Xiao Guo

Date of Award

5-2011

Level of Access Assigned by Author

Campus-Only Thesis

Degree Name

Master of Science (MS)

Department

Food Science and Human Nutrition

Advisor

Vivian Chi-Hua Wu

Second Committee Member

Benildo G. de los Reyes

Third Committee Member

Chih-Sheng Lin

Abstract

Escherichia coli O157: H7 is a significant pathogenic microorganism responsible for foodborne illnesses, yet the current rapid detection methods which based on biosensor setting are not mature enough to detect viable E. coli 0157: H7 in low concentration for routine usage. Time-consuming culture remains the routinely used method for detection. Piezoelectric biosensors, which monitor targets according to frequency shifts caused by "mass changes" on the chip surface, have been used for pathogen detection. However, the unique function has not been developed in a setting for real-time enrichment and detection of viable bacteria or effectively applied for the microbial analysis in food samples. The objective of this research was to develop a sensitive real-time enrichment and detection system for viable E. coli 0157: H7 by a piezoelectric biosensor-quartz crystal microbalance (QCM) with gold nanoparticles (AuNPs) amplification. Capture antibodies for E. coli O157: H7 were immobilized on the QCM chip by glutaraldehyde in a circulating-flow system. Zero to one log colony-forming unit (CFU)/ml or g of E. coli O157: H7 were circulated through the system with the presence of brain heart infusion (BHI) broth for the following 18 hours, in order to enrich and confirm the viability of the E. coli O157: H7. The enriched cells were captured by the immobilized capture antibodies for E. coli O157: H7 on the QCM chip surface. After the enrichment, detection antibodies for E. coli O157: H7 which were conjugated with AuNPs (detection antibody-conjugated AuNPs) were added as signal amplifier and target verifier. The detection was monitored through frequency changes. Listeria monocytogenes and Salmonella Typhimurium were used as negative controls. Last, the simultaneous enrichment and detection nanoparticle-functionalized piezoelectric biosensor-QCM system was applied in the detection of real food and environment samples, which were Maine wild blueberries and local lake water samples. The results indicated the developed simultaneous enrichment and detection nanoparticle-functionalized piezoelectric biosensor-QCM system showed promising sensitivity and specificity in the detection of viable E. coli O157: H7 in Maine wild blueberries and local lake water samples. The application of the detection antibody-conjugated AuNPs provided a 15-fold improvement in the frequency change, compared with the signal obtained without its addition. Moreover, the use of BHI enrichment had further enhanced the sensitivity of the developed system with the detection limit of 0-1 log CFU/ml or g. Frequency changes were observed during the enrichment, indicating the growth of the bacteria. The specificity of the developed system was demonstrated by a lack of significant frequency change (p < 0.05) when 1 log CFU/ml or g of L. monocytogenes and S. Typhimurium were tested. The real-time detection method of viable E. coli O157: H7 using the simultaneous enrichment and detection nanoparticle- functionalized piezoelectric biosensor-QCM system developed in this study can be used to enrich and detect viable cells simultaneously within 24 hours using a comparable large sample size of 10 ml. The unique advantages of the developed system give it great potential in the microbial analysis of food and environmental samples in the routine settings.

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