title: Electrogenerated chemiluminescence biosensing method for the discrimination of DNA hydroxymethylation and assay of the β-glucosyltransferase activity.
abstract: An electrogenerated chemiluminescence (ECL) biosensing method for highly sensitive discrimination of DNA hydroxymethylation and assay of the β-glucosyltransferase (β-GT) activity was developed. The ECL biosensing electrode was fabricated by gold nanoparticles (AuNPs)/Nafion film, and then, tris(2, 2'-ripyridine) dichlororuthenium(II) (Ru(bpy)3(2+)) was electrostatically adsorbed into the AuNPs/Nafion film, finally, the hydroxymethylated double-stranded DNA (ds-DNA)-tagged with ferrocene was self-assembled onto the surface of the AuNPs. When β-GT and uridine diphosphoglucose (UDP-Glu) were introduced, the hydroxymethylcytosine residues within 5'-CCGG-3' of ds-DNA on the biosensing electrode were glucosylated. After the glucosylated biosensing electrode was treated by MspI endonuclease, the unglucosylated hydroxymethylcytosine was cleaved, leading to the quencher leaving the electrode, resulting in an increased ECL signal. For the ECL biosensing method, it showed an extremely low detection limit of 0.04 U/mL for β-GT, and offered a good discrimination toward cytosine, 5-methylcytosine, and 5-hydroxymethylcytosine. This work demonstrates that the combination of the enzyme-linkage reactions with the highly sensitive ECL method is a promising strategy for the discrimination of DNA hydroxymethylation, assay of the activity of β-GT, and evaluation of the capability of inhibitors for the β-GT.
authors.affiliation: Institute of Analytical Science, Northwest University, Shaanxi Provincial Key Laboratory of Electroanalytical Chemistry, Xi'an, Shaanxi 710069, China.
authors.forename: Huiping
authors.initials: H
authors.lastname: Sun
authors.affiliation: Institute of Analytical Science, Northwest University, Shaanxi Provincial Key Laboratory of Electroanalytical Chemistry, Xi'an, Shaanxi 710069, China.
authors.forename: Shangxian
authors.initials: S
authors.lastname: Ma
authors.affiliation: Institute of Analytical Science, Northwest University, Shaanxi Provincial Key Laboratory of Electroanalytical Chemistry, Xi'an, Shaanxi 710069, China. Electronic address: yanli@nwu.edu.cn.
authors.forename: Yan
authors.initials: Y
authors.lastname: Li
authors.affiliation: Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi'an 710062, China.
authors.forename: Honglan
authors.initials: H
authors.lastname: Qi
authors.affiliation: Institute of Analytical Science, Northwest University, Shaanxi Provincial Key Laboratory of Electroanalytical Chemistry, Xi'an, Shaanxi 710069, China.
authors.forename: Xiaohui
authors.initials: X
authors.lastname: Ning
authors.affiliation: Institute of Analytical Science, Northwest University, Shaanxi Provincial Key Laboratory of Electroanalytical Chemistry, Xi'an, Shaanxi 710069, China.
authors.forename: Jianbin
authors.initials: J
authors.lastname: Zheng
mesh_terms: D015082:2,2'-Dipyridyl / Q000031:analogs & derivatives / Q000737:chemistry; D017122:Bacteriophage T4 / Q000201:enzymology*; D015374:Biosensing Techniques / Q000379:methods*; D056831:Coordination Complexes; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D019175:DNA Methylation; D055664:Electrochemical Techniques / Q000379:methods; D057075:Enzyme Assays / Q000379:methods*; D005964:Glucosyltransferases / Q000378:metabolism*; D006046:Gold / Q000737:chemistry; D006900:Hydroxylation; D057230:Limit of Detection; D008163:Luminescent Measurements / Q000379:methods*; D053768:Metal Nanoparticles / Q000737:chemistry; D017975:Ruthenium Compounds
chemical_list: D015082:2,2'-Dipyridyl; D056831:Coordination Complexes; D004247:DNA; D005964:Glucosyltransferases; D006046:Gold; C547232:tris(2,2-bipyridine)-ruthenium(II); D017975:Ruthenium Compounds
keywords: DNA hydroxymethylation; Electrogenerated chemiluminescence; Tris(2, 2′-bipyridyl)ruthenium; endonuclease; β-glucosyltransferase