B Cell-based Biosensors For Rapid And Specific Detection Of Escherichia Coli O157:H7

Since current food processing and distribution systems operate at very high speed and the contamination dosage of some foodborne pathogens is very low, it is extremely important to develop more rapid, specific and sensitive methods for the detection of pathogens in food products, a critical step in ensuring the safety of our food supply. In this research, a B cell-based biosensor was studied based on cellular biology, immunology, photology and biological engineering. Using Escherichia coli O157:H7 as a model bacterium, the feasibility of the B cell to be the bioreceptor was proved, and two types of B cell-based biosensors were designed, fabricated, and evaluated. 1. The interaction between E. coli 0157:H7 and B cells. The property of the bio-receptor of the B cell-based biosensor was studied, such as the growth characteristics and the activity of B cell receptor (BCR) on the surface of B cells. Then the flow cytometry was used to determine the attachment of B cells to E. coli O157:H7 at different concentrations, and the viability of B cells cultured with E. coli O157:H7 was confirmed. Thus a preliminary evaluation of the bio-receptor of the B cell-based biosensor was conducted. 2. A B cell-based biosensor based on chemical calcium indicator. A rapid and sensitive detection technology is highly desirable for specific detection of E. coli O157:H7, one of the leading bacterial pathogens causing foodborne illness. In this study, we reported the rapid detection of E. coli O157:H7 using calcium signaling of the B cell upon cellular membrane anchors anti-E. coli O157:H7 IgM. The binding of E. coli O157:H7 to the IgM on the B cell surface activated the B cell receptor (BCR)-induced Ca2+signaling pathway and resulted in the release of Ca2+ within seconds. The elevated intracellular Ca2+triggers Fura-2, a fluorescent Ca2+ indicator, for reporting the presence of target pathogens. The Fura-2 was transferred to B cells before detection. The study demonstrated that the developed B cell-based biosensor was able to detect E. coli O157:H7 at low concentrations within 10 min. The specificity of the biosensor was confirmed using three non-target bacteria. Finally, the B cell-based biosensor was used for the detection of E. coli O157:H7 in ground beef samples. With its short detection time and high sensitivity for the low concentration of the target bacteria, this B cell biosensor shows promise in future applications of the high throughput and rapid detection of bacterial pathogens in food safety, biosafety and environmental monitoring. 3. A B cell-based biosensor based on genetically engineered calcium indicator.A Forster resonance energy transfer (FRET)-based B cell biosensor was developed in this study using a genetically encoded calcium indicator (GECI) as the reporter. The principles underlying the development of this biosensor are that the binding of E. coli O157:H7 to the IgM on the B cell surface activates the B cell receptor (BCR)-induced Ca2+signaling pathway to induce the release of Ca2+ within seconds and that this elevated intracellular Ca2+ triggers GECI TN-XXL to report the presence of target bacteria. The results demonstrated that TN-XXL and FRET-based approaches could be employed in the B cell biosensor to detect the target bacteria with suitable sensitivity and specificity within 10 min without sample pre-enrichment. The FRET efficiencies indicated that the monoclonal antibodies (MAbs) on the surface of B cells are specific to the target bacteria, E. coli O157:H7. The experimental result provided the first evidence that the average FRET value based on the intracellular Ca2+concentration increased with an increase in the bacteria concentration (101-108 CFU/mL). This study also provides a general approach for observing the activation of the BCR by target bacteria, and this approach can be used to detect any target bacteria that activate the BCR on the surface of specific B cells.The results indicated that those B cell-based biosensors were able to detect E. coli O157:H7 rapidly, sensitively and specifically, which established theoretical and technological bases for the future research on cell-based biosensors for food safety.