Date of Graduation

12-2010

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

Rhoads, Douglas D.

Second Committee Member

Erf, Gisela F.

Third Committee Member

Slavik, Michael F.

Fourth Committee Member

Tian, Z. Ryan

Keywords

Biological sciences; Impedance immunosensors; Interdigitated microelectrodes; Listeria monocytogenes; Microfluidics; Nanoparticles

Abstract

Listeria monocytogenes continues to be a major foodborne pathogen that causes food poisoning and sometimes death in immunosuppressed people and abortion in pregnant women. Nanoparticles have recently drawn attentions for use in immunomagnetic separation techniques due to their greater surface area/volume ratio and better stability against sedimentation in the absence of a magnetic field. Interdigitated microelectrodes and microfluidics make material transfer more efficient and biological/chemical interaction between the surface and solution phase much quicker. Magnetic nanoparticles (Fe3O4) with a 30 nm diameter were functionalized with rabbit anti-L. monocytogenes antibodies via biotin-streptavidin bonds and then amalgamated with target bacterial cells to capture them during a 2 h immunoreaction. A magnetic field was applied to capture the nanoparticle-L. monocytogenes complexes and the supernatant was removed. After a washing step, L. monocytogenes was separated from a food sample and could be ready for detection by a microfluidics and interdigitated microelectrode based impedance biosensor. Capture and separation efficiency of 75% was obtained with the magnetic nanoparticles for L. monocytogenes in phosphate buffered saline (PBS) solution. When combined with the microfluidics and interdigitated microelectrode, the lower detection limits of L. monocytogenes in pure culture and food matrices were 10^3 and 10^4 CFU/ml, respectively, which were equivalent to several bacterial cells in 34.6 nl volume of a sample injected into the microfluidic chamber. A linear correlation was found between the impedance change and target bacteria in a range of 10^3-10^7 CFU/ml. Equivalent circuit analysis indicated that the impedance change was mainly due to the decrease in medium resistance when L. monocytogenes cells attached to the magnetic nanoparticle-antibody conjugates in mannitol solution. The separation and detection of L. monocytogenes were not affected by presence of other foodborne bacteria. A specific, sensitive, and reproducible method using the microfluidics and interdigitated microelectrode based impedance immunosensor in couple with antibody conjugated magnetic nanoparticles was able to detect L. monocytogenes as low as 10^3 CFU/ml in 3 h.

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