Date of Graduation

8-2014

Document Type

Dissertation

Degree Name

Doctor of Philosophy in Cell & Molecular Biology (PhD)

Degree Level

Graduate

Department

Cell & Molecular Biology

Advisor/Mentor

Li, Yanbin

Committee Member

Erf, Gisela F.

Second Committee Member

Hargis, Billy M.

Third Committee Member

McNabb, David S.

Fourth Committee Member

Kong, Byung-Whi

Keywords

Aptamer; Biodetection; Biosensor; E. coli; Influenza

Abstract

This research investigated impedance biosensors for the rapid detection of viral and bacterial pathogens using avian influenza virus (AIV) subtypes H5N1 and H7N2 and Escherichia coli O157:H7 as the model targets, which were chosen due to their impact on the agricultural and food industries. For the detection of AIV H7N2, a single stranded DNA aptamer was selected using systematic evolution of ligands by exponential enrichment (SELEX). The selected aptamer and a previously selected aptamer against AIV H5N1 were used in a microfluidics chip with an embedded interdigitated array microelectrode to fabricate an impedance biosensor for specific detection of AIV H7N2 and H5N1. The developed label-free biosensor was capable of detecting AIV H7N2 and H5N1 at a concentration down to 27×10-4 hemagglutinination units (HAU) in 30 min without sample pre-treatment, comparable to previously designed biosensors though with the advantage of DNA aptamers. Two impedance biosensors based on the use of screen-printed interdigitated electrodes were developed for the detection of E. coli O157:H7. The first was a label-free biosensor based on magnetic separation and concentration of target bacteria using antibody-labelled magnetic nanobeads and Faradic impedance measurement. It was capable of detecting 1400 cells or more of E. coli O157:H7 in a total detection time of 1 h. COMSOL Multiphysics software was used to analyze the biosensor using a simplified model and determine the role of the magnetic nanobeads in the impedance measurement. The second biosensor for detection of E. coli O157:H7 was based on aptamer-labeled magnetic nanobeads and glucose oxidase/Concanavalin A-coated gold nanoparticle labels. This biosensor was capable of detecting 8 cells or more of E. coli O157:H7 in 1.5 h. The lower detection limit of the developed impedance biosensor was comparable to the most sensitive biosensors published for the detection of E. coli O157:H7 and was also more rapid and more practical for in-field tests.

Multiple impedance biosensor designs were developed in this research. The developed biosensor for AIV could conceivably be adapted for detection of other AIV subtypes and the developed E. coli O157:H7 biosensors could easily be adapted to detect different bacterial pathogens.

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