Go with the Flow: An Updated Tool for Detecting Molecules

Article excerpt

To date, some 4,000 species of bacteria have been identified, 200 of which are pathogenic to humans. Some are a double-edged sword--in the case of Escherichia coli, for example, some strains exist as benign and beneficial occupants of the human intestinal system, and others cause potentially life-threatening illnesses. In the event of a bacterial disease outbreak, it's vital for public health officials to know which strain they're dealing with as quickly as possible to be able to track the outbreak's source and limit its extent. (Similar knowledge at the clinical level, usually obtained through a process known as culture and sensitivity, allows for selection of appropriate antibiotics for treatment, as many bacterial strains have developed a resistance to some antibiotics.)

A recent analytical advancement at Los Alamos National Laboratory in New Mexico may have a great influence on rapid bacterial strain identification. A group of Los Alamos scientists led by Richard Keller, Babetta Marrone, and James Jett has built upon earlier flow cytometry technology to create a device that allows public health officials and others to study bacteria at the molecular level, differentiating between individual strains more quickly and with greater accuracy than was possible before.

In the original flow cytometer, developed at Los Alamos in the early 1970s, the substance being tested is broken down into individual cells, and each cell passes individually in a continuous flow through a laser beam, scattering the light in a characteristic manner. Dyes bound to different parts of the cell emit light, or fluoresce, when passed through the laser.

Sensors within the cytometer measure several parameters, including "low-angle forward scatter intensity," which is approximately proportional to cell diameter, and fluorescence intensities at several wavelengths, which allows for the study of cell components such as total DNA per cell, specific nucleotide sequences, and, by labeling with monoclonal antibodies, specific cellular proteins and other molecules. Flow cytometers are now common in hospitals and public health labs across the country.

A few years ago, the Los Alamos group began refining the capabilities of the flow cytometer so that it could analyze not just a single cell but a single molecule. This development makes the term "cytometer" somewhat of a misnomer, as the new device deals with molecules rather than cells.

Flow Chart

"Single molecule detection is the Holy Grail of analytical chemistry," Jett says. "In addition to instrumental developments, one of the things that helped us ... was the creation of a whole new family of DNA-binding dyes that showed a tremendous leap in fluorescence when they bonded with DNA."

The Los Alamos group has used several different nucleic acid stains, including PicoGreen, POPO-3, and TOTO-1, all of which show a 600-fold or larger enhancement in fluorescence when they bind to DNA. But even with the increased fluorescence, relatively little light is emitted when the individual molecules pass through the laser beam. So the group slowed the flow rate from 10 meters per second to 10 millimeters per second, keeping the fragments and the dye bound to them in the light source for a longer period so more photons could be emitted, collected, and measured using a solid-state photon-counting detector.

As stained DNA fragments are run through the flow cytometer, they trigger brief bursts of fluorescence. The size of the burst is directly proportional to the number of dye molecules that bind to the DNA and thus reveals the size of the DNA fragment as measured in base pairs. These bursts are recorded, producing a histogram, a DNA "fingerprint," which is exactly analogous to the electrophoretogram produced by gel electrophoresis. In that technique, DNA-containing samples are purified, then treated with enzymes that cut the DNA at specific points, creating a collection of "clippings" that is characteristic of the organism that produced it. …