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Northeastern's Label-free Optical Detection of DNA Nanopore Transport Promises Higher Pore Density

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NEW YORK (GenomeWeb) – Researchers at Northeastern University have developed an optical method for recording ion current changes by DNA travelling through a solid-state nanopore.

The approach, which requires no labeling of the DNA and allows several nanopores to be measured in parallel, might enable sequencers with greater nanopore density than those that rely on electrical measurements, such as Oxford Nanopore Technologies' MinIon.

Earlier this month, the scientists, led by Meni Wanunu, an assistant professor in the department of physics at Northeastern, published a proof-of-principle study in ACS Nano in which they showed that optical and electrical measurements of the nanopore ion current yield similar information, though they have not demonstrated sequence-specific signals yet.

"Their technique offers a solid-state nanopore readout method that is entirely different from the many others currently under development," said Amit Meller, a professor of biomedical engineering at Boston University, who did not participate in the study.

Though many details still need to be worked out for sequencing, the approach could be useful because it greatly simplifies the parallel readout from an array of nanopores, and because the signal is generated near the nanopore itself, he said.

The approach, which might have applications other than DNA sequencing, has been patented by the Northeastern group but has not been licensed out yet, Wanunu said.

The idea for measuring nanopore ion current blockage by optical means goes back to a 2009 publication by a group at the University of Oxford, which demonstrated the principle using alpha-hemolysin protein pores. "It's exactly the same idea, but we had to transfer it to our solid-state platform, so we had to develop a slightly different configuration," Wanunu said.

For their experiments, the researchers used a thin silicon nitride membrane in which they drilled a small number of nanopores.

To measure the ion current, they flow in calcium ions on one side of the membrane and calcium-sensitive fluorophores on the other side. After applying a voltage, calcium ions are pulled through the pore and bind to the dye molecules, resulting in a fluorescent spot underneath the pore that is proportional to the calcium concentration, which reflects the ion current, and can be recorded by a camera.

When DNA enters the pore, the calcium current decreases and the intensity of the fluorescence goes down, and when the DNA leaves, the fluorescence increases again. To prevent the signal from decaying over time by bleaching, the researchers maintain a continuous flow of fluorophores. They also place electrodes in each of the two chambers, allowing them to make simultaneous electrical and optical measurements of the ion current.

In one experiment, they fed 1-kilobase double-stranded DNA through single nanopores, ranging in size from 2.5 to 3 nanometers, and measured the current both optically and electrically. In another experiment, they detected 153-base single-stranded oligonucleotides translocating through three sub-2-nanometer pores in parallel, again recording both optical and electrical traces.

Both types of measurements showed similar dwell times for the DNA and reproducible amounts of current blockage. "We can characterize molecules optically the same way that we can characterize them electrically," said Robert Henley, a graduate student in Wanunu's group and an author of the study.

The experiments also showed that the optical method can record current for several individual nanopores simultaneously. For electrical detection, on the other hand, each nanopore needs to be electrically independent, which requires microfluidics. "With electrical readout, you would need fluidics to separate all the chambers and embedded electrodes. This requires a lot more real estate per pore. With optical methods, you don't need any of this, you can pack them as tightly as you can resolve them," Henley said, meaning the pores could be as close as a micrometer.

For sequencing, a higher pore density would translate to greater throughput and require smaller amounts of DNA, which could lower overall sequencing costs and enable studies of limited samples, Wanunu said.

However, in contrast to electrical nanopore detection systems like Oxford Nanopore's, an optical system requires a camera, which is larger and more expensive. But according to Wanunu, cameras are not the only type of light detector and could be replaced by others, such as avalanche photodiodes, which are much smaller.

The main advantage of the Northeastern group's approach over other nanopore sequencing schemes with optical readout — for example Quantapore's or NobleGen's — is that it does not require the DNA or its components to be labeled, which Wanunu said increases cost and can lead to problems because single molecules are difficult to measure.

So far, the researchers have not demonstrated that they can discriminate bases from the optical signals. This would require improvements in sensitivity, for example by reducing the thickness of the pores, Wanunu said, noting that his group has explored promising new materials, such as hafnium oxide.

Also, the resolution of the optical signal is currently worse than that of the electrical signal, meaning the DNA would need to be slowed down. The calcium ion concentration in the experiments is compatible with motor enzymes, such as polymerases, that are used to put a break on the DNA, the researchers noted.

According to Meller, adapting the optical readout method for sequencing would require "a couple of key technological advances that have yet to be demonstrated," including a better illumination scheme that would restrict the area of excitation and improve the signal-to-noise ratio; an improved readout bandwidth to allow sub-millisecond measurements; and confining the sensing volume to the vicinity of the nanopore.

Going forward, the Northeastern team will focus on improving the signal-to-noise ratio and exploring "to what limits we can push the technology," Henley said, for example by trying different fluorescent dyes, working on ways to reduce the background signal, using a faster camera, and enzymatically slowing down the DNA.

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