Arrays of nanopores help detect individual molecular events en masse
Lynn M. Savage
As scientists continue to pull information out of the human genome, two desires are coming into apposition: that for the rapid separation and quantification of DNA and that for performing analysis on nanofluidic-based devices. To achieve high sensitivity, DNA should be examined on a single-molecule basis. However, recent methods — measuring changes in ionic current as DNA translocates through channels or pores in thin membranes — permit detection of individual DNA strands at a rate of only ~104 per minute.
Now researchers at Imperial College London and at Drexel University in Philadelphia have developed a technique that permits detection of DNA translocation events at a rate of up to 106 molecules every 6 ms. “The primary motivation for this study was to use optical methods, which allow us to probe multiple holes simultaneously,” said Andrew J. deMello, professor of chemical nanosciences at the college.
Researchers milled 300-nm-diameter holes into a membrane of SiN and Al, then placed the membrane between analyte and buffer reservoirs. When they initiated a current between the reservoirs, individual DNA molecules were forced from the analyte reservoir into the buffer reservoir (upper right) and imaged with an electron-multiplying CCD camera as they emerged from the holes (lower right). Courtesy of Dr. Jongin Hong, Imperial College London.
The scientists built a membrane with a 200-nm-thick layer of silicon nitride that was coated with a 100-nm-thick layer of aluminum. Through this they milled a three-by-three array of ~300-nm-diameter holes spaced 5 μm apart, using a Leica focused-ion beam system. They mounted the membrane onto a glass substrate and added a coverslip coated with indium tin oxide to act as an electrode, using spacers to form an analyte reservoir and a buffer reservoir that gave the mobile DNA room to move between the pores and the electrode.
They labeled DNA strands with YOYO-1 fluorescent dye made by Invitrogen Corp. of Carlsbad, Calif., and placed the duplexes into solution at a concentration of 10 pM inside the analyte reservoir. They began DNA translocation by applying a voltage between the analyte and buffer reservoirs, causing the molecules to corkscrew through the holes one at a time.
As the DNA emerged through the nine pores, it was imaged using a custom inverted confocal fluorescence microscope comprising a mercury lamp filtered for 488-nm excitation, an Olympus 60× objective, a pair of avalanche photodiodes made by EG&G (now part of PerkinElmer Inc. of Fremont, Calif.) and an electron-multiplying CCD camera made by Photometrics of Tucson, Ariz. Importantly, the aluminum layer blocked much of the background fluorescence emitted from the DNA in the analyte reservoir, increasing the sensitivity of the imaging.
The investigators are working to decrease the pore size and to increase the array size, which will further increase throughput and the amount of data acquirable by the technique.
The research “will result in single-molecule detection becoming a realistic tool within the medical diagnostics and analytical communities,” said team leader Joshua B. Edel.
Nano Letters, September 2007, pp. 2901-2906.
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