Fabrication And Analytical Application Of Efficient Enzyme Immobilization Based On Metal-Organic Frameworks And Their Derivative Materials

The high selectivity,excellent catalytic efficiency and specificity of enzymes under relatively mild conditions made it valuable for applications in the detection of complex substance.However,the three-dimensional structural state of enzymes is maintained by thermodynamically unstable interactions（such as intermolecular hydrogen bonds,hydrophobic and electrostatic interactions）,resulting in sensitivity to the external environment and thus greatly limiting their practical application.Enzyme immobilization,especially the use of carriers,could stabilize the dynamic structure of the enzyme and minimize external interference.Comparing with the low specific surface area and poor binding capacity of conventional materials,metal-organic backbone materials（MOFs）with flexible and adjustable graded porous structures were considered ideal carriers for enzyme immobilization.Regrettably,MOFs-based enzyme immobilization methods also exhibited shortcomings,such as enzyme shedding in surface immobilization strategy,diffusion through macroporous pores,irreversible damage of enzyme structure by tight domain-limiting and hydrophobic interaction in pore channels,which in turn led to low enzymatic efficiency and negative enzyme stability.Therefore,how to construct an efficient immobilization system with high loading capacity and friendly to enzyme structure was crucial in the practical application of enzymes.This thesis focused on the design of immobilization strategy and the development of ideal carrier for enzyme immobilization,aiming to solve the major issues with MOFs immobilized enzyme technology,including easy enzyme detachment,poor maintenance of enzyme structure,and low enzyme reaction efficiency,realizing the performance improvement of immobilized enzyme system and reducing the cost of enzyme usage.The main research contents are as follows:（1）A simple and efficient cascade enzyme co-immobilization strategy was proposed to address the problems of enzyme shedding and low enzyme loading in the surface immobilization of MOFs.This strategy tightly co-immobilized glucose oxidase（GO_X）and horseradish peroxidase（HRP）on a dual carrier consisting of PCN-222 and graphene oxide（GO）by introducing single-stranded DNA（ssDNA）with Janus properties as a linker.Mechanistic studies have shown that the enzyme is immobilized byπ-πstacking between the nucleic acid bases of ssDNA and GO and by the Zr-O-P coordination of the sugar-phosphate backbone with the metal zirconium cluster in PCN-222.The introduction of ssDNA reduced enzyme leaching and increased the affinity of the immobilized enzyme for the substrate;the use of dual carriers provided more enzyme binding sites;and the excellent electrical properties of GO increased the electron transfer rate in the enzymatic reaction.Therefore,the cascade enzyme co-immobilized on the dual carrier by ssDNA showed strong physicochemical stability and reusability.The prepared immobilized enzyme was successfully used for highly selective detection of glucose with a wide linear range（50-750μM）.Moreover,the immobilization strategy was versatile and flexible adjustable,and could be widely used in different fields such as biosensing and industrial biocatalysts by changing the type of MOFs and enzymes.（2）A MOFs-derived hollow layered double hydroxide（ZnCo-LDH）-based encapsulation enzyme strategy was developed to address the problem of low enzymatic activity due to low degrees of freedom,high mass transfer resistance and structural changes in MOFs encapsulated enzyme.Under mild and controlled reaction conditions,the enzyme-DNA conjugates were pre-encapsulated in ZIF-L using biomineralization to synthesize a digestible self-sacrificing template,and then the hollow ZnCo-LDH was formed by etching the template with Co（NO₃）₂ to trap the biological enzyme inside,achieving in situ encapsulation of the enzyme.Benefiting from the protective and friendly microenvironment constructed by the hydrophilic hollow structure of ZnCo-LDH,the encapsulated enzyme maintained the biological structure of the nearly natural enzyme and obtained enhanced stability and reusability,retaining about85%of the initial enzymatic activity after 10 cycles.In addition,ZnCo-LDH had excellent electrical properties and an abundant mesoporous structure,which greatly facilitated the electron and mass transfer in the cascade reaction.Therefore,ZnCo-LDH@DNA@GO_X&HRP showed nearly 5-fold higher catalytic efficiency than free and other porous materials（e.g.,COF,ZIF）encapsulated enzymes.This work broadened the options of enzyme encapsulation methods and carrier types and provided a reference for the design and development of controlled synthesis of enzyme-friendly encapsulation systems under mild conditions.（3）To address the problem of low catalytic efficiency of the nanozyme-natural enzyme cascade system,a nanomaterial with desirable carrier properties and mimicking enzyme activity was designed for enzyme immobilization.Based on the concept of"pre-encapsulation-post-etching"in the previous chapter,ZIF-67 and Cu（NO₃）₂ were used as the sacrificial template and etchant to provide variable-valence transition metals,respectively.Enzyme-friendly CuCo-LDH nanocage with mimetic enzymatic activity was fabricated as the encapsulation carrier for cholesterol oxidase（Ch O_X）to construct a natural enzyme-nanozyme cascade system.This cascade system exhibited higher enzymatic activity than the ZIF-67 encapsulated Ch O_X-HRP cascade system.The effects of CuCo-LDH nanozyme on the enzymatic activity were investigated by various characterization techniques in terms of nanozyme properties,the conformational relationship between enzyme and carrier,and enzymatic reaction kinetics.The results showed that CuCo-LDH nanozyme,with high stability,excellent substrate affinity and specificity,was a good substitute for natural enzyme;In addition,CuCo-LDH possessed has large cage-like cavities,suitable mesoporous structure and good hydrophilicity,which was beneficial for the retention of enzyme structure and substrate/product transfer,and its excellent electrical properties can accelerate electron transfer in enzymatic reactions,making it an ideal carrier for immobilized enzymes.Therefore,a dual-mode colorimetric and electrochemical detection platform for cholesterol was established based on the cascade catalysis of Ch O_X and CuCo-LDH with a wide detection range（colorimetric:0.09-1.0 m M;electrochemical:5-500μM）and low detection limits（colorimetric:0.01 m M;electrochemical:2.55μM）,which can be used for highly sensitive analysis and detection of cholesterol in human samples with good recoveries.This study provided a new perspective for the construction of efficient natural enzyme-nanozyme cascade catalytic system.