Sensitive electrogenerated chemiluminescence biosensing method for the determination of DNA hydroxymethylation based on Ru(bpy) 3 2+ -doped silica nanoparticles labeling and MoS 2 -poly(acrylic acid) nanosheets modified electrode.

5-Hydroxymethylcytosine (5-hmC), an oxidation product of 5-mC (5-methylcytosine), is presented in DNA of most mammalian cells and play an important role in the alteration of cancer-related genes. Herein, a sensitive electrogenerated chemiluminescence (ECL) biosensing method for the determination of 5-hmC in DNA (5-hmC DNA) was established on the basis of chemical modification and nanomaterial amplification. First, electrochemically reduced molybdenum disulfide-poly(acrylic acid) (rMoS2 -PAA) nanosheets were used to modify glassy carbon electrode (GCE) to form an ECL biosensing electrode (rMoS2 -PAA/GCE) which has large accessible surface area to immobilize more DNA. Then, a capture probe with amino group was hybridized with the target 5-hmC DNA and immobilized on the surface of rMoS2 -PAA/GCE via amido bond. When cysteamine was introduced, the M.HhaI methyltransferase (M.HhaI) was used as specific recognition element to replace the hydroxyl group of 5-hmC by thiol and generated the amine-derivated DNA. Finally, surface chemically activated Ru(bpy)3 2+ -doped silica (Ru@SiO2 ) nanoparticles, carriers of ECL reagents, were employed as signal amplification unit which covalently bonded to the amine-derivated DNA resulting in an increased ECL intensity. The increased ECL intensity was linearity to the 5-hmC DNA concentration in a range from 5.0 × 10-14 M to 1.0 × 10-11 M, with a lower detection limit of 1.2 × 10-14 M. Besides, the proposed method also displayed a good selectivity to 5-hmC in the presence of 5-C and 5-mC. Moreover, the developed biosensing method was successfully employed to monitor human urine sample.