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New Fluorescein Precursors for Live Bacteria Detection
journal contribution
posted on 2015-09-01, 00:00 authored by Celia Guilini, Corinne Baehr, Etienne Schaeffer, Patrick Gizzi, Frédéric Rufi, Jacques Haiech, Etienne Weiss, Dominique Bonnet, Jean-Luc GalziSwiftness,
reliability, and sensitivity of live bacteria detection
in drinking water are key issues for human safety. The most widespread
used indicator of live bacteria is a caged form of carboxyfluorescein
in which 3′ and 6′ hydroxyl groups are masked as acetate
esters (CFDA). This derivatization altogether abolishes fluorescein
fluorescence and renders the molecule prone to passive diffusion through
bacterial membranes. Once in the cytoplasm, acetate groups from CFDA
are removed by bacterial hydrolases and fluorescence develops, rendering
live but not dead cells detectable. Yet the reagent, carboxyfluorescein
diacetate, still possesses a free carboxyl group whose ionization
constant is such that the majority of the probe is charged at physiological
pH. This unfavors probe permeation through membranes. Here, we prepare
several chemical modifications of the carboxyl moiety of CFDA, in
order to neutralize its charge and improve its passive diffusion through
membranes. We show that the ethylamido derivative of the 5-carboxyl
group from 5-carboxy-fluorescein diacetate or from Oregon green diacetate
or from Oregon green diacetoxymethylester are stable molecules in
biological media, penetrate into bacterial cells and are metabolized
into fluorescent species. Only live bacteria are revealed since bleached
samples are not labeled. Other derivatives with modification of the
5-carboxyl group with an ester group or with a thiourea-based moiety
were almost inefficient probes. The most interesting probe, triembarine
(5-ethylaminocarboxy-oregon green, 3′,6′diacetoxymethyl
ester) leads to 6–10 times more sensitive detection of bacteria
as compared to CFDA. Addition of contrast agents (trypan blue or brilliant
blue R) improve the signal-to-noise ratio by quenching extracellular
fluorescence while bromophenol blue quenches both intracellular and
extracellular fluorescence, allowing standardization of detections.
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bacteria detectionextracellular fluorescenceabolishes fluorescein fluorescencecontrast agentsmembranedrinking watercarboxyfluorescein diacetateester groupacetate groupsquenching extracellular fluorescenceacetate estersLive Bacteria DetectionSwiftnesschemical modificationsNew Fluorescein PrecursorsCFDAcarboxyl moietyOther derivativescarboxyl groupunfavors probe permeation
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