A Study in Capture Capability of E2 Phage, Thiolated E2 Phage, and Anti-Salmonella Antibody for Salmonella Using Magnetoelastic Biosensor Platform
Type of DegreeMaster's Thesis
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The magnetoelastic biosensor platform is a superior method that can improve food safety and protect from possible bioterrorism versus conventional culture, nucleic acid-, and immunology-based methods due to low cost, time-savings, convenience, and portability. Although magnetoelastic biosensors have been proven to be one of the most promising methods in the rapid detection of food pathogens, many needs remain such as studying different immobilization methods of bio-molecular recognition elements and the effects of immobilization method on the capture efficiency. This thesis will investigate different methods of immobilizing E2 phage, thiolation E2 phage, and anti-Salmonella antibody to improve the performance of the magnetoelastic biosensor platform. This paper also investigates the effect of different washing techniques on the detection capabilities ME biosensors. Washing is used to remove food particles and salts that are in the food being analyzed and become attached to the biosensor surface. However harsh washing may remove bound target bacteria from the ME biosensor surface, resulting in a lower measured population of the bacteria. Thee different washing methods to maintain captured pathogens on the E2 phage-coated magnetoelastic (ME) biosensors surfaces are investigated. ME biosensors with pre-determined resonance frequencies were exposed to Salmonella Typhimurium of 5x104 cfu/mL for 1 h. E2 phage immobilized on the biosensors was responsible for specifically capturing the bacteria. After this capture step, the biosensors were washed using thee different washing methods, pipette washing, magnetic bar washing, and dilution washing. The dilution method was found to result in the highest capture efficiency of the bacteria and the greatest resonance frequency shift of the sensors, which means the lowest loss rate of Salmonella Typhimurium. On the other hand, a milli-scale, free-standing, and wireless biosensor has been prepared using Metglas alloy magnetoelastic (ME) particles. The new coil detector was used to detect the resonant frequency of ME biosensors by placing biosensors outside the coil boundaries under an external magnetic field. Here, the size of the ME biosensor is 1 x 0.2 x 0.03 mm3, which is easy to control and has good sensitivity. Biosensors were loaded with filamentous E2 phage using Tris-buffer solution. In this study, Salmonella solution was loaded uniformly on the surface of the ME biosensor with population ranging from 5x105 to 5 x102 cfu/mL. The control sensor was a standard ME biosensor without E2 phage. The resonance frequency shift decreased with population of Salmonella ranging from 5x105 to 5 x102 cfu/mL. The capture efficiency was detected using plate counting, which demonstrated that the capture efficiency decreased with the population of Salmonella, which was positively correlated with the change of the resonance frequency shift. Atomic Force Microscope (AFM) was used to study the binding between filamentous E2 phage or thiolation E2 phage and Salmonella. AFM showed that filamentous E2 phages and thiolation E2 phages overlay on the surface of the ME biosensor, and several E2 phages or thiolation E2 phages bound a single Salmonella cell simultaneously.