Detection of bar gene encoding phosphinothricin herbicide resistance in plants by electrochemical biosensor

An electrochemical biosensor for the detection of bar gene coding phosphinothricin herbicide resistance is presented. The detection was based on hybridization reaction between the specific to bar gene 19-mer probe immobilized on the electrode surface and complementary DNA in a sample. Single-stranded DNA probe specific to bar gene was covalently attached by 5'-phosphate end to the surface of carbon paste electrode. Outer layer of a conventional CPE was provided with carboxyl groups of stearic acid. ssDNA was coupled to the electrode through ethylenediamine with the use of water-soluble 1-ethyl-3(3'-dimethylaminopropyl)carbodiimide and N-hydroxy-sulfosuccinimide as activating reagents. Hybridization reaction at the electrode surface was detected via Co(bpy)(3)(3+) which posses a much higher affinity to the resulting DNA duplex compared to ssDNA probe. Detection limit of the sensor was 0.1 mu M of target DNA fragments and its response was linear from 5 to 20 mu M. Hybridization event was also detected by measuring guanine peak but this approach presented distinctly higher detection limit (1 mu M) and lower reproducibility. Complete time of one measurement with the use of the biosensor including covalent attachment of ethylenediamine (linker) and ssDNA probe to the electrode, hybridization with target and interaction with electroactive indicator was about 70 min. (C) 2008 Elsevier B.V. All rights reserved.