Analytical Sciences

Abstract − Analytical Sciences, 32(11), 1245 (2016).

A Label-free Time-resolved Luminescent Platform for Sensitive Endonuclease V Detection Based on Exonuclease III Regulated DNA-Tb3+ Luminescence
Huaijun NIE,* Huifen HUANG,** Wang LI,*** and Tao YANG**
*State Environmental Protection Key Laboratory of Drinking Water Source Management and Technology, Shenzhen Key Laboratory of Drinking Water Source Safety Control, Shenzhen Research Academy of Environmental Sciences, Shenzhen 518001, P. R. China
**College of Food Science & Engineering, Central South University of Forestry & Technology, Changsha 410004, P. R. China
***State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, P. R. China
Endonuclease V (EndoV) plays the important role of nucleotide excision repair (NER) in the maintenance of genomic stability. Highly sensitive detection of EndoV was achieved through an oligonucleotides sensitizing Tb3+ luminescent technique. We found that although both guanine-rich (G-rich) single-stranded DNA and dGMP could enhance the time-resolved luminescence of Tb3+, their efficiencies of enhancement were considerably different. Employing such interesting phenomenon, a label-free and time-resolved luminescent strategy for the sensitive detection of EndoV activity was developed based on DNA-enhanced time-resolved luminescence (TRL) of Tb3+. The EndoV was used to cut off the deoxyinosine site (dI) and convert the 3′-protruding termini to a recessed end, and Exonuclease III (Exo III) was used to enhance the signal contrast via digestion of G-rich DNA to dNTP. Combining with the natural advantages of the TRL, the proposed method exhibited a good linear response to EndoV ranging from 0.005 to 0.4 U/mL, with a low limit of detection of 0.005 U/mL.