title: An enzyme-amplified amperometric DNA hybridisation assay using DNA immobilised in a carboxymethylated dextran film anchored to a graphite surface.
abstract: This report describes a simple methodology for preparation of an enzyme-amplified amperometric DNA hybridisation assay using solution-phase ferrocenemethanol mediation of glucose oxidase oxidation of glucose. The recognition layer consists of amine-terminated ssDNA (designed for binding of the sequence ssrA gene of Listeria monocytogenes) bound within a carboxymethylated dextran film that is anchored to a graphite electrode. Anchoring sites are provided by electrochemical grafting of an arylamine to the carbon surface following in situ diazotisation of p-phenylenediamine. Hybridisation between the immobilised probe ssDNA and a biotin-labelled target ssDNA sequence is detected by following the oxidation of glucose upon addition of a glucose oxidase-avidinD conjugate and ferrocenemethanol. The stability of the anchored film permits washing and blocking steps to discriminate between hybridisation and non-specific binding. The signal current, measured by cyclic voltammetry and constant potential amperometry, scales with biotin-complementary DNA concentration, from 2.5x10(-6) M to 3x10(-7) M and a detection limit of 0.2 nmol in the 500 microL sample at the 3-mm diameter graphite electrode is estimated. Approaches to improve upon the analytical performance of this simple assay are highlighted.
authors.affiliation: School of Chemistry, National University of Ireland, Galway, Galway, Ireland.
authors.forename: Joanna
authors.initials: J
authors.lastname: Hajdukiewicz
authors.forename: Susan
authors.initials: S
authors.lastname: Boland
authors.forename: Paul
authors.initials: P
authors.lastname: Kavanagh
authors.forename: Dónal
authors.initials: D
authors.lastname: Leech
mesh_terms: D015374:Biosensing Techniques / Q000295:instrumentation; D020099:Coated Materials, Biocompatible / Q000737:chemistry; D004247:DNA / Q000032:analysis / Q000737:chemistry* / Q000235:genetics*; D003911:Dextrans / Q000737:chemistry*; D004563:Electrochemistry / Q000295:instrumentation*; D004566:Electrodes; D004867:Equipment Design; D019544:Equipment Failure Analysis; D005949:Glucose Oxidase / Q000737:chemistry*; D006108:Graphite / Q000737:chemistry*; D017403:In Situ Hybridization / Q000295:instrumentation*; D008745:Methylation; D020411:Oligonucleotide Array Sequence Analysis / Q000295:instrumentation; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D020099:Coated Materials, Biocompatible; D003911:Dextrans; D006108:Graphite; D004247:DNA; D005949:Glucose Oxidase