Date of Award

Spring 5-12-2018

Level of Access Assigned by Author

Open-Access Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Food and Nutrition Sciences

Advisor

Vivian C.H. Wu

Second Committee Member

Benildo de los Reyes

Third Committee Member

Chih-Sheng Lin

Additional Committee Members

Paul Millard

Jianjun Hao

Abstract

A fast and reliable on-site foodborne pathogens screening can reduce the incidence of foodborne illnesses, hospitalizations and economic loss. It can also circumvent conventional laboratory-based tests with minimal sample treatments and shorter turnaround time. Rapid detection of biological hazards has been largely dependent on immunological agents (ie antibodies). Antibodies are expensive to manufacture and experience cross-reactivity, instability with shorter shelf life. Our aim was to improve the screening process of Shiga-toxin producing Escherichia coli (STEC) strains in food and environmental matrices by developing a novel, inexpensive handheld bacteriophage-based amperometric biosensor that can directly detect live STEC cells.This biosensor development began by isolating STEC-specific bacteriophages from natural environmental samples (ie cow manure and surface water) hence, constructing a comprehensive bacteriophage isolates collection targeting an array of significant STEC serogroups. As an alternative to antibodies, purified bacteriophages could be easily and inexpensively propagated in a standard laboratory. Isolated bacteriophages were morphologically characterized while its physiologic behavior and specific host interactions were also investigated. The results indicated that majority of STEC-specific bacteriophages belong to Myoviridae and Siphoviridae families. Suitable bacteriophages for biosensor purposes were selected on the basis of the presence of head and tail and absence of virulence genes (stx1/stx2). Chemical modification via site-specific biotinylation of bacteriophage heads was performed prior to its biosensor incorporation. The results showed that biotinylation of bacteriophages did not reduce biofunctionality. Representative STEC O26, O157, and O179-specific biotinylated bacteriophages were immobilized onto the surface of streptavidin-modified screen-printed carbon electrodes (SPCE) to capture their target STEC cells. After STEC cells were bound to the capture elements, another set of biotinylated bacteriophages labeled with streptavidivin-horseradish peroxidase were added forming stable binding complexes which were then subjected to amperometric detection. The sandwich-type bacteriophage-based detection approach allowed live STEC cells rapid detection in microvolume samples (50 µL) via amperometric readouts (∆ current) between target and non-target bacteria in pure culture setup and complex matrices. With its simplicity and reliability, this technology can immensely assist the food industry and regulatory inspectors to efficiently maintain food safety in a fraction of the cost of traditional method.

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