Electrogenerated chemiluminescence biosensing method for methyltransferase activity using tris(1, 10-phenanthroline) ruthenium-assembled graphene oxide

A novel electrogenerated chemiluminescence (ECL) biosensing method for highly sensitive detection of DNA methylation and assay of the methyltransferase (MTase) activity was developed by using the methylation-sensitive restriction endonuclease Dpn I and methylation-responsive hairpin-capture DNA probe to improve selectivity and by employing signal amplification of graphene oxide (GO) to enhance the assay sensitivity. The ECL biosensing electrode was fabricated by self-assembling a design of 5'-thiol modified methylation-responsive hairpin-capture DNA probe on the surface of a gold electrode. When DNA adenine methylation (Dam) MTase and S-adenosylmethionine were introduced, all adenines in the symmetric tetranucleotide 5'-GATC-3' of hairpin-capture DNA probe on the biosensing electrode were methylated. After the methylated biosensing electrode was treated by Dpn I endonuclease, the methylated adenines were cleaved, methylation-induced scission of hairpin-capture DNA probe would displace the hairpin section and remain the capture DNA probe section on the gold electrode. Subsequently, the remained capture DNA probe on the gold electrode can hybridize with the 5'-amino modified DNA probe. Finally, tris(1, 10-phenanthroline) ruthenium (Ru(phen)(3)(2+))-assembled GO composites were conjugated to the electrode surface via EDC NHS coupling, a strong ECL response was electrochemically generated. The increased ECL intensity was proportion to Dam MTase activity in the range from 0.05U/mL to 40 U/mL with a detection limit of 0.02 U/mL. The present work demonstrates that the combination of the enzyme-linkage and GO as a platform for signal probe is a great promising approach for MTase activity and evaluation of the capability of inhibitors for the MTase. (C) 2014 Elsevier B.V. All rights reserved.