Graduation Year

2004

Document Type

Thesis

Degree

M.S.

Degree Granting Department

Biology

Major Professor

Valerie Harwood.

Co-Major Professor

Andrew Cannons.

Keywords

sonication, enrichment, evanescent, fiber-optic, immunoassay

Abstract

Biosensor development has the potential to meet the need for rapid, sensitive, and specific detection of pathogenic bacteria from natural sources. An antibody-based fiber-optic biosensor assay to detect low levels of Vibrio vulnificus in estuarine waters following an enrichment step was developed. The principle of the sensor is based on an immuno-sandwich assay where an anti-V. vulnificus polyclonal capture antibody preparation was first immobilized on a polystyrene fiber-optic waveguide using a biotin-avidin association. The capture antibody is responsible for binding the target cells to the waveguide. Cyanine-5-conjugated anti-V. vulnificus polyclonal antibodies are subsequently allowed to bind to immobilized cells, and detection occurs when a photodetector collects emitted light (670-710 nm) from the fluorophore, which is excited with 635-nm laser light produced by the Analyte 2000 biosensor.

Any detection signal greater than a pre-determined threshold signal is considered to be a positive detection event, while any signal lower than the threshold is considered no detection. This immunosensor assay proved highly specific when tested against whole cells and cell extracts from V. cholerae, V. parahaemolyticus, V. alginolyticus, and E. coli. isolates. Following a four hour enrichment in PNCC broth, and in a total of less than seven hours, the assay was able to detect cell extracts from as few as 100 V. vulnificus colony forming units suspended in sterile water. This method holds promise for detection of low numbers V. vulnificus and other autochthonous pathogens in estuarine waters.

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