Design Of Integrated Nanoenzyme For Colorimetric Sensing Applicationy

Enzymes are powerful biocatalysts and play an important role in industrial processes,biosensors and biofuel cells.However,the fragility and the relatively narrow application range severely limit the wide application of enzyme systems.These disadvantages can usually be overcome by enzyme immobilization.The immobilized enzyme provides a convenient method to isolate the enzyme from the product and improve the stability of the enzyme,thereby helping to efficiently recover and reuse expensive enzymes and reduce production costs.Many effective carriers have been used to immobilize natural enzymes to construct artificial multi-enzyme systems and have achieved excellent success.However,the natural enzymes are still very expensive.In recent decades,nanozymes with the characteristics of high stability and low cost,have become substitutes for natural enzymes.However,the selectivity of most nanozymes is very limited.By encapsulating natural enzymes and nanozymes in a nanoscale framework,the design of integrated nanozymes?INAzymes?can overcome the limited selectivity of nanozymes.Many INAzymes have been developed and used for multiple purposes.Among them,metal-organic frames?MOFs?are porous crystalline materials with large specific surface area,uniform cavities and high enzyme encapsulation capacity,and some iron-based/copper-based MOFs have been proven to have good peroxidase activity.Therefore,MOFs are used to design and synthesize a variety of INAzymes with excellent properties through adsorption,covalent bonding,pore encapsulation and co-precipitation.Despite the wide range of applications,there are still some problems to be solved.For example,zeolite imidazolate frames?ZIF?-base INAzymes have poor chemical stability;chemical covalent immobilization synthesis method is cumbersome and enzymes are easily inactivated;micropore MOFs result in low substrate mass transfer efficiency and long response time.Therefore,it is still meaningful to design more novel INAzymes with excellent stability,simple synthesis method and high cascade catalytic efficiency.In this thesis,we use different methods to immobilize glucose oxidase?GOx?in enzyme-like activity MOFs carriers,design three different INAzymes,and investigate and evaluate their performance.Based on the cascade reaction,the INAzymes colorimetric sensor detection platform was constructed,which was applied to the one-step rapid detection of glucose content in serum.1.ZIF-8 has become one of the most typical examples of nanostructures for multi-enzyme immobilization due to its economical,mild and easy synthesis process.However,ZIF-8nanocrystals are easily decomposed under acidic conditions.To solve this problem,the Fe-polydopamine?Fe-PDA?was bonded with ZIF-8 surface to form ZIF-8@Fe-PDA hybrid shell with good stability.GOx@ZIF-8 was synthesized by one-pot method,then GOx@ZIF-8was combined with Fe-PDA to synthesize GOx@ZIF-8@Fe-PDA INAzymes.Fe-PDA not only prevents acid degradation,but also acts as a simulated HRP.In the INAzymes system,glucose is converted to gluconic acid by GOx in the presence of oxygen to produce H₂O₂ as an intermediate.The H₂O₂ reacts rapidly with Fe-PDA to generate·OH,which oxidizes3,3',5,5'-tetramethylbenzidine?TMB?.The UV absorbance of oxidized TMB is directly proportional to the glucose concentration,and has a good linear relationship in the range of5.0-100.0?M glucose with detection limit of 1.1?M.The INAzymes system has been successfully applied to rapid colorimetric detection of blood glucose levels.The INAzymes system exhibits high catalytic activity,excellent sensitivity,and enhanced chemical stability,playing great promise in clinical diagnosis and biosensing.2.A MOFs of type Fe-BTC?where BTC is 1,3,5-benzenetricarboxylic acid?was utilized to construct an integrated system for cascade colorimetric determination of glucose.The MOFs performs a dual function in acting?a?as a horseradish peroxidase?HRP?mimic,and?b?as a solid support for immobilization of GOx.The MOFs was prepared by a one-pot method.Glucose is consumed while H₂O₂ is produced during the enzymatic oxidation by GOx.In the presence of H₂O₂,the HRP mimic catalytically oxidizes TMB to form a blue-green product.The absorbance of oxidized TMB?measured at 652 nm,UV-Visible?increases linearly in the of 5.0–100?M glucose concentration range,and the detection limit is 2.4?M.The GOx@Fe-BTC was successfully applied to the determination of glucose in blood.3.MOFs of uniform porosity and large surface areas,play an important role as carriers of immobilized enzymes.However,drawbacks,such as long response times or enzyme leakage have hindered applicability.Herein,we report on a boronic acid-functionalized hierarchically porous MIL-88B?HP-MIL-88B-BA?as an efficient immobilization carrier for GOx.HP-MIL-88B-BA features a hierarchical,porous structure with sufficient recognition sites that facilitate GOx immobilization and prevent enzyme leakage.The hierarchical porosity increases the mass transfer efficiency of the substrate,thereby reducing the response time.Moreover,HP-MIL-88B-BA can be used as a HRP mimic.Based on these factors,an integrated nanozyme was constructed for rapid one-step detection of glucose.GOx@HP-MIL-88B-BA has a rapid response to glucose?10 min?and displays a good linear relationship in the range of 2-100?M with a detection limit of 0.98?M.This methodology provides a straightforward,rapid,and efficient strategy for the synthesis of an integrated nanozyme for the purpose of biosensing.