| Summary: | 碩士 === 國立暨南國際大學 === 應用化學系 === 91 === Although Escherichia coli serotype O157:H7 was identified as a human pathogen in the ninth decade of the twentieth century, it has become recognized a major foodborne pathogen. The severity of E. coli O157:H7 infection in the young and the elderly has had a tremendous impact on human health, the food industry, and federal regulations regarding food safety in the United States. Traditional diagnosis of this pathogen often relies on a single phenotype to selectively isolate it by microbiological assays. However, the process is labor and time consuming. It is important eventually to develop new assay procedures to detect them. The purpose of this study is to develop low-cost, rapid, easy-to-perform, and sensitive biosensing devices for the detection of E. coli O157:H7 using anti-E. coli O157:H7 antibody-tagged liposomes, encapsulating sulforhodamine B dye (so called immunoliposomes) as signal amplifiers. Two types of devices were successfully demonstrated in the present study, which were based on a direct sandwich assay format.
The preparation of immunoliposomes is described as follow. The N-succinimidyl-S-acetylthioacetate (SATA) derivative of the antibodies (anti-E. coli O157:H7) was first conjugated through the reactive N-(k-maleimidoundecanoyloxy)sulfosuccinimide ester (sulfo-KMUS) derivative of dipalmitoylphosphatidylethanolamine (DPPE) and subsequently incorported into liposomes to form the immunoliposomes. In liposome-based strip assay system, a plastic-backed nitrocellulose strip with two immobilized zones is the basis for a sandwich assay to detect E. coli O157: H7. The first zone is the antigen capture zone (AC zone), which is used in a sandwich (noncompetitive) assay format; the other is the biotin capture zone (BC zone), which is used as a positive control for the strip. During the capillary migration of the wicking reagent containing 50μL of immunoliposomes and 90μL of the test sample, E. coli O157:H7 with surface-bound immunoliposomes is captured at the AC zone, while the unbound immunoliposomes migrate and bind to the anti-biotin antibodies coated on BC zone. The color density of the AC zone were directly proportional to the amount of E. coli O157:H7 in the test sample. The detection limit of the current assay with heat-killed E. coli O157:H7 was ~2500 cells. An assay can be performed within 5 min without enrichment. The selectivity of this biosensor system was investigated, and pathogens including Salmonella typhimurium and Listeria genus specific, were proven to have no interference with the detection of E. coli O157:H7.
Part II of this study was to develop a novel micro microcapillary flow injection liposome immunoanalysis (mFILIA) system for detecting heat-killed Escherichia coli O157:H7. A silica-fused microcapillary with anti-E. coli O157:H7 antibodies chemically immobilized on the internal surface via protein A was served as the immunoseparator/immunoreactor in the mFILIA system. After binding of the sample E. coli O157:H7 onto inner surface coated antibody of capillary column, SRB dye-encapsulating, anti-E. coli O157:H7 antibodies-tagged liposomes were introduced into the system, and subsequently bound to the antibody-captured sample E. coli O157:H7. The sandwich complexes (immobilized antibodies—E. coli O157:H7—SRB encapsulating, antibodies tagged-liposome) were then formed, and the detection of the SRB signal was generated after lysis of bound liposomes by 30 mM n-octyl- -D-glucopyranoside. The collected signal was directly proportional to the amount of E. coli O157:H7 in the test sample. The FILIA system was successfully demonstrated its feasibility to detect E. coli O157:H7 for as low as 275 cells/mL. One assay can be performed within 45 min without enrichment procedure. Thirty percent MeOH is used for the regeneration of antibody binding sites after each measurement, which allows the immunoseparator/immunoreactor to be used for at least 50 repeated assays. The calibration curve for heat-killed Escherichia coli O157:H7 had a working range of 6×103 to 6×107 cells.
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