Date of Graduation

5-2012

Document Type

Thesis

Degree Name

Master of Science in Biological Engineering (MS)

Degree Level

Graduate

Department

Biological and Agricultural Engineering

Advisor

Yanbin Li

Committee Member

Kaiming Ye

Second Committee Member

Gisela F. Erf

Abstract

Rapid and specific detection of avian influenza (AI) virus is urgently needed with the concerns over the outbreaks of highly pathogenic H5N1 avian influenza in animal and human infection. Aptamers are artificial oligonucleic acid that can specifically bind to target molecules. They show comparable affinity for target virus and better thermal stability than monoclonal antibodies. Those advantages make aptamers promising candidates in diagnostic and detection applications. The goal of this research was to use DNA&ndashaptamer as the specific recognition element in a portable surface plasmon resonance (SPR) biosensor for detection of AI H5N1 virus in poultry.

A SPR biosensor was fabricated using the selected aptamers based on streptavidin&ndashbiotin method. Streptavidin was directly adsorbed onto the surface of a gold waveguide in the SPR biosensor. Then, biotinylated aptamers were immobilized on the sensor surface via streptavidin&ndashbiotin binding. The immobilized aptamers captured AI H5N1 virus in a sample solution, causing an increase in refraction index (RI). Performances of the aptamer&ndashbased SPR biosensor were studied in streptavidin modification, aptamer immobilization and virus detection. The optimal concentrations of streptavidin and aptamers were determined to improve the sensitivity of the biosensor. The response of the aptamer&ndashvirus interaction was shown to be virus titer&ndashdependent, and a linear range for the titers of AI H5N1 was found between 0.128 and 1.28 HA unit. The aptamer&ndashbased SPR biosensor could detect the H5N1 virus at a titer greater than 0.128 HA unit within 1.5 h. No significant interference was observed from non&ndashtarget subtypes such as AI H7N2, H9N2, H2N2, H1N1 and H5N2. The aptamer&ndashbased SPR biosensor was further evaluated for detection of AI virus in poultry swab samples. All of the AI viruses used in this study were killed ones to ensure biological safety.

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