Homogeneous detection of 5-hydroxymethylcytosine based on electrochemiluminescence quenching of g-C

A sensitively homogeneous electrochemiluminescence (ECL) method was developed for 5-hydroxymethylcytosine (5hmC) detection using TiO2/MoS2/g-C3N4/GCE as substrate electrode, where g-C3N4 was employed as the ECL active material, the MoS2 nanosheets were used as co-catalyst, and TiO2 was adopted as phosphate group capture reagent. To achieve the specific recognition and capture of 5hmC, the covalent reaction between -CH2OH and -SH was employed under the catalysis of HhaI methyltransferase, in which, -SH functionalized ferrocenedicarboxylic acid polymer (PFc-SH) was prepared as 5hmC capture reagent and ECL signal quencher. Then, based on the interaction between TiO2 and phosphate group of 5hmC, the target was recognized and captured on electrode, resulting in a decreased ECL response due to the quenching effect of PFc-SH. Under optimal conditions, the biosensor presented the linear range from 0.01 to 500 nM with the detection limit of 3.21 pM (S/N = 3). The steric effect on electrode surface is a bottle-neck issue restricting devised biosensors advancement. In this work, the reaction between 5hmC and PFc was carried out in the solution, which can avoid steric effect on electrode surface to keep the high activity of enzyme. In addition, the biosensor was successfully applied to detect 5hmC in genomic DNA of chicken embryo fibroblast cells and different tissues of rice seedlings.