| The objective of this research was to develop a rapid detection system for Salmonella Typhymurium using, standard computer-controlled fluorescence microscopy, image processing, and immobilization of antibodies on glass slides. This system takes advantage of the strength of microscopy (visual proof, morphology assessment, and information about the viability of the bacterial cells) and inmmunosensors (specificity and speed). Biotinylated capture antibodies, specific to the target bacterial cell, were immobilized on patterned areas where avidin-coated beads formed a capture matrix on glass slides using a thiol-terminal silane and a heterobifunctional crosslinker. Deep ultraviolet irradiation (DUV) and a photo-mask were used to alter the silane layer on the glass slide. Avidin-coated beads selectively attached via covalent linkage at locations protected from DUV light. A focused magnetic field forced 50 run diameter magnetic beads and attached cells into precisely located capture areas. A second fluorescently labeled antibody (Method 1) bound the bacterial cell, forming a sandwich complex, and propidium iodide (method 2) stained the cells allowing also detection by using the optics of motorized fluorescence microscope. Upon excitation of the fluorescent label by the microscope's excitation light, a high resolution-cooled CCD camera was used to detect the captured bacteria on the glass slide. A computer program using image processing and pattern recognition techniques will be used to discriminate them from debris and to enumerate them. Results showed that method 2 had better capture performance (60%) than method 1 (30%), the sensitivity of the system was 103 CFU/ml in approximately 90 min (including 40min for sample preparation and 30 min for data collection and 20 min for image processing). |
