Target-catalyzed assembly formation of metal-ion dependent DNAzymes for non-enzymatic and label-free amplified ATP detection

Abstract In this work, on the basis of a dual recycling amplification means with the involvement of toehold-mediated strand displacement reaction (TSDR) and metal-ion dependent DNAzymes, we describe the establishment of a non-enzymatic and label-free method for sensitively detecting ATP in human serums. The target ATP binds the corresponding aptamer recognition probes and causes structure switching of the aptamers to expose the toehold regions for TSDR-based recycling of ATP and subsequent catalytic assembly formation of many DNAzymes with the assistance of DNA fuel strands. The corresponding metal ions further cyclically catalyze the cleavage of the G-quadruplex containing hairpin substrate strands of the DNAzymes, releasing lots of free G-quadruplex segments, which associate with the thioflavin T dye to result in drastically enhanced fluorescent signals for detecting ATP with high sensitivity. Such an ATP sensing method shows a dynamic range of 50–600 nM and a detection limit of 20 nM. The developed sensing system also has a high discrimination capability between ATP and other interfering analogue molecules, and can realize the detection of human serum ATP molecules, demonstrating its potential for convenient monitoring of low levels of ATP for the diagnosis of different diseases.