Self-assembled DNA/enzyme Functional Complex Based On Rolling Circle Amplification Technology And Its Application In Photoelectric Biosensor

As an isothermal signal amplification technique,rolling circle amplification（RCA）has the advantages of simple operation,strong amplification ability and so on,and provides new idea and method for the design and preparation of DNA self-assembly materials.In this paper,RCA is applied to the self-assembly of DNA flower structure.Then the DNA flowers were used as a signal amplification probe by encapsulating enzyme molecules to achieve high sensitivity detection of biomolecules such as thrombin and glucose,combined with colorimetric and electrochemical method.The RCA-based enzyme-encapsulated DNA flower has broad application prospects in molecular encapsulation,signal amplification analysis and biosensor construction.This thesis carried out the following three works:1.Rolling circle amplifying self-assembled horseradish peroxidase-encapsulated DNA flower-based colorimetric biosensor for amplified detection of thrombinThe self-assembled micron-sized DNA flowers were formed via RCA in this work and encapsulated a large number of horseradish peroxidase（HRP）molecules in the RCA process.The geometry,morphology,encapsulation efficiency and catalytic activity of DNA flowers were systematically studied.Since the RCA product contains a large number of repeated thrombin aptamer（Apt29）sequences,magnetic microparticles were used as a carrier to form an aptamer-thrombin-DNA flower sandwich structure by aptamer recognition.The colorimetric biosensor was constructed by HRP catalytic etching of gold nanorods（AuNRs）encapsulated in DNA flowers to achieve high sensitivity and high specificity detection of thrombin.In addition,the detection background value was greatly reduced by magnetic separation and the detection limit of thrombin was 0.22 fM.The colorimetric biosensor showed good selectivity and was applied to the analysis and detection of thrombin in actual samples.2.Rolling circle amplifying self-assembled HRP-encapsulated DNA flower-based electrochemical biosensor for the detection of thrombinThe submicron HRP-encapsulated DNA flower structure was prepared by RCA reaction and the complementary sequence of Apt29 was designed into a circular template to realize linear amplification of Apt29.Another thrombin aptamer Apt15 was immobilized on the surface of electrode and therefore thrombin and HRP-encapsulated DNA flowers were captured on the surface of electrode by specific recognition between thrombin and aptamer to form a sandwich structure of aptamer-thrombin-DNA flower.In the presence of H₂O₂,HRP catalyzes the oxidation of 3,3’,5,5’-tetramethylbenzidine（TMB）to produce electron transfer and electrical signals.Due to the high encapsulation efficiency of the DNA flower for the HRP molecule,the detection sensitivity was greatly improved and the detection limit of thrombin was 1 fM.The DNA flower-based electrochemical biosensor also showed good selectivity to thrombin.3.Rolling circle amplifying self-assembled HRP and glucose oxidase-encapsulated DNA flower-based electrochemical biosensor for the detection of glucoseHRP and glucose oxidase（GOx）were simultaneously added during the RCA reaction to obtain DNA flower encapsulating the two enzymes（HRP-GOx-DFs）.The internal composition,morphology,encapsulation efficiency and catalytic activity of the HRP-GOx-DFs were systematically studied.Due to the high-density phosphate group and the enzyme-catalyzed cascade reaction,the catalytic activity of HRP-GOx-DFs is significantly improved compared to the free enzymes.Further,the glucose electrochemical biosensor was prepared by modifying the electrode with HRP-GOx-DFs,which greatly improved the detection sensitivity of glucose,and the detection limit was3.87μM.In addition,the resulting electrochemical biosensor had good selectivity for glucose.