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Highly selective and sensitive electrochemical biosensor for ATP based on the dual strategy integrating the cofactor-dependent enzymatic ligation reaction with self-cleaving DNAzyme-amplified electrochemical detection.

Research paper by Lu L Lu, Jing Cao JC Si, Zhong Feng ZF Gao, Yu Y Zhang, Jing Lei JL Lei, Hong Qun HQ Luo, Nian Bing NB Li

Indexed on: 23 Jul '14Published on: 23 Jul '14Published in: Biosensors and Bioelectronics



Abstract

A dual strategy that combines the adenosine triphosphate (ATP)-dependent enzymatic ligation reaction with self-cleaving DNAzyme-amplified electrochemical detection is employed to construct the biosensor. In this design, the methylene blue-labeled hairpin-structured DNA was self-assembled onto a gold electrode surface to prepare the modified electrode through the interaction of Au-S bond. In the procedure of ATP-dependent ligation reaction, when the specific cofactor ATP was added, the two split oligonucleotide fragments of 8-17 DNAzyme were linked by T4 DNA ligase and then released to hybridize with the labeled hairpin-structured DNA substrate. The linked 8-17 DNAzyme catalyzes the cleavage of the hairpin-structured substrate by the addition of Zn(2+), causing the methylene blue which contains high electrochemical activity to leave the surface of the gold electrode, therefore generating a dramatic decrease of electrochemical signal. The decrease of peak current was readily measured by square wave voltammetry and a relatively low detection limit (0.05 nM) was obtained with a linear response range from 0.1 to 1000 nM. By taking advantage of the highly specific cofactor dependence of the DNA ligation reaction, the proposed ligation-induced DNAzyme cascades demonstrate ultrahigh selectivity toward the target cofactor ATP. A catalytic and molecular beacons strategy is further adopted to amplify the electrochemical signal detection achieved by cycling and regenerating the 8-17 DNAzyme to realize enzymatic multiple turnover, thus one DNAzyme can catalyze the cleavage of several hairpin-structured substrates, which improves the sensitivity of the newly designed electrochemical sensing system.