Fabrication And Application Of Artificial Multi-Enzyme System Based On DNA Surface Technology And Magnetic Particles

Artificial multi-enzyme systems are immobilized enzymes which combines different enzymes in vitro for cascade catalysis by simulating the metabolic pathway in vivo.Artificial multi-enzyme systems not only inherit the high selectivity,catalytic efficiency and atomic economy of enzymatic cascade reaction systems,but also possess great stability and reusability.Up to now,they have been broadly applied in biochemical analysis,diagnosis,manufacturing and ecological improvement.Nevertheless,there are still some problems in the synthesis of artificial multi-enzyme systems based on traditional methods.Thus,it is urgent to develop new methods to fabricate artificial multi-enzyme systems with stable activity,high versatility and low cost.In this paper,the features of DNA surface technology and magnetic particles are fully combined to develop new strategies for constructing artificial multi-enzyme systems with improved performance.Five aspects are mainly included as follows:(1)Glucose oxidase(GOx)was anchored on magnetic N-doped graphene quantum dots by DNA directed immobilization(DDI)technology to construct a nanozyme-enzyme multi-catalytic system.Magnetic graphene quantum dots not only served as the enzyme support,but also exhibited excellent peroxidase-activity,establishing a tandem catalysis network with GOx.The system demonstrated satisfying bioactivity,renewability and operation stability.Compared with adsorbed enzymes and free enzymes,the multi-catalytic system had better substrate affinity(Km=1.069 mM)and faster reaction rate(Vmax=11.2×10-8 M/s).The multicatalytic system was applied to the detection of glucose in commercial beverages with excellent sensitivity and selectivity.This research suggests an innovative way to construct chemo-enzyme systems which is promosing in biological applications.(2)GOx functionalized with oligo-DNA was immobilized on the surface of magnetic layered double hydroxides(mLDHs)by chemisorption,and a multimodal catalytic platform was constructed for realizing divergent reactions with heterogeneous and biocatalytic steps.The flower-like mLDHs served both as an enzyme support and a peroxidase mimic cooperating with enzymes for tandem catalysis.DNA not only connected GOx with mLDHs stably,but also improved the bioactivity of the whole catalytic platform.The multimodal catalytic platform was used for colorimetric and electrochemical sensing of glucose with broad linear range(5-250 μM for colorimetry and 0.05-2 mM for electrochemistry)and low limit of detection(LOD,0.42 μM for colorimetry and 2.47 μM for electrochemistry).The practical analysis capability of the ultrasensitive sensor was evaluated by detecting glucose in human serum and sweat,showing reliable results,satisfactory recovery and excellent stability.The strategy of combining mLDHs and enzymes with DNA for cascade catalysis provides a universal approach to prepare chemo-enzyme hybrids with high performance.(3)To enhance the stability,thickness-manageable nucleotide coordinated polymers(NCPs)were used to encapsulate GOx which was linked to magnetic particles by DDI technology.By employing magnetic microspheres as the "anchor" of immobilized enzymes and NCPs as the"shield" for enzyme protection,a multi-enzyme catalytic system with"anchor-shield" structure was constructed.The supramolecular layer of NCPs not only reduced the interference of external environment on the catalytic performance of enzymes,but also acted as a peroxidase mimic to cooperate with GOx for cascade reaction.On the premise of good biocactivity,the multienzyme system had excellent physical,chemical and biological stability with great storage stability and reusability.Based on the above properties,a biodegradation method for the removal of phenolic pollutants was established,which showed the broad application prospect of this magnetic "anchor-shield" system in the field of environmental treatment.(4)Based on the previous work,a "flexible segregation" strategy was developed to construct an exquisite multi-compartment multi-enzyme system(MMS)using polyphenol-coated GOx/horseradish peroxidase(HRP)which was orientedly anchored on Janus magnet spheres by DNA.The MMS provided a bionic catalytic network containing a cascade reaction model in confined space for simulating cell activities,which had great bioactivity and stability.In addition,by taking the MMS as a nanomotor,it was found that the asymmetrical distribution of enzymes on Janus particles could enhance the diffusion capability of the multienzyme system by accelerating the enzyme-directed self-driving motion,thus improving the overall catalytic performance(30%and 96%higher than free enzymes and adsorbed enzymes,respectively).The remarkable bioactivity,steadiness and bioavailable of the MMS made it prospective for extracorporeal and endocellular glucose detetction.This work provides a new idea for the design and preparation of high-performance multienzyme systems with multi-compartment structure.(5)On the basis of the above work,a new strategy for the preparation of artificial multi-enzyme system was reported by encapsulating GOx in hemin-bridged metal-organic framework(HMOF)in situ through DNAinduced biomimetic mineralization,which constructed an integrated multizyme mimc combining both nanozymes and natural enzymes.DNA not only accelerated the nucleation of MOF on enzyme surface to improve the encapsulation efficiency and loading amount,but could also regulate the morphology and structure of MOF through competitive coordination.Based on the peroxidase-like activity of hemin and the confinement effect of HMOF on the enzyme,compartmentalization and substrate channelization were realized simultaneously in the integrated multienzyme mimic,showing great biological activity and stability.The integrated multizyme mimc was use for glucose analysis.The range of linearity was 5-500 μM and the LOD was 0.72 μM.This study opens up a new way for the preparation of artificial multi-enzyme systems based on MOF nanozymes.