A Electrochemical Sensor Based On DNA Walker For Determination Of Telomerase Activity And Copper Ion

DNA walkers are a class of molecular machines,which move autonomously along the designed DNA track driven by cleavage and strand displacement reactions.They realize the transport of cargo in a eertain direction,leading to considerable signal amplification eventually.Deoxyribozymes(DNAzymes)specifically interact with metal ions or otherl biological molecules and catalyze cleaving deoxyribonucleic acid.DNAzymes have many advantages such as good specificity,high catalysis activity,easy to functionalization,strong stability and so on.Thus,we combined Cu2+ specific DNAzyme with DNA walker to design an electroehemical sensor based on DNAzyme walker.This sensor can achieve signal amplification and without additional protease.Human telomerase is a unique ribonucleoprotein which catalyze repeated units TTAGGG to the ends of telomeres introducing the primer,and thus protect the telomere from erosion during cell division.Due to the high expression in human cancer cells and low expression in normal cells,telomerase has a significant impact on early cancer diagnosis and the evaluation of cancer therapy.Therefore,it is necessary to develop new electrochemical method for ultrasensitive detection of telomerase activity.Copper ion(Cu2+),one of the necessary microelements of human beings,participates in various life activities of the human body,such as electronic transduction,enzyme catalysis and oxygen transportation.The improper concentration of Cu2+ cause serious damage to the kidneys,liver and neurological systems,leading to Parkinson's disease and Alzheimer's disease,etc.Therefore,it is crucial to construct new sensor for ultrasensitive detection of copper ions.This paper designed electrochemical sensors based on DNAzyme walker for the detection of telomerase activity and copper ions.The contents are as follows:Chapter 2:An electrochemical sensor based on Cu2+ specific DNAzyme walker was constructed to detect telomerase activity.The Y1,Y2 could hybridize with synthetic telomerase product to form a Y-shape DNAzyme walker due to the proximity effect.On account of the specific recognition and cleavage ability of the Cu2+ specific DNAzyme,the Y-shape DNAzyme walker could move autonomously and achieve remarkable electrochemical signal.Hairpin capture(HC)was attached a mercapto group(SH)at the 5'-end and a ferrocene(Fc)at the 3'-end,so HC was self-assembly on the surface of the gold electrode(GE).The anodic peaks of Fc molecule on the modified electrode were used to reflect the surface chemistry.The loops of HC was the substrate of Cu2+specific DNAzyme.In normal HeLa cells,telomerase activity was high expression and it could recognize the primer and elongate D-TSP with DNA sequence repeats("TTAGGG")at the 3' ends.The synthetic telomerase products hybridized with core sequences of Y1 and Y2 probes to form Y-shape DNAzyme walker with three catalytic enzymes of Cu2+ specific DNAzyme due to the proximity effect.These enzymes could bind to a DNA substrate through Watson-Crick base-pairing.With the assist from Cu2+,DNAzyme cleaved HC substrates on GE surface into two fragments.A large amount of released Fc-labeled fragemnts left on GE surface and lead to considerable electrochemical signal amplification eventually.In heated HeLa cells,telomerase activity with low expression,D-TSP kept its short strand with low affinity to form DNA machines inintroducing Y1 and Y2.The efficiency of cleavage of HC probes was low.In consequence,weak signals were produced from a few of Fc-labeled cleaved fragments.Under optimized experimental conditions,a good nonlinear correlation(R2=0.9939)between relative peak current and the logarithm of the number of HeLa cells was showed ranging from 25-2000 HeLa cells with a detection limit of 23 HeLa cells.The sensor also could detect the telomerase activity of MCF-7 cells and A549 cells.This method broadens the research of DNA walker and can be applied to the early diagnosis of cancer,having potential for the practical application of clinical detection.Chapter 3:In this chaper,an electrochemical sensor based on Cu2+ specific DNAzyme walker was constructed to detect copper ions.The enzyme of Cu2+ specific DNAzyme could cleave the substrate with the aid of copper ions.The Y-shaped DNAzyme walker containing three catalytic enzyme could move autonomously along designed DNA track and achieve remarkable electrochemical signal.Hairpin capture(HC)was attached a mercapto group(SH)at the 5'-end and a ferrocene(Fc)at the 3'-end,so HC was self-assembly on the surface of the gold electrode.The anodic peaks of Fc molecule on the modified electrode were used to reflect the surface chemistry.The loops of HC was the substrate of Cu2+specific DNAzyme In the presence of the Cu2+,the Y-shaped DNAzyme walker cleaved HC substrates on GE surface into two fragments.A large amount of released Fc-labeled fragemnts left on GE surface and lead to considerable electrochemical signal amplification eventually.In the absence of the Cu2+,the activity of the DNAzyme walker was extremely low,leading to weak signals.Under optimized experimental conditions,a good nonlinear correlation(R2=0.9942)between relative Peak current and the logarithm of Cu2+ ions concentration was showed ranging from 5-500 nM with a detection limit of 3.834 nM.This sensor allow to specifically determine Cu2+ with simple operation and without additional protease.Therefore,this method is valuable in practical application on the detection of environmental pollutants.