An enzyme-amplified amperometric DNA hybridisation assay using DNA immobilised in a carboxymethylated dextran film anchored to a graphite surface

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 2.5 x 10(-6) M to 3 x 10(-1) M and a detection limit of 0.2 nmol in the 500 mu L sample at the 3-mm diameter graphite electrode is estimated. Approaches to improve upon the analytical performance of this simple assay are highlighted. (C) 2009 Elsevier B.V. All rights reserved.