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ANALYTICAL CURRENTS Fast bacterial detection To speed up the identification of pathogenic bacteria, Peter Seeberger and colleagues at the Swiss Federal Institute of Technology Zürich (Switzerland) and the Massachusetts Institute of Technology have developed an assay based on carbohydrate-functionalized fluorescent polymers. The new assay, which provides results in minutes, can be adapted to detect a variety of harmful microbes. In the initial stages of infection, many pathogens bind to multiple carbohydrate molecules displayed on the surfaces of human cells. The binding specificity of various pathogens to certain carbohydrates is well known. E. coli, for example, binds to mannose. Seeberger and colleagues coated a fluorescent polymer with mannose to detect this bacterium. Two strains of E. coli, a wild-type strain and a mutant strain that cannot bind mannose, were incubated in sepa-
rate tubes with mannose-functionalized polymer. After washing the cell pellets with buffer, the researchers observed that only the wild-type bacteria bound to the polymer and fluoresced. In another set of experiments, the researchers imaged wild-type bacteria after mannose- and galactose-functionalized polymers were added to the cultures. Bacteria aggregated around mannose-coated polymers but not around galactose-coated polymers. As few as 104 E. coli cells were detected with this new assay. This detection limit is similar to that obtained with fluorescent antibodies, which bind to more than one molecule. To determine the enhancement of mannose-functionalized polymer binding due to multivalency, or multiple binding, Seeberger and colleagues performed a competition experiment. D-Mannose was added until binding of the polymer was completely
(a)
(b)
When incubated with mannose-functionalized polymer, (a) a wild-type E. coli strain fluoresces, whereas (b) a mutant strain that cannot bind mannose does not fluoresce.
inhibited. The researchers found that multivalency increased binding by a factor of 3.5 104. (J. Am. Chem. Soc. 2004, 126, 13,343–13,346)
Detecting a single bacterium Weihong Tan and colleagues at the University of Florida have developed an assay sensitive enough to find a single bacterial cell within 20 min. To illustrate the sensitivity of their assay, they detected 1–400 bacterial cells of a dangerous E. coli strain in spiked ground beef samples. The investigators used fluorescent silica nanoparticles (~60 nm in diameter) in their assay. An antibody that is highly specific to a particular strain of E. coli bacteria was conjugated to the nanoparticles. Thousands of these fluorescent, antibodyconjugated nanoparticles were able to bind to a single bacterial cell. Because multiple nanoparticles bound to a single
© 2004 AMERICAN CHEMICAL SOCIETY
cell, the fluorescent signal was amplified. Tan and colleagues developed two detection methods. In one, they measured the nanoparticles’ fluorescence with a spectrofluorometer. In another, they used a simple optical flow cytometry device for detection. The investigators also demonstrated that the assay could be expanded into a 384-well plate format to increase its throughput. The new assay could have applications in food inspection, water quality testing, and bioterrorism monitoring. Different kinds of antibodies can be conjugated to the nanoparticles, so the assay could be used for the detection of a wide range of
A scanning electron micrograph shows an E. coli bacterium bound with antibodyconjugated nanoparticles. (Adapted with permission. Copyright 2004 National Academy of Sciences, U.S.A.) bacterial strains, viruses, and cell types. (Proc. Natl. Acad. Sci., U.S.A. 2004, 101, 15,027–15,032)
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