Phage-Based Magnetoelastic Sensor for the Detection of Salmonella typhimurium
Type of DegreeDissertation
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In recent years, food-borne illness have garnered the attention of mainstream America with calls now coming from the media for more inspections to ensure the safety of our food supply. Food borne illness from the ingestion of S. typhimurium has been of great concern due to its common occurrence in food products of daily consumption. Annually approximately 80 million cases of food poisoning are reported in the United States alone. The ever growing need for rapid detection of pathogenic microorganisms present in food, environmental and clinical samples has invoked an increased interest in research efforts towards the development of novel diagnostic methodologies. Currently, the detection of bacteria in contaminated food relies on conventional microbiological methods that are time consuming and manpower intensive. This study presents the results of the characterization of a phage-based magnetoelastic biosensor for the detection of Salmonella typhimurium. This affinity- based biosensensor is comprised of a magnetoelastic material as the transducer and filamentous phage as the bio-recognition element. Magnetoelastic materials are ferromagnetic amorphous alloys that change dimensions in the presence of a magnetic field. This effect in combination with the reverse effect (inverse magnetostriction) is utilized in a typical sensor application. A time varying magnetic field causes these sensors to oscillate at a characteristic resonance frequency. The characteristic resonance frequency is dependent on the initial dimensions and physical properties of the material. These materials are of particular interest owing to their unique capability to perform remote (without direct wire contacts to the sensor) sensing, making in-vivo detection and detection in closed containers possible. The phage-immobilized magnetoelastic biosensor was characterized for specificity; dose response in water, spiked apple juice and in spiked milk; selectivity; and longevity. The sensor’s sensitivity is known to be higher for sensors with smaller dimensions. Hence sensors of different dimensions were studied to obtain better detection limits. The sensors’ sensitivity increased from 98 Hz/decade to 1150 Hz/decade (decade of S. typhimurium concentrations) for a decrease in length from 5 mm to 500 ?m. The responses of 2 mm (length) sensors were studied in spiked fat free milk and in mixtures with other bacteria. Binding assays of tests conducted in water showed Kd values of 149±76 cfu/mL with a binding valency of 2.42±0.02, whereas in fat free milk tests showed a Kd value of 136±42 with a binding valency of 2.50±0.03. The similar responses obtained in two dissimilar liquids demonstrates the consistent performance of the sensor even in complex matrices. The effect of phage aggregation using varying counterion concentrations on the sensor performance was also studied. It was established that the formation of phage aggregates at higher counterion concentrations (>420 mM Na+) was realizable. Its effect on the binding numbers was, however, contrary to the expectations. A sharp decline in the binding numbers was observed for higher counterion concentrations (>420 mM Na+) owing to localized accumulation of these aggregates upon immobilization. Visual verification of bacterial binding to the phage-immobilized sensor was achieved through Scanning Electron Microscopy (SEM) studies of the sensor surfaces. High magnification SEM also provided an insight into the distribution characteristics of immobilized phage. In summary, specific and selective biosensor with a magnetoelastic transducer and filamentous phage was investigated and demonstrated to be suitable for the detection of S. typhimurium in liquid foods.