The Construction Of Aptamer-fluorescent Sensors Based On New Type Of Enzyme Assisted Signal Amplification Strategy And The Research Of Its Application

Aptamer-fluorescent sensor is a sensitive,simple and cost-effective detection method.The principle of that is:(1)The aptamer recognizes and binds with the target,which induce the conformational change of itself.(2)The conformational change of the aptamer induces the downstream signal amplification and the production of fluorescence signal.(3)The fluorescence signal has a positive correlation with the concentration of the target and it is easy to calculate the concentration of the target.The sensors can be used to detect a serious of target such as ion,small molecule,protein and even the whole cell.These sensors are widely used in disease diagnosis,environment monitoring and life science research.The aptamer-fluorescent sensor consists of aptamer,fluorescence signal and signal amplification strategy.The aptamer can be divided into intact aptamer and split aptamer.The intact aptamer have a stronger binding force with the target while the split aptamer can lower the background signal due to it has fewer secondary structure.As for the fluorescence signal,it can be divided into labeled strategy and label-free strategy.The labeled strategy can simple the design of the probe,and the label-free strategy can forbid the decline of amplification's efficiency causing by the decrease of binding ability about the labeled probe and target.The amplification can be split into enzyme assisted signal amplification and non-enzyme assisted signal amplification.The enzyme assisted signal amplification has higher amplification efficiency than non-enzyme assisted signal amplification's.In this assay,we focus on the label-free,enzyme assisted amplification strategy and try to use the same enzyme push the operation of target recycling and downstream signal amplification.In the chapter one,we introduce the every part of the aptamer-fluorescent sensor,summarize the problems that the enzyme-assisted signal amplification faced with and find ways to solve that.In the chapter two,we describe the problems,ignoring the target recycle and the huge size of the aptamer-adenosine complex decrease the downstream signal amplification efficiency,which exist in the designing of aptamer-fluorescence sensor for adenosine detection,and we design a target and inhibit recycle strategy to solve these problems.First,we prepared a capture/inhibit complex,when the adenosine exists,the capture recognize and bind with that,causing the separated of capture/inhibit complex and the release of inhibit.Then we use T7 Exo to recognize the 5' terminal of capture-adenosine complex and hydrolyze them at the same time,which reduce the affinity of capture and adenosine,causing the divide of capture-adenosine complex and the recycle of the adenosine.In the meantime,the inhibit hybrid with H1,and based on the same theory,we achieve the recycle of the inhibit.Finally,we manage to the detection of adenosine.In the chapter three,based on the problems such as unstable structure of the split aptamer-target complex,which we faced with in the construction of sensor platform based on split aptamer,we come up with a new type of target recycle strategy.By using the target to induce the binding of two ssDNA,we finally get a split aptamer-target complex.With the help of KF polymerase and Nt.BbvcI,we realize the operation of strand displacement amplification(SDA),and products of the SDA can hybrid with the split aptamer-adenosine complex.With the help of KF polymerase,the products begin to extend,and we finally succeed in replacing the unstable aptamer-target into stable DNA helix and use it to induce the downstream signal amplification.In the meantime,the split aptamer-target get divided and the target get recycled.We use this strategy to realize the detection of thrombin.