title: A label-free electrochemical assay for methyltransferase activity detection based on the controllable assembly of single wall carbon nanotubes.
abstract: A sensitive label-free "signal-on" electrochemical approach for detection of methyltransferases (MTase) activity is developed based on the signal transduction and amplification of single wall carbon nanotubes (SWCNTs). In this method, the oligonucleotide I is first self-assembled on the electrode via Au-S bonding. After hybridization with its complement ssDNA (oligonucleotide II), duplex strand DNA (dsDNA) probes containing specific recognition sequence of Dam MTase and methylation-sensitive restriction endonuclease Dpn I is then formed on the electrode. In the presence of Dam MTase and Dpn I, the dsDNA probes are methylated and subsequently cleaved into two dsDNA fragments. After heating, the remained dsDNA fragments on the electrode melted into ssDNA fragments. Then the SWCNTs can be controllably assembled on the ssDNA fragments remained on the electrode, mediating efficient electron transfer between the electrode and electroactive species. It generates measurable current signal (eT ON), which is related to the concentration of the Dam MTase. The resulting change in electron transfer efficiency is readily measured by differential pulse voltammetry at Dam MTase concentrations as low as 0.04 U/mL. This method does not need electroactive molecules labeling on the methylation-responsive DNA probes. The linear response of the developed facile signal-on electrochemical sensing system for Dam MTase is in the range of 0.1-1.0 U/mL. In addition, such a SWCNTs based electrochemical assay also has the ability to screen inhibitors for Dam MTase.
authors.affiliation: State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Biology, Hunan University, Changsha 410082, PR China.
authors.forename: Yonghong
authors.initials: Y
authors.lastname: Wang
authors.forename: Xiaoxiao
authors.initials: X
authors.lastname: He
authors.forename: Kemin
authors.initials: K
authors.lastname: Wang
authors.forename: Jing
authors.initials: J
authors.lastname: Su
authors.forename: Zhifeng
authors.initials: Z
authors.lastname: Chen
authors.forename: Genping
authors.initials: G
authors.lastname: Yan
authors.forename: Yudan
authors.initials: Y
authors.lastname: Du
mesh_terms: D015374:Biosensing Techniques / Q000295:instrumentation*; D003217:Conductometry / Q000295:instrumentation*; D003460:Crystallization / Q000379:methods; D015254:DNA Modification Methylases / Q000032:analysis* / Q000737:chemistry*; D015342:DNA Probes / Q000737:chemistry* / Q000235:genetics*; D004789:Enzyme Activation; D004867:Equipment Design; D019544:Equipment Failure Analysis; D036103:Nanotechnology / Q000295:instrumentation*; D037742:Nanotubes, Carbon / Q000737:chemistry / Q000648:ultrastructure; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013194:Staining and Labeling
chemical_list: D015342:DNA Probes; D037742:Nanotubes, Carbon; D015254:DNA Modification Methylases