Construction Of Fluorescent Aptamer Sensor Based On Shared Components

As an energy currency molecule in cells,adenosine triphosphate（ATP）plays an important role in many physiological and biochemical activities.Therefore,accurate measurement of ATP is of great biological significance.A variety of biosensing strategies had been developed for the measurement of ATP up to now,which demonstrate good detection performance with the aid of signal amplification technology.However,the introduction of signal amplification often resulted in the problems of multi-step operation or system complexity.Therefore,a fluorescence aptamer sensing strategy based on shared components had been proposed in this study.This strategy enabled the combination of a self-driven catalytic hairpin assembly circuit（S-CHA）and a multicomponent nuclease（MNAzyme）in order to achieve effective amplification of the signal and reduction of complexity of the aptamer sensor.The main research contents and conclusions are as follows:1.Design of programmable ATP aptazymesATP aptamer and Mg²⁺dependent DNAzyme were programmed by engineering the functional components of nucleic acid aptamer and RNA cutting DNAzyme,and then three groups of ATP aptazymes were successfully constructed.Nucleic acid analysis tool NUPACK was used to simulate the secondary structure and substrate binding state of the aptazymes system.The designed aptazymes could activate the catalytic activity of DNAzyme by binding to the target to cause conformational change,and then catalyzed the hydrolysis of the substrate chain（Ds）cleavage site with the assistance of Mg²⁺.Active aptazymes could cut the substrate chain to produce large amounts of ss DNA（c Ds）periodically.In this process,not only could the target detection be converted into signal nucleic acid output,but also the output signal could be effectively amplified.2.The establishment of signal amplification system based on CHAA self-actuated catalytic hairpin assembly circuit（S-CHA）based on aptazymes shared component strategy was designed and embedded in MNAzyme to achieve efficient signal amplification.Firstly,the hairpin chain of CHA was designed with c Ds as initiator for assembling three-way DNA nanostructures（3WJ）.3WJ could hybridized with aptazymes（sharing components AZ1 and AZ2）to assemble an active 3WJ-MNAzyme,which could also hydrolyze the substrate chain Ds and release more c Ds to produce S-CHA circuits.S-CHA was recognized and activated by primary c Ds,and a large number of c Ds were hybridized with double-modified（FAM/BHQ2）DNA probe P to produce a high fluorescence signal for detection when the target ATP was presented.3.Sensitive detection of ATP by fluorescent aptamer sensors based on shared componentsThe biosensor can achieve highly sensitive and specific detection of ATP,and has a good linear relationship between 10 n M and 100μM,and the detection limit was 4.7 n M.And this method successfully detected the content of ATP in fetal bovine serum,which proved that this method could realize the detection of target objects in complex samples.This method could also detect extracellular ATP（e ATP）levels of different locations before and after Arabidopsis leaf injury,and achieve the determination of e ATP released from Arabidopsis leaf.In summary,a shared component and cascaded fluorescent aptamer sensor for ATP detection was proposed in this study.This strategy could accurately detect the content of ATP in fetal bovine serum and realize the detection of plant extracellular ATP level.This method could be applied to various targets in the fields of biological detection and environmental analysis because of the diversity of aptamers.