title: A rapid and sensitive fluorimetric β-galactosidase assay for coliform detection using chlorophenol red-β-D-galactopyranoside.
abstract: 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.
authors: Biointerfaces Institute and Department of Chemistry & Chemical Biology, McMaster University, Hamilton, ON, L8S 4L8, Canada.
authors: Clémence
authors: C
authors: Sicard
authors: Norman
authors: N
authors: Shek
authors: Dawn
authors: D
authors: White
authors: Raymond J
authors: RJ
authors: Bowers
authors: R Stephen
authors: RS
authors: Brown
authors: John D
authors: JD
authors: Brennan
mesh_terms: D001681:Biological Assay; D002021:Buffers; D002733:Chlorophenols / Q000737:chemistry*; D003124:Colorimetry; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000201:enzymology* / Q000302:isolation & purification; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry; D005470:Fluorometry; D005690:Galactose / Q000737:chemistry; D005697:Galactosides / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D010455:Peptides / Q000737:chemistry; D010637:Phenolsulfonphthalein / Q000031:analogs & derivatives* / Q000737:chemistry; D011073:Polyamines / Q000737:chemistry; D013234:Stem Cells; D014466:Ultraviolet Rays; D001616:beta-Galactosidase / Q000032:analysis* / Q000737:chemistry
chemical_list: D002021:Buffers; D002733:Chlorophenols; D005456:Fluorescent Dyes; D005697:Galactosides; D010455:Peptides; D011073:Polyamines; C015462:polyarginine; C007016:chlorophenol red; C074184:chlorophenol red galactopyranoside; D001616:beta-Galactosidase; D010637:Phenolsulfonphthalein; D005690:Galactose