Studies On Electrochemical Biosensor Based On Efficient Enzyme Cascade Catalytic Amplification

Electrochemical biosensor is a new analysis model combining biosensing and electrochemical analysis technology.It has the characteristics of high sensitivity,good selectivity,low cost,strong stability,and rapid detection in complex systems,so it has been widely used in clinical analysis and disease diagnosis.Currently,in order to meet the trace analysis for targets in clinical analysis and diagnosis,a variety of signal amplification techniques have been developed and applied to amplify the output response of electrochemical biosensors and improve the performance of the biosensors.Among them,enzyme catalytic amplification is one of the most mature technologies for its well specificity,high efficiency and mild reaction conditions.In addition,for the enzyme cascade reaction,the former enzymatic reaction can produce the substrate of the latter enzymatic reaction.In this case,it can reduce the invalid diffusion of intermediates and improve the efficiency of enzyme cascade catalysis.In this paper,a series of enzyme cascade catalytic amplification systems are designed,which are applied to the construction of highly sensitive electrochemical biosensor to detect a variety of tumor markers.The specific works are as follows:1.Target-Initiated Enzyme Cascade Reaction on a DNA Dimer Nanodevice with High Catalytic Efficiency for Sensitive Electrochemical BiosensingThe traditional immobilization mode of cascade enzymes usually relies on nano matrix materials,such as carbon nanotubes,graphene and other carbon materials,metal and its oxides,or metal organic framework materials.However,in this case,cascade enzymes are randomly adsorbed or covalently modified to the surface of nonomaterials,which may limit the catalytic efficiency of enzyme cascade reaction.In order to solve this problem,a simple DNA dimer nanodevice was prepared in this work to precisely assembly the model enzymes glucose oxidase(GOx)and horseradish peroxidase(HRP),which could overcome the problem of disorder arrangement for cascade enzymes from traditional support materials.Here,target induced the assembly of a simple DNA dimer nanodevice to fix glucose oxidase(GOx)and horseradish peroxidase(HRP).Compared with the free GOx/HRP catalytic system,the catalytic efficiency of the enzyme cascade system controlled by DNA dimer nanodevice exhibits significantly improved catalytic efficiency.By monitoring the changed enzyme cascade electrocatalytic signal,a highly sensitive electrochemical biosensor is constructed for microRNA-21 detection with a low detection limit of 0.03 pmol/L.In summary,this work developed a novel and simple DNA dimer nanodevice to arrange cascade enzymes on the nanoscale for high enzyme cascade catalytic efficiency,which paved an avenue for arrange other biomolecules system with ultimate biological analysis and assay applications.2.Precise Regulation of Enzyme Cascade Catalytic Efficiency with DNA Tetrahedron as Scaffold for Ultrasensitive Electrochemical Detection of DNAIn living system,the reaction efficiency of enzyme cascade reaction relies on the appropriate distance regulation of participating enzymes on a scaffold(e.g.,cytoskeleton or cell membrane).Recently,some traditional scaffold materials,such as MOF materials,metal nanoparticles or DNA origami are applied for regulating the interenzyme distance to improve the efficiency of enzyme cascade reactions.However,they inevitably have the disadvantages of poor material controllability and biocompatibility,or complex preparation.In this paper,a rigid DNA tetrahedron(TDN)scaffold was synthesized to precisely control the interenzyme distance by randomly anchoring two pairs of horseradish peroxidase(HRP)/glucose oxidase(GOx)at the vertices of TDN.Impressively,by varying the side length of TDN scaffold,the interenzyme distance was precisely regulated,thus,an optimal TDN scaffold with highest catalytic efficiency was acquired and subsequently applied for constructing an ultrasensitive biosensor for DNA detection with a low detection limit of 3 fmol/L,which provides a new idea for the construction of a highly efficient enzyme cascade amplification system with ultimate applications in bioanalysis and clinical diagnosis.3.Lattice-like DNA Tetrahedron Nanostructure as Scaffold to Locate GOx and HRP Enzymes for Highly Efficient Enzyme Cascade ReactionSome traditional rigid DNA nanostructures,including DNA tetrahedron,DNA tweezers and DNA origami,have been used as reliable scaffolds to precisely control the arrangement and location of cascade enzymes,and efficient enzyme cascade reaction systems have been constructed.Despite these successes,there are also inevitable defect:the mutual independence between any two scaffolds enables only one or two pairs of cascade enzymes are simultaneously manipulated by a single scaffold,which could not guarantee the orderly arrangement of all cascade enzymes,thus some ineffective diffusion of intermediate between cascade enzymes still exists,limiting enzyme cascade catalytic efficiency.In this work,a lattice-like tetrahedron nanostructure was prepared by alternate and equidistant self-assembly of two types of DNA tetrahedron(TDN)decorated with different model enzymes of GOx and HRP to achieve the array arrangement of all cascade enzymes.Under this circumstance,the proposed enzyme cascade system enabled intermediates produced by any GOx catalyzing substrate glucose concurrently diffuse towards four adjacent HRP enzymes surfaces,thus avoiding invalid diffusion effect of intermediates between cascade enzymes and exhibiting much higher catalytic efficiency than that of randomly arranged cascade enzyme system.As a proof of concept,the array arrangement of cascade enzymes with high cascade catalytic efficiency was further applied in the construction of a sensitive electrochemical detection platform for biomarker thrombin detection with a low detection limit of 0.32 pmol/L,which also provided a promising strategy for ultrasensitive monitoring of other biomolecules in sensing analysis and disease diagnosis.4.Fe-N-Doped Carbon Material as Nanoenzyme to Construct Novel Enzyme Cascade Reaction for Sensitive Electrochemical BiosensingIn the previous work,we explored different ways to improve the efficiency of cascade reaction between two natural enzymes.Although natural enzymes have high catalytic activity,most of them are unstable to heat,acid and alkali,and their structure is easy to change with reduced activity.At the same time,the content of natural enzymes in organism is very low,which is difficult to purify,and the production cost is high,so they will be limited in practical application.In order to solve this problem,a Fe-N-doped carbon material as a nanoenzyme was prepared in this work to construct a novel enzyme cascade system for biosensing application.Fe-N-doped carbon materials has shown good properties of horseradish peroxidase.Combining DNA signal amplification technology with DNA walker on the electrode interface,Fe-N-doped carbon materials was fixed on the electrode surface,which enabled Fe-N-doped carbon materials was closed to the glucose oxidase on the modified electrode,and finally obtaining a high enzyme cascade electrocatalytic signal for sensitive thrombin analysis with a low detection limit of 30 fmol/L.In summary,the cascade reaction betweennatural enzyme and mimic enzyme designed in this work combining the advantages of natural enzyme and mimic enzyme,promoting the application of enzyme cascade reaction and opening up a new idea for the construction of efficient enzyme cascade system.