Novel Homogeneous Biosensor Based On DNA-Templated Click Chemistry And Its Application

Electrochemical DNA biosensors have always been highly regarded by researchers because they are low cost,easy to use,hihly sensitive and have the potential to be miniaturization.With the development of electronic technology and smart flexible materials,electrochemical DNA biosensors have been improved constantly and can be developed into an effective and cheap detection technology which will be very useful wearable biosensors and mobile healthcare analysis.Click chemistry was a kind of synthetic reaction introduced by chemist Sharpless in 2001 and has always been appreciated in organic synthesis area because of its available raw materials,simple operations,mild reaction conditions and so on.However,its application potential is more than above.Click chemistry has great application value in biosensors and now a number of papers related to its application in biosensors have been published.But its application in biosensors'signal hasn't been reported yet.In this paper,we have developed a novel approach to indicate electrochemical biosensors'signal based on click chemistry and this approach has been successfully applied into novel homogeneous electrochemical DNA biosensors used to detect miRNA and ARP.Chapter1.IntroductionIn this chapter,the work principles,classification and development trends of biosensors have been briefly introduced firstly.Then the basic composition,construction method,classification and application potentials of electrochemical DNA biosensors are summarized shortly.Finally,the characteristics,advantages,classification and current development of homogeneous electrochemical DNA biosensors are generalized.Chapter2.In this chapter,a novel homogeneous electrochemical biosensor for miRNA-21detection based on DNA-templated click chemistry and catalytic hairpin assembly has been proposed.According to the target miRNA-21,two probes of hairpin structure,probe1andprobe2,havebeendesignedandlinkedto5-amino-2,3-dicyano-1,4-naphthoquinone(ADNQ)and 2-furanpropanoic acid(FPA),respectively.When miRNA-21 is present,the catalytic hairpin assembly between the two probes can be triggered.Therefore,ADNQ and FPA linked to the probes get very close to each other and can react by a Diels-Alder reaction to change the quinone structure of ADNQ intoto ketone,making it no longer electro-active.Thus,the current difference(?I)can be used to indicate the concentration of miRNA-21.The feasibility and detecting performance of the proposed biosensor are also examined by cyclic voltammetry(CV),differential pulse voltammetry(DPV)and polyacrylamide gel electrophoresis(PAGE).The experimental results show the biosensor can be successfully applied to sensitively detect miRNA-21 in a linear range from0.1~1.0×10~4 pmol/L with a detection limit of 0.037pmol/L.The biosensor is suitable for the detection of miRNA-21 in blood.Chapter3.In this chapter,a novel homogeneous electrochemical biosensor for ATP detection based on DNA-templated click chemistry has been developed.Two splitting aptamer probes,probe1 and probe2,have been designed according to the ATP aptamer sequence and linked to 5-amino-2,3-dicyano-1,4-naphthoquinone(ADNQ)and 2-furanpropanoic acid(FPA),respectively.When ATP is present,the two probes can be triggered by ATP molecules to form double-stranded DNA.Therefore,ADNQ and FPA linked to the probes get very close to each other and can react by a Diels-Alder reaction to change the quinone structure of ADNQ intoto ketone,making it no longer electro-active.Thus,the current difference(?I)can be used to indicate the concentration of ATP.The feasibility and detecting performance of the proposed biosensor are also examined by differential pulse voltammetry(DPV).The experimental results show the biosensor can be successfully applied to sensitively detect ATP in a linear range from 0.1~1.0×10~2 nmol/L with a detection limit of0.40nmol/L.The biosensor is suitable for the detection of miRNA-21 in serum.