DNA Chips, Nanopores, Bio Assays
After sequencing the humane genome, biomedical research and development is en route to elucidate functions of genes that are closely associated with common ailments and disease states and apply the obtained genomic information for diagnosis, prognosis, and the screening for DNA-targeting therapeutic drugs. Designed to advance molecular functional genomics, DNA microarrays (“DNA chips”) are libraries of gene-specific nucleic acid recognition entities gritted in systematic order on a solid support. They emerged as a powerful microanalytical tool for studying the activity of many genes at a time and are valuable for rapid gene expression profiling, sequence mapping and genotyping of mutations and polymorphisms or pharmacogenomics. The ability of immobilized single-strands of DNA (capture probes) to interact with complementary sequences of free DNA fragments in solution (targets), to hybridize and form a double helix through base pairing provides the fundamental principle of nucleic acid hybridization techniques for the analysis of DNA variations. Accordingly, the reliable conversion of the biological recognition process into a detectable analytical signal is the key element for successful application of DNA chips. Presently, optical methods using fluorescence-labeled target DNA are state-of-the-art for the detection of the formation of the double helix. However, they require a rather complex pre-treatment of the target DNA during PCR amplification and expensive and bulky high-tech optical instrumentation. On the other hand, electrochemical (EC) detection schemes as reviewed in were explored and considered promising cost-effective alternatives to the well-established fluorescence-based read-outs of hybridization as they offer a high sensitivity in combination with simplicity of instrumentation and compatibility with microfabrication technology. So far, EC detection schemes are taking advantage of inherent electrochemical properties of single (ss) and double (ds) stranded DNA and/or electrochemically active hybridization indicators, intercalating compounds, and redox labels. In our group, electrochemical methodology for a truly label free visualizing of the status of surface-bound DNA probes was developed. With the proposed electrostatic approach the detection of complementary DNA hybridization becomes assessable through coulomb interactions between a negatively charged, free-diffusing redox mediator and the phosphate groups at the backbone of immobilized DNA strands. Diffusion of, for instance, [Fe(CN)6]3-/4- towards the surface of a DNA chip is hindered at DNA-modified regions due to localized repelling forces. Obviously, these charge interactions will modulate the mediator’s electron transfer kinetics with electron transfer rates expected to be significant lower at DNA spots as compared to the surrounding Au areas. The formation of double helices by complementary recognition processes is raising the density of negative charge inside spots of the capture probe, and detection of hybridization becomes possible due to a further decrease of the redox cycling rate. Taking this into consideration, a DNA microarrays is a sample with considerable local variations in superficial redox activity, at least for anionic electroactive species. For imaging these regions and for verifying the above-mentioned concept of electrostatic detection of hybridization scanning electrochemical microscopy (SECM) was the tool of choice because of its exceptional capability to visualize microscopically small local variations in (electro)chemical reactivity with high spatial resolution. The modulation of the amperometric SECM feedback current due to electrostatic repelling forces between negatively charged mediator molecules and the phosphate groups of DNA strands was used to establish a simple electrochemical assay that allows a label-free imaging of single-stranded capture probes and their double-stranded aggregates with complementary targets. Presently, novel signal amplification schemes are evaluated in nanopore structures aiming on the design of high-sensitive biorecognition assays.