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ORNL, Yale Team Advances Virtual Nanopore Method with an Eye to Third-Gen Sequencing System

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A group of researchers from Oak Ridge National Lab and Yale University is developing a method for creating virtual pores that can immobilize very small objects in an aqueous system — an approach that they hope can eventually be applied in the third-generation sequencing realm.

The approach uses a so-called quadrupole trap, also called a "Paul trap," (IS 9/23/2012) to make virtual pores from gradients of radiofrequency electric field potential in water within a larger device — a departure from most nanopore approaches that must fashion pores with physical walls that are small enough to contain DNA or other bioparticles of interest.

"The real walls could be 100 nanometers or 50 nanometers, is our prediction, and you still can have that virtual pore that can confine the DNA at the size of a couple of nanometers," project leader Predrag Krstic told In Sequence.

"That has unparalleled advantages for the fabrication because it relaxes the requirements of fabrication of very small traps and reproducible size of that trap," added Krstic, a physics researcher who was formerly at ORNL and is now based at the University of Tennessee-ORNL Joint Institute for Computational Sciences.

"On the other hand," he said, "it removes the possibly negative effect of 'gluing,' or internal interactions of translocating DNA with the physical walls."

Together with postdoctoral researcher Jae Hyun Park and researchers from collaborator Mark Reed's Yale University experimental physics lab, Krstic pubSmalllished a study in the March issue of the journal Small, looking at how the properties of experimentally created aqueous virtual micropores corresponded to their theoretical predictions.

In that paper, researchers demonstrated that they could nab charged polystyrene beads using the parameters of the system itself to stabilize the virtual pores and tune their size. Fluctuations of particles in the pore depended, in part, on charge, study authors added, hinting at a "novel method for accurate experimental estimation of effective charge of a single biomolecule or generally a particle in a liquid."

While the team has yet to create virtual pores small enough to contain DNA, its predictions suggest that it should be possible to come up with such a system.

"Currently, we are trying to trap the charged biomolecules (e.g., DNA, protein) using [aqueous quadrupole traps] by reducing the trap size in electrolyte environment for the third-generation DNA sequencing applications," Krstic, Reed, and the co-authors of the study explained.

If and when that goal is achieved, the next step would be to come up with a third-generation sequencing system that can read electrical signals generated as DNA or other charged particles move through the virtual pore.

The group already holds patents related to using a liquid-based Paul trap to ensnare charged microparticles, but Krstic said it has not yet taken additional steps toward commercializing the approach.

The Paul trap, devised by German physicist Wolfgang Paul, has been used for dynamical trapping in a number of devices, including the quadrupole mass spectrograph, Krstic noted. But certain features of the traditional quadrupole trapping devices made them less than ideal for DNA sequencing-related applications, he explained.

For instance, the trap typically works in a vacuum, where DNA can't migrate in a stable way, he said. Another issue that needed to be overcome was trying to create a Paul trap small enough to deal with the genetic material.

To get around both these problems, the researchers decided to try making a Paul trap in water — an approach that they have now shown to be theoretically and experimentally feasible.

For example, in a study published in the Proceedings of the National Academy Sciences last year, Reed, Krstic, and colleagues presented proof-of-principle experiments showing that they could create aqueous Paul traps and use them to immobilize charged, 481 nanometer and 982 nanometer plastic beads.

As it turned out, creating the traps in water had certain advantages, too, according to Krstic, who said the viscosity of water helps to focus and contain the charged particles of interest, enhancing the trapping process.

The minimum pore diameter that can be achieved with the virtual aqueous pore system still needs to be determined experimentally and the team has not yet reached the dimensions necessary to confine something as small as DNA, though that appears to be possible.

"Our predictions are saying that it is possible to reach a virtual trap radius [of a few nanometers] that would be compatible with DNA or other biomolecules that are charged in the water environment," Krstic said.

The diameter of the virtual pore is controlled by the parameters of the trap itself, including the voltage and frequency of the radiofrequency- or DC-based electric field used, the physical diameter of the trap, and viscosity of the medium, which can also include liquid solvents besides water, he explained. "All these parameters can be optimized so that you get the smallest diameter of the trap confinement and the best stability."

That stability is important, since researchers have to contend with interference from the thermal fluctuations of water molecules when creating Paul traps in water. Researchers have found that they can minimize the negative effects of this random, Brownian movement by tuning the dimension parameters of the trap, Krstic explained, allowing for a small and stable system.

The next step will be to work on ways to further decrease the size of the virtual pore so that it's small enough to localize and control DNA-sized charged molecules. Further down the road, the researchers hope to combine this virtual aqueous pore strategy with methods such as electron tunneling or ion blockade-based detection methods to detect and decode the sequence of DNA bases in the system.

"We have not reached a real device," Krstic emphasized. "We have proven the principle that the Paul trap can be fabricated to the size of hundreds of nanometers and that it can work in an environment that is water rather than air."

The team hopes to secure sufficient funding to see the project through to the sequencing device stage.

In 2008, Krstic's former ORNL lab, in collaboration with Reed's Yale lab, received a two-year, $720,000 grant through the National Human Genome Research Institute's $1,000 genome initiative to support the development of a quadrupole gating method that could be used for transporting and sequencing DNA (IS 8/20/2012).

Krstic secured a two-year, $279,000 American Recovery and Reinvestment Act-funded supplemental award for the project in 2009 (IS 10/13/2009).

Krstic is also continuing to work with researchers at Arizona State University on another independent strategy for developing a nanopore sequencer based on a recognition tunneling method (IS 2/16/2010).

That project, which began as a collaboration between ASU and ORNL, is supported by a four-year, $4.1 million grant that was awarded to ASU researcher Stuart Lindsay as part of NHGRI's $1,000 genome program late last summer (IS 8/23/2011).

The effort is ongoing and expected to continue for at least a few more years, according to Krstic, who said the recognition tunneling method is "currently really blooming." Last fall, ASU's Lindsay told IS that the group aims to have a nanopore sequencing prototype by the end of this year and a demonstration device by around 2015 (IS 11/8/2011).

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