Investigation of binding of Bacteriophage and Staphylococcus aureus using Magnetoelastic Biosensor

Abstract

Consumption of bacteria-contaminated food products results in illness, hospitalization and even deaths and researchers have been making efforts in finding a way for early detection of pathogenic bacteria, such as Staphylococcus aureus, with low-cost, highly specific devices. However, traditional methods, such as culture-based and PCR-based assays, not only are expensive but also take hours-to-days to detect and identify the bacteria present in the contaminated food. Hence, there is a need for low-cost, rapid analytical devices in conjunction with highly specific biomolecular-recognition elements for detection and identification of food borne pathogens. This thesis presents the development and characterization of a lytic phage-based magnetoelastic (ME) biosensor for the detection of Staphylococcus aureus. ME biosensors are mass sensitive devices composed of a ME material as the transducer platform and phage-based biomolecular-recognition element for specific target species detection. A lytic bacteriophage 12600, with high binding affinity for the Staphylococcus aureus bacterium, was immobilized on gold-coated ME biosensors via physical adsorption with various phage concentrations and immobilization times. To maximize bacteria capture on a ME biosensor, optimization of mean free length and uniform distribution of the biomolecular-recognition element is necessary. The maximum surface density of the phage physically bound on the sensor surface was calculated with the assistance of SEM images for five different concentrations, ranging from 108 pfu/mL to 1012 pfu/mL, as a function of immobilization time. In addition, the mean free length (MFL) between successive bound phages was calculated for the surfaces exposed to the two highest concentrations of phage, 1011 pfu/mL and 1012 pfu/mL, and compared with the size of the bacterium. Experiments were conducted to confirm the maximum bacteria capture between the two highest surface densities of phage immobilized sensors. The maximum bacteria capture was achieved with 1011 pfu/mL phage concentration, surface density of 1.68 × 107 phage particles/mm2 of sensor surface and MFL of 0.92 µm. In addition, from the bacteriophage-characterized biosensors, the specificity and sensitivity of the lytic phage immobilized ME biosensors to detect Staphylococcus aureus in the presence of high concentration of masking bacteria was carried out. The sensors were exposed to different types of bacteria such as Salmonella typhimurium, E. coli O157:H7 and Listeria monocytogenes to determine the specificity of biomolecular-recognition element towards S. aureus. The effect of the presence of one masking bacteria (L. monocytogenes), two masking bacteria (L. monocytogenes and E. coli) and three masking bacteria (L. monocytogenes, E. coli and S. typhimurium) in a mixture with S. aureus upon the response of the ME biosensor was studied. In response, the sensors had the similar trends; however the sensitivity and frequency differences were slightly lower for the masked mixtures.