| Immobilized enzymes not only possess the advantages of high efficiency and specificity of free enzymes,but also have many characteristics,such as good stability,easy separation and recovery,and reusability,which have attracted wide attention in the fields of life science,chemistry and clinical medicine.However,the traditional immobilization methods still have some problems,which make it difficult to give full play to the high catalytic and stability of the enzyme.Therefore,how to construct an immobilized enzyme system with simple and efficient immobilization process,mild reaction conditions,high enzymatic activity,good reusability,and reversible immobilization has become an urgent problem in the field of immobilized enzymes.Therefore,this work focuses on the construction of the novel immobilized enzyme strategies to significantly improve the overall performance of the immobilized enzyme based on magnetic nanoparticles and DNA directed immobilization(DDI)technology.This work mainly carries on the research from the following aspects,(1)Trypsin was immobilized on the surface of three generation PAMAM modified magnetic microspheres by DDI technology,and the immobilized trypsin reactor was applied to the digestion of standard protein.The effects of enzymatic reaction conditions,generation of polyamide dendrimer and DNA length on the activity of immobilized enzyme were investigated.Under optimized conditions,the prepared immobilized trypsin has good substrate affinity and better enzymatic reaction rate,excellent performance of surface regeneration,good reusability and excellent stability.The prepared immobilized trypsin was then applied to the digestion of proteins in different practical samples,and fast digestion speed and high digestion efficiency were achieved by the immobilized enzyme.The immobilized strategy can be extended to prepare the novel immobilized enzyme system with excellent reusibility and high economic value.(2)Under mild conditions,the model enzymes,alkaline phosphatase,horseradish peroxidase(HRP)and trypsin,were successfully immobilized and released on the surface of functional magnetic particles by DNA strand displacement reaction.Compared with the target enzymes released by strong alkali and high temperature,the enzyme activity could be signifieantly retained by DNA strand displacement reaction.At the same time,the immobilized enzymes prepared by DNA strand displacement reaction exhibited good substrate affinity,high enzymatic activity,and excellent stability and reusability.The prepared immobilized trypsin was applied to the analysis of protease digestion,and the high digestion efficiency was achieved in a short time compared with free enzyme.The immobilized strategy developed in this study provides a new way for continuous immobilization of multiple enzymes on the same carrier.(3)Based on the previous research work,the surface modifieation method of magnetic particles was further optimized,and the zwitterion-functionalized magnetic particles with amino,phosphonate,and thiol functional groups were prepared to coimmobilize glucose oxidase(GOx)and HRP by DDI.The hydrated layer formed by ionic solvation of the zwitterionic surface on the surface of magnetic particles could efficiently suppress non-specific adsorption of different kinds of enzymes.The immobilized multienzyme catalyst without non-specifically adsorbed enzymes exhibited excellent performance of kinetic parameters,enzymatic activity,stability,and reusability compared with those of free and non-specifically adsorbed enzymes.The prepared immobilized multienzyme was used for the determination of glucose and exhibited good sensitivity and,selectivity.The prepared strategy provides a new way for designing and preparing a high performance immobilized multienzyme reactor,which can efficiently suppress non-specific adsorption enzyme.(4)A novel magnetic DNA hydrogel encapsulation enzyme system was prepared by self-assembly of GOx,HRP and magnetic nanoparticles using the prepared biotin-functionalized DNA supramolecular structure as a monomer,and the encapsulation enzyme was applied to detect glucose in real samples.Compared with traditional hydrogel encapsulation enzymes,the prepared magnetic DNA hydrogel encapsulation enzymes can be easily separated and recovered from the reaction system under the control of magnetic field,and can better inhibit the leakage of enzymes and magnetic nanoparticles.Because of its good proximity and outer DNA protection,the encapsulated enzymes exhibited better cascade reaction efficiency and kinetic parameters,as well as better stability and reusability.The encapsulated enzymes were used to detect glucose in buffer solution and human serum,respectively,which had the advantages of low detection limit,good selectivity and wide linear range.The developed encapsulated enzyme strategy provides a new way to design and prepare highly controllable DNA encapsulation enzymes.(5)Based on above research,the strategy of enzyme immobilization was further optimized and developed.ZIF-8 metal organic framework material(MOFs)was used as encapsulation enzyme carrier,and Y-DNA scaffold was applied to cross-link GOx and HRP multienzymes.The prepared cross-linked enzymes were then encapsulated into ZIF-8 to prepare encapsulation multienzyme system.Compared with traditional MOFs encapsulation enzymes,the prepared encapsulation enzymes can significantly inhibit the leakage of enzymes from MOFs structure and maintain the overall enzymatic activity of encapsulation enzymes.Compared with free enzymes and encapsulated enzymes without DNA scaffold cross-linking,the well-tailored proximity and colocalization of cascade enzymes by the DNA scaffold network obviously improved the overall catalytic efficiency,enzymatic activity,and kinetic performance.Furthermore,because of the protective effect of extermal MOFs,the encapsulated enzymes exhibited better stability and reusability.This study provides a reference for the denzymes,which can significantly inhibit enzyme leakage and have good esign and preparation of encapsulated enzymatic performance. |
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