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Wake Forest, Quantapore Tweak Array Production Method In Pursuit of Solid State Nanopores with Optical Detection

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This article was originally published May 23.

NEW YORK (GenomeWeb) – Researchers from Wake Forest University and the California-based nanopore sequencing company Quantapore have demonstrated the feasibility of using helium ion beams to not only form nanopores in solid-state silicon nitride material, but also to dial down the fluorescence of membrane material between the pores.

That dip in background fluorescence is sought after by those interested in coming up with ways of optically detecting DNA bases and other molecules passing through solid-state nanopores, explained Wake Forest University nanoscience, nanoengineering, and bioengineering researcher Adam Hall, a member of Quantapore's scientific advisory board.

Hall and his colleagues introduced the helium ion beam-based membrane fluorescence quenching approach in a study published online recently in Nanoscale. There, they showed that they could treat silicon nitride membrane with low intensity helium ion beams and subsequently see short stretches of fluorescently-labeled, single-stranded DNA moving through nanopores in the material.

The Wake Forest team is continuing to work with Quantapore on the various components required to come up with a complete solid-state nanopore sequencing system using optical base detection.

In particular, the researchers believe that coming up with ways of accurately tracking nucleotide-specific fluorescent signals — rather than ionic or current signals — as DNA moves through solid-state nanopores will ultimately offer an advantage in developing massively parallel solid-state sequencing schemes.

"If you were trying to do this with more conventional ionic current measurements or even try to use some other approaches like tunneling current … you'd basically have to have each one of these pores individually addressable," Hall noted.

"Here, you basically could take a wide-field image of all [of the pixels] at once and then focus on pixels that represent one pore and only analyze those," he added.

Hall and his team have focused much of their attention so far on developing the helium ion beam for pore production and membrane fluorescence reduction, though they are also considering various fluorescent tagging approaches for DNA.

In both solid-state and protein nanopore sequencing systems, the goal is to decipher DNA sequences as charged molecules are prompted through the pore by electric fields applied in the presence of ionic solution.

Efforts to distinguish one DNA base from another in such systems have largely centered on ways of interpreting the ionic current produced as translocation occurs. But there are potential downsides to the current-based detection scheme, authors of the new study argued, particularly when dealing with some common solid-state nanopore materials.

"The major hitch is that the [ionic] sensing is not entirely confined to the inside of the nanopore," Hall said. "You're actually measuring over a distance that sweeps outside of the nanopore and into the solution."

In practice, that means the current signals created as DNA molecules move through the solid-state nanopores often represents several bases instead of one, he explained.

In an effort to circumvent that issue, some solid-state nanopore researchers have started looking at ways of doing optical detection of individual bases in a single-molecule, solid-state nanopore sequencing system.

While that approach could make it possible to collect and interpret signals from a multitude of pores in a single solid-state membrane, though, it has been fraught with its own complications — namely, background fluorescence from the membrane itself. Such membrane autofluorescence is not an insurmountable problem, though it impacts the nature of the nanopore fabrication approaches teams can take.

In a 2012 study published in Nanotechnology, for example, Boston University's Amit Meller and his colleagues described a method for producing silicon nitride-based nanopore arrays coated with a titanium dioxide layer that not only narrowed pore sizes but also diminished background fluorescence.

For their part, Hall and his colleagues noticed that the background fluorescence of silicon nitride membranes tended to diminish around pores that had been drilled with a helium ion beam approach they described in Nanotechnology in 2011.

After noticing lower-than-usual background fluorescent halos around pores that had been produced in silicon nitride membranes by helium ion beam milling, the team started investigating this phenomenon in more detail.

As it turned out, Hall explained, training a concentrated helium ion beam in one spot ablates membrane material as shown previously, forming pores as small as 2.5 nanometers in diameter. At slightly lower intensity, the same type of beam can be used to carefully shave off layers of the membrane.

But when the beam's intensity is dialed down far enough, no membrane material is actually removed. Instead, the beam seems to cause internal damage to the material that diminishes its autofluorescence, which are thought to be caused by small quantum dots in membrane material.

"We went back to study this a little more systematically and we found that we could control it very accurately," Hall said.

In their Nanoscale study, for example, the researchers tried out various helium ion doses on a 30 nanometer thick silicon nitride membrane using a commercially available helium ion microscope.

After finding a suitable set of conditions to decrease the fluorescence of this material to 10 percent or less of that seen with the original membrane, the team focused the ionic beam for drilling pores with diameters smaller than 3 nanometers.

Generally speaking, Hall said the group has had success getting rid of autofluorescence over a 20 micron piece of silicon nitride by applying a low intensity helium beam for around 10 seconds. For smaller membrane areas, the same treatment takes just a fraction of a second.

The group hasn't tested the helium ion beam approach on other types of material, since it's currently focused on silicon nitride-based solid-state nanopores. Still, Hall noted that background fluorescence is "common to most materials" being used as membranes for solid-state nanopore fabrication and said that there's "no reason I can see that would prevent it from working on materials like silicon oxide."

Additional research is needed to build from the reduced fluorescence membrane step and flesh out various features of the group's optical detection approach to solid-state nanopore sequencing, Hall noted.

Still, he said the general optical detection scheme that he and his collaborators are pursuing may make it possible to do solid-state sequencing on DNA molecules, RNA molecules, modified nucleotides, and more.

"In the far future, there's really no reason to think that this couldn't expand to things like proteomics, where you could sequence proteins as they're translocated," Hall said. "It's a wide open field at the moment."

In their proof-of-principle study in Nanoscale, he and his colleagues demonstrated that silicon nitride membranes with diminished autofluorescence did not interfere with the detection of translocating single-stranded DNA that had been labeled with Cy3 fluorescent tags. Rather than attempting to identify individual bases as they moved through the silicon nitride pores, though, those fluorescent labels were "really just used as beacons to represent translocation through the pore," Hall noted.

The researchers have filed for intellectual property related to the use of helium ion beam-based quenching of background fluorescence on solid-state membranes.

Hall and his team are continuing to collaborate with Quantapore, which is exploring options for developing a more complete solid-state nanopore sequencing device. Earlier this week, the company announced that it had nailed down around $35 million in Series B financing in support of its quest to come up with and commercialize a nanopore sequencing platform using an optical read out system.

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