Preparation of a new electrochemical biosensor for single base mismatch detection in DNA

Abstract

In this study, a sandwich-type electrochemical DNA hybridization biosensor for the detection of single base mismatches (SBMs) has been described. 2,5-Dihydroxybenzoic acid (DHBA) is employed as a new redox probe in this study. For construction of the biosensor, thiolated capture DNA is self-assembled on a gold electrode surface and hybridized with one region of a target DNA sequence. Subsequently, a signaling amino-labeled probe DNA is hybridized to a second region of the target DNA to complete the DNA double-helix self-assembled monolayer (SAM). Finally DHBA is covalently attached to the signaling probe DNA using the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysulfosuccinimide (NHS) cross-linking reaction. Electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) are employed to characterize the modification steps. The electrochemical signal of DHBA was followed for studying different types of mismatches (GA, GT, TT, CA), both near the electrode surface (N-type) and far from it (F-type). The presence of a SBM leads to a considerable decrease in the electrochemical signal of DHBA compared to that of complementary DNA (cDNA), which makes the detection of mismatches (including thermodynamically stable GA mismatches) possible. The redox signal of DHBA was compared with that of methylene blue (MB) as a well-known and standard redox reporter in DNA biosensors. The results showed that for an N-type SBM, which is located between the electrode and the site of intercalation, the redox signal of MB switches off, whereas for an F-type SBM, which is located above the site of intercalation, electrons flow from the Au electrode to the intercalated MB, and hence the biosensor could not distinguish the presence and absence of an SBM. The proposed biosensor is straightforward with no need for target labeling, and is sensitive enough to detect SBMs independent of their position in DNA double helix.