Sequence Specific Electrochemical DNA Detection Based on Solution-Phase Competitive Hybridization

Abstract

We report a novel electrochemical method for detecting sequence-specific DNA based on competitive hybridization that occurs in a homogeneous solution phase instead of on a solution-electrode interface as in previously reported competition-based electrochemical DNA detection schemes. The method utilizes the competition between the target DNA (t-DNA) and a ferrocene-labeled peptide nucleic acid probe (Fc-PNA) to hybridize with a probe DNA (p-DNA) in solution. The neutral PNA backbone and the electrostatic repulsion between the negatively-charged DNA backbone and the negatively-charged electrode surface are then exploited to determine the result of the competition through measurement of the electrochemical signal of Fc. Upon the introduction of the t-DNA, the stronger hybridization affinity between the t-DNA and p-DNA releases the Fc-PNA from the Fc-PNA/p-DNA hybrid, allowing it to freely diffuse to the negatively charged electrode to produce a significantly enhanced electrochemical signal of Fc. Therefore, the presence of the t-DNA is indicated by the appearance or enhancement of the electrochemical signal, rendering a signal-on DNA detection, which is less susceptible to false positive and can produce more reliable results than signal-off detection methods. All the competitive hybridizations occur in a homogeneous solution phase, resulting in very high hybridization efficiency and therefore extremely short assay time. This simple and fast signal-on solution-competition-based electrochemical DNA detection strategy has promising potential to find application in fields such as nucleic acid-based point-of-care testing.