Amperometric determination of live Escherichia coli using antibody-coated paramagnetic beads


Boyaci I., Aguilar Z., Hossain M., Halsall H., Seliskar C., Heineman W.

ANALYTICAL AND BIOANALYTICAL CHEMISTRY, cilt.382, sa.5, ss.1234-1241, 2005 (SCI-Expanded) identifier identifier identifier

  • Yayın Türü: Makale / Tam Makale
  • Cilt numarası: 382 Sayı: 5
  • Basım Tarihi: 2005
  • Doi Numarası: 10.1007/s00216-005-3263-8
  • Dergi Adı: ANALYTICAL AND BIOANALYTICAL CHEMISTRY
  • Derginin Tarandığı İndeksler: Science Citation Index Expanded (SCI-EXPANDED), Scopus
  • Sayfa Sayıları: ss.1234-1241
  • Hacettepe Üniversitesi Adresli: Hayır

Özet

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.