A novel electrochemiluminescence biosensor for the detection of 5-methylcytosine, TET 1 protein and β-glucosyltransferase activities based on gold nanoclusters-H2O2 system

5-Methylcytosine (5 mC) is considered to play a key regulating role in gene transcription and can be oxidized by ten-eleven translocation (TET) dioxygenases to 5-hydroxymethylcytosine (5 hmC). In order to better understand the function of 5 mC in gene transcription and the mechanism of demethylation, sensitive and selective methods must be developed for detecting 5 mC, the TET 1 protein and T4 beta-glucosyltransferase (beta-GT). In this work, a novel electrochemiluminescence (ECL) biosensor was fabricated for the quantification of 5 mC, TET 1 protein and beta-GT activities, as well as inhibitor screening, based on the interaction of chemically excited gold nanoclusters (AuNCs) with H2O2. The ECL biosensor was constructed on an Au nanoparticle (AuNP) decorated glassy carbon electrode, to which BSA coated AuNCs and thiol-containing probe DNA was subsequently attached. Hybridization with target 5 mC-DNA, was followed by demethylation by TET 1 and glycosylation by beta-GT. After reaction of the glycosyl group with 4-carboxyphenylboronic acid, horseradish-peroxidase was then grafted to the DNA backbone to catalyze H2O2 reduction and the oxidation of hydroquinone to benzoquinone. The ECL of the chemically excited AuNCs was attenuated in the presence of H2O2, with more H2O2 being decomposed in the presence of 5 mC DNA, TET-1 or beta-GT, leading to an enhanced ECL response. The proposed assay demonstrated high selectivity for 5 mC-DNA detection in the concentration range 0.01 nM-50 nM, with a detection limit of 3.46 pM. The applicability of this biosensor was confirmed by the evaluation of blood serum, with the developed biosensor having great potential in drug discovery.