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A miniaturized fiber optic biosensor has been fabricated for rapid analysis of bilirubin. Bilirubin is detected indirectly, based on fluorescence quenching of tris(4,7-diphenyl-1,10-phenanthroline) ruthenium chloride, [Ru(dpp)₃], by dissolved molecular oxygen. Oxygen is consumed in the oxidation of bilirubin to biliverdin, catalyzed by the enzyme bilirubin oxidase (BOX). Both Ru(dpp)₃ and bilirubin oxidase are physically immobilized in an acrylamide polymer matrix that is attached covalently to a silanized optical fiber tip surface via controlled photopolymerization. Leaching of the ruthenium dye is minimized by the optimization of the ratio between the acrylamide monomer and the cross-linker (N,N-methylene bisacrylamide). An 8% isobutyl alcohol in a phosphate buffer at pH 6 is used for storing the fiber optic biosensor. This solution maintains the enzymatic activity and the sensor response while minimizing the leaking …