APPLICATION OF CAPILLARY COLUMN BIOSEPARATOR/BIOREACTOR TO DETECTION OF E. COLI O157:H7

Abstract

3 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” immuno-complexes (immobilized antibody-E. coli O157:H7-enzyme-labeled antibody) after the sample and the enzyme-labeled antibody went through the column, and detecting the absorbance of the product produced in the bioreactor with an optical detector. Firstly, a capillary column was modified with g- aminopropyltriethoxysilane and anti-E. coli O157:H7 antibodies were immobilized onto the inner wall of the capillary column with a cross linking agent (glutaraldehyde). The antibody-immobilized column was used as a bioseparator for separating the target bacteria while the sample was pumped through the column, and the “sandwich” complexes were formed on the inner wall of the column while the alkaline phosphatase (EC 3.1.3.1)-labeled antibodies went through the column. The column with the immuno-complexes acted as a bioreactor for p-nitrophenyl phosphate hydrolysis while the substrate for the enzymatic reaction was pumped into the column. Finally, p-nitrophenol from pnitrophenyl phosphate hydrolysis in the bioreactor was detected with an optical detector and the cell number of E. coli O157:H7 was indirectly measured with the absorbance of p-nitrophenol at 400 nm. The effects of blocking agent, flow rate of the sample and substrate, buffer, MgCl2 and pH on the detection of E. coli O157:H7 were investigated. The parameters, 0.5 ml/h of the sample flow rate, 1.0 ml/h of the substrate flow rate, 2% BSA in 1.0 ´ 10-2 M PBS (pH 7.4), the substrate composed of 1.0 ´ 10-2 M MgCl2 and 2.0 ´ 10-4 M p-nitrophenyl phosphate in 1.0 M Tris buffer (pH 9.0), 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 detection of E. coli O157:H7. Its working range was from 5.0 ´102 to 5.0 ´105 CFU/ml without any enrichment. The relative standard deviation was 2.0~7.3%. The total assay time was less than 1.5 h.