Single molecules of a fluorescent dye in microscopic droplets of solution have been detected by ORNL researchers. The laser-based technique enables target molecules to be counted in very small volumes of liquid, providing far greater sensitivity than bulk analysis schemes. The technique could be used to characterize gene-containing material that may be responsible for inherited diseases or to track flow of pollutants in rivers.

The group at ORNL has conducted a "needle-in-the-haystack" detection experiment in which the fluorescence signal from a single dye molecule is distinguished from light emitted by about one trillion solvent molecules. In the experiment, a "microdroplet" produced by a device similar to an ink-jet printer head is suspended in the center of the analysis chamber. The droplet is then illuminated by the green light of a continuous-wave argon-ion laser.

The dye molecule repeatedly absorbs the green laser light and then relaxes by emitting red light in individual packets called photons. The photons emitted from the dye molecules are then counted using a sensitive light detector, and the number of molecules in the drop is determined roughly by the number of photons detected.

The technique was developed by Mike Barnes, Bill Whitten, and Mike Ramsey, all of the Analytical Spectroscopy Section of ORNL's Chemical and Analytical Sciences Division. Other collaborators in the project have been Steve Arnold of Polytechnic University of New York (who developed the droplet generator), Burt Bronk of the U.S. Army's Edgewood Research and Development Engineering Center, and Kin Ng of California State University at Fresno.

"Our technique for detecting the presence or absence of a single fluorescent molecule (or two or three such molecules) could revolutionize ultrasensitive chemical measurement," Ramsey says. "The ultimate goal of liquid chemical analysis is to measure trace chemical concentrations by counting molecules in a very small sample of solution. We have shown that one, two, or three--or no--molecules can be detected in a tiny drop of solution."

The ORNL scientists say that ultrasensitive analysis can also be performed on materials that are not normally fluorescent by attaching dye molecules as fluorescent tags. For example, detection of dye molecules attached to DNA molecules could provide information on the sizes of DNA fragments. If the dye molecules are attached at intervals along the DNA strand, then the total fluorescence signal can be related to strand size. Such information could be useful for the national Human Genome Project for understanding the genetic makeup of humans and the origins of genetic disorders.

Another important use of this technique is tracking pollutant flow in rivers and locating sources. "As the pollutant spreads out from the source, the number of molecules concentrated in one place becomes smaller and smaller," says Ramsey. "With our technique, we could detect a few molecules in a tiny drop of river water and work upstream to determine the origin of the contamination."

Ramsey says the technique shows promise for carrying out fundamental chemical experiments. "We may be able to study chemical reactions between single molecules in a microdroplet," he notes. "Chemistry in a drop has the advantage of fewer impurities, no container, and a very small sample. With this technique, we could have the world's smallest chemical test tube."

The key to the technique's success is the extremely small size of the liquid drop. "It is important to reduce the number of solvent molecules as much as possible to decrease background emission from the solvent molecules," Whitten says. Filters, he explained, separate light emission of one color (fluorescence from the molecule of interest) from that emitted by the drop's solvent molecules (Raman scattering). As a result, the signal from the target molecules is easily detected over the noise of the solvent molecules, just as the solo of a trumpet player standing at a microphone is heard over the background of numerous accompanying stringed instruments.

The persistence of the droplet is also important. Whitten says that glycerin was selected as a droplet solvent because its low evaporation rate causes it to exist a long time in the measurement apparatus. As a result, the target molecules remain in the laser light long enough for the maximum number of signal photons to be extracted.

The fundamental aspects of this work were supported by the Department of Energy's Office of Basic Energy Sciences. The U.S. Army supported a project at ORNL to determine whether single-molecule detection can be used to detect minute traces of chemical or biological warfare agents. Practical applications of single-molecule detection for assisting in detection of nuclear weapons proliferation are now being supported by DOE's Office of Nonproliferation and National Security.

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