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A rapid and sensitive fluorimetric β-galactosidase assay for coliform detection using chlorophenol red-β-d-galactopyranoside
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A rapid and sensitive fluorimetric β-galactosidase assay for coliform detection using chlorophenol red-β-d-galactopyranoside
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Journal Article
A rapid and sensitive fluorimetric β-galactosidase assay for coliform detection using chlorophenol red-β-d-galactopyranoside
2014
Overview
We report on a new fluorimetric assay for β-galactosidase (β-gal) and faecal coliform bacteria that utilizes a long-wavelength dye, chlorophenol red-β-
d
-galactopyranoside (CPRG), that has been widely used for colorimetric assays. The novel feature of this new assay is the unexpected development of a large fluorescence response from liberated chorophenol red (CPR) upon complexation with poly-
l
-arginine (pR) in solution. The binding of CPR to pR occurs through the sulphonate group of CPR, causing formation of a charge-transfer complex and up to a 70-fold increase in emission intensity. A major advantage of the assay is the ability to utilize excitation and emission wavelengths in the red end of the spectrum, which avoids common interferences obtained when using UV-absorbing dyes such as 4-methylumbelliferyl-β-
d
-galactopyranoside. We provide data on the utility of CPRG as a fluorimetric reporter for both β-gal and
Escherichia coli
ATCC 25922 and demonstrate optimized reaction conditions for rapid and sensitive detection of
E. coli
at a level of 1 colony-forming unit (cfu)/10 mL after 12 h of culture followed by a 1-h assay, which is below the regulatory limit for testing of recreational water.
Figure
Beta-galacosidase an coliforms can be assayed with CPRG by for mina a fluorescent complex between chlorophenol red and polyarginine
Publisher
Springer Berlin Heidelberg,Springer
Subject
/ arginine
/ Assaying
/ assays
/ Bacteria
/ beta-Galactosidase - analysis
/ beta-Galactosidase - chemistry
/ Buffers
/ Characterization and Evaluation of Materials
/ Chemistry and Materials Science
/ Culture
/ Dose-Response Relationship, Drug
/ Dyes
/ Emission
/ Escherichia coli - enzymology
/ Escherichia coli - isolation & purification
/ Fluorescent Dyes - chemistry
/ Monitoring/Environmental Analysis
/ Phenolsulfonphthalein - analogs & derivatives
