Tag Archives: nanopores

Physics, nanopores, viruses, and DNA

A June 17, 2014 news item on Azonano describes a project which could help scientists decode strands of DNA at top speeds,

Nanopores may one day lead a revolution in DNA sequencing. By sliding DNA molecules one at a time through tiny holes in a thin membrane, it may be possible to decode long stretches of DNA at lightning speeds. Scientists, however, haven’t quite figured out the physics of how polymer strands like DNA interact with nanopores. Now, with the help of a particular type of virus, researchers from Brown University have shed new light on this nanoscale physics.

“What got us interested in this was that everybody in the field studied DNA and developed models for how they interact with nanopores,” said Derek Stein, associate professor of physics and engineering at Brown [Brown University, US] who directed the research. “But even the most basic things you would hope models would predict starting from the basic properties of DNA — you couldn’t do it. The only way to break out of that rut was to study something different.”

A June 16, 2014 Brown University news release (also on EurekAlert), which originated the news item, describes the problems with nanopores,

The concept behind nanopore sequencing is fairly simple. A hole just a few billionths of a meter wide is poked in a membrane separating two pools of salty water. An electric current is applied to the system, which occasionally snares a charged DNA strand and whips it through the pore — a phenomenon called translocation. When a molecule translocates, it causes detectable variations in the electric current across the pore. By looking carefully at those variations in current, scientists may be able to distinguish individual nucleotides — the A’s, C’s, G’s and T’s coded in DNA molecules.

The first commercially available nanopore sequencers may only be a few years away, but despite advances in the field, surprisingly little is known about the basic physics involved when polymers interact with nanopores. That’s partly because of the complexities involved in studying DNA. In solution, DNA molecules form balls of random squiggles, which make understanding their physical behavior extremely difficult.

For example, the factors governing the speed of DNA translocation aren’t well understood. Sometimes molecules zip through a pore quickly; other times they slither more slowly, and nobody completely understands why.

One possible explanation is that the squiggly configuration of DNA causes each molecule to experience differences in drag as they’re pulled through the water toward the pore. “If a molecule is crumpled up next to the pore, it has a shorter distance to travel and experiences less drag,” said Angus McMullen, a physics graduate student at Brown and the study’s lead author. “But if it’s stretched out then it would feel drag along the whole length and that would cause it to go slower.”

The news release then goes on to detail a possible solution to the problem of why DNA translocation varies in speed. Answering this question about DNA translocation could lead to faster and more accurate nanopore sequencing,

The drag effect is impossible to isolate experimentally using DNA, but the virus McMullen and his colleagues studied offered a solution.

The researchers looked at fd, a harmless virus that infects e. coli bacteria. Two things make the virus an ideal candidate for study with nanpores. First, fd viruses are all identical clones of each other. Second, unlike squiggly DNA, fd virus is a stiff, rod-like molecule. Because the virus doesn’t curl up like DNA does, the effect of drag on each one should be essentially the same every time.

With drag eliminated as a source of variation in translocation speed, the researchers expected that the only source of variation would be the effect of thermal motion. The tiny virus molecules constantly bump up against the water molecules in which they are immersed. A few random thermal kicks from the rear would speed the virus up as it goes through the pore. A few kicks from the front would slow it down.

The experiments showed that while thermal motion explained much of the variation in translocation speed, it didn’t explain it all. Much to the researchers’ surprise, they found another source of variation that increased when the voltage across the pore was increased.

“We thought that the physics would be crystal clear,” said Jay Tang, associate professor of physics and engineering at Brown and one of the study’s co-authors. “You have this stiff [virus] with well-defined diameter and size and you would expect a very clear-cut signal. As it turns out, we found some puzzling physics we can only partially explain ourselves.”

The researchers can’t say for sure what’s causing the variation they observed, but they have a few ideas.

“It’s been predicted that depending on where [an object] is inside the pore, it might be pulled harder or weaker,” McMullen said. “If it’s in the center of the pore, it pulls a little bit weaker than if it’s right on the edge. That’s been predicted, but never experimentally verified. This could be evidence of that happening, but we’re still doing follow up work.

The new approach using a virus answered questions while leading to new insights and possibilities (from the news release),

A better understanding of translocation speed could improve the accuracy of nanopore sequencing, McMullen says. It would also be helpful in the crucial task of measuring the length of DNA strands. “If you can predict the translocation speed,” McMullen said, “then you can easily get the length of the DNA from how long its translocation was.”

The research also helped to reveal other aspects of the translocation process that could be useful in designing future devices. The study showed that the electrical current tends to align the viruses head first to the pore, but on occasions when they’re not lined up, they tend to bounce around on the edge of the pore until thermal motion aligns them to go through. However, when the voltage was turned too high, the thermal effects were suppressed and the virus became stuck to the membrane. That suggests a sweet spot in voltage where headfirst translocation is most likely.

None of this is observable directly — the system is simply too small to be seen in action. But the researchers could infer what was happening by looking at slight changes in the current across the pore.

“When the viruses miss, they rattle around and we see these little bumps in the current,” Stein said. “So with these little bumps, we’re starting to get an idea of what the molecule is doing before it slides through. Normally these sensors are blind to anything that’s going on until the molecule slides through.”

That would have been impossible to observe using DNA. The floppiness of the DNA molecule allows it to go through a pore in a folded configuration even if it’s not aligned head-on. But because the virus is stiff, it can’t fold to go through. That enabled the researchers to isolate and observe those contact dynamics.

“These viruses are unique,” Stein said. “They’re like perfect little yardsticks.”

In addition to shedding light on basic physics, the work might also have another application. While the fd virus itself is harmless, the bacteria it infects — e. coli — is not. Based on this work, it might be possible to build a nanopore device for detecting the presence of fd, and by proxy, e. coli. Other dangerous viruses — Ebola and Marburg among them — share the same rod-like structure as fd.

“This might be an easy way to detect these viruses,” Tang said. “So that’s another potential application for this.”

Here’s a link to and a citation for the paper,

Stiff filamentous virus translocations through solid-state nanopores by Angus McMullen, Hendrick W. de Haan, Jay X. Tang, & Derek Stein. Nature Communications 5, Article number: 4171 doi:10.1038/ncomms5171 Published 16 June 2014

This paper is behind a paywall.

“It is more important to have beauty in one’s equations than to have them fit experiment” and nano protection against nerve agents

Michael Berger’s Nov. 7, 2012 Nanowerk Spotlight article about nanoporous adsorbents and protection against toxic nerve agents features Dr. Piotr Kowalczyk, a Senior Research Fellow at the Nanochemistry Research Institute at Curtin University of Technology in Australia, quoting English theoretical physicist, Paul Dirac,

“Some of my colleagues asked me if I believe in our theoretical results” says Kowalczyk. “The great physicist Paul Dirac used to say: ‘This result is too beautiful to be false; it is more important to have beauty in one’s equations than to have them fit experiment’.”

“And I truly believe that our theoretical results have to be correct – within the assumed model of nanopores – because they are so simple and beautiful” he concludes.

Kowalczyk is discussing some of  his latest work on protection against toxic nerve agents (Note: I have removed a link),

In a paper published in the October 31, 2012 online edition of Physical Chemistry Chemical Physics (“Screening of Carbonaceous Nanoporous Materials for Capture of Nerve Agents”), an international team led by Kowalczyk and Alexander V Neimark, a professor at Rutgers University, together with scientists from the Physicochemistry of Carbon Materials Research Group at Nicolaus Copernicus University in Poland, is shedding new light on the selection of an optimal nanomaterial for capturing highly volatile nerve agents.

Berger’s article gives some context for this research,

Protection against nerve agents – such as tabun, sarin, soman, VX, and others – is a major terrorism concern of security experts. Nerve agents, which attack the nervous system of the human body, are clear and colorless or slightly colored liquids and may have no odor or a faint, sweetish smell. They evaporate at various rates and are denser than air. Current methods to detect nerve agents include surface acoustic wave sensors; conducting polymer arrays; vector machines; and the most simple: color change paper sensors. Most of these systems have have certain limitations including low sensitivity and slow response times.

You can find more detail about nanopores and toxic nerve agents in Berger’s article.

Nanopore instruments, femtomolar concentrations, Ireland, and New Zealand

It was the word femtomolar that did it for me. While I have somehow managed to conceptualize the nanoscale, the other scales (pico, femto, atto, zetto, and yocto) continue to  elude me. If my experience with the ‘nanoscale ‘ is any guide, the only solution will be to find as much information as I can on these other ones and immerse myself in them. With that said, here’s more from the July 19, 2012 Izon press release,

Researchers at the Lee Bionanosciences Laboratory at UCD [University College Dublin] School of Chemistry and Chemical Biology in Dublin have demonstrated the detection and measurement of biological analytes down to femtomolar concentration levels using an off the shelf qNano instrument. This ultra low level biodetection capability has implications for biomedical research and clinical development as trace amounts of a biological substance in a sample can now be detected amd quantfied using standard commercially available equipment.

Platt [Dr Mark Platt] and colleagues’ [Professor Gil Lee and Dr Geoff Willmott] method for femtomolar-level detection uses magnetic particle systems and can be applied to any biological particle or protein for which specific aptamers or antibodies exist. Resistive pulse sensing, the underlying technology of the qNano [Izon product], was used to monitor individual and aggregated rod-shaped nanoparticles as they move through tunable pores in elastomeric membranes.

Dr Platt says, “The strength of using the qNano is the ability to interrogate individual particles through a nanopore. This allowed us to establish a very sensitive measurement of concentration because we could detect the interactions occurring down to individual particle level.

”The unique and technically innovative approach of the authors was to detect a molecule’s presence by a process that results in end on end or side by side aggregation of rod shaped nickel-gold particles. The rods were designed so that the aptamers could be attached to one end only.

“By comparing particles of similar dimensions we demonstrated that the resistive pulse signal is fundamentally different for rod and sphere-shaped particles, and for rod shaped particles of different lengths. We could exploit these differences in a new agglutina¬tion assay to achieve these low detection levels,” says Dr Platt.

In the agglutination assay particles with a particular aspect ratio can be distinguished by two measurements: the measured drop in current as particles traverse the pore (∆ip), which reveals the particle’s size; and the full width at half maximum (FWHM) duration of the resistive pulse, which relates to the particle’s speed and length. Limits of detection down to femtomolar levels were thus able to be demonstrated.

I’m a little unclear as to what qNano actually is. I did find this description on the qNano product page,

qNano uses unique nanopore-based detection to enable the physical properties of a wide range of particle types to be measured with unsurpassed accuracy.

Detailed Multi-Parameter Analysis.

Particle-by-particle measurement through qNano enables detailed determination of:

Nanopore-based detection allows thousands of particles to be measured individually, providing far greater detail and accuracy than light-based techniques.

Applications & Particle Types

A wide range of biological and synthetic particle types, spanning 50 nm – 10 μm, can be measured, across a broad range of research fields.

qNano Package

qNano is sold as a full system ready for use including the base instrument, variable pressure module, fluid cell and a starter kit of nanopores, buffer solution and standard particle sets.

Here’s what the product looks like,

qNano (from the Izon website)

As for what this all might mean to those of us who exist at the macroscale (from the Izon press release),

Izon Science will continue to work with Dr Platt at Loughborough, and with University College Dublin and various customers to develop a series of diagnostic kits that can be used with the qNano to identify and measure biomolecules, viruses, and microvesicles.“This is a real milestone for Izon’s technology as being able to measure biomolecules down to these extremely low levels opens up new bio-analysis options for researchers. 10 femtomolar was achieved, which is the equivalent dilution to 1 gram in 3.3 billion litres, or 1 gram in 1300 Olympic sized swimming pools,” says Hans van der Voorn, Executive Chairman of Izon Science.

For those interested in finding out about nanopores, these were mentioned in my July 18, 2012 posting while aptamers were discussed in my interview (Oct. 25, 2011 posting) with Dr. Maria DeRosa who researches them in her Carleton University laboratory (Ottawa, Canada).

Self-assembling, size-specific nanopores or nanotubes mimic nature

I guess you can call this biomimicry or biomimetics as it’s also known. From the  State University of New York at Buffalo  July 17, 2012 news releaseby Charlotte Hsu,

Inspired by nature, an international research team has created synthetic pores that mimic the activity of cellular ion channels, which play a vital role in human health by severely restricting the types of materials allowed to enter cells.

The pores the scientists built are permeable to potassium ions and water, but not to other ions such as sodium and lithium ions.

This kind of extreme selectivity, while prominent in nature, is unprecedented for a synthetic structure, said University at Buffalo chemistry professor Bing Gong, PhD, who led the study.

Here’s how they did it (from the news release),

To create the synthetic pores, the researchers developed a method to force donut-shaped molecules called rigid macrocycles to pile on top of one another. [emphasis mine] The scientists then stitched these stacks of molecules together using hydrogen bonding. The resulting structure was a nanotube with a pore less than a nanometer in diameter.

The July 17, 2012 media advisory by Tona Kunz from the Argonne National Laboratory (one of the partners in this research) describes why creating consistently sized nanopores/nanotubes has been so difficult and offers more information about the macrocycles,

Nanopores and their rolled up version, nanotubes, consist of atoms bonded to each other in a hexagonal pattern to create an array of nanometer-scale openings or channels. This structure creates a filter that can be sized to select which molecules and ions pass into drinking water or into a cell. The same filter technique can limit the release of chemical by-products from industrial processes.

Successes in making synthetic nanotubes from various materials have been reported previously, but their use has been limited because they degrade in water, the pore size of water-resistant carbon nanotubes is difficult to control, and, more critically, the inability to assemble them into appropriate filters.

An international team of researchers, with help of the Advanced Photon Source at Argonne National Laboratory, have succeeded in overcoming these hurdles by building self-assembling, size-specific nanopores. This new capability enables them to engineer nanotubes for specific functions and use pore size to selectively block specific molecules and ions.

Scientists used groupings of atoms called ridged macrocycles that share a planar hexahenylene ethynylene core that bears six amide side chains. Through a cellular self-assembly process, the macrocycles stack cofacially, or atom on top of atom. Each layer of the macrocycle is held together by bonding among hydrogen atoms in the amide side chains. This alignment creates a uniform pore size regardless of the length of the nanotube. A slight misalignment of even a few macrocycles can alter the pore size and greatly compromise the nanotube’s functionality.

Here’s an image of the macrocycles supplied by the Agronne National Labortory,

A snapshot of a helical stack of macryocycles generated in the computer simulation.

The size specificity is  important if  nanopores/nanotubes are going to be used in medical applications,

The pore sizes can be adjusted to filter molecules and ions according to their size by changing the macroycle size, akin to the way a space can be put into a wedding ring to make it fit tighter. The channels are permeable to water, which aids in the fast transmission of intercellular information. The synthetic nanopores mimic the activity of cellular ion channels used in the human body. The research lays the foundation for an array of exciting new technology, such as new ways to deliver directly into cells proteins or medicines to fight diseases.

The research group’s paper has appeared in Nature Communications as of July 17, 2012, from Hsu’s news release,

The study’s lead authors are Xibin Zhou of Beijing Normal University; Guande Liu of Shanghai Jiao Tong University; Kazuhiro Yamato, postdoctoral scientist at UB; and Yi Shen of Shanghai Jiao Tong University and the Shanghai Institute of Applied Physics, Chinese Academy of Sciences. Other institutions that contributed to the work include the University of Nebraska-Lincoln and Argonne National Laboratory. Frank Bright, a SUNY Distinguished Professor of chemistry at UB, assisted with spectroscopic studies.