Ratiometric and fluorescence lifetime-based biosensors incorporating cytochrome c′ and the detection of extra- and intracellular macrophage nitric oxide

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 (less than or equal to 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 mu M nitric oxide. The lifetime-based measurements are made using free, dye-labeled cytochrome c' in solution and have a limit of detection of 30 mu M 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 mu M for activated macrophages, while an average intracellular concentration of 160 +/- 10 mu M 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.