Nucleic acid analysis is frequently sought for diagnosis of disease, microbial contamination, forensics, biosecurity and basic research and development. These analyses often use surface capture methods to identify a soluble DNA or RNA target analyte by selective or specific, complementary complexation with a matching probe nucleic acid oligomer immobilized on a capture surface. One popular commercialized method—nucleic acid microarray analyses—use rapid robotic printing methods to place micrometer-sized spots of DNA target libraries onto activated solid supports in cataloged arrays. Exposure of these surfaces to samples containing unknown nucleic acid analytes permits simultaneous screening of up to tens of thousands of different complementary DNA or mRNA analytes or their single nucleotide polymorphisms (SNPs) in complex biological samples. While many modes of analyte detection of bound nucleic acid signal on the support are possible, measurement usually exploits image analysis of fluorescent emission from tags placed specifically on the captured analyte (typically using polymerase chain reaction). The fluorescent intensity of each microarray spot on the slide is then collected (via surface fluorescence scanning) and analyzed.
Fluorescence-based nucleotide microarray assays are technically attractive, currently the focus of a billion-dollar commercial effort, but exhibit significant limitations: (1) fluorescence has only relative correlation with natural DNA target abundance without absolute reference to actual target concentrations, (2) fluorescence is relatively insensitive, requiring either pre-purification or amplification of DNA target to achieve reasonable assay detection limits (picomolar), (3) probe printing and target dye-labeling processes are expensive and time consuming, and (4) detection requires highly sophisticated optical scanners, laser excitation sources and post-assay image processing. As one alternative, electrochemical DNA detection has received substantial attention due to its intrinsically rapid response, ease of handling, compatibility with miniaturization technology and relatively low cost. By combining electrochemistry with the selectivity or specificity of biological recognition processes, electrochemical biosensors occupy an important analytical position.
Electrochemically based DNA analysis based on nucleic acid hybridization are prepared by immobilizing single-stranded DNA (ssDNA) oligomer probe sequences on electrode surfaces and using electroactive indicators to measure hybridization events between immobilized probes and their complementary DNA (cDNA) target fragments. Such electrochemical assays can be classified into two categories depending on their signal transduction mode on the electrode. Direct signal transduction sensors rely on electro-oxidation of nucleotide guanine or adenine residues in DNA target chains hybridized to electrode surfaces. See, for example, Wang, et al., Anal. Chim Acta, 1998, 375, 197-2003. To amplify weak oxidative signals from guanine or adenine oxidation to improve detection limits, Ru(bpy)32+ ion has been employed as a redox mediator, producing electrocatalytic oxidation. See, for example, Johnston, et al., J. Am. Chem. Soc., 1995, 117, 8933-8938; and Gore, et al., Anal Chem., 2003, 75, 6586-6592. One of the drawbacks of this sensor design is the inherent destruction of the electrode-captured DNA probe and target, preventing further measurement. Indirect detection of hybridization, on the other hand, exploits enzyme labels, or other redox mediators such as Os(bpy)32+, Co(phen)33+ and Co(bpy)33+, or intercalating organic compounds including daunomycin, methylene blue or acridine orange. It is believed that these electroactive sensitizers, some of which are chosen for their stability and reversibility in redox reactions, interact with ssDNA probes and hybridized double stranded DNA (dsDNA) via electrostatic binding to DNA phosphate groups, hydrophobic binding to dsDNA's minor groove and/or intercalation (hydrophobic partitioning) into base pairs. In some of these cases, DNA target hybridization is detected by measuring the redox current generated by redox mediators associated with dsDNA on the electrode. For sensitive, accurate and reliable determination of DNA target hybridization, these redox species must interact more efficiently with dsDNA than with ssDNA targets.
Accordingly, there is a continuing need for redox species that can interact more efficiently with dsDNA than with ssDNA targets in order to provide more sensitive, accurate and reliable determination of DNA target hybridization.