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

Joanna Hajdukiewicz, Susan Boland, Paul Kavanagh, Dónal Leech*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

22 Citations (Scopus)

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 2.5 × 10-6 M to 3 × 10-7 M and a detection limit of 0.2 nmol in the 500 μL sample at the 3-mm diameter graphite electrode is estimated. Approaches to improve upon the analytical performance of this simple assay are highlighted.

Original languageEnglish
Pages (from-to)1037-1042
Number of pages6
JournalBiosensors and Bioelectronics
Volume25
Issue number5
DOIs
Publication statusPublished - 15 Jan 2010

Keywords

  • Amperometric
  • Carboxymethylated dextran
  • Diazonium salts
  • DNA hybridisation
  • DNA sensor
  • EDC/NHS
  • Mediator

ASJC Scopus subject areas

  • Biophysics
  • Biomedical Engineering
  • Biotechnology
  • Electrochemistry

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