Liposome-based microcapillary immunosensor for detection of Escherichia coli O157:H7.
Our group has previously reported a sandwich-based strip immunoassay for rapid detection of Escherichia coli O157:H7 [Anal. Chem. 75 (2003) 4330]. In the present study, a microcapillary flow injection liposome immunoanalysis (mFILIA) system was developed for the detection of heat-killed E. coli O157:H7. A fused-silica microcapillary with anti-E. coli O157:H7 antibodies chemically immobilized on the internal surface via protein A served as an immunoreactor/immunoseparator for the mFILIA system. Liposomes tagged with anti-E. coli O157:H7 and encapsulating a fluorescent dye were used as the detectable label. In the presence of E. coli O157:H7, sandwich complexes were formed between the immobilized antibodies in the column, the sample of E. coli O157:H7 and the antibody-tagged sulforhodamine-dye-loaded liposomes. Signals generated by lysing the bound liposomes with 30 mM n-octyl-beta-D-glucopyranoside were measured by a fluorometer. The detected signal was directly proportional to the amount of E. coli O157:H7 in the test sample. The mFILIA system successfully detected as low as 360 cells/mL (equivalent to 53 heat-killed bacteria in the 150 microL of the sample solution injected). MeOH (30%) was used for the regeneration of antibody binding sites in the capillary after each measurement, which allowed the immunoreactor/immunoseparator to be used for at least 50 repeated assays. The calibration curve for heat-killed E. coli O157:H7 has a working range of 6 x 10(3)-6 x 10(7)cells, and the total assay time was less than 45 min. A coefficient of variation for triplicate measurements was < or =8.9%, which indicates an acceptable level of reproducibility for this newly developed method.
Ho
Ja-an Annie
JA
BioAnalytical Lab, Department of Applied Chemistry, National Chi-Nan University, Puli, Nantou, 545 Taiwan, ROC. jah@ncnu.edu.tw
Hsu
Hsiu-Wen
HW
Huang
Ming-Ray
MR
D000907:Antibodies, Bacterial; D019453:Escherichia coli O157 / Q000302:isolation & purification*; D005456:Fluorescent Dyes; D007118:Immunoassay / Q000379:methods*; D008081:Liposomes; D015203:Reproducibility of Results
D000907:Antibodies, Bacterial; D005456:Fluorescent Dyes; D008081:Liposomes