Fluorescence detection of DNA-ethidium complexes in the diffusion layer of a modified electrode

An extremely rapid and highly sensitive technique for the quantitative determination of bulk (total) deoxyribonucleic acid (DNA) samples at the picogram level is described. The method is based on the release of ethidium cation (2,7-Diamino-10-ethyl-9-phenyl-phenanthridinium), Et⁺, into a DNA solution via oxidation of a tetracyanoquinodimethane (TCNQ-) acceptor anion at a carbon paste/Et⁺TCNQ^- composite electrode surface. Detection of the DNA was performed fluorometrically by exciting the DNA-ethidium complex in the diffusion layer using the 514.5 nm line of an argon ion laser aligned parallel to the electrode surface. Detection occurred in less than 20 seconds after application of +0.45 V potential. Sample preparation was quickly and easily done by dissolving calf thymus DNA in HEPES (N-[2-Hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid]) buffer solution, pH 7.0 without the need for sample denaturization by heat. Experimental parameters which have been optimized include (i) electrode composition, (ii) applied electrode potential, (iii) distance of the excitation source from the electrode surface, (iv) electrolysis time, and (v) laser power. Although various ratios of EtTCNQ to carbon proved acceptable for data collection, a 1:20 ratio enabled complex measurement over 6 orders of magnitude of DNA concentration. It was determined that the optimum applied potential for ethidium release was +0.45 V. Varying the distance of the excitation source from the electrode surface provided a means of monitoring the diffusion front as it traversed the containment cell. A very small distance of the source from the electrode resulted in short analysis times and reasonable reproducibility. Because the surface of a carbon paste electrode had to be renewed after the chemical concentration was exhausted, a method was developed so that the electrode could be pulsed several times before the ethidium supply was depleted. Various electrolysis times were compared for peak reproducibility and sufficient signal to noise ratio. A 5 seconds pulse time met the criteria. Laser power was examined in attempts to further improve signal to noise ratios, but high excitation power resulted in rapid and uneven diffusion from heating and increased background signal.