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Novel Technique Detects Toxin That Causes Cholera

Date:
November 18, 1998
Source:
Los Alamos National Lab
Summary:
Researchers have stolen a page from Mother Nature to develop a technique for detecting the toxin that causes cholera. The technique should work equally well at detecting other protein-based toxins potentially used in biowarfare or terrorism and at detecting early signs of infection in clinical settings.

LOS ALAMOS, N.M., Nov. 10, 1998 -- Researchers have stolen a page from Mother Nature to develop a technique for detecting the toxin that causes cholera. The technique should work equally well at detecting other protein-based toxins potentially used in biowarfare or terrorism and at detecting early signs of infection in clinical settings.

The researchers at the Department of Energy's Los Alamos National Laboratory now aim to convert their technique, which mimics how cells naturally communicate with each other, from a successful lab demonstration to a device suitable for use in the field.

"This technique offers a promising new approach to detecting terrorist activity involving biological agents, and exemplifies how the tremendous science base of the Department of Energy's laboratories can be brought to bear on a wide range of national security challenges," DOE Secretary Bill Richardson said.

The method's high sensitivity, rapid results and ability to pluck out specific proteins against a sea of background molecules would be especially vital for first responders, who must act quickly to identify and minimize the impact of a terrorist attack, the researchers say.

In addition, Los Alamos chemist Basil Swanson, who leads this effort, said, "Although we developed this technique for use in counter- terrorism it would have great value in the early diagnosis of infections. We anticipate this technique could be adapted to a simple field assay that could spot infections before most symptoms appear."

The method could also be used in the food industry, pharmaceutical development or other fields requiring quick and easy identification of pathogens.

The research appears in the Nov. 11 issue of the Journal of the American Chemical Society.

The Los Alamos approach mimics natural cell signaling processes, such as used in the sense of smell, in which a single recognition event triggers a cascade of signals. The technique also involves an artificial membrane that acts similarly to a real cell membrane.

To generate a detection signal, the Los Alamos scientists links receptor molecules designed to latch onto the cholera toxin with reporting molecules that fluoresce when struck with a pulse of laser light. The assemblage is lodged in a double layer membrane formed from lipids, a class of insoluble organic compounds that are important constituents of living cells.

"The process we use is similar to that of a real biosystem," said Xuedong Song of Los Alamos' Advanced Chemical Diagnostics and Instrumentation Group. "Nature has evolved the best surface for this type of protein recognition."

The cholera toxin receptors protrude from the surface of the bilayer membrane and the fluorescent group resides within the bilayer. When a cholera protein comes into contact with the surface, the protruding receptor grabs one of several available docking sites on the toxin, sites the toxin normally uses to establish a toehold when trying to penetrate a cell.

The lipid bilayer allows molecules to migrate around the surface, just as natural cell membranes do. When one receptor nabs a cholera toxin, other receptors aggregate around it and grab onto the remaining toxin docking sites.

This aggregation has two effects: First, by binding onto more than one site it holds the toxin tightly; second, as the receptors crowd together it changes the fluorescent signal from the reporting molecules. Monitoring the fluorescent signal for this change indicates when a toxin has been detected.

Burying the fluorescent probes within the bilayer isolates them from proteins or other molecules that are not of interest but that might otherwise affect their fluorescent response. The researchers use two different fluorescent probes to produce a double signal that minimizes extraneous effects from temperature and other changes.

The fluorescent response is easily measured in a flow cytometer, a well-established analytical tool in biological research.

Los Alamos researchers have developed a field-portable flow cytometer in a project for the U.S. Army. But Swanson and his colleagues are trying to adapt their technique to devices called "wave guides," which are like fiber optic sheets.

Karen Grace of Los Alamos' Space Engineering Group is leading the effort to apply the biomimetic films to wave guides while preserving the fluorescent response. "Wave guides are easy to handle and offer

multiple channels," Grace said, so the researchers could design a device that is small overall but features components that target a number of different toxins.

Song, Swanson and John Nolan, co-authors on the JACS paper, are exploring ways to add other elements to the fluorescent probes to boost their optical response, which would increase sensitivity. The researchers have already achieved sensitivities down to less than 50 parts per trillion.

The technique also has advantages over existing detection methods, such as immunoassays -- which require various reagents and aren't easily used in the field -- and DNA sequencing, which can be used to detect the organism that produces the toxin but not the toxin itself. This work was supported through Los Alamos' Laboratory Directed Research and Development Program, which applies a small portion of the Laboratory's operating budget to support innovative research.

Los Alamos National Laboratory is operated by the University of California for the U.S. Department of Energy.


Story Source:

The above story is based on materials provided by Los Alamos National Lab. Note: Materials may be edited for content and length.


Cite This Page:

Los Alamos National Lab. "Novel Technique Detects Toxin That Causes Cholera." ScienceDaily. ScienceDaily, 18 November 1998. <www.sciencedaily.com/releases/1998/11/981118081143.htm>.
Los Alamos National Lab. (1998, November 18). Novel Technique Detects Toxin That Causes Cholera. ScienceDaily. Retrieved July 31, 2014 from www.sciencedaily.com/releases/1998/11/981118081143.htm
Los Alamos National Lab. "Novel Technique Detects Toxin That Causes Cholera." ScienceDaily. www.sciencedaily.com/releases/1998/11/981118081143.htm (accessed July 31, 2014).

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