Researchers from the University of Arkansas have developed a nanopositioning system that makes use of a tuning fork, probe-bound DNA, and a solid-state nanopore to control translocation of the DNA through the pore.
The team demonstrated in a study published in ACS Nano that they could thread DNA through the pore at a rate 100 microseconds per base, a rate that is slow enough to detect each base as it passes through the pore, according to the authors.
Jiali Li, senior author of the study, told In Sequence that the team is now working on using the system to detect different types of molecules and is also looking to collaborate with groups working on graphene nanopores to develop a sequencing device. Additionally, she said that the group has a pending patent application on the technique and hopes to eventually commercialize the technology.
Li said that the team developed the technique after trying to use probe-bound DNA to control translocation through the pore. "In [the] process of doing that, we found that we had a hard time controlling the tip of the probe, and a lot of times the tip would break the membrane," she said.
The tuning fork is able to control the position of the probe's tip and also provides a way of measuring the distance of the tip from the pore. The tuning fork is attached to a piezo actuator and nanopositioner, so that as the tip moves closer, the piezo actuator and nanopositioner work to keep it in a precise location such that the probe tip is near enough to the pore without piercing the membrane.
"The nanopositioning system can control the position of the tip in the X, Y, and Z direction … with Angstrom resolution," Li said.
As the tip reaches the nanopore, DNA molecules are then captured by the electric field pulled into the nanopore. The tethering force of the probe-bound DNA stretches the DNA, holding it in the pore, blocking the ionic current flowing through the pore.
"By observing these current drops and recoveries, the process of capturing and releasing tethered DNA molecules by a nanopore can be measured," the authors wrote.
Li said that while the pore can detect the presence of DNA in the pore, the resolution is currently not high enough to detect each individual base, which would be important for developing a sequencing device.
The next steps along those lines would be to demonstrate that the system can detect not only double-stranded DNA, but that it can detect the difference between double-stranded and single-stranded DNA, and single-stranded DNA with "large pieces of hybridized, 60-mer DNA," Li said. "The ultimate goal is to tell the difference between G bases relative to the other three," she said.
One strategy for increasing the resolution to detect single bases is to change the type of pore used, Li said. In the most recent study, her team used a silicon nitride solid-state pore, but she said that switching to a graphene nanopore could increase resolution because a sheet of graphene is so thin that only one base could fit inside a pore at one time.
Other groups, such as a team from Harvard and Slaven Garaj who started a laboratory in Singapore, have been working on graphene nanopores, which are promising because they are so thin (IS 11/6/2012). But, while graphene provides a greater spatial resolution, the main hurdle in working with graphene has been controlling the temporal resolution, or the speed of translocation through the pore, Li said.
Combining the Arkansas group's nanopositioning system with a graphene nanopore could potentially couple the spatial resolution benefits of graphene with the nanopositioning system's ability to control the rate of translocation to create a sequencing device with single-base resolution, Li said.
Moving forward, Li plans to reach out to groups working with graphene to do those experiments.
While protein nanopores have been leading the nanopore sequencing field, researchers are interested in developing solid-state nanopore sequencers because they may be more stable than proteins, Li said.
Groups like Marija Drndic's team from the University of Pennsylvania have been making advances in solid-state nanopores. Her team recently demonstrated that its solid-state nanopore could distinguish between short DNA homopolymers. They used a silicon nitride nanopore combined with an amplifier specifically developed for nanopore studies with collaborators at Columbia University and Brown University (IS 5/14/2013). The amplifier enables the researchers to measure the signal without slowing down the rate of translocation.
Other groups are working on developing mechanical methods like the Arkansas team's nanopositioning system for controlling DNA translocation, such as a DNA hairpin method developed by French and American researchers (IS 3/13/2012).
"Protein nanopores, right now, have advantages over solid state nanopores," Li said. "They have the resolution and people have figured out ways to slow DNA translocation. But, the protein is not stable. Solid-state nanopores are stable and can last for a long time," she said.