Analytical Sciences

Abstract − Analytical Sciences, 29(2), 193 (2013).

A Highly Sensitive Electrochemical Platform for the Assay of Uracil-DNA Glycosylase Activity Combined with Enzymatic Amplification
Hua ZHANG, Liangliang ZHANG, Jianhui JIANG, and Ruqin YU
State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, P. R. China
Uracil-DNA glycosylase (UDG) plays a crucial role in DNA lesion repair because it is one of the most important base excision repair (BER) enzymes. Quantitative analysis of UDG activity is of fundamental importance in bioanalysis. Here, an electrochemical sensing platform combined with enzymatic amplification was developed for simple and sensitive assay of UDG activity and its inhibition. This strategy relies on the release of a biotinylated signal probe from the electrode surface, due to the lowered melting temperature of the duplex UDG substrate after UDG treatment. A biotin modification was used as a tracer in the signal probe and streptavidin-alkaline phosphatase (SA-ALP) was taken as a reporter molecule. Upon reacting with UDG, the loss of biotin label led to a decrease in the amount of bound SA-ALP on the electrode surface, resulting in a weaker electrochemical signal. This strategy allowed for a simple, cost-effective, sensitive and selective assay for UDG with a wide linear response range from 0.01 to 10 U/mL and a low detection limit of 0.0079 U/mL. In addition, the effects of drugs on UDG activity have also been investigated. The proposed strategy not only provides a universal platform for the assay of BER enzymes, but also shows potential application for drug screening.