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Sensors 2008, 8(1), 193-210; doi:10.3390/s8010193
Article

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

1
,
1
,
2
 and
1,*
1 Institute of Biophysics, v.v.i., Academy of Sciences of the Czech Republic, Kralovopolska 135, CZ-612 65 Brno, Czech Republic 2 Department of Analytical Chemistry, University of Pardubice, Nam. Cs. Legii 565, CZ-53210, Czech Republic
* Author to whom correspondence should be addressed.
Received: 28 December 2007 / Accepted: 7 January 2008 / Published: 21 January 2008
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Abstract

Electrochemical enzyme-linked techniques for sequence-specific DNA sensingare presented. These techniques are based on attachment of streptavidin-alkalinephosphatase conjugate to biotin tags tethered to DNA immobilized at the surface ofdisposable screen-printed carbon electrodes (SPCE), followed by production andelectrochemical determination of an electroactive indicator, 1-naphthol. Via hybridizationof SPCE surface-confined target DNAs with end-biotinylated probes, highly specificdiscrimination between complementary and non-complementary nucleotide sequences wasachieved. The enzyme-linked DNA hybridization assay has been successfully applied inanalysis of PCR-amplified real genomic DNA sequences, as well as in monitoring of planttissue-specific gene expression. In addition, we present an alternative approach involvingsequence-specific incorporation of biotin-labeled nucleotides into DNA by primerextension. Introduction of multiple biotin tags per probe primer resulted in considerableenhancement of the signal intensity and improvement of the specificity of detection.
Keywords: electrochemical detection; enzyme-linked assay; DNA hybridization; primer extension; PCR; gene expression electrochemical detection; enzyme-linked assay; DNA hybridization; primer extension; PCR; gene expression
This is an open access article distributed under the Creative Commons Attribution License (CC BY 3.0).

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Horaková-Brazdilova, P.; Fojtova, M.; Vytras, K.; Fojta, M. Enzyme-Linked Electrochemical Detection of PCR-Amplified Nucleotide Sequences Using Disposable Screen-Printed Sensors. Applications in Gene Expression Monitoring. Sensors 2008, 8, 193-210.

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