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Improved Method for Optical Nanopore Readout Promises Better Signal than Electrical Detection

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NEW YORK (GenomeWeb) — A US-Israeli team has developed an improved optical method for reading out the ion current through solid-state nanopores, providing an alternative to present approaches for nanopore sequencing that use electrical measurements.

Though it requires more equipment to record, the optical signal appears to be less noisy than electrical signals, and the approach may enable nanopore sequencing from larger numbers of pores in parallel.

The researchers, led by Amit Meller, a professor of biomedical engineering with appointments at both Boston University and the Technion – Israel Institute of Technology in Haifa, published their method online earlier this month in ACS Nano.

Their approach is similar in nature, though different in a number of key details, to a method published recently by researchers at Northeastern University. Both teams built on research by scientists at the University of Oxford, who first demonstrated that nanopore ion currents can be measured by optical means.

Meller and his colleagues "have pushed the limits of detection of this technique by employing a more sophisticated illumination/detection approach, and their traces do show lower noise than our [recent] work," said Meni Wanunu, who led the Northeastern University group's work. "In principle, this approach should yield a better time resolution than our approach."

"The development of label-free optical detection of the passage of DNA through nanopores is of interest since it potentially allows [scientists] to detect DNA molecules in parallel, in a way much easier than for electrical detection," said Cees Dekker, a professor and nanopore researcher at the Kavli Institute of Nanoscience at Delft University of Technology in the Netherlands, who was not involved in either work.

The basic idea of the approach is to have a high concentration of calcium ions on one side of a membrane with a solid-state nanopore, and a low concentration on the other side. Under a voltage, calcium ions travel through the pore and bind to a calcium-sensitive dye, which results in a fluorescent signal that can be recorded by a photon sensor. DNA or other analytes going through the nanopore partially block the calcium ion current.

For their ACS Nano study, the researchers drilled solid-state nanopores of about 4 nanometer diameter into a silicon nitride chip, which they mounted in a custom cell with a calcium gradient, calcium-activated fluorophores, and a calcium chelator on one side of the pores, and electrodes on both sides. The cell was placed onto a custom microscope, and two lasers were used to excite the dyes. The emitted light was collected using either an EM-CCD camera in Total Internal Reflection Fluorescence, or TIRF, mode, or photon-by-photon using an avalanche photo diode, APD, in confocal mode. In parallel, the researchers also measured the ion current electrically. For their DNA translocation experiments, they used double-stranded DNA ranging in size from 1 kilobase to 10 kilobases.

According to Meller, one novel aspect of their approach is the use of APDs, which allow for the digital counting of photons and faster measurements. "With that, we could enhance the bandwidth of our measurements by a few orders of magnitude compared to what you could do with the CCD camera," he said.

This enabled them to gauge the noise spectrum, or the contribution of noise to any frequency component of the signal. "What we found is that the optical noise is completely flat," Meller said, unlike the noise in the electrical measurements, which is "really the main thing that stops us from getting real sequences."

"The fact that we see a flat noise response means that we might be able to use this optical sensing in a much more effective way than the electrical sensing," he added.

At the low frequency end, for example, electrical measurements have what is called a "flickering noise," which probably results from either charge fluctuations on the membrane or from fluctuations in the number of ion carriers. Whatever the source of the noise, "the end result is that it really disturbs your signals if you try to read sequences," Meller said. However, there was no such flickering noise in the optical signal. "We believe this is a very promising sign," he said.

In addition, the researchers showed in simulations and experiments that by adjusting the concentrations of calcium ions and chelator, as well as the voltage, they can create a very small volume around the nanopore in which the fluorophore is activated, so the optical signal is spatially confined. "We think this might be the main reason the noise spectrum is so flat, because if the signal comes only from the very close vicinity of the pore, that means we're insensitive to all contributions far away from the pore," Meller explained.

Unlike the Northeastern group, he and his colleagues also did not need to flow in fresh fluorophores during the experiment, because they did not observe any bleaching of the dyes — probably because they used a lower laser intensity.

The next step will be to increase the optical signal by a factor of five or 10. With that, "our signal is going to be already superior to the electrical signal in terms of the signal-to-noise ratio," Meller said. A larger signal could be achieved by using more sensitive fluorescent dyes and increasing their concentration, and by improving the optical system, he said.

For DNA sequencing applications, other than increasing the signal strength, it will be important to move to smaller nanopores, in the range of 1 to 2 nanometer diameter, and thinner membranes, and to work with single-stranded DNA.

The downside of the approach is that it requires an optical system, including a light source and a sensor. "Right now, the system still looks more complicated and more sophisticated than the electrical measurements that are so elegant and simple," Meller said.

His group has not filed any patents on the method yet, but "there will be some intellectual property coming out of this," he said. Other than DNA sequencing, the approach might be useful for characterizing proteins with nanopores.

Whether or not there will be any commercial interest in optical nanopore detection for DNA sequencing remains to be seen. "We will see in a year how much traction and how much attention this approach gets," Meller said. "I think it's maybe a little bit too early."

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