Development and Cellular Applications of Fiber Optic Nitric Oxide Sensors Based on a Gold-Adsorbed Fluorophore Susan L. R. Barker and Raoul Kopelman* Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109-1055 A new design for optochemical sensors has been applied to the development of a nitric oxide selective fiber optic sensor. This sensor is composed of a fluorescein deriva- tive dye attached to colloidal gold. The fluorescein dye rearranges as nitric oxide adsorbs onto the gold, inducing a decrease in the fluorescence intensity of the dye. This mechanism has allowed preparation of fiber optic dye- based nitric oxide sensors, which have been made ratio- metric by addition of reference dye microspheres. Pre- viously developed fast, selective optical sensors for detection of aqueous nitric oxide involved a protein, such as cytochrome c′. The new fluorescein derivative chemi- cal sensors have characteristics similar to those of the protein-based biosensors, including fast response times, excellent selectivity, and complete reversibility. In addi- tion, the chemical sensors have advantages such as greater stability and commercially available components. These sensors were utilized to measure nitric oxide production by BALB/ c mouse macrophages. The ability to selectively measure nitric oxide in vitro is crucial to a further understanding of the numerous biological roles of nitric oxide. 1-6 A number of electrodes and optical sensors have been developed in attempts to accomplish this task. 7-16 Recently two methods 17,18 involving fluorescein derivatives have been used for optical detection of nitric oxide in solution and intracellularly. The first 17 measured nitric oxide by monitoring the oxidation of nonfluorescent 2,7-dichlorofluorescin to fluorescent dichlorofluo- rescein. Unfortunately, both nitric oxide and reactive oxygen species oxidized the dichlorofluorescin. More recently, 18 diami- nofluoresceins were synthesized which exhibit an increase in quantum efficiency upon exposure to nitric oxide. These com- pounds do not react directly with NO, but react with N 2 O 3 , formed when nitric oxide reacts with oxygen, to produce the triazole form of the dye. These dyes were utilized for cellular measurements but were not incorporated into a sensor. Therefore, the only currently working optic sensors 9 for fast, selective detection of aqueous nitric oxide are based on cytochrome c′. Sensors generally incorporate molecular recognition elements that are biomolecules, such as an enzyme, resulting in a biosensor, or a nonbiological molecule, such as an indicator dye, giving a chemical sensor. We present here a novel kind of molecule- specific sensor, based on analyte adsorption to a metal surface reported by fluorescence changes of an attached dye molecule. Specifically, we have developed nitric oxide selective sensors prepared with a difluorofluorescein derivative on gold. In solution, this dye is optically insensitive to nitric oxide. However, the dye absorbance and fluorescence have been determined to be sensitive to nitric oxide when attached directly to colloidal gold. Gold surfaces (which do not fluoresce) have been used to detect gaseous nitric oxide by measuring the reversible increase in electrical resistance of the Au film upon nitric oxide adsorption. 19 However, in the sensors described here, the fluorescence intensity of the fluorescein dye decreases as nitric oxide adsorbs on the gold. This mechanism has allowed preparation of fiber optic dye- based nitric oxide sensors, which have been made ratiometric by the addition of reference dye microspheres. These chemical sensors have several advantages over nitric oxide selective biosensors, including greater stability and commercially available components. 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Chem. 1998, 70, 4902-4906 4902 Analytical Chemistry, Vol. 70, No. 23, December 1, 1998 10.1021/ac981016z CCC: $15.00 © 1998 American Chemical Society Published on Web 11/04/1998