DNA Directed Immobilization Of Enzyme On Functionalized Magnetic Nanoparticles And Their Applications In Biological Analysis

As an effective and selective biocatalyst,enzyme has already been used in various fields such as diagnosis,food industry and biosensors.But the applications of free enzymes are limited because of some significant drawbacks,including instability and difficulties in reuse and recovery.Immobilization of enzymes is a promising strategy to improve the operability of enzymes and offer an opportunity to establish the continuous enzyme reactors.Different immobilization protocols have been studied,such as adsorption,entrapment,covalent bonding,and cross-linking method.Given that there are several advantages and disadvantages associated with the current immobilization methods,plenty of scope remains for the development of effective and reversible techniques for the immobilization of enzymes.DNA directed immobilization(DDI)is a facile and versatile chemical method for specific immobilization of biological molecules based on the specific Watson-Crick base pairing(A-T,C-G)of DNA molecules,which has high selectivity,great reversibility and can reduce the activity loss to enzyme.Although DDI was used for the immobilization of enzyme in pervious reports,the researches about anchoring enzyme on the surface of magnetic nanoparticles through DDI are rare.In addition,further investigations remain to be explored for improving the reusability and fabricating a multienzyme system that allows control of the stoichiometric ratio of the enzymes.In this thesis,we focus on the fabrication of single enzyme or multienzyme system on magnetic nanoparticles through DDI strategy,and study on how to improve the activity and resusibility of immobilized enzyme.This will lay the foundation for preparing the magnetic controlled immobilized enzyme reactors with improved performance,high reusability and great reversibility based on the combination of DDI technique and functionalized magntic nanoparticles.(1)Alkaline phosphatase(ALP)was immobilized on magnetic nanoparticles through DDI strategy.The anchored ALP was confirmed by confocal laser scanning microscopy and several other characterization methods,and the results showed ALP was anchored through DNA hybridization.The strand length of the DNA affected the enzymolysis efficiency of the ALP-DNA-MNPs,with a strand length of 24 bases providing the optimal results.Furthermore,the immobilized ALP exhibited good enzymolysis and high reversibility.The biological activity of anchored ALP was evaluated in an enzyme inhibition assay.The results revealed that theophylline exhibited greater inhibitory activity than L-phenylalanine.The proposed protocol demonstrates a mild,effective and versatile pathway for fabricating enzyme modified nanomaterial and can be applied in the high-throughput screening of inhibitors.(2)We described a mild and versatile method for the preparation of immobilized enzyme reactors by anchoring trypsin on polydopamine(PDA)functionalized magnetic nanoparticles through DDI strategy.This reactor exhibited prominent reusability and enhanced catalytic efficiency.The specificity constant(kcat/Km,where kcat is the catalytic rate constant)of the reactor was 1.82 s-1 ?M-1,which was approximately 4.7-fold higher than free trypsin.Notably,the immobilized trypsin reactors maintained more than 55%of their initial activity after 70 cycles.In addition,the performance of the immobilized trypsin was further investigated by digesting the protein solution of myoglobin and results indicated that the immobilized trypsin could offer higher activity and faster digestion than free trypsin.Given the excellent reusability and high catalytic efficiency of this immobilized trypsin reactors,we believe that this study paves the way for preparing low-cost enzyme reactors for a wide range of applications in biocatalysis,biological diagnosis,and biotechnology.(3)We introduced a versatile strategy for fabricating a multienzyme system by coimmobilizing horseradish peroxidase(HRP)and glucose oxiase(GOx)on magnetic nanoparticles multifunctionalized with dopamine derivatives through DNA-directed immobilization.This multienzyme system exhibited precise enzyme ratio control,high catalytic efficiency,magnetic retrievability,and enhanced stability.The enzyme ratio was conveniently adjusted,as required,by regulating the quantity of functional groups on the multifunctionalized nanoparticles.The optimal mole ratio of GOx/HRP was 2:1.The Michaelis constant Km and specificity constant of the multienzyme system were 1.41 mM and 5.02 s-1 mM-1,respectively,which were approximately twice the respective values of free GOx&HRP.Given the wide variety of possible enzyme associations and the high efficiency of this strategy,we believe that this work provides a new route for the fabrication of artificial multienzyme systems and can be extended for a wide range of applications in diagnosis,biomedical devices,and biotechnology.