Study On Fluorescence Biosensor Based On DNA Enzyme And Nucleic Acid Cleavage Signal Amplification

Rapid, accurate and sensitive detection of target biomolecules has been attracted attention in modern biochemical analysis chemistry field, such as biological small molecules, nucleic acid, protein and enzyme activity. With the rapid development of economic society, it’s necessary to develop sensitive and selective analysis techniques to meet the higher requirements for biomolecules detection. With the low cost, analysis speed, selectivity, high sensitivity, and the ability of detection in complex systems, biosensors have been applied to chemistry, biomedical, food, medicine, and environmental monitoring and other fields.At present, fluorescent biosensor is one of the most rapid development and extensive used biosensors. With the rapid development of analysis techniques, it’s urgent to improve the selectivity and sensitivity of fluorescence biosensor to satisfy the demands of reality. Sensitivity is one of the important parameters of fluorescence biosensor. Currently, two main strategies have been used to improve the sensitivity of biosensors: using super quenchers to achieve lower background fluorescence and applying various enzymes to achieve signal amplification for targets detection. Due to the significant sensitive improvement, signal amplification technology has been attracted more and more attentions from researchers. Among the various signal amplification technologies, enzymatic signal amplification technology has been becoming one of the research hotspot of fluorescent biosensors.Based on the reported literatures about the detection of target biomolecules, such as biological small molecules, nucleic acids and proteins, in this thesis, super-quenchers have been used to achieve lower background fluorescence, and various enzymes to achieve signal amplification of detection at the same time. The specific contents are summarized as follows:(1) Study on the biosensors based on DNAzyme recycling signal amplification. In this chapter, wehave developed an amplified sensing system for fluorescence turn-on detection of ALP activity based on PPi-regulated 17 E DNAzyme activity. In this sensing system, PPi was chosen as the substrate of ALP, and the 17 E DNAzyme was chosen as the catalytic unit. A CAMB strategy was further employed to amplify the detection signal by cycling and regenerating the DNAzyme to realize true enzymatic multiple turnover of catalytic beacons. These designs together allow a high sensitivity for ALP in the range from 0 to 800 p M with a detection limit of 20 p M. The proposed sensing system also exhibits high selectivity to ALP due to the high speci?city of the ALP-catalyzed hydrolyzation of PPi.(2) Contribute to the super quenching ability of GO and the efficient digestion capacity of ExoIII, we have developed a novel amplified fluorescence sensing system for sensitive detection of T4 PNK activity and inhibition. The proposed method provides a linear range from 0 to 0.2 U mL-1 and a low detection limit of 0.003 U m L-1. The proposed method may provide a new platform for monitoring the DNA phosphorylation related process, drug discovery and clinical diagnostics.(3) Study on the biosensors based on the aggregated cationic perylene diimide(PDI). In this chapter, we have developed a novel fluorescence sensing system for sensitive and selective detection of bleomycin(BLM) in complex biological environments. Contribute to the super quenching ability of PDI, the fluorescent background signal has been successfully reduced. The linear range of the proposed method is from 0 to 1 μM with a detection limit of 0.2 n M. The proposed method may provide a new platform for the clinical use of bleomycin.