Rapid detection of Staphylococcus aureus using a membrane fiber-optic biosensor.

摘要

A membrane chemiluminescent assay was developed for rapid detection of Staphylococcus aureus. This one step liquid phase immuno-recognition procedure was achieved by incubating samples containing S. aureus cells with horseradish peroxidase (HRP) labeled monoclonal antibody, followed by recovery of the cells on a membrane, and measurement of the HRP catalyzed chemiluminescent signal on the membrane surface with a luminometer. Optimum pH, antibody binding temperature and time, and sensitivity of the assay were determined. The optimum pH for the chemiluminescent reaction was at 8.5. No significant difference was found between ambient temperature (22°C--25°C) and 37°C incubation. Binding time of 60 min had a higher signal than 10 or 20 min. Chemiluminescent signal due to non-specific binding was tested with various membrane materials, and DuraporeRTM polyvinylidene fluoride (PVDF) had the lowest background signal. Different chemiluminescent reagents were studied to achieve the highest sensitivity combined with a low reagent background. SuperSignalRTM LBA produced a significantly higher signal output than Luminol/Enhencer and LumiGLOTM reagents. Two different diameter membranes, 25 mm and 13 mm, were first tested in a luminometer tube format assay. A hand-operated syringe filtration unit was used to capture cells and the membrane was then transferred to a luminometer tube for the chemiluminescent reaction. The sensitivity of the immunoassay was approximately 104 CFU/ml of S. aureus. Efforts had been made to refine the immunoassay into a simple, sensitive and rapid chemiluminescent fiber optic biosensor. An improved system utilized a simple but efficient microwell plate vacuum filtration unit with an 8 mm membrane sealed at the bottom of the sample well. The sample was concentrated on the membrane and positioned directly in front of a fiber optic light guide to effectively collect and transmit the signal to the luminometer. Labeling S. aureus in solution proved to be much more effective than on the membrane surface. Using the microwell plate filtration system resulted in less sample handling, better reproducibility, and dramatically reduced assay time. The average variability for 25 mm and 13 mm assays were 24.7% and 13.3%, while the microwell plate assay reduced this to only 4.0%. The ability of the fiber optic probe to effectively collect the signal meant the sensitivity of the assay was not compromised with smaller membrane and sample size. The sensitivity of the biosensor was 3.8 x 104 CFU/ml, adequate to detect the organism at concentrations lower than the level that could result in food poisoning. The biosensor demonstrated specificity to S. aureus cells in the presence of sample food materials as well as in buffer.