Research Information System
ANALYTICAL AND BIOANALYTICAL CHEMISTRY, vol.382, no.5, pp.1234-1241, 2005 (SCI-Expanded)
Detecting and enumerating fecal coliforms, especially Escherichia coli, as indicators of fecal contamination, are essential for the quality control of supplied and recreational waters. We have developed a sensitive, inexpensive, and small-volume amperometric detection method for E. coli beta-galactosidase by bead-based immunoassay. The technique uses biotin-labeled capture antibodies (Ab) immobilized on paramagnetic microbeads that have been functionalized with streptavidin (bead-Ab). The bead-Ab conjugate captures E. coli from solution. The captured E. coli is incubated in Luria Bertani (LB) broth medium with the added inducer isopropyl beta-D-thiogalactopyranoside (IPTG). The induced beta-galactosidase converts p-aminophenyl beta-D-galactopyranoside (PAPG) into p-aminophenol (PAP), which is measured by amperometry using a gold rotating disc electrode. A good linear correlation (R-2=0.989) was obtained between log cfu mL(-1) E. coli and the time necessary to product a specific concentration of PAP. Amperometric detection enabled determination of 2x10(6)supercript stop cfu mL(-1) E. coli within a 30 min incubation period, and the total analysis time was less than 1 h. It was also possible to determine as few as 20 cfu mL(-1) E. coli under optimized conditions within 6-7 h. This process may be easily adapted as an automated portable bioanalytical device for the rapid detection of live E. coli.