Amperometric determination of live Escherichia coli using antibody-coated paramagnetic beads.
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 (R2 = 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) 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.
Department of Food Engineering, Hacettepe University, Ankara 06532, Turkey.
Ismail H
IH
Boyaci
Zoraida P
ZP
Aguilar
Mahmud
M
Hossain
H Brian
HB
Halsall
Carl J
CJ
Seliskar
William R
WR
Heineman
D000627:Aminophenols / Q000032:analysis; D000906:Antibodies / Q000737:chemistry*; D015374:Biosensing Techniques / Q000295:instrumentation / Q000379:methods; D001710:Biotin / Q000737:chemistry; D002138:Calibration; D004563:Electrochemistry; D004566:Electrodes; D004926:Escherichia coli / Q000201:enzymology / Q000276:immunology / Q000302:isolation & purification*; D007118:Immunoassay; D008280:Magnetics; D008863:Microspheres; D012680:Sensitivity and Specificity; D001616:beta-Galactosidase / Q000737:chemistry*
D000627:Aminophenols; D000906:Antibodies; D001710:Biotin; D001616:beta-Galactosidase