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BU Team Behind NobleGen's Sequencing Tech Advances Fabrication Method as Firm Seeks Series A Funding

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The research team behind the Optipore sequencing strategy adopted by NobleGen Biosciences has published a new proof-of-principle study outlining the steps for fabricating nanopore arrays from solid-state material, as the company itself works to secure Series A financing.

In the Sept. 28th issue of the journal Nanotechnology, Boston University researcher Amit Meller and his colleagues reported that they had successfully produced and tested 6x6 and 8x8 nanopore arrays containing 36 and 64 nanopores apiece, respectively.

To do this, the team turned to a method known as focused ion beam milling to bore 30-nanometer pores into silicon nitride membranes before using a technique called atomic layer deposition to coat the arrays with titanium dioxide. As described in the paper, this titanium dioxide overlay served to narrow the size of the nanopores, while simultaneously curbing the background signal associated with the system's optical readout.

"Now that we know that it's working and we have a process in place, we can start looking at larger and larger arrays," Meller told In Sequence.

Meller's Boston University lab is already working with a prototype instrument. And in its internal studies, the group has started moving to array membranes with 20x20 nanopores or more — a size that would theoretically be sufficient to tackle some small genomes.

Larger arrays on the order of 50x50 or 100x100 nanopores will likely be needed to take on more complex genomes, Meller predicted. But, he added, producing and reading arrays of that size "will not require any qualitative leap" in technology development.

"It's really a matter of linear progression in the R&D," he said.

NobleGen, the company that holds an exclusive license to the intellectual property behind Meller's Optipore sequencing strategy (IS 5/25/2010), is eventually shooting for instruments that can read signals from as many as 160,000 nanopores at once from a 400x400 nanopore array.

With arrays that large, the company estimates that the Optipore system will be able to sequence a human genome to a depth of 30-fold coverage in just 20 minutes or so.

The earliest commercial sequencing devices from the company will likely come to market before reaching those full product specifications, according NobleGen CEO Frank Feist, although the timeline for such an instrument has not been nailed down.

The company is in the midst of securing funding. Feist said that after that is finalized, the R&D cycle to produce a beta-prototype instrument is projected to be two years.

"We are in the process of raising an A-round of capital," Feist told IS. "Once we have that we will build up a team, including an engineering team, that will then industrialize both the instrument as well as the consumables side."

"The new results that Amit [Meller] has published on the consumable are a very important milestone that we can then take on the commercial side and translate into a commercial-grade consumable that will work with the Optipore instrument," he added.

Optimizing Optical Readout

The Optipore method differs from nanopore sequencing strategies being pursued by other groups in that it is based on an optical readout system that revolves around the fluorescence emitted as genetic material moves through solid-state nanopores, Meller explained.

"The reason we have shifted to an optical readout is because we anticipate the need to record signals not from one or two or ten nanopores," he said. "Probably in the future for whole-genome sequencing we will require thousands of nanopores be read simultaneously at very high speed."

In the Optipore system, researchers first replace every nucleotide in a stretch of DNA with longer 15-nucleotide sequences that are specific to each base through a process known as conversion. Then, the genetic material is hybridized with four colored probes, each binding one of the four longer sequences that were introduced during the conversion step.

From there, DNA gets channeled through a nanopore constructed in a solid material by applying an electronic voltage. And because the physical pore itself serves as a barrier to the hybridized fluorescent probe, each probe gets turfed as DNA goes through the pore, producing optical signals that can be detected and used to discern the original DNA sequence.

"As the DNA is moved through the pore by the electrical voltage, those probes that are hybridized to the converted DNA are removed one after the other," Meller explained, "because the pore is acting as a physical barrier that can only allow one strand of the DNA to move through but not the other."

By combining multiple nanopores in an array platform with charge-coupled device cameras and wide-field detectors, it's possible to pick up fluorescence patterns for many nanopores at once without using anything but the pore itself to slow DNA progression through each of the pores.

Another advantage to this system, according to Meller, is that the overall pore size can remain relatively large, since the main function of the pore is to knock off fluorescent probes rather than to interact with the DNA itself.

In the current study, for example, the team constructed 6x6 and 8x8 nanopore arrays by drilling silicon nitride membranes using a speedy process known as focused ion beam milling. That step produced pores that were initially around 30 nanometers in diameter. Using titanium dioxide applied by atomic layer deposition, the nanopores in these arrays were then narrowed to 7 or 8 nanometers.

Besides bringing the pore diameter down, Meller explained, results of the new study suggest that the titanium dioxide significantly reins in the background noise associated with optical detection in the Optipore system. That reduced noise is expected to be important for ramping up the speed of the system, by making each fluorescent molecule easier to read.

"We always look for ways to increase the speed of readout," Meller said. "We are reading, already, quite fast — over 100 bases per second — but if we want to go up it means that we need lower background."

"So we're looking for ways to improve the membrane," he added. "And at the same time, looking for ways to make larger and larger arrays. Both of those research goals were done in parallel."

For their Nanotechnology study, members of Meller's lab showed that it was possible to sequence short synthetic pieces of DNA using the fairly modest 6x6 and 8x8 nanopore arrays.

The researchers have now started doing conversion and sequencing experiments using genomic DNA rather than synthetic molecules.

"What we've been focusing our efforts on in the last couple of months is really starting with genomic DNA and going all the way to the readout," Meller said. "That's a process that we're still undertaking, but I think we're making very, very good progress there."

In addition to making ever-larger nanopore arrays, the team is exploring other ways of improving the system and speeding up the sequencing process, including additional tweaks to the array fabrication process. For instance, Meller noted that there have been further improvements to the ALD process used to apply the titanium dioxide material.

The team is particularly interested in finding ways of further staunching any background signal in the system, though the robustness of the array membrane is also a concern, Meller noted.

"We want a membrane that is very robust mechanically, with nothing that you can move around," he explained. "Because in the end it will be some sort of consumable that you use for sequencing — you don't want it to break."

The group is also continuing to automate the biochemical process behind the DNA conversion step that precedes probe hybridization, Meller noted, and is exploring options for quickly converting an entire genome's worth of DNA in parallel.

Last October, NobleGen received a one-year, $182,000 phase I Small Business Innovation Research Grant from the National Human Genome Research Institute to support optimization of the circular DNA conversion process used in Optipore library preparation (IS 10/11/2011). NobleGen's Feist said the group has met the goals set out in that grant.

"It really helped us advance the level of results that we had in the chemistry," Feist said. "We're totally on track with regard to our own internal milestones and we've taken it orders of magnitude beyond the initial published results on the [circular DNA conversion] process."

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