Enzyme-Linked Electrochemical Detection of PCR-Amplified Nucleotide Sequences Using Disposable Screen-Printed Sensors. Applications in Gene Expression Monitoring.docVIP

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Enzyme-Linked Electrochemical Detection of PCR-Amplified Nucleotide Sequences Using Disposable Screen-Printed Sensors. Applications in Gene Expression Monitoring.doc

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Enzyme-Linked Electrochemical Detection of PCR-Amplified Nucleotide Sequences Using Disposable Screen-Printed Sensors. Applications in Gene Expression Monitoring

Sensors 2008, 8, 193-210 sensors ISSN 1424-8220 ? 2008 by MDPI /sensors Full Research Paper Enzyme-Linked Electrochemical Detection of PCR-Amplified Nucleotide Sequences Using Disposable Screen-Printed Sensors. Applications in Gene Expression Monitoring Petra Horakova-Brazdilova 1,2, Miloslava Fojtova 1, Karel Vytras 2 and Miroslav Fojta 1,* 1 Institute of Biophysics, v.v.i., Academy of Sciences of the Czech Republic, Kralovopolska 135, CZ- 612 65 Brno, Czech Republic; E-mail: petrahor@ibp.cz (P. H.); fojtova@ibp.cz (M. Fojtova); fojta@ibp.cz (M. Fojta) 2 Department of Analytical Chemistry, University of Pardubice, Nam. Cs. Legii 565, CZ-53210 Pardubice, Czech Republic; E-mail: karel.vytras@upce.cz (K. V.) * Author to whom correspondence should be addressed. Received: 28 December 2007 / Accepted: 7 January 2008 / Published: 21 January 2008 Abstract: Electrochemical enzyme-linked techniques for sequence-specific DNA sensing are presented. These techniques are based on attachment of streptavidin-alkaline phosphatase conjugate to biotin tags tethered to DNA immobilized at the surface of disposable screen-printed carbon electrodes (SPCE), followed by production and electrochemical determination of an electroactive indicator, 1-naphthol. Via hybridization of SPCE surface-confined target DNAs with end-biotinylated probes, highly specific discrimination between complementary and non-complementary nucleotide sequences was achieved. The enzyme-linked DNA hybridization assay has been successfully applied in analysis of PCR-amplified real genomic DNA sequences, as well as in monitoring of plant tissue-specific gene expression. In addition, we present an alternative approach involving sequence-specific incorporation of biotin-labeled nucleotides into DNA by primer extension. Introduction of multiple biotin tags per probe primer resulted in considerable enhancement of the signa