Electrochemical Biosensing Techniques Based On Nucleic Acid Probes For The Detection Of Enzyme Activity And Adenosine

Electrochemical biosensors have played important roles in chemical and biological basic research, clinical diagnosis and environmental analyses for their accessibility of minimization, integration and operation.A variety of flexible signal transformation strategies can be conceived based on the specific binding of nucleic acid probes to their targets.In the thesis,by taking the advantages of nucleic acid probes,electrochemical biosensors for simple,high-speed, and sensitive detection of enzyme activity and small molecule were developed. The main contents are as followed:1.Synthesis and electrochemical characterization of ferrocene modified nucleic acid probeThe covalent link of ferrocene and 3'NH2 of DNA was achieved by modifying the DCC-NHS method,which decreased the effect of water on the active intermediate, and the products were purified by HPLC and characterized by electrochemical techniques.Compared with the traditional dialysis method to purify ferrocene modified nucleic acid probes,the products got by HPLC were much purer and their electrochemical signal was higher. It served as the foundation for other similar functional nucleic acid probe synthesis.2.Electrochemical detection of APE1 activity based on ferrocene modified nucleic acid probesAPE1 is an essential protein for DNA damage repair. Though the high sensitivity of the traditional radiolabelling analysis, the assay entails time-consuming, complicated operation and radioactive contamination.Herein, electrochemical detection of APE1 activity was achieved by attaching a single strand sulfhydryl-terminal DNA on gold electrode and hybridizing it with its complement AP site-containing ferrocene modified DNA,which was cut by APE1.The method is simple and sensitive with a detection range of 0.02-2 U/ml and a detection limit of 0.02 U/ml.It has potential application in DNA damage and repair analyses in biological samples.3 Highly sensitive adenosine detection based on split aptamer combined with gold nanoparticle amplification.In spite of aptamer's specific recognition of small molecules,the binding constant is relatively low and it always needs signal amplification systems to enhance sensitivity. Herein, an electrochemical adenosine biosensor was constructed based on split aptamer combined with gold nanoparticle amplification.The approach was highly sensitive and specific,with a detection range of 0.1 pM-1 nM and a detection limit of 0.1 pM.It has the potential to be applied in other small molecule detection.