An Antibody-Immobilized Capillary Column as a Bioseparator/Bioreactor for Detection of Escherichia coli O157:H7 with Absorbance Measurement

A capillary-column-based bioseparator/bioreactor was developed for detection of Escherichia coli O157:H7 by chemically immobilizing anti-E. coli O157:H7 antibodies onto the inner wall of the column, forming the "sandwich" immunocomplexes (immobilized antibody-E. coli O157:H7-enzyme-labeled antibody) after the sample and the enzymelabeled antibody passed through the column and detecting the absorbance of the product in the bioreactor with an optical detector. The effects of the blocking agent, flow rate of samples and substrates, buffer, MgCl2, and pH on the detection of E. coli O157:H7 were investigated. The parameters, 2% BSA in 1.0 × 10-2 M, pH 7.4, PBS as the blocking agent, 0.5 mL/h as the sample flow rate, 1.0 × 10-2 M MgCl2, and 2.0 × 10-4 M p-nitrophenyl phosphate in 1.0 M, pH 9.0 Tris buffer as the substrate for the enzymatic reaction, and 1.0 mL/h as the substrate flow rate, were used in the bioseparator/bioreactor system for detection of E. coli O157:H7. The selectivity of the system was checked, and other pathogens, including Salmonella typhimurium, Campylobacter jejuni, and Listeria monocytogenes, had no interference with the detection of E. coli O157:H7. Its working range was from 5.0 × 102 to 5.0 ×106 cfu/mL, and the total assay time was <1.5 h without any enrichment. The relative standard deviation was ~2.0-7.3%.