Ratiometric and Fluorescence-Lifetime-Based Biosensors Incorporating Cytochromec ‘and the Detection of Extra- and Intracellular Macrophage Nitric Oxide

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Abstract
Ratiometric and lifetime-based sensors have been designed for cellular detection of nitric oxide. These sensors incorporate cytochrome c ‘, a hemoprotein known to bind nitric oxide selectively. The cytochrome c ‘ is labeled with a fluorescent reporter dye, and changes in this dye's intensity or fluorescence lifetime are observed as the protein binds nitric oxide. The ratiometric sensors are composed of dye-labeled cytochrome c ‘ attached to the optical fiber via colloidal gold, along with fluorescent microspheres as intensity standards. These ratiometric sensors exhibit linear response, have fast response times (≤0.25 s), and are completely reversible. The sensors are selective over numerous common interferents such as nitrite, nitrate, and oxygen species, and the limit of detection is 8 μM nitric oxide. The lifetime-based measurements are made using free, dye-labeled cytochrome c ‘ in solution and have a limit of detection of 30 μM nitric oxide. The use of these two techniques has allowed measurement of intra- and extracellular macrophage nitric oxide. Employing the ratiometric fiber sensors gave a multicell culture average extracellular nitric oxide concentration of 210 ± 90 μM for activated macrophages, while an average intracellular concentration of 160 ± 10 μM was determined from the lifetime-based measurements of dye-labeled cytochrome c ‘ in the macrophage cytosol. Microscopic adaptation of the lifetime-based methods described here would allow direct correlation of intracellular nitric oxide levels with specific cellular activities, such as phagocytosis.