Agatha Feltus

Peer Reviewed: Applications of Reporter Genes

Reporter proteins can be used in bioanalytical methods to produce signals indicating the presence of a target analyte.

Bioluminescence competitive binding assays for biotin based on photoprotein aequorin

Publisher Summary This chapter describes competitive binding assays for biotin that use the bioluminescent protein aequorin as the label. Heterogeneous and homogeneous competitive binding assays using aequorin bioluminescence have been developed for detection of biotin. In the heterogeneous system, an aequorin–biotin conjugate competes with unlabeled biotin for binding to immobilized avidin, followed by a separation step to remove the unbound conjugate. Bioluminescence assays are best performed on luminometers operated in a photon-counting mode. A variety of such instruments is commercially available. In addition, modern scintillation counters may be used to perform bioluminescence assays. The relative amounts of avidin and biotinylated aequorin (conjugate) influence the response characteristics of the assay. Therefore, the amounts of avidin and biotinylated aequorin must be optimized in order to enhance assay performance. The development of a homogeneous assay for biotin using aequorin is based on the ability of avidin in solution to inhibit the bioluminescence of biotinylated aequorin. To establish the optimal amount of avidin to use in the assay, a binder dilution curve is constructed by varying the amount of avidin in the presence of a fixed amount of biotinylated aequorin, and measuring the degree of signal inhibition.

Genetic Engineering of Signaling Molecules

Publisher Summary To expand the capabilities of biosensors, there is a need to develop new signaling molecules. This chapter focuses on molecules, produced through genetic engineering, that combine the recognition element with a signaling element (such as a fluorophore) in an effort to optimize the signal caused by the binding of the analyte to the recognition element. These systems, while not necessarily originally developed for an optical fiber, can be immobilized at the tip of the fiber either through chemical attachment or entrapment behind a membrane. Three different systems will be examined: fluorophore-labeled binding proteins, FRET-based systems, and bacteria-based sensors. These systems use optical signaling methods to reveal the binding event, taking advantage of molecular biological techniques to optimize the signal. This chapter discusses the advantages and disadvantages of each system as well as the current state of the art of these biosensors.

Post-capillary reaction detection in capillary electrophoresis based on the streptavidin-biotin interaction : Optimization and application to single cell analysis

A class-selective post-capillary reaction detection method for capillary electrophoresis is described in which a streptavidin-fluorescein isothiocyanate (streptavidin-FITC) conjugate is used to detect biotin moieties. The selective binding of biotin moieties to the streptavidin-FITC conjugate causes an enhancement of fluorescence proportional to the concentration of biotin present. After capillary electrophoresis the separated analytes react with streptavidin-FITC in a coaxial reactor and are then detected either by a benchtop spectrofluorometer (2.5 microM detection limit) or by an epi-fluorescence microscope (1 x 10(-7) M detection limit). The method is used to examine biotinylated species in a crude mammalian cell lysate which was found to contain 83+/-3 fmol in 3600 cell volumes. In addition, it is used to examine the uptake of biotin by individual sea urchin oocytes. The results indicate that, in the oocytes, biocytin is the prevalent form of biotin and its concentration varies widely between cells (mean=2+/-2 microM).