Multipotential Electrochemical Detection of Primer Extension Reactions on DNA Self-Assembled Monolayers

摘要

Electrochemical detection of nucleic acids is an attractive alternative to established mass- and fluorescence-based methodologies, with advantages including cost, sensitivity, and direct electronic readout. The development of self-assembled monolayers (SAMs) of DNA oligonucleotides on gold electrodes has facilitated assay development, coupling the specificity of nucleic acid hybridization to a bioelectronic interface. Target nucleic acids can be captured on these DNA SAMs, especially for single-nucleotide polymorphism (SNP) identification. One potential SNP genotyping strategy has relied upon differential charge conduction from electroactive intercalators through matched or mismatched duplexes to the SAM electrode surface. The capture of labeled electroactive probes near the SAM surface has also been described. While these methods use Watson-Crick hybridization to discriminate between very similar nucleic acids, it should also be possible to achieve more robust identification by utilizing the intrinsic fidelity of enzymes. With these applications in mind, electroactive nucleoside triphosphates have recently been introduced for incorporation by polymerases or terminal transferases. These species have first been applied in HPLC-ECD, in capillary gel electrophoresis (CE)-based primer extension, and in the detection of viral DNA following redox enzyme amplification. In this communication we describe a range of electroactive nucleoside triphosphates ("electrotides") and their use in polymerase-mediated single base extension (SBE) assays with specific detection on DNA SAMs in several alternative formats.