Taking nano-fluidics a mega-step forward, researchers from Princeton University have created chips with hundreds of thousands of channels, each with a width as narrow as 10 nanometers. The researchers also managed to thread individual DNA molecules inside, opening the possibility for applications like genotyping or DNA sequencing without the need for cloning or amplification.
No wonder that Han Cao, lead author of a paper in last week’s Applied Physics Letters that describes the channel fabrication, is planning to found a company with his colleagues in the next half-year to commercialize the patent-pending technology.
Cao, a postdoctoral fellow in Stephen Chou’s nanostructure laboratory at Princeton, knows there is stiff competition: US Genomics of Woburn, Mass., and Nanofluidics of Ithaca, NY, are already developing similar methods based on stretching the DNA out on a chip and scanning it with an optical system. But Cao believes nobody can make channels smaller, faster, and cheaper than his group.
The key method in this process is nanoimprint lithography. The technique, which was invented and patented by Chou, involves making a mold, creating the channels by laser interference patterns, then transferring these nanochannels onto a wafer substrate. Cao first used this method to make half a million channels between 50 and 100 nanometers wide on a four-inch quartz or silicon wafer. In a second step, he narrowed these trenches by depositing silicon dioxide inside of them. Doing this at an angle made the side walls of the channels thicken more quickly near the top than the bottom, creating a vaulted roof and sealing the channel on top. This step was important because liquids would rapidly evaporate from open trenches, Cao said.
The hardest part of this process was getting down to 10 nanometers, he said: “In nanotechnology, the last nanometer gets extremely difficult, and can be very costly.”
After creating these channels, Cao inserted fluorescently-stained DNA molecules inside them through hydrodynamic force or by applying an electric field, and visualized them using an inverted optical microscope coupled to a CCD digital camera.
These DNA-stuffed nanochannels could have a number of applications, including genomic mapping: Digesting the DNA with restriction enzymes inside the channel would retain the order of the fragments, which could then be sized using microscopy. This and other “detection and labeling schemes” could be used to scan for deletions, mutations, or translocations, Cao said, and might translate into diagnostic applications that do not require PCR amplification of the genetic material.
Kalev Kask, CEO of EGeen International, an Estonian company specializing in studying genetic markers, said DNA amplification is the main cost for genotyping and whole-genome sequencing today. “Whatever is less expensive is going to be the method to go [with]” he said. A possible alternative, genotyping microarrays, could be difficult to use, he said.
Other applications of the nanochannel chips include anal-
yzing transcription factors bound to the DNA, separating very long DNA fragments, as well as direct sequencing — reading the DNA bases like a ticker tape. The channel size is important for these applications, because it allows the DNA to stretch out entirely, Cao said. “If the tunnel is bigger than 50 nanometers, the DNA will fold back. Any scanning will become less meaningful if the tunnel is huge.”
Eugene Chan, chairman and CEO of US Genomics, agreed that smaller channels “offer greater control over the DNA molecules and hence a more accurate and precise reading of individual DNA molecules.” However, his company uses channels “nowhere near as small” as those used by Cao, in combination with upright posts that feed the DNA into them for stretching the DNA out.
US Genomics is developing its chip-based technology for high-speed DNA sequencing, with the aim of scanning an entire human genome in one hour or less someday at a fraction of today’s costs. Instead of reading the “Book of Life” in random chunks of 500 letters, like current sequencers, then piecing it together later, the new technique would read each of the 46 chapters from beginning to end, Chan has said in previous interviews. So far, Chan’s machine can scan 200,000 base pairs in a single run within a few minutes, he said, detecting fluorescently labeled bases, and the company is hoping to come out with an instrument within three to five years.
Cao said he is interested in collaborating, not competing with companies like US Genomics. “Strategic partnerships with other startups are common these days,” he said, adding that “for now, we will position ourselves as a nanobiotech company with unique powerful fabrication tools.”