymes.At present,Rh-complexes are usually fixed on the electrode surface by polymer bonding,π-πinteraction,covalent bond etc.However,above immobilization methods still have problems such as low grafting amount or uncontrolled loading of catalyst on the electrode surface.Although the loading can be increased by using porous carbon nanotubes,the diffusion limitation caused by the irregular porous structure will also limit the regeneration efficiency of cofactor NADH.Thus,fixation of Rh-complex in 3D ordered porous electrode structure is expected to rationally regulate the loading concentration of catalyst and achieve efficient and controllable regeneration of cofactor NADH.As a kind of Zr-based Metal-organic frameworks（MOFs）,NU-1000 has attracted the attention of researchers due to its advantages such as ordered mesopore structure,abundant chemical grafting sites and high stability in aqueous phase.NU-1000 is an ideal carrier for constructing ordered functional electrodes in enzyme electrocatalysis system.However,the limited electrical conductivity of NU-1000 limits its application in the field of electrochemistry.In order to solve the above problems,NU-1000 thin film is growing perpendicular on the surface of glassy carbon（GC）electrode was made.it’s feasibility in electrochemical application is verified by using redox molecule ferrocene as the model,and the loading of Rh-complex in 3D ordered porous electrode structure is fine-tuned to achieve efficient and controllable electrochemical regeneration of cofactor NADH.The specific research content includes the following two aspects:（1）Preparation and electrochemical characterization of ferrocene functionalized NU-1000 thin film electrode（GC-NU-1000-Fc）Firstly,the carboxyl group（-COOH）functionalization of glassy carbon（GC）electrode was realized by electrochemical oxidation,and GC-NU-1000 thin films were grown on the electrode surface by solvothermal method.Secondly,the GC-NU-1000 thin film electrode was activated with hydrochloric acid to remove the template agent and expose the Zr₆ nodes which could be chemically grafted.Finally,the activated NU-1000 films were functionalized with ferrocene by solvent-assisted ligand incorporation（SALI）.The electrochemical characterization of GC-NU-1000-Fc thin film electrode was made by cyclic voltammetry.The results show that the electrons are transferred in GC-NU-1000-Fc thin film by redox-hopping,and the electrochemical signal is stable.This indicates that the prepared GC-NU-1000 thin film electrode grown perpendicular to the electrode surface has the feasibility of application in electrochemical test,which lays a foundation for the subsequent work.（2）Preparation of Rh-complex functionalized NU-1000 thin film electrode（GC-NU-1000-Rh）for efficient electrochemical regeneration of cofactor NADHGC-NU-1000 thin film electrode was prepared by the method described in Chapter 2.The regeneration performance of coenzyme NADH can be fine-tuned by precise control the Rh catalyst loading.At the highest grafting ratio,the saturated catalytic current reaches 9μA/cm²,which is 7.7 times better than that of the film electrode prepared based on the unmodified bare GC electrode using the same method.At the same time,the GC-NU-1000-Rh thin film electrode with the highest graft ratio was used to regeneration of cofactor NADH for a long time by chronoamperometry（i-t）.The results show that the Faraday efficiency of the regeneration of cofactor 1,4-NADH is 97%,and the turnover frequency（TOF）is～1400 h^-1.This is significantly higher than the highest value of 164 h^-1 in previous studies of this kind.