Luminescence sensing systems for biomolecules: Applications in cellular analysis

In this work, we have explored the used of luminescence sensing systems for the analysis of cellular compounds. In the first part of this work, two assays that have been fully developed on the large scale (μL volumes) are miniaturized to nL or pL volumes. The characteristics of these assays at reduced volumes are evaluated and they are used to study the biotin composition of individual sea urchin oocytes. The first system described is a post-capillary assay in which a fluorescence binding assay for biotin is merged with the effluent stream of a capillary electrophoresis (CE) separation. This system uses a streptavidin-fluorescein isothiocyanate (streptavidin-FITC) that shows increased fluorescence upon biotin binding. The second system is a bioluminescence binding assay for biotin that depends upon competition between an aequorin-biotin conjugate (AEQ-biotin) and free biotin within the oocytes for binding sites on the protein avidin. The assay is performed by microinjecting each component into the oocytes and following the resulting bioluminescence within the oocyte upon triggering of aequorin. This is the first reported multi-component binding assay to be performed inside an intact single cell.; In the second half of this work, two new sensing systems for cAMP are developed that have characteristics that make them amenable to small-volume analysis. Namely, they consist of a single reagent and they have sensitivities sufficient for such analyses. The first described is a bacteria-based sensing system in which the fluorescent protein GFPuv is produced in a concentration-dependent manner upon addition of exogenous cAMP. The system is also shown to be selective for cAMP over other nucleotides. The system is compared to a commercial immunoassay in the detection of cAMP in real samples. The other system is based on a recombinant cAMP receptor protein (CRP) that is covalently labeled with a cysteine-selective environment-sensitive fluorophore. Changes in the conformation of CRIP upon cAMP binding are reflected as changes in fluorescence of the fluorophore. Studies of CRIP mutants, labeled at positions 178 and 189 and of their binding to a consensus DNA sequence indicate that cAMP binding causes changes in the region around helix F of the protein. Anisotropy measurements indicate that these changes are due to cAMP-induced dimerization of the protein.