Application of tetrahedral -deoxyribonucleic acid electrochemistry platform coupling aptazymes and hybridized hairpin reactions for the measurement of extracellular adenosine triphosphate in plants

Highlights: • Novel T-DNA-based electrochemical aptazyme sensor for detecting ATP was designed. • Signal amplification was achieved by coupling aptazymes with HCR and SNAzymes. • The method showed high selectivity and detected extracellular ATP in plants. Extracellular ATP (eATP) is an important biological signal transduction molecule. 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. Here, an eATP measurement strategy based on tetrahedral DNA (T-DNA)-modified electrode sensing platform and hybridization chain reaction (HCR) combined with G-quadruplex/Hemin (G4/Hemin) DNAzyme dual signal amplification is proposed. In this strategy, ATP aptamer and RNA-cleaving DNAzyme were combined to form a split aptazyme. In the presence of ATP, this aptazyme hydrolyzes the cleaving substrate strand with high selectivity, releasing cleaved ssDNA, which are captured by the T-DNA assembled on the electrode surface, triggering an HCR on the electrode surface to form numerous linker sequences of the HCR dsDNA product. When G-quadruplex@AuNPs (G4) spherical nucleic acid enzymes (SNAzymes) with other linkers are used as nanocatalyst tags, they are captured by HCR dsDNA through sticky linkers present on the electrode surface. An amplified electrochemical redox current signal is generated through SNAzyme-mediated catalysis of H₂O₂, enabling easy detection of picomole amounts of ATP. Using this strategy, eATP levels released by tobacco suspension cells were accurately measured and the distribution and concentration of eATP released on the surface of an Arabidopsis leaf was determined.