The immunoimmobilization of living bacteria on solid surfaces and its applications.

摘要

This thesis focuses on immobilizing living bacteria on material surfaces in their physiological environment without jeopardizing cell viability or replication. The ability to immobilize an individual bacterium opens up new research frontiers, as it not only enables us to conduct fundamental research on an individual living bacterium but also creates new opportunities for biosensor applications. A number of pathogenic organisms from Salmonella enterica and Escherichia coli strains are used to develop bacterial immobilization, herein referred to as immunoimmobilization. Our findings suggest that the most efficient, specific and reliable immunoimmobilization hinges on the use of affinity-purified antibodies raised against bacterial surface antigens such as fimbriae, lipopolysaccharides, and flagella. This approach produces a full monolayer of densely packed living bacteria (limited only by steric hindrance) on an antibody-activated area on a flat surface. Immunoimmobilization does not influence the viability or cell replication of the bacteria. To the best of our knowledge, to this day this is the highest packing density of immobilized bacteria reported in the literature. For successful immobilization, a monolayer of antibodies with full freedom of motion must be covalently linked to a flat surface (typically a silicon wafer). This has been achieved through a carefully optimized series of chemical reactions. More importantly, it was essential to monitor the activated surface using surface-sensitive analytical techniques to verify each step of the linker chemistry.;A new technique was developed for quantifying the initial rate of cell capture in terms of the number of bacteria immobilized per unit time per unit area per cell concentration in the physiological environment of the bacteria. This rate turned out to be (7.2 +/- 0.3) x 10 -6 cells/(min·(100 mum)2)/(cells/mL) for S. Typhimurium, and (1.1 +/- 0.1) x 10 -6 cells/(min·(100 mum)2)/(cells/mL) for E. coli, both expressing fimbriae. The lowest detection limit was ∼3 x 104 cells/mL in 30 minutes of incubation.;For the first time, immunoimmobilization studies were conducted in a number of Navy fuels, including biofuels. It was discovered that antibodies subjected to the fuel environment preserve their structure, functionality and stability. The immunoimmobilization process works as efficiently in fuels as in aqueous environments.