Doctor of Philosophy (PhD)
Biological and Agricultural Engineering
The development of biosensors may assist for the on-site detection of foodborne pathogens. The overall goal of this study was to develop a biosensor system for detecting Listeria innocua (non-pathogenic surrogate bacteria used as a model for pathogenic Listeria monocytogenes) in food systems. The study was divided into three main parts: (1) development of a sample collection and interface system for Listeria innocua from food samples, (2) development of a sample concentration system for the collected bacteria prior detection, and (3) development of a detection system based on a carbon nanotube potentiometric biosensor for a quantitative detection of Listeria innocua. In the second chapter, we discussed a sample collection protocol and delivery system developed for bacteria from food surfaces. Listeria innocua was used for testing and illustration. For this purpose, the surface of meat samples was inoculated with Listeria innocua at different concentrations from 10^1-10^5 CFU/mL. Then, cellulose membranes were applied to the surface of products for different times: 5, 10, 15, 20, 25, and 30 min sampling. The cellulose membranes were analyzed for their suitability for bacteria enumeration using a plating method for Listeria innocua. It was observed that sampling times between 5-10 min were the best and collection of >80% of bacteria from the food’s surface was achieved. In the third chapter we discussed a microfluidic device for concentration of biological samples based on removal of liquids by hydrogel films. The performance of the device was demonstrated by concentrating 1-5 µm fluorescent beads followed by concentration of bacteria samples such as Listeria innocua. Results showed that fluorescence intensity of the beads was increased by 10 times at the end of concentration. Recovery efficiencies of 85.60 and 91.75 % were obtained for initial bacteria concentrations of 1x10^1 and 1x10^2 CFU/mL. Moreover, cell counts were observed to increase by up to 10 times at the end of concentration. This study showed that the concentrator device successfully concentrated the samples and no significant loss of living cells was observed for most of the bacteria concentrations. A carbon nanotube potentiometric biosensor for the detection of bacteria from food samples was demonstrated in the fourth chapter. The biosensor was constructed by depositing carboxylic acid (–COOH) functionalized single walled carbon nanotubes (SWCNTs) on a glassy carbon electrode (GCE), followed by the attachment of anti-Listeria antibodies to the SWCNTs between the amine groups and the –COOH by covalent functionalization using EDC/Sulfo-NHS chemistry. The performance of the biosensor was evaluated at various concentrations of L. innocua, for factors such as limit of detection, sensitivity, response time, linearity, and selectivity. In addition, the application of the complete detection system based on sample collection, concentration and detection of bacteria from food samples such as meat and milk was evaluated. Results showed that the system could successfully detect L. innocua with a linear response between electromotive force (EMF/voltage) and bacteria concentrations and a lower limit of detection of 11 CFU/mL. Additionally, similar results were obtained from the biosensor system for L. innocua from food samples.
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Alfaro Sanabria, Luis Alonso, "Development of a biosensor system to detect bacteria in food systems" (2016). LSU Doctoral Dissertations. 4472.