Isothermal Cycle Reaction Mediated Fluorescent Signal Amplification And Highly Sensitive Biochemical Sensing Applications

In recent years,due to nucleic acid isothermal cycle reaction possesses the characteristics of low equipment requirements,fast detection speed,good selectivity,high stability and efficiency,it has attracted the attention of many researchers.Fluorescence signal amplification technology also has a series of advantages,such as high sensitivity,simple operation,small sample amount,which has become an important means of scientific research,widely used in biomedicine,disease diagnosis,environmental monitoring fields.However,traditional fluorescence signal amplification technology often depends on the luminescence properties of fluorescent materials to achieve signal amplification,which limits its application in biochemical sensing analysis.Moreover,the vast majority of fluorescent materials have the advantage of good biocompatibility,which could be combined with nucleic acid isothermal cyclic reaction to effectively avoid the shortcomings of traditional fluorescence signal amplification technology limited sensitivity.On the basis of above considerations,to develop nucleic acid isothermal cycle reaction-based biochemical sensing strategies with high sensitivity,low cost and simplicity,we have made some efforts mainly in the following aspects:1.The homogenous Hg²⁺sensing based on enzymatic cycle amplification reaction and DNA templated copper nanoparticle signal outputBased on the target-triggered isothermal cyclic reaction and the label-free DNA templated Cu NPs,we successfully constructed a novel fluorescence sensor for Hg²⁺ultrasensitive homogeneous detection.The sensor induced the configuration transformation of the hairpin substrate through the ExoⅢassisted target cyclic signal amplification reaction,and formed the synthesis template of Cu NPs.Then,the reduction of Cu²⁺by ascorbic acid is used to obtain Cu NPs,generating the fluorescence signal.Under optimized conditions,the sensor strategy achieved accurate detection of Hg²⁺in the linear range of 10 p M to 1.0μM,the limit of detection as low as 3.9 p M.The sensor strategy has high stability and anti-interference ability,and has been successfully applied to the detection of Hg²⁺in actual water samples.In addition,the high dependence of Cu NPs on the DNA template and the specific recognition function of T-Hg²⁺-T structure make the sensor show high sensitivity and good selectivity.2.Construction of closed-loop DNA machine and its application in molecular logic operationBased on the closed-loop isothermal reaction activated by hybridization chain reaction（HCR）and catalytic hairpin assembly（CHA）,we constructed a closed-loop DNA machine with high sensitivity,good specificity,simple operation and enzyme-free involvement,which can be used for highly sensitive molecular logic calculation.Once the primer was triggered,the DNA machine ran continuously in a fully closed loop,achieving infinite exponential amplification of the signal.On the basis of the above DNA machine,a variety of convenient logic gates were constructed via rational design of input elements and computing elements,such as OR,NAND,NOR,YES,NAND and INHIBIT.In addition,this work verified the signal amplification effect of DNA machine by changing the input signal concentration of OR logic gate.The results showed that the synergistic effect of HCR and CHA can accelerate signal amplification,and the DNA machine can realize the signal transduction of"weak input-strong output".3.Label-free and highly sensitive APE1 detection based on closed-loop DNA machineIn this work,by combining the target recognition module with the closed cycle circuit（CCC）in DNA machine,we constructed a biosensor platform which can be used for the label-free highly sensitive detection of cancer marker APE1.APE1 first specifically recognized and cleaved the APE1 probe to release the primer T1 of the DNA machine,which then triggered the fluorescence resonance energy transfer of the HCR,producing significant fluorescence signals.At the same time,HCR products can trigger CHA,obtaining T1 analogues with the same sequence as initiator T1.The T1analogues reversely triggered HCR and started the DNA machine again,forming CCC.Target substances can generate exponentially amplified fluorescence signals through the autocatalytic feedback loop between HCR and CHA systems,which can be applied to the highly sensitive and specific detection of intracellular APE1.Under optimized conditions,the DNA machine can accurately determine APE1 in the linear range of 0.0001 to 1.0 U/m L,and the limit of detection is 0.078 U/L.The method had also been successfully applied to the detection of APE1 in human cell lysates,and could clearly distinguish the concentration of APE1 in cancer cells and normal cells.Therefore,by integrating the target recognition module with the CCC module,the DNA machine can accurately identify the target and be applied to the highly specific and sensitive detection of intracellular APE1.