Studies On Electrochemical Biosensor Based On Nanozymes Cascade Catalytic Amplification

The electrochemical biosensor that combines biosensing and electrochemical analysis technology has been widely used in disease diagnosis,environmental monitoring,food analysis and other fields due to the advantages of high sensitivity,good selectivity,strong stability,low cost,and rapid detection in complex systems.The introduction of a variety of signal amplification technologies in electrochemical biosensors is an indispensable means to improve the sensitivity of biosensors and realize the sensitive detection of low-content biomolecules.Among them,nanozymes with unique advantages such as low cost,high stability,reusability and adjustable catalytic activity,has became an ideal substitute for natural enzymes,which make the nanozymes cascade catalytic amplification become an effective way to fabricate electrochemical biosensors with high catalytic efficiency.In this paper,the enzyme cascade catalytic activity is regulated from two aspects:the spatial distribution of cascade enzymes(such as enzyme spacing and enzyme ratio)and substrate/intermediate mass transfer,and combines multiple nucleic acid signal amplification strategies to further improve the detection sensitivity.The experimental results show that the constructed electrochemical biosensors possess high sensitive and catalytic efficiency for biomolecules detection.The specific works are as follows:1.DNA nanopillar as scaffold to regulate ratio and distance of nanozymes for efficient cascade catalytic platformThe cascade enzymes in multi-enzyme system with rational spatial arrangement is crucial to obtain highly efficient cascade catalysis.However,some traditional DNA scaffolds,such as DNA origami,DNA tetrahedrons and DNA nanotweezers,only focused on single factor regulation of enzyme ratio or enzyme spacing,in which optimal enzyme ratio and spacing couldn't be obtained at the same assay system,resulting in ineffective diffusion of intermediate in enzyme cascade processes with limited catalytic efficiency.To solve above-mentioned problems,using AuNPs(glucose oxidase-like activity)and hemin/G-quadruplex DNAzyme(peroxidase-like activity)as model enzymes,DNA nanopillar was acted as scaffold to control enzyme ratio and continuously regulate of enzyme spacing in the same analysis system,thereby significantly improving the enzyme cascade catalytic efficiency.Specifically,the ratio of AuNPs and hemin/G-quadruplex DNAzyme was adjusted by changing the number of edges in a DNA polyhedron,resulting in an optimal enzymes ratio of 1:4 with a quadrangular prism(QP₄)as the scaffold.In addition,the DNA nanopillar formed by quadrangular prism layer-by-layer assembly was acted as a track for directional and controllable movement of bipedal DNA walker based on toehold-mediated strand displacement reaction,which endowed the assay system with continuous regulation of enzyme spacing compared with traditional enzyme spacing regulation strategies that need to re-construct the biosensing interface for different enzyme spacings,with the advantages of reduced batch-to-batch variation,flexible operation,strong controllability and saving time.As a proof of concept,the proposed dual regulation strategy of cascade enzymes was applied in the sensitive electrochemical detection of Pb²⁺with a wide linear range of 20 nmol·L^-1 to 10 mmol·L^-1 and a low detection limit of 5.5 nmol L^-1,which provides a new route to fabricate high-performance artificial enzyme cascade platform for ultimate applications in bioanalysis and biodiagnostics.2.Electrochemical biomolecule detection based on the regeneration of high-efficiency cascade catalysis for bifunctional nanozymesCommonly,the reported nanozymes are used to mimic the individual enzyme activity,and integrated with other coenzymes to construct cascade reaction to improve the sensitivity of the assay system.However,the intermediates produced by the first enzyme needs to be transported to the coenzyme,and this process inevitably produces ineffective diffusion and side reactions,resulting in limited enzyme cascade catalytic efficiency.To avoid the above-mentioned challenges,In this work,nanozymes with two catalytic functions(?-cyclodextrin functionalized gold nanoparticles,?-CD@AuNPs)was used as the sole catalyst to trigger one-step cascade reaction in the presence of glucose,and the intermediates produced in this process didn't need to transport to the coenzyme but was consumed by itself in situ,leading to effective cascade catalytic amplification for ultrasensitive 8-hydroxy-2?-deoxyguanosine(8-OHd G)detection with a linear range of 20 nmol·L^-1to 10 mmol·L^-1 and a detection limit as low as 30 fmol·L^-1.Interestingly,the MB-L₂-Azo could be released from the hydrophobic cavity of?-CD under10 min UV light irradiation,which endowed the assay system with more desirable regeneration ability compared with traditional regenerated strategies that introduced foreign substances with inflexible and time-consuming operation.Overall,the proposed strategy provides a new reusable approach with eco-friendly and simply operation for a wide range of applications in biocatalysis and bioassays.3.A high-efficiency enzyme cascade catalytic platform for miRNA-21 electrochemical detection based on the COF capsule with adjustable pore size to encapsulate nanozymeEncapsulation of proteases in the uniform pores of metal-organic frameworks(MOFs)can avoid them being exposed to harsh environments.This strategy provides a suitable microenvironment to protease with enhanced stability.However,the free conformation of the protease is hampered due to the small pore size of MOF,which limits the diffusion efficiency of substrates and intermediates,thereby resulting in reduced enzyme catalytic efficiency.In this work,MOFs were used as sacrificial templates to construct hollow covalent organic framework(COF)capsules for bifunctional nanozyme(hemin-AuNPs)encapsulation,improving the stability and catalytic activity of encapsulated enzymes simultaneously.The cascade reaction started when AuNPs catalyzed glucose to gluconic acid with the concomitant formation of hydrogen peroxide(H₂O₂).The produced H₂O₂ was confined in the cavity of COF capsule for in-situ oxidation reaction of hemin,which accelerated the mass transfer of substrate/intermediates,leding to enhanced enzyme cascade activity.More importantly,the catalytic efficiency of the enzyme cascade reaction could be effectively controlled by changing the pore size of COF capsule benefiting from improved mass transfer rate of the substrate,thereby realizing the regulation of the catalytic efficiency of the enzyme cascade reaction.In addition,COF capsules showed good stability,avoiding the disadvantages of MOF structural collapse in an acid or alkali environment.Under the optimal nanozymes cascade catalysis,a novel electrochemical biosensor was constructed for sensitive detection of miRNA-21,with a detection range of 2.5 pmol·L^-1?25 nmol·L^-1 and a detection limit of 0.8 pmol·L^-1.As a proof of concept,this research provides impetus for the development of a new generation of high-performance nanozyme cascade platform,which hold great potential in biotechnology,bioanalysis and disease diagnosis.