When a patient is admitted to the hospital with signs of a dangerous systemic bacterial infection, or when a post-office worker finds white powder in a suspicious-looking envelope, the ability to quickly identify potential pathogens is important. To accomplish that, a team of Massachusetts researchers is developing a microfluidic chip that performs fast DNA sequencing to rapidly identify bacteria. The goal is a device simple enough to use in airport and other security screening.
In order to identify the bacteria in a blood sample or in a building’s ventilation system, researchers or clinicians usually must start by coaxing it to grow in culture in the lab. This takes about 14 to 48 hours. In the meantime, a patient with a drug-resistant infection may be given the wrong antibiotic, or emergency medical workers may miss the signs of a potential bioterror attack.
Researchers at U.S. Genomics, in Woburn, MA, and Draper Laboratory, in Cambridge, MA, are working together to improve technology that allows for the sequencing and identification of bacteria and other pathogens without culturing. The researchers don’t read every single base on a strand of DNA, but they look for distinctive patterns of repetition of a single, very short sequence. There are only four elements to the genetic code, so six to eight base lengths like GTAGCC occur many times within all genomes. But in each species, such a sequence will occur in a unique pattern. Even different strains of the same kind of bacteria will have unique identifying patterns of a given short sequence.
U.S. Genomics has built a database of these patterns, which it calls bar codes, for many bacterial species and strains. Because the work is being funded by the U.S. Department of Homeland Security, the company will not disclose how many pathogens are in its database or what they are. Staff scientist Jeff Krogmeier does say that the government agency is interested in an instrument “that would sit in an airport, mall, or other public area and continuously monitor the air.” Its compact analysis chip is a step in this direction in that it can identify bacteria based on analysis of long strands of DNA that don’t need to be extensively treated. “We just need a few molecules [of DNA],” says Krogmeier.
First, DNA must be extracted from the sample and labeled with a fluorescent tag that attaches only to places along the strand where the short sequence of interest occurs. (U.S. Genomics is working to simplify this step, which currently must be done in the lab.) Single long molecules of DNA are then fed into a microfluidic chip, where hydrostatic pressure pulls them at a constant speed through a narrow channel. As the labeled DNA flows through the channel, it passes over a very narrowly focused beam of light. When the DNA pass over the beam, the labels fluoresce. Flashes of light from the labels are recorded like a bar code and compared with the U.S. Genomics database to identify the organism that the DNA came from.
In the current incarnation of the company’s DNA chips, the bar-code patterns are read at a resolution of 0.5 micrometers. The version being developed by U.S. Genomics and Draper Laboratory uses a waveguide and nanoantennas to focus the light to a spot size much smaller than half its wavelength, giving far higher resolution and allowing the device to read shorter strands with greater accuracy. What’s more, the light from these antennas is 10 times as intense, which means a stronger signal, says Jonathan Bernstein, a Draper researcher working on the project. And focusing the light with the nanoantennas instead of with a lens means the chips are more compact and rugged.
Krogmeier says the U.S. Department of Homeland Security is interested not only in identifying pathogens, but also in identifying whether they’ve been tampered with. A bioterrorist trying to make anthrax or E. coli more deadly or more easily dispersed would often attempt to do so by adding long stretches of DNA from another organism. The U.S. Genomics chip, says Krogmeier, would be able to detect such tampering.
Bernstein says that the microfluidic channels could also be useful for looking at molecules besides DNA. Common lab techniques like PCR, the process used to make many copies of a single strand of DNA, simply do not exist for studying RNA and proteins; as a result, they are harder to identify and manipulate. “Most of the cell is not DNA,” says Bernstein. Something like the microfluidic chip he has developing for U.S. Genomics, he says, could be very helpful for studying other biological molecules.
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