Preliminary evaluation for a novel voltammetric analysis of targeted nucleic acid by combining electrochemical DNA chip and digital loop-mediated isothermal amplification

Abstract

We propose a novel voltammetric analysis method for the targeted nucleic acid quantification using electrochemical DNA chip and digital loop-mediated isothermal amplification (LAMP), which is based on the most probable number (MPN) method. We designed the position of electrodes (30 regions) on the DNA chip surface at 2 mm intervals in the flow channel. Since the LAMP reaction is carried out at constant temperature, it is supposed that the motions of both the targeted nucleic acid and amplicon in the LAMP solution are diffusion-limited. So an independent volume is formed around the electrode (like a test tube). MPN for a single dilution with any positive electrodes was given by MPN (μL−1)=1/me×2.303×log ((number of total electrode)/(number of negative electrode)). The me denotes the amount of sample in each volume covered by an electrode on the DNA chip. When different concentrations of targeted nucleic acid were amplified, all electrodes on the DNA chip were positive above 2×101copies/μL. On the other hand, positive ratios of detection decreased to 25% and 88% at 2×10−1 and 2×100copies/μL, respectively. From these results, the volume covered by an electrode in the flow channel was calculated to be 1.2±0.2μL. The MPN method is applicable to nucleic acid analysis using electrochemical DNA chip and several copies of targeted nucleic acid are quantified precisely without calibration curve.