Fabrication And Analytical Application Of Immobilized Enzymes Based On Metal-organic Frameworks

The three-dimensional structure of the enzyme molecule endowed the enzyme with extremely high specificity and catalytic activity,so the enzyme-based analytical method can achieve high selectivity and high sensitivity detection of the substrate under mild conditions.The cost severely restricted its application in the field of analysis.Metal-organic frameworks（MOF）,composed of organic linkers and metal ions or clusters,exhibit high surface area,large yet adjustable pore sizes,functionalisable pore walls,and diverse architectures.which greatly made MOF an ideal platform to host enzymes.Encapsulation of enzymes in MOF protected the enzyme from external stimuli and allowed selective delivery of substrates and products through the regular pore structure of the MOF,thereby realizing the stable enzymatic catalysis and expanding the application of enzyme-based catalysts.However,there were still some problems in the study of MOF-enzyme composites,such as the enzyme leakage in the traditional separation method of MOF-enzyme composites,the unclear interaction mechanism between MOF and enzymes,relatively low enzymatic activity,and the fragility of enzyme molecules originated from protein backbone,etc.This disertation aimed at addressing the challenges and difficulties in the development of MOF-enzyme composites.Based on the structural characteristics of MOF and enzymes,a series of novel MOF-enzyme composites have been constructed by combining doping,modification and self-assembly methods to achieve the detection of a variety of targets.This disertation provided theoretical support and practical exploration for the design and construction of high-performance MOF-enzyme composites.The main research contents are as follows:（1）A general method for loading MOF-enzyme composites onto magnetic particles was proposed to address the problems of low controllability and reusability of conventional MOF-enzyme composites.This method combined DNA-mediated immobilization（DDI）technology with a"post-encapsulating"strategy.The specific recognition function of DNA and its negative charge properties were used to immobilize the enzyme and modulate the surface charge of the magnetic carrier,inducing in situ encapsulation of the enzyme molecule on the surface of the magnetic carrier by zeolite imidazole ester framework-8（ZIF-8）.The mechanism of in situ encapsulation was discussed in detail,and it was found that the enzyme molecule was the key factor for the nucleation of ZIF-8 on the carrier surface,while the DNA molecule mainly played the role of accelerating the growth of ZIF-8.The prepared immobilized horseradish peroxidase（HRP）showed the superior thermal,chemical and physical stability.After 10 cycles of catalysis,the in situ encapsulated HRP retained84%of its initial activity.The glucose oxidase（GOx）-HRP cascade immobilized enzyme system was further constructed to optimize the cascade enzyme ratio using the programmability of DNA and applied to glucose assay,showing high selectivity and wide linear range（25-500μM）for glucose.This work realized the combination of MOF-enzyme material with magnetic material.（2）A simple method to enhance the catalytic activity of MOF-enzyme composites was proposed based on the understanding of protein-MOF interactions for two major reasons for the reduced activity of MOF-enzyme composites:high mass transfer resistance caused by the smaller pore size of MOF and enzyme conformational changes caused by the interaction between MOF interface and enzyme.Short-chain polyacrylic acid（PAA）was modified on the surface of the enzyme molecule,and the obtained enzyme-PAA composite was encapsulated with MOF in situ.The effects of PAA modification on MOF crystals in terms of morphology,crystalline shape,coordination state,pore structure and the conformational changes experienced by the modified enzyme molecules during the encapsulation process were investigated by various characterization techniques.The experimental results showed that PAA not only generated mesopores（pore size:15 nm）inside the MOF through competitive coordination,but also suppressed the effect of MOF interfacial interaction on the enzyme conformation.This method was used for the immobilization of HRP,GOx,and cytochrome c（Cyt c）,and proved to be a universal method for immobilizing enzymes.Loading（GOx-PAA&HRP-PAA）@ZIF-L on fiber paper to construct a paper-based sensor for immediate glucose detection was able to achieve naked-eye resolution of glucose droplets in the range of 1-11 m M within 5 min.This work deepened the understanding of MOF-enzyme interactions and provided theoretical basis for constructing highly active MOF-enzyme composites.（3）In order to overcome the fragility of the protein backbone of the enzyme molecule,a hemin-based peroxidase mimetic enzyme（ZIF-L-Hemin）was constructed based on the understanding of the chemical structure of the natural HRP active site.The mimic enzyme was assembled from ZIF-L and hemin in aqueous solution,and the hemin molecule was embedded in ZIF-L crystals in an intercalated manner and encapsulated in the internal environment of ZIF-L imidazole-rich.During the catalytic process,the imidazole molecule can play the role of ligating hemin and providing hydrogen bonding sites instead of histidine in the protein backbone,thus enhancing the catalytic activity of hemin.Meanwhile,the ZIF-L-Hemin structure exhibited stable catalytic activity under different environmental conditions due to its better stability than the natural enzyme.Based on the coordination ability of uranium(UO₂²⁺)and imidazole,ZIF-L-Hemin was applied to colorimetric sensing of UO₂²⁺in aqueous solution with a wide linear range（0.25-40μM）and low detection limit（0.079μM）.In the spiked recovery detection of UO₂²⁺in four actual water samples,the spiked recoveries ranged from 90.6%to 105.3%with RSD≤7.0%,showing good accuracy and sensitivity.The novel mimetic enzyme is expected to be applied to the detection of radioactive elements in environmental water samples.