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.

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The Potential and Challenges of Nanopore Sequencing

The passage of individual molecules through nanosized pores in membranes is central to many processes in biology. Previously, experiments have been restricted to naturally occurring nanopores, but advances in technology now allow artificial solid-state nanopores to be fabricated in insulating membranes. By monitoring ion currents and forces as molecules pass through a solid-state nanopore, it is possible to investigate a wide range of phenomena involving DNA, RNA and proteins. The solid-state nanopore proves to be a surprisingly versatile new single-molecule tool for biophysics and biotechnology.

Solid-state nanopores

This thesis explores transport phenomena in nanochannels on a chip. Fundamental nanofluidic ionic studies form the basis for novel separation and preconcentration applications for proteomic purposes. The measurements were performed with 50-nm-high 1D nanochannels, which are easily accessible from both sides by two microchannels. Nanometer characteristic apertures were manufactured in the bonded structure of Pyrex-amorphous silicon – Pyrex, in which the thickness of the amorphous silicon layer serves as a spacer to define the height of the nanochannels. The geometry of the nanometer-sized apertures is well defined, which simplifies the modeling of the transport across them. Compared to biological pores, the present nanochannels in Pyrex offer increased stability. Fundamental characteristics of nanometer-sized apertures were obtained by impedance spectroscopy measurements of the nanochannel at different ionic strengths and pH values. A conductance plateau (on a log-log scale) was modeled and measured, establishing due to the dominance of the surface charge density in the nanochannels, which induces an excess of mobile counterions to maintain electroneutrality. The nanochannel conductance can be regulated at low ionic strengths by pH adjustment, and by an external voltage applied on the chip to change the zeta potential. This field-effect allows the regulation of ionic flow which can be exploited for the fabrication of nanofluidic devices. Fluorescence measurements confirm that 50-nm-high nanochannels show an exclusion of co-ions and an enrichment of counterions at low ionic strengths. This permselectivity is related to the increasing thickness of the electrical double layer (EDL) with decreasing salt concentrations, which results in an EDL overlap in an aperture if the height of the nanochannel and the thickness of the EDL are comparable in size. The diffusive transport of charged species and therefore the exclusion-enrichment effect was described with a simple model based on the Poisson-Boltzmann equation. The negatively charged Pyrex surface of the nanometer characteristic apertures can be inversed with chemical surface pretreatments, resulting in an exclusion of cations and an enrichment of anions. When a pressure gradient is applied across the nanochannels, charged molecules are electrostatically rejected at the entrance of the nanometer-sized apertures, which can be used for separation processes. Proteomic applications are presented such as the separation and preconcentration of proteins. The diffusion of Lectin proteins with different isoelectric points and very similar compositions were controlled by regulating the pH value of the buffer. When the proteins are neutral at their pI value, the diffusion coefficient is maximal because the biomolecules does not interact electrostatically with the charged surfaces of the nanochannel. This led to a fast separation of three Lectin proteins across the nanochannel. The pI values measured in this experiment are slightly shifted compared to the values obtained with isoelectric focusing because of reversible adsorption of proteins on the walls which affects the pH value in the nanochannel. An important application in the proteomic field is the preconcentration of biomolecules. By applying an electric field across the nanochannel, anionic and cationic analytes were preconcentrated on the cathodic side of the nanometer-sized aperture whereas on the anodic side depletion of ions was observed. This is due to concentration polarization, a complex of effects related to the formation of ionic concentration gradients in the electrolyte solution adjacent to an ion-selective interface. It was measured that the preconcentration factor increased with the net charge of the molecule, leading to a preconcentration factor of > 600 for rGFP proteins in 9 minutes. Such preconcentrations are important in micro total analysis systems to achieve increased detection signals of analytes contained in dilute solutions. Compared to cylindrical pores, our fabrication process allows the realization of nanochannels on a chip in which the exclusion-enrichment effect and a big flux across the nanometer-sized aperture can be achieved, showing the interest for possible micro total analysis system applications. The described exclusion-enrichment effect as well as concentration polarization play an important role in transport phenomena in nanofluidics. The appendix includes preliminary investigations in DNA molecule separation and fluorescence correlation spectroscopy measurements, which allows investigating the behavior of molecules in the nanochannel itself.

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Transport phenomena in nanofluidics

Nanopore analysis is an emerging technique that involves using a voltage to drive molecules through a nanoscale pore in a membrane between two electrolytes, and monitoring how the ionic current through the nanopore changes as single molecules pass through it. This approach allows charged polymers (including single-stranded DNA, double-stranded DNA and RNA) to be analysed with subnanometre resolution and without the need for labels or amplification. Recent advances suggest that nanopore-based sensors could be competitive with other third-generation DNA sequencing technologies, and may be able to rapidly and reliably sequence the human genome for under $1,000. In this article we review the use of nanopore technology in DNA sequencing, genetics and medical diagnostics.

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Nanopore sensors for nucleic acid analysis

In nanopore analytics, individual molecules pass through a single nanopore giving rise to detectable temporary blockades in ionic pore current. Reflecting its simplicity, nanopore analytics has gained popularity and can be conducted with natural protein as well as man-made polymeric and inorganic pores. The spectrum of detectable analytes ranges from nucleic acids, peptides, proteins, and biomolecular complexes to organic polymers and small molecules. Apart from being an analytical tool, nanopores have developed into a general platform technology to investigate the biophysics, physicochemistry, and chemistry of individual molecules (critical review, 310 references).

Nanopore analytics: sensing of single molecules

Reducing a DNA molecule's translocation speed in a solid-state nanopore is a key step toward rapid single molecule identification. Here we demonstrate that DNA translocation speeds can be reduced by an order of magnitude over previous results. By controlling the electrolyte temperature, salt concentration, viscosity, and the electrical bias voltage across the nanopore, we obtain a 3 base/μs translocation speed for 3 kbp double-stranded DNA in a 4−8 nm diameter silicon nitride pore. Our results also indicate that the ionic conductivity inside such a nanopore is smaller than it is in bulk.

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Slowing DNA translocation in a solid-state nanopore

In this work, we report on how salt concentration and cation species affect DNA translocation in voltage-biased silicon nitride nanopores. The translocation of dsDNA in linear, circular, and supercoiled forms was measured in salt solutions containing KCl, NaCl, and MgCl(2) . As the KCl concentrations were decreased from 1 to 0.1 M, the time taken by a DNA molecule to pass through a nanopore was shorter and the frequency of the translocation in a folded configuration was reduced, suggesting an increase in DNA electrophoretic mobility and DNA persistence length. When the salt concentration was kept at 1 M, but replacing K(+) with Na(+) , longer DNA translocation times (t(d) ) were observed. The addition of low concentrations of MgCl(2) with 1.6 M KCl resulted in longer t(d) and an increased frequency of supercoiled DNA molecules in a branched form. These observations were consistent with the greater counterion charge screening ability of Na(+) and Mg(2+) as compared to K(+) . In addition, we demonstrated that dsDNA molecules indeed translocated through a ∼10 nm nanopore by PCR amplification and gel electrophoresis. We also compared the dependence of DNA mobility and conformation on KCl concentration and cation species measured at single molecule level by silicon nitride nanopores with existing bulk-based experimental results and theoretical predictions.

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Brian Thomas

Papers

Slowing DNA translocation in a solid-state nanopore

K+, Na+, and Mg2+ on DNA translocation in silicon nitride nanopores