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Intel Team Developing Electrical Detection Method with Eye Toward Single-Molecule Sequencing

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By Molika Ashford

A group from Intel's integrated biosystems lab is developing a label-free electrical detection method to detect DNA polymerase reactions on a chelator-modified field-effect transistor.

The researchers described the work this month in the journal Analyst. While it is early stage, the approach holds promise for massively parallel sensor arrays for DNA sequencing among other applications, according to the researchers.

The method is one of several avenues Intel is investigating with the aim of advancing biodetection for potential use in single-molecule sequencing and other biological applications, company investigators told In Sequence this week, though they stressed that the work is still in the exploratory stage and the company has no definite plans to develop or commercialize a sequencing platform on its own.

In the paper, the researchers argued that electronic sensing offers several advantages for detection of pyrophosphate release over older optical techniques, including better scalability, simplified instrumentation, and the potential for seamless data acquisition located on a single device.

Other groups, most notably by Life Technologies' Ion Torrent, have also achieved electrical detection of base extension using field-effect transistors, but the Intel group's approach appears to differ from Ion Torrent's in its focus on the detection of inorganic pyrophosphate rather than protons generated by the DNA base incorporation reaction.

In its paper, the Intel researchers — working in partnership with researchers from the University of Illinois and Purdue University — describe their effort to chemically modify silicon-on-insulator field-effect transistor devices. This modification included covering the sensor surface with DNA amplified through rolling-circle amplification along with immobilized chelators to allow detection of diffusible reaction by-products.

Madoo Varma, head of Intel's integrated biosystems lab and one of the study's lead authors, told In Sequence this week that Intel's interest in this research area grew out of a realization that Intel's semiconductor technology could play a "key role in a lot of bio areas," and a desire to leverage that potential.

"We [have] looked a several sensing approaches," she said. "Typically, we are doing a lot of very early exploratory research, such as in figuring out, for example for sequencing, what kinds of sensing schemes would work best in the context of customer needs."

Ultimately, Varma said she believes those needs are moving in the direction of single-molecule sequencing. The company's work with this particular project is among many approaches it is investigating toward such ends, she explained.

Intel is not the first company with experience in semiconductors to set its sights on the sequencing market. IT giant IBM has been developing a so-called "DNA transistor" for several years and is collaborating with Roche to bring the system to market, though the partners have not yet disclosed a commercialization timeline for the system (IS 7/6/2010).

And sequencing startup Genia was founded by several veterans of the semiconductor industry with the aim of developing a "single-molecule electrical detection sequencing platform," according to CEO Stefan Roever (IS 1/17/2012).

Modified SOI-FET

In their paper, the Intel researchers described how they modified silicon-on-insulator field-effect transistor devices in order to measure "specific and static" electrical response to DNA polymerase reactions in both bulk solution and on the device surface using pyrophosphate as the target signaling molecule.

In previous work, published last year in Chemical Communications, the team reported synthesizing a chelator that could be surface-immobilized, demonstrated strong binding affinity for PPi, and showed "reversible SOI-FET sensing of PPi standard solutions."

In the more recent study, the group was able to functionalize a chip containing multiple devices with surface coverage of both DNA and chelator molecules to capture the pyrophosphates, the researchers wrote, through a set of surface modifications that enabled the co-attachment of the chelator in close proximity to the surface-bound DNA reaction sites.

The team used rolling circle amplification of the immobilized DNA to "increase the number of parallel reactions occurring at each binding site," the authors reported.

They tested their modified SOI-FET sensor using both DNA polymerase reactions conducted off of the chip in a reaction tube, and those on surface-immobilized DNA colonies, as a well as an initial test using a simple PPi solution.

In the first test, the group observed results consistent with an expected positive shift in voltage dictated by charge effects induced by the negatively-charged PPi.

The group then progressed to testing PPi detection from off-chip DNA reactions, finding another positive voltage shift after exposing the FET sensor to a mixture containing hairpin DNA2 and a matching base (dGTP) with T4 polymerase, the authors reported.

In a final set of experiments, the researchers added a reaction solution containing two bases, dGTP and dCTP, to the chelator and DNA colony-modified chip, and measured both before and after incubation. While measuring the effect on voltage was more complicated, the authors reported that the shift measured after reactions of the on-chip colonies was in the same consistent direction, though only "approximately half that of the other experiments."

The researchers wrote that this report represents the first time their chelator has been used for "label-free (non-fluorescent, non-luminescent) electrical detection of enzymatic DNA base incorporation reactions." This demonstrates the approach's potential DNA analysis applications, the authors argued.

By coupling the chelator-modified field-effect sensor with rolling circle amplification of DNA to generate a larger number of PPi signaling molecules, the approach represents a "relatively new approach to electrical detection of enzymatic DNA base incorporation reactions," the researchers wrote.

Although the team's results were from PCR-amplified DNA, they reported that they have also demonstrated RCA colony formation from total genomic DNA in other "parallel studies."

The work suggests RCA and SOI-FET detection methods can be used for complex DNA samples from wide sources without pre-amplification or selection, and a label-free approach using PPi-sensitive sensors could be optimized for variety of applications, including massively parallel arrays for DNA applications such as genotyping or sequencing, the authors wrote.

Varma declined to discuss Intel's ultimate plans for the approach. However, the researchers highlight in their report that the detection method could be integrated into a sample-to-answer system with data acquisition and database mining all co-located on a single chip. This suggests that if the approach were to be furthered toward DNA sequencing; it would likely be as part of a small, closed system.

Varma said that if Intel were to move into the sequencing space it would most likely be through "partnering with the right people."

"We're [in the] early exploratory stage," she said. "So it's not that we don't think about these things, but obviously we collaborate very heavily with outsiders … definitely partnership is what we would be looking for even at a research stage."

"We will continue in this area, as we have funded research in the nanopore area as well. But our focus is [more broad than this particular sensing approach] because we would potentially want to [move toward] single-molecule sequencing," she said.


Have topics you'd like to see covered in In Sequence? Contact the editor at mashford [at] genomeweb [.] com.

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