A fluorescent aptasensor for ATP based on functional DNAzyme/walker and terminal deoxynucleotidyl transferase-assisted formation of DNA-AgNCs

The development of sensitive adenosine triphosphate (ATP) sensors is imperative due to the tight relationship between the physiological conditions and ATP levels in vivo. Herein, a fluorescent aptasensor for ATP is presented, which adopts a strategy that combines a split aptamer and a DNAzyme/walker with terminal deoxynucleotidyl transferase (TDT)-assisted formation of DNA-AgNCs to realize fluorescence detection of ATP. A multifunctional oligonucleotide sequence is rationally designed, which integrates a split aptamer, a DNAzyme and a DNA walker. Both multifunctional oligonucleotide and its substrate strand are connected to the surface of Fe3O4@Au nanoparticles via Au-S bonds. The existence of ATP can induce the formation of the complete aptamer, and then activate the DNAzyme to circularly cleave the substrate strand, leaving 2 ',3 '-cyclophosphate at the 3 ' end of the strand. This blocks the polymerization of dCTP to form poly(C) even in the presence of TDT and dCTP, due to the lack of free 3 '-OH. In contrast, when ATP is absent, the DNAzyme/walker cannot work and then TDT catalyzes the formation of poly(C) at the free 3 '-OH of the substrate strand, which is subsequently utilized as the template to prepare DNA-AgNCs. The fluorescence response derived from AgNCs thus reflects the ATP concentration. Under the optimum conditions, the aptasensor shows a linear response range from 5 nM to 10 000 nM, with a detection limit of 0.27 nM. The level of ATP in human serum can be effectively measured by the aptasensor with good recovery, indicating its application potential in medical samples.