Duplex featured polymerase-driven concurrent strategy for detecting of ATP based on endonuclease-fueled feedback amplification

Abstract

We have developed a novel amplification strategy termed Endo IV-assisted feedback amplification (EFA) taking advantages of rolling circular amplification (RCA) and Endo IV-assisted signal amplification (ESA) biosensing platform for detecting of adenosine triphosphate (ATP). Two kinds of specially programmed DNA complexes were employed into EFA system, one composed of a split aptamer fragment and a circular template, and the other composed of AP probe and the same circular template. Hence, ATP as a target induced the self-assembly of spilt aptamer fragments and initiated RCA reaction generating a linear DNA, which consists of hybridization elements with Complex II and formation elements of G-quadruplex. More importantly, the addition of endonuclease IV can cut the Complex II into two parts, and one of which can be trimming by phi29 DNA polymerase initiating the new round of RCA reaction producing more RCA products. Thus significantly enhanced fluorescent signal can be measured for ATP as expected, and our proposed strategy exhibits improved performances toward ATP ultrasensitive detection with a limit of detection (LOD) as low as 0.09 nM. Additionally, our developed biosensor demonstrates high selectivity and the superiority of simplicity towards ATP. Above these significant aspects, our proximity binding-induced RCA reaction-based fluorescent assay and Endo IV-fueled feedback signal amplification strategy presents an optimal detection performance towards ATP for potential application in related research and clinical diagnosis.