Three decades of nanopore sequencing.

A long-held goal in sequencing has been to use a voltage-biased nanoscale pore in a membrane to measure the passage of a linear, single-stranded (ss) DNA or RNA molecule through that pore. With the development of enzyme-based methods that ratchet polynucleotides through the nanopore, nucleobase-by-nucleobase, measurements of changes in the current through the pore can now be decoded into a DNA sequence using an algorithm. In this Historical Perspective, we describe the key steps in nanopore strand-sequencing, from its earliest conceptualization more than 25 years ago to its recent commercialization and application.

Fracture faces of frozen membranes: 50th anniversary.

In 1961, the development of an improved freeze-etching (FE) procedure to prepare rapidly frozen biological cells or tissues for electron microscopy raised two important questions. How does a frozen cell membrane fracture? What do the extensive face views of the cell's membranes exposed by the fracture process of FE tell us about the overall structure of biological membranes? I discovered that all frozen membranes tend to split along weakly bonded lipid bilayers. Consequently, the fracture process exposes internal membrane faces rather than either of the membrane's two external surfaces.

Nanopatterning on nonplanar and fragile substrates with ice resists.

Electron beam (e-beam) lithography using polymer resists is an important technology that provides the spatial resolution needed for nanodevice fabrication. But it is often desirable to pattern nonplanar structures on which polymeric resists cannot be reliably applied. Furthermore, fragile substrates, such as free-standing nanotubes or thin films, cannot tolerate the vigorous mechanical scrubbing procedures required to remove all residual traces of the polymer resist.

An ice lithography instrument.

We describe the design of an instrument that can fully implement a new nanopatterning method called ice lithography, where ice is used as the resist. Water vapor is introduced into a scanning electron microscope (SEM) vacuum chamber above a sample cooled down to 110 K. The vapor condenses, covering the sample with an amorphous layer of ice.

Ice lithography for nanodevices.

We report the successful application of a new approach, ice lithography (IL), to fabricate nanoscale devices. The entire IL process takes place inside a modified scanning electron microscope (SEM), where a vapor-deposited film of water ice serves as a resist for e-beam lithography, greatly simplifying and streamlining device fabrication. We show that labile nanostructures such as carbon nanotubes can be safely imaged in an SEM when coated in ice.

Graphene as a subnanometre trans-electrode membrane.

Isolated, atomically thin conducting membranes of graphite, called graphene, have recently been the subject of intense research with the hope that practical applications in fields ranging from electronics to energy science will emerge. The atomic thinness, stability and electrical sensitivity of graphene motivated us to investigate the potential use of graphene membranes and graphene nanopores to characterize single molecules of DNA in ionic solution. Here we show that when immersed in an ionic solution, a layer of graphene becomes a new electrochemical structure that we call a trans-electrode.

Base dependent DNA-carbon nanotube interactions: activation enthalpies and assembly-disassembly control.

We quantify the base dependent interactions between single stranded DNA and single walled carbon nanotubes (SWNTs) in solution. DNA/SWNT hybrids hold the promise of applications ranging from nanoscale electronics and assembly of nanotube based materials, to drug delivery and DNA sequencing. These applications require control over the hybrid assembly and disassembly.

The potential and challenges of nanopore sequencing.

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.

DNA conformation and base number simultaneously determined in a nanopore.

When dsDNA polymers containing identical number of base pairs were electrophoresed through a nanopore in a voltage biased silicon nitride membrane, the measured time integral of blocked ionic current (the event-charge-deficit, ecd, Fologea, D., Gershow, M., Ledden, B.

Focused ion beam induced deflections of freestanding thin films.

Prominent deflections are shown to occur in freestanding silicon nitride thin membranes when exposed to a 50 keV gallium focused ion beam for ion doses between 10(14) and 10(17) ions/cm(2). Atomic force microscope topographs were used to quantify elevations on the irradiated side and corresponding depressions of comparable magnitude on the back side, thus indicating that what at first appeared to be protrusions are actually the result of membrane deflections. The shape in high-stress silicon nitride is remarkably flattopped and differs from that in low-stress silicon nitride.

Eddies in a bottleneck: an arbitrary Debye length theory for capillary electroosmosis.

Using an applied electrical field to drive fluid flows becomes desirable as channels become smaller. Although most discussions of electroosmosis treat the case of thin Debye layers, here electroosmotic flow (EOF) through a constricted cylinder is presented for arbitrary Debye lengths (kappa(-1)) using a long wavelength perturbation of the cylinder radius. The analysis uses the approximation of small potentials.

Nanometer patterning with ice.

Nanostructures can be patterned with focused electron or ion beams in thin, stable, conformal films of water ice grown on silicon. We use these patterns to reliably fabricate sub-20 nm wide metal lines and exceptionally well-defined, sub-10 nanometer beam-induced chemical surface transformations. We argue more generally that solid-phase condensed gases of low sublimation energy are ideal materials for nanoscale patterning, and water, quite remarkably, may be among the most useful.

PROBING SINGLE DNA MOLECULE TRANSPORT USING FABRICATED NANOPORES.

Nanopores can serve as high throughput, single molecule sensing devices that provide insight into the distribution of static and dynamic molecular activities, properties, or interactions. We have studied double stranded DNA electrophoretic transport dynamics through fabricated nanopores in silicon nitride. A fabricated pore enables us to interrogate a broader range of molecules with a wider range of conditions than can be investigated in a self-assembled protein pore in a lipid membrane.

DNA heterogeneity and phosphorylation unveiled by single-molecule electrophoresis.

Broad-spectrum analysis of DNA and RNA samples is of increasing importance in the growing field of biotechnology. We show that nanopore measurements may be used to assess the purity, phosphorylation state, and chemical integrity of nucleic acid preparations. In contrast with gel electrophoresis and mass spectrometry, an unprecedented dynamic range of DNA sizes and concentrations can be evaluated in a single data acquisition process that spans minutes.

Atomic Layer Deposition to Fine-Tune the Surface Properties and Diameters of Fabricated Nanopores.

Atomic layer deposition of alumina enhanced the molecule sensing characteristics of fabricated nanopores by fine-tuning their surface properties, reducing 1/f noise, neutralizing surface charge to favor capture of DNA and other negative polyelectrolytes, and controlling the diameter and aspect ratio of the pores with near single Ångstrom precision. The control over the chemical and physical nature of the pore surface provided by atomic layer deposition produced a higher yield of functional nanopore detectors.

Unzipping kinetics of double-stranded DNA in a nanopore.

We studied the unzipping of single molecules of double-stranded DNA by pulling one of their two strands through a narrow protein pore. Polymerase chain reaction analysis yielded the first direct proof of DNA unzipping in such a system. The time to unzip each molecule was inferred from the ionic current signature of DNA traversal.

Characterization of nucleic acids by nanopore analysis.

Single-stranded DNA and RNA molecules in solution can be driven through a nanoscopic pore by an applied electric field. As each molecule occupies the pore, a characteristic blockade of ionic current is produced. Information about length, composition, structure, and dynamic motion of the molecule can be deduced from modulations of the current blockade.

Single molecule measurements of DNA transport through a nanopore.

We examined the voltage-driven movement of single-stranded DNA molecules in a membrane channel or "nanopore". Using single channel recording methods and a statistical analysis of many single molecule events, we determined how voltage influences capture and translocation in the nanopore. We verified that the mean time between capture events follows a simple exponential distribution, whereas the translocation times follow a unique distribution that is partly Gaussian and partly exponential.

Ion-beam sculpting at nanometre length scales.

Manipulating matter at the nanometre scale is important for many electronic, chemical and biological advances, but present solid-state fabrication methods do not reproducibly achieve dimensional control at the nanometre scale. Here we report a means of fashioning matter at these dimensions that uses low-energy ion beams and reveals surprising atomic transport phenomena that occur in a variety of materials and geometries. The method is implemented in a feedback-controlled sputtering system that provides fine control over ion beam exposure and sample temperature.

Voltage-driven DNA translocations through a nanopore.

We measure current blockade and time distributions for single-stranded DNA polymers during voltage-driven translocations through a single alpha-hemolysin pore. We use these data to determine the velocity of the polymers in the pore. Our measurements imply that, while polymers longer than the pore are translocated at a constant speed, the velocity of shorter polymers increases with decreasing length.

Characterization of PDZ-binding kinase, a mitotic kinase.

hDlg, the human homologue of the Drosophila Discs-large (Dlg) tumor suppressor protein, is known to interact with the tumor suppressor protein APC and the human papillomavirus E6 transforming protein. In a two-hybrid screen, we identified a 322-aa serine/threonine kinase that binds to the PDZ2 domain of hDlg. The mRNA for this PDZ-binding kinase, or PBK, is most abundant in placenta and absent from adult brain tissue.