Electrogenerated chemiluminescence biosensing method for DNA hydroxymethylation detection via glycosylation and a new multi-functional ECL signal compound

•An ECL biosensing method for the detection of DNA hydroxymethylation was developed.•A new multi-functional ECL signal compound was synthesized the first time.•The multi-functional ECL signal compound worked as ECL signal reagent, bridge reagent, and anchorage.•The ECL biosensing method could be easily extended to detect various analytes with 1,2- or 1,3-diols. A simple electrogenerated chemiluminescence (ECL) biosensing method was proposed for specific detection of 5-hydroxymethylcytosine (5-hmC) in double-stranded DNA (5-hmC-dsDNA) on the basis of glycosylation and a new multi-functional ECL signal compound (N-(phthalhydrazide-4-yl)-thiophen-2-amino-5-boronic acid, PTAB). 5-hmC-dsDNA was chosen as the target, T4-β-Glucosyltransferase (β-GT) was as a molecular recognition element, and PTAB was as ECL signal reagent. PTAB includes the three main modules: 4-aminophthalhydrazide (isoluminol module), borinic acid module, and thiophene module. 5-hmC-dsDNA was glycosylated to glucosyl-5-hydroxymethylcytosine dsDNA (5-ghmC-dsDNA) by β-glucosyltransferase (β-GT); then, PTAB (boronic acid module) acted as a bridging agent to couple with 5-ghmC-dsDNA, yielding PTAB-5-ghmC-dsDNA; finally, the PTAB-5-ghmC-dsDNA (thiophene module) anchored to the gold surface via Au-S bond, the ECL signal parts (isoluminol module) was brought close to the biosensing electrode interface and the ECL enhanced simultaneously. The ECL intensity positively correlated with 5-hmC-dsDNA content in the range of 0.0090 % to 0.5761 %, with a detection limit of 0.0058 %. The proposed method was verified by detecting 5-hmC-dsDNA content in mice tissues. In addition, due to the multi-functional ECL signal compound, the presented strategy could be easily extended to ECL detection of various analytes with 1, 2- or 1, 3-diols sample, which provides a great potential for clinical applications.