ELECTROCHEMICAL DNA BIOSENSOR FOR DETECTION OF Ganoderma boninense Pat. PATHOGEN OF OIL PALM TREE
Electrochemical DNA biosensors (ssDNA / AgNPs / PEDOT-PSS / AuE; ssDNA / AuNPs / PEDOT-PSS / AuE and ssPNA / AuNPs / PEDOT-PSS / AuE) are made based on the recognition of 20-mer single-stranded oligonucleotide and peptide nucleic acid (ssDNA and ssPNA probes) sequences specific to Ganoderma boninense gene immobilized onto a modified gold electrode. The electrodes were modified using nanocomposite film of poly(3,4-ethylenedioxythiopen) poly(styrenesulfonate) (PEDOT-PSS) with silver (AgNPs), and gold (AuNPs) nanoparticles respectively. The nanoparticles and their nanocomposites with PEDOT-PSS were characterized where the particles size, shape, distribution of the nanoparticles and nanocomposite surface modification morphology, elemental compositions were investigated. Cyclic voltammetry (CV) and differential pulse voltammetry (DPV) systems of measurements were used throughout the detection analysis. Ruthenium [Ru(dppz)2(qtpy)]2+ and [Ru(phen)2(qtpy)]2+ complexes were used as redox indicators in monitoring the hybridization events. The complexes belongs to the family of quarterpyridyl of dipyrido and phenanthroline ligands respectively and have exhibited higher electrical conductivity to bare and modified gold electrode. A 5’-NH2-ends of the DNA/PNA probes were used to form covalent amide bond immobilization as shown in Figure 1 for hybridization. The hybridization events
Schematic representation of electrode modification and covalent attachment of DNA probe for hybridization events
were carried out with synthesised complementary, mismatched and genomic DNA of Ganoderma boninense fungi extracted using DNeasy Plant Maxi Kit Qiagen protocol. Hybridization temperature and time were optimized at 45 °C for 35 minutes and the effect of target DNA concentrations (in the range 1.0 x 1015 M to 1.0 x 109 M) in the hybridizations were performed. An increased DNA concentration was found to correspond with a higher electrochemical response due to high amount of redox intercalation by the hybridized DNA (on DNA-DNA or PNADNA helix). The biosensors have allowed the recognition of labeled DNA/PNA probes with full complementary and mismatched target sequences. However the sensors offer good selectivity, high sensitivity (LoD = 3.3/S mol L1 between 1.55 x 1018 to 7.90 x 1019) in a wider detection range and simple method for routine detection of Ganoderma boninense DNA.