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Commercial Nanopore Sequencer Remains 'Top Goal' for Electronic BioSciences after Nanopatch Launch

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Electronic BioSciences is developing an electronic nanopore sequencing platform that promises high speed and low per-run costs.

Late last year, the company launched a version of its hardware platform, called Nanopatch, for electrophysiology measurements while continuing to optimize alpha-hemolysin nanopores for DNA sequencing.

Developing a commercial electronic nanopore sequencing system remains the firm's "top goal," according to Eric Ervin, EBS' vice president of research and development.

EBS' plan is to develop a sequencing system that reads DNA at a faster rate than platforms developed by Oxford Nanopore and others and requires fewer nanopores in parallel, he said. "Instead of reading tens of bases per second, we would like to reads tens of thousands per second per nanopore."

EBS, which has about 15 employees at its San Diego and Salt Lake City offices,
was founded in 2002 by Andew Hibbs, a physicist and entrepreneur who remains the firm's CEO and has been focusing on low-noise measurements of electrical current through biological and synthetic nanopores.

The company has been working on nanopore-based DNA strand sequencing since at least 2007, when it won a $1.2 million grant from the National Human Genome Research Institute. This was followed by a $4.3 million grant under the NHGRI's "$1,000 Genome" program in 2009, and several other grants for sequencing technology development from the National Institute of Standards and Technology, the Department of Homeland Security, the National Science Foundation, and the Defense Advanced Research Projects Agency.

Overall, the company is primarily funded through research grants but is currently pursuing new funding opportunities to finance its sequencing efforts, including private capital and collaborations, Ervin told In Sequence.

The company licenses its core technology, glass nanopore membranes, or GNMs, from the University of Utah, where it was invented by Ervin and others in the laboratory of Henry White. Essentially, a GNM consists of a nanometer-diameter conical hole in a thin glass or quartz membrane at the tip of a capillary.

A lipid bilayer can be put across the hole and a single protein nanopore inserted into it so small ion currents on the order of picoamperes through the pore can be measured.

According to EBS, GNMs allow for those currents to be measured "with exceptionally low noise and high bandwidth." Also, because the holes in the glass membrane are so small, the bilayers are stable and robust, so they can resist higher voltages and pressure than bilayers on other supports. EBS has developed methods to form the bilayers "automatically and reproducibly" and can insert a protein nanopore in a controlled manner by adjusting the internal pressure of the GNM.

At the end of last year, EBS launched a commercial platform called Nanopatch that contains a single, reusable quartz nanopore membrane and allows users to make high-bandwidth, low-noise measurements of ion channels and other nanopores. While the company does not disclose the list price of the Nanopatch, which it sells as an alternative to the patch-clamp method for electrophysiology experiments, it already has several customers.

In order to develop the platform into an electronic sequencing system, EBS has been working on improving the alpha-hemolysin nanopore to make it more suitable for distinguishing the bases of DNA.

Over the last few years, the company has made and tested approximately 400 alpha-hemolysin varieties with unique mutations, Ervin said. Some of these mutations aimed at creating a single, focused sensing zone within the pore, instead of the naturally occurring three sensing zones. Others focused on increasing the contrast between the nucleotides, so they can be more easily distinguished. A third type of mutation was geared at reducing the noise that is associated with DNA rattling and twisting inside the pore as it translocates through it.

"You put all of those together, and hopefully, eventually, you have a pore that will allow for simple electronic sequencing," Ervin said, noting that favorable mutations are not always compatible with each other.

Earlier this month, the company published a paper in Bionanoscience detailing its early efforts on creating an alpha-hemolysin pore with a single sensing zone. According to the article, they were able to remove one sensing region and enhance another one.

In the meantime, EBS researchers have increased the contrast between A and C and between C and T more than six-fold compared to the wildtype protein, and have reduced the sensing zone to a single one with a width of three to five nucleotides, Ervin said. It has also developed a proprietary method to determine the sequence of translocating DNA when reading this many bases at a time.

This has allowed the company to read 5-mer repeats of identical bases in synthetic DNA that is freely traversing the pore under a voltage, without the use of molecular motors to control the rate of translocation, an approach Oxford Nanopore is using.

According to Ervin, he and his colleagues probably need to slow the DNA somewhat for sequencing, but because they want to maintain a fast translocation rate, they are pursuing approaches other than motor proteins, such as chemical methods.

Going forward, EBS will try to shorten the repeats it can read to 2-mers or 3-mers and increase the resolution of the four bases. However, due to the length of the electric field in the sensing zone, it might not be possible to read single bases, Ervin said, so the signal will always come from a few nucleotides at a time.

This would be sufficient, though, he said, citing an article published two years ago in the Biophysical Journal that showed the DNA sequence can be reconstructed with 98-percent accuracy from electrical nanopore measurements from three bases at a time. Oxford Nanopore, for its part, has been generating signals from 6-mers, according to a presentation by one of its collaborators at the Advances in Genome Biology and Technology conference in February.

EBS is also working on a "flossing procedure," Ervin said, to re-read the same segment of DNA by threading it back and forth through the pore, which would reduce sequencing errors.

In terms of coming to market, "we believe that we're a few years out from commercializing a single nanopore system for electronic sequencing of single-stranded DNA," Ervin said. While the company is currently focused on technology development, it is "open to collaboration at any time in the future," he added.

An advantage of the platform would be its high throughput with just a small array of nanopores. "The rate of sequencing per nanopore would determine how large the array would be," Ervin said. "Based on the throughput per pore we're anticipating, we would like our single nanopores to provide about 35 megabases per hour," so an array of 100 nanopores could deliver on the order of 3.5 gigabases per hour.

In addition, he said, EBS' platform, the price of which has yet to be determined, would be reusable and have per-run costs as low as $10, including consumables, automated bilayer formation, and protein incorporation.

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