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Researchers Combine Nanopores and Optical Sensing in Single-molecule Detection Device

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NEW YORK (GenomeWeb) – Researchers at the University of California, Santa Cruz and Brigham Young University have combine nanopore and optical sensing in a single electrical-optical nanoparticle sensing platform.

In a study published last month in NanoLetters, the researchers used the device for the identification of single viruses. The platform can also be modified for single-molecule detection of smaller analytes, including DNA and proteins, UCSC researcher Holger Schmidt, senior author on the paper, told ProteoMonitor.

While groups have previously combined nanopores with optical detection, these efforts have typically used confocal microscopy to make the optical measurements. "So even if you had a little fluidic chip for the electrical [nanopore-based] measurement, the optical hardware was not integrated at all," Schmidt said.

By combining these two components on a single chip, the researchers enabled higher throughput and better integration of the two data forms, allowing for improved analyses of target molecules, he noted.

A number of companies and research groups have targeted nanopores as potential devices for detecting biomolecules. On the protein side of things, for instance, Oxford Nanopore has said it aims to apply its nanopore-based GridION and MinION DNA sequencing platforms to protein detection. Likewise, Oxford Nanopore Co-founder Hagan Bayley has done work independent of the company pursuing nanopore-based protein detection.

In February 2013, several of Schmidt's UCSC colleagues published a paper in Nature Biotechnology in which they used the protein unfoldase ClpX to unfold three differentially modified Smt3 proteins and pull them through an α-HL nanopore.

With their platform, Schmidt and his co-authors have taken a somewhat different tack, using the nanopore for gating to ensure that only a single molecule makes it through at a time and following that with optical detection.

In addition to controlling the delivery of the molecules, the nanopore collects electrical information generated by their passage through the pore. This information, Schmidt noted, can be useful in identification of the target molecule – sometimes on its own and sometimes in combination with the subsequently collected optical signal.

For instance, he said, in the NanoLetters paper, the researchers fluorescently labeled two populations of nanobeads – one larger and one small – and sent them through the device to determine if it could distinguish between the two groups.

"Just based on the optical signal, you could see that there were two different types of beads," Schmidt said. "But you couldn't actually decide based on just the optical signal if [a given bead] was a big bead or a small one."

"The small ones were generally not as bright, but not always," he said.

By adding the electrical data from the nanopore, though, the two populations were as clear as "day and night," he noted. "So now you can correlate the two measurements, saying that one is a small bead for sure and one is a big bead, and so you can use the electrical signal to learn more about the optical signal."

In another experiment, in which the researchers aimed to identify virus particles amidst a population of similarly sized nanobeads, the situation was reversed, with the electric information provided by the nanopore similar for all particles but the optical information distinct.

In addition, Schmidt said, while it was initially difficult to distinguish between the two types of analytes using the nanopore, the information provided by the optical sensing then allowed the researchers to better interpret the nanopore data.

"What we were able to do with the optical signal was to distinguish between these same size [nanobead and virus] particles and determine that the viruses took longer to move through the nanopore than the beads," he said. "So you can really learning something about the electrical signal by having the optical one in addition to it."

Additionally, Schmidt said, the combination allows the researchers to ensure that they are looking at only one analyte at a time.

"It could be [in cases] accurate enough to just have the optical signal… but [in the past] every time we have given a talk or published, people ask how do we know that it really is one particle, that they are not clumped together?" he said, noting that by using the nanopore to control the flow of the particles they could be sure they were looking at single molecules.

Key to the integration of the nanopore and optical sensing was developing an optical technology that could guide light on and off the chip in a beam narrow enough that it would light only single particles at a time, Schmidt said.

"Light crossed the [chip's] liquid channel right in the middle [in a stream] small enough that you are only lighting a small part of the channel – small enough to get down to the single molecule, single virus level," he said. "That is what gives us high enough resolution to see single molecules."

Schmidt and his colleagues are now working to modify the device so that the nanopore can be set to turn off after a molecule passes through, allowing them to hold that molecule in the optical chamber for longer analysis if desired. The current device passes molecules through the nanopore and the optical detection component in a steady stream. According to Holger, a single channel can process around 1 microliter of sample per minute.

The researchers are currently looking into using the device to quantify the levels of different virus particles in a sample, by, for instance, labeling them with antibodies to surface proteins.

"You can make different color [fluorescent labels] for different viruses and then distinguish them optically, and then also sometimes viruses come in different shapes, which would make different electrical signals," he said. "So you can use either one of these two signal modalities to pick them apart while at the same time you are counting them."

For protein analysis, the device could help reduce problems with background and non-specific binding, he noted, as the nanopore could help distinguish between target analytes bound to detection antibodies versus non-specifically bound antibodies or free fluorescent markers.

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