Study On The Measurement Of Plants Extracellular Adenosine Triphosphate Based On DNA Tetrahedron Coupled With Aptazymes And Hybridization Chain Reaction

Adenosine triphosphate(ATP)is one of the indispensable energy-carrying molecules and genetic building blocks of living organisms,plays an important role in a variety of physiological and pathophysiological processes.In plants,ATP can be released from cells and into the extracellular matrix in response to a variety of environmental stresses.Extracellular ATP(eATP)is now recognized as an important extracellular signaling molecule in plants.Although a variety of detection methods have been extensively used in ATP sensing and analysis,accurate detection of eATP remains difficult due to its extremely low concentration and spatiotemporal distribution.Therefore,it has practical significance to develop a novel method that can quickly and accurately quantify changes in eATP.Based on the above research background,an eATP measurement strategy based on DNA tetrahedral modified electrode sensing platform and hybridization chain reaction(HCR)combined with G-quadruplex/Hemin(G4/Hemin)DNAzyme dual signal amplification is proposed.The main contents and conclusions are as follows:1.Design of ATP-specific aptazymes cleavage to trigger HCRThe NUPACK analysis tool was used to theoretically simulate the secondary hairpin structure of the aptazymes system probe and the design of the hybridization chain reaction triggered by substrate cleavage.The DNAzyme enzyme chain sequences containing two splits of ATP aptamer and a substrate chain including ribonucleic acid cleavage sites were designed.Through specific recognition and complementary binding of the target ATP,in the presence of Zn²⁺,a stable active aptazymes was formed,which triggers the cleavage of substrate chain(ST)to produce multiple trigger chains,thereby converting the input ATP molecule into the output signal chain.The research results showed that the target ATP can bind to its aptamer and induce the activity of the aptazymes to release the substrate chain,and this signal chain can trigger HCR in solution.2.Detection of ATP by DNA tetrahedral platform coupled hybridization chain reactionBased on the previous part of the research,the DNA tetrahedral was used as a platform to construct HCR on the electrode.Subsequently,when Au NPs@G-quadruplex(G4)spherical nucleases(SNAzymes)with linker sequences were used as nanocatalyst tags,they were captured by the ds DNA polymer of HCR through the adhesive linkers present on the electrode surface.Finally,through the combination of G4 and hemin,a G4-DNAzyme was formed,which is a simulated peroxidase activity that catalyzes hydrogen peroxide(H₂O₂)an identifiable amplified electrochemical redox current signal.The biosensor has high sensitivity and specificity to the target.In electrochemical detection,linear sweep voltammetry(LSV)was used to detect ATP,and it has a good linear relationship between 10-10000 p M(R²=0.9982).The limit is 5 p M.3.Monitoring of plant extracellular ATP(eATP)by electrochemical measurement platformThe method successfully and accurately measured the level of ATP released by tobacco suspension cells,and the recovery rate was between 97.59%and 106.34%.Therefore,the developed sensor has high sensitivity,wide linear range,low detection limit,and it has high reliability in practical samples.The sensor also monitored the content of eATP released before and after wounding the leaves of Arabidopsis,and its distribution and concentration showed obvious positional effects.In summary:This study proposes a new type of electrochemical aptasensor to detect ATP.This strategy can accurately measure the extracellular ATP in tobacco suspension and the eATP released from Arabidopsis leaves.Therefore,this design provides a promising strategy for the measurement of extracellular ATP levels.Since a variety of aptamers can be obtained through in vitro selection,the platform can be further used to accurately detect various targets encountered in bioanalysis and clinical environments.