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NIST Team Aims to Develop Graphene Nanopore Sequencing Tech


NEW YORK (GenomeWeb) – Researchers at the National Institute of Standards and Technology have come up with a method for graphene nanopore sequencing that could theoretically have a raw read accuracy as high as 90 percent.

The method, which the team described in the journal Nanoscale last month, takes advantage of the electrical properties of graphene and Watson-Crick base pairing.

"This is essentially a hybrid solid-state and biological nanopore," senior author Alex Smolyanitsky, a materials scientist at NIST, told GenomeWeb.

In the study, the NIST researchers simulated their idea to test its feasibility. Smolyanitsky said the next steps would be more in-depth simulations and ultimately experimental tests, although an eventual prototype is a long way off, he said.

The researchers proposed creating a graphene nanoribbon with a nanopore that is chemically functionalized with a nucleobase attached as an overhang into the pore. As single-stranded DNA translocates through the pore, the complementary base will bind to the nucleobase. In the study, the researchers model a pore that is functionalized with cytosine, so as guanine passes through, it binds. The binding induces strain on the graphene, which can be converted into an electrical signal, and when the nucleobase bond is broken, the graphene snaps back into its original place. By stacking nanoribbons functionalized with each nucleobase, a device could be made to detect each of the four bases.

Smolyanitsky, who has a background in solid-state physics and water-based systems, was working on a project at NIST studying the effects of strain on graphene when he wondered whether the strain could be used as essentially a sensor.

Nanopore sequencing has been a promising but challenging field, Smolyanitsky said. While graphene has been touted as an ideal material for a solid-state nanopore sequencing device due to the fact that it is only one atom thick, most experiments involving graphene nanopore sequencing have been done at temperatures close to zero Kelvin, Smolyanitsky said. So, one goal of his research was to make it functional at ambient temperatures. 

The NIST team's method also differs from others in its sensing mechanism. While many nanopore sequencing methods use ion current blockage to detect bases as they pass through the pore, Smolyanitsky's team measures the strain on graphene.

The researchers attach a nucleobase to the pore in order to take advantage of Watson-Crick base pairing, in which cytosine binds to guanine and adenine binds to thymine by making three or two hydrogen bonds, respectively. In the study, the team modeled a cytosine-functionalized pore.

An advantage of using Watson-Crick base pairing to inform detection is that "the probability of forming a spurious pair is quite low," Smolyanitsky said.

In the simulation, the researchers moved two six-base sequences through the pore for 300 nanoseconds, which allowed the molecules to translocate approximately 3.3 times. The researchers observed no false base pairs. Out of 10 times that guanine should have bound to the cytosine-functionalized pore, they detected nine events, Smolyanitsky said, giving the device a theoretical raw read accuracy around 90 percent, although he noted that this finding was not statistically significant since they only measured 10 events.

Although the research is still far from being developed into a prototype, Smolyanitsky said he thinks the sensing method could have advantages over other nanopore sequencing sensing methods. For instance, one well-known approach in nanopore sequencing, which is used by Oxford Nanopore Technologies, is to measure the ionic current that passes through the nanopore. Different bases cause slightly different current blockages when they are in the pore, which can be recorded. However, Smolyanitsky said, the current changes "are not very specific to the particular nucleobase," but more related to the size of the base that is blocking the current, so accurate sequencing involves numerous repeated reads to overcome the noise. Because Watson-Crick base pairing is so specific, it has the potential to be a more accurate single-measurement sensor, he said. Most recently, researchers comparing the reproducibility of Oxford Nanopore's MinIon device found an average error rate of 12 percent for 2D base calls. Users of the MinIon have nevertheless still had success in using the device for a number of applications, including bacterial genome assembly and identifying structural variations in cancer genomes.

Smolyanitsky said there are still numerous challenges to overcome in making a prototype of the NIST method.

The main one will be in designing the pore correctly so that the nucleobase attached to the pore is positioned properly, enabling it to form a hydrogen bond with the DNA molecule. Inserting the DNA is another major challenge, Smolyanitksy said, especially because graphene is very hydrophobic, so DNA has a tendency to stick to it unless it is positioned and inserted extremely precisely. The "proper insertion and translocation" is the "major issue," he said.

One way to get around the problem of graphene's hydrophobicity would be to apply a coating to the membrane that is more hydrophilic, or to use a different material that exhibits similar strain and electrical properties as graphene, such as molybdenum disulfide, the authors wrote. "The idea doesn't hinge on graphene," Smolyanitsky said.

In order to create a device that could detect all four bases, Smolyanitsky said that four graphene nanoribbons, each with a pore functionalized by one of the four bases, would have to be vertically stacked.

In addition, Smolyanitsky said, researchers would have to figure out the proper dimensions and lattice orientation for the nanoribbon. In their simulations, the team tested a nanoribbon that was 4.5 nanometers by 15.5 nanometers. They will also have to figure out the ideal lattice that would elicit the strongest signal, as well as how vertically stacking the different sensors would affect that signal.

The next step is to do more detailed simulations of the signal generated by the four different base-functionalized pores, and ultimately, to start testing the method experimentally. Smolyanitsky said the team would be looking to collaborate with other groups for the experimental tests.