Phage at the Air Liquid Interface for the Fabrication of Biosensors
Type of DegreeDissertation
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Food borne diseases cause an estimated 76 million illnesses, accounting for 325,000 hospitalizations and more than 5000 deaths in the United States each year. Currently, there are more than 250 known food borne diseases caused by different pathogenic microorganisms, including viruses, bacteria, fungi. Conventional methods of detecting pathogens entail a minimum of 24-48 hours of investigation, only after which results can be obtained. Apart from the urgent need of detection of foodborne pathogens, there is an even urgent need for the development of biosensors for the specific, sensitive and rapid detection of probable bio-terror agents. The general working principles of molecular recognition using thickness shear mode (TSM) sensors have been studied by employing different techniques such as formation of monolayer, and self assembled monolayers (SAM). But, the specific mechanisms of molecular interaction between the probe-analyte that provides the sensitivity and specificity to the biosensor have not been thoroughly investigated. As a part of a project for environmental monitoring of biothreat agents, this work was done to determine if filamentous phage could be used as a recognition molecule on a sensor. E.coli obtained from ß-galactosidase (ß-gal) was used as a model threat agent. Binding of ßgal to the selected landscape phage was characterized by enzyme linked immunosorbent assay (ELISA), thickness shear mode (TSM) and a surface plasmon resonance (SPR-SPREETA ™) sensors and responses obtained were compared. The landscape phage was immobilized through physical adsorption. The characteristics of the gold surfaces of both the TSM and SPR sensors were investigated using an atomic force microscope (AFM). The orientation of phage on formvar, carbon coated copper grids was also studied using a transmission electron microscope (TEM). Results obtained from 52 independent experiments showed a dose dependency in a range of 0.013 to 210 nM. The results of this work provided evidence that phage can be used as a recognition element on biosensors instead of antibodies and achieve detection in nanomolar ranges. Dose response curves indicated a stronger binding on a biosensor than that seen in ELISA. The sensitivity and specificity of phage peptide binding to an analyte envisages future applications of phage for the detection of bio-threat agents in bio-sensors. The sensitivity of both SPR and QCM sensor show similarities. The binding valences were 3.1 and 1.4 for the TSM and SPR sensor respectively. The apparent dissociation constants (Kd) are not significantly different It was observed that apparent Kd of the phage/ß-gal complex was 2.8 nM ± 1.1 (S.D.) in TSM quartz sensor. The affinity valences of 2.3 ± 0.8 (S.D.) were estimated. AFM studies were conducted using a The SPM-100™ (Nanonics Imaging Ltd, Jerusalem Israel) NSOM & SPM System for studying the effect of the cleaning procedures used for both the TSM and SPR sensors. While the control set showed an Rq (average roughness) of 45.9 nm, the treated TSM samples showed an Rq of 31.2 nm. The values obtained from the SPR sensors on the other hand, showed a much smaller difference in Rq values.