Development of electrochemical DNA biosensor for “DG74 primer” using modern FFT voltammetry

“DG74 primer” is a universal bacterial primer to amplify and detect DNAs from phylogenetically divergent bacteria by targeting conserved regions of the 16S rRNA gene [1, 2]. In this study, a simple and rapid fast Fourier transform square-wave voltammetry (FFT-SWV) method for development of an electrochemical DNA biosensor for “DG74 primer”, using a gold electrode as transducer and methylene blue (MB) as electroactive label is described. The sensor relies on covalent attachment of the 19-mer thiolated DNA probe on the gold electrode. Covalently selfassembled probe could selectively hybridize with a complementary sequence (DG74 primer) in solution to form double-stranded DNA on the surface [3]. The hybridization between the probe and its complementary sequence as the target was studied by modern FFT-SWV signal of MB accumulated on the modified electrode by scanning the electrode potential between 0.1 and -0.5 V. In this approach the extent of hybridization is evaluated on the basis of the difference between FFTSWV signals of MB accumulated on the probe modified Au electrode before and after hybridization. Some hybridization experiments with noncomplementary oligonucleotides were carried out to assess whether the suggested DNA sensor responds selectively to the target. Furthermore, signal-to-noise ratio was significantly increased by application of discrete fast Fourier transform (FFT) method [4]. Some experimental variables affecting the performance of the biosensor including: MB accumulation time, probe SAM formation time and concentration of probe and method and required time for hybridazition were investigated. Furthermore effects of square-wave frequency, step potential and pulse amplitude were examined for the optimization of instrumental conditions. Diagnostic performance of the biosensor is described and the calibration graph is linear between 0.1 and 3 nM. The detection limit was found to be 33 pM and the relative standard deviation over five independently probe-modified electrodes measured at 1 nM Target, was 1.7%, indicating a remarkable reproducibility of the detection method.