No abstract

A nanopore-based device provides single-molecule detection and analytical capabilities that are achieved by electrophoretically driving molecules in solution through a nano-scale pore. The nanopore provides a highly confined space within which single nucleic acid polymers can be analyzed at high throughput by one of a variety of means, and the perfect processivity that can be enforced in a narrow pore ensures that the native order of the nucleobases in a polynucleotide is reflected in the sequence of signals that is detected. Kilobase length polymers (single-stranded genomic DNA or RNA) or small molecules (e.g., nucleosides) can be identified and characterized without amplification or labeling, a unique analytical capability that makes inexpensive, rapid DNA sequencing a possibility. Further research and development to overcome current challenges to nanopore identification of each successive nucleotide in a DNA strand offers the prospect of `third generation' instruments that will sequence a diploid mammalian genome for ~$1,000 in ~24 h.

The electrical noise characteristics of ionic current through organic and synthetic nanopores have been investigated. Comparison to proteinaceous alpha-Hemolysin pores reveals two dominant noise sources in silicon nitride nanometre-scale pores: a high-frequency noise associated with the capacitance of the silicon support chip (dielectric noise), and a low-frequency current fluctuation with 1/fα characteristics (flicker noise). We present a technique for reducing the dielectric noise by curing polydimethylsiloxane (PDMS) on the nanopore support chip. This greatly improves the performance of solid-state nanopore devices, yielding an unprecedented signal-to-noise ratio when observing dsDNA translocation events and ssDNA probe capture for force spectroscopy applications.

Figure 4. Comparison of single-mismatch detection with gold-quenched beacons versus DABCYL-quenched beacons. Titration of 5 µM of random target mixed with 4.2 nM of gold-DNA-rhodamine 6G conjugate and 0.6 µM of gold (A), and 5 µM of random target mixed with 10 nM of molecular beacon (B), with the perfect target (target 2) and the mismatch one (target 3). Target concentrations vary from 67 pM to 13 µM. For both probes, the perfect target (solid line) produces a faster and sharper increase of fluorescence than the target containing the mismatch (dashed line). Fluorescence intensities due to the buffer and the gold have been subtracted. The inset graphs in (A) and (B) show the evolution of the fluorescence as a function of time when the probe is mixed with 5 µM of random targets. In both cases, the random targets do not induce any change of fluorescence of the probe during the time of the titration. The hybridization is thus very specific to the matched or the mismatched targets. (C) Ratio between the titration curve with the perfect target (target 2) and the titration curve with the mismatched one (target 3). (D) Resolution of a matched and a mismatched target, competing for hybridization. Molecular beacon (dashed line), gold-DNA-dye conjugate (solid line). α is the population ratio of match to mismatch targets. The concentration of perfect target is fixed at 0.2 µM. D

We have engineered a nanosensor for sequence-specific detection of single nucleic acid molecules across a lipid bilayer. The sensor is composed of a protein channel nanopore (alpha-hemolysin) housing a DNA probe with an avidin anchor at the 5' end and a nucleotide sequence designed to noncovalently bind a specific single-stranded oligonucleotide at the 3' end. The 3' end of the DNA probe is driven to the opposite side of the pore by an applied electric potential, where it can specifically bind to oligonucleotides. Reversal of the applied potential withdraws the probe from the pore, dissociating it from a bound oligonucleotide. The time required for dissociation of the probe-oligonucleotide duplex under this force yields identifying characteristics of the oligonucleotide. We demonstrate transmembrane detection of individual oligonucleotides, discriminate between molecules differing by a single nucleotide, and investigate the relationship between dissociation time and hybridization energy of the probe and analyte molecules. The detection method presented in this article is a candidate for in vivo single-molecule detection and, through parallelization in a synthetic device, for genotyping and global transcription profiling from small samples.