Label-free chemiluminescent strategy for highly selective and sensitive detection of adenosine triphosphate by cofactor-dependent enzymatic ligation-triggered polymerase chain reaction

In this work, a novel label-free chemiluminescent (CL) assay system was developed for highly sensitive and selective detection of adenosine triphosphate (ATP). The strategy relies on the powerful signal amplification capability of rolling circle amplification (RCA), the preferential binding ability of graphene oxide (GO) to single-stranded DNA (ssDNA), and instantaneous derivatization reaction between phenylglyoxal (PGO) and guanine nucleobases (G). In the presence of ATP, the T4 DNA ligase catalyzes the ligation reaction between the two ends of the padlock probe producing a closed circular DNA template that initiates the RCA reaction with phi29 DNA polymerase and dNTP. Therein, many complementary copies of the circular template can be generated. The formed long single-stranded DNA which contained an amount of guanine bases could be adsorbed on the surface of GO forming DNA-GO complexes. And the CL signal of DNA-GO complexes can be obtained via the instantaneous derivatization reaction between PGO and guanine bases. The CL signal increased linearly with the concentration of ATP from 0.1 to 2.5 nM with a detection limit of 0.03 nM. The system also showed high specificity to ATP against its analogues such as CTP, GTP, UTP, AMP, ADP, BSA and CaM. In addition, ATP has been determined in diluted serum indicating the applicability of this assay. (C) 2016 Elsevier B.V. All rights reserved.