Development of anion- and nitric oxide-selective chemical sensors and biosensors.

Abstract

The biological roles of chloride, nitrite, and nitric oxide create the need for techniques which can provide fast, sensitive, and selective detection of these analytes. Small sensor size is advantageous in biological applications, and the coupling of fluorescence transduction with optical fiber technology has allowed the preparation of micrometer and submicromter sized chemical sensors and biosensors with good selectivity, fast response times, and excellent signal to noise ratios, which are utilized for <italic>in vitro</italic> and cellular applications. Micrometer and submicrometer size fiber optic nitrite and chloride sensors have been prepared, based on immobilized metalloporphyrins, using the ion correlation principle, and characterized with respect to selectivity, sensitivity, and reproducibility. The chloride sensors were applied <italic>in vitro</italic> to rat conceptuses. The hemoprotein cytochrome c<super>'</super> and the heme domain of soluble guanylate cyclase (sGC) have been labeled with a fluorescent dye and utilized for intensity and fluorescence lifetime-based nitric oxide sensing. Ratiometric fiber optic sensors have been prepared by attaching the dye-labeled cytochrome c<super>'</super> or heme domain of sGC to the fiber along with reference dye spheres. In addition, the fluorescence lifetime of the dye-labeled cytochrome c<super>'</super> in solution has been monitored. A second class of nitric oxide sensors has also been developed. These are dye-based chemical sensors with a response based on the interaction of nitric oxide with a fluorophore adsorbed on a gold surface. Such chemical sensors have the advantage of commercially available components and long-term stability. The nitric oxide bio- and chemical sensors have excellent signal to noise ratios and linear responses down to low micromolar nitric oxide. The various sensors show minimal interference from numerous other chemicals that are commonly found in the cellular environment. In addition, the sensors have low micromolar limits of detection, subsecond response times and complete reversibility, making these sensors applicable to dynamic measurements of cellular nitric oxide. Extra- and intracellular nitric oxide were measured in unactivated and activated macrophages. The macrophages were activated with interferon-gamma (IFN-gamma) and lipopolysaccharide (LPS), known stimulants of macrophage nitric oxide production. Both protein and dye-based fiber optic ratiometric sensors have been used to determine the macrophage produced extracellular nitric oxide concentration. For intracellular measurements, the dye-cytochrome c<super>'</super> complex was scrape-loaded into the cytoplasm of the macrophages.