Representative procedure for the synthesis of hydrocyanines: Cy3 (100 mg, 0.2 mmol) was dissolved in methanol (5 mL), placed in a four-drum vial, and covered with aluminum foil. NaBH4 (3 mg, 0.08 mmol in 0.5 mL methanol) was added dropwise to the purple Cy3 solution, which was stirred for 10 min to generate a colorless solution. The reaction mixture was then stirred for an additional 10 min, and the solvent was removed under reduced pressure. The resulting solid was dissolved in 10 mL dichloromethane and 5 mL water and vigorously shaken. The organic layer was extracted with additional dichloromethane (5 mL × 2), dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The reduced dye thus obtained was used without further purification. See the Supporting Information for detailed characterization of the products.
Detection of the hydroxyl radical (Figure 3): The hydroxyl radical was generated in situ by treating hydrogen peroxide with Fe2+ for the following experiments. To a solution (1 mL) of hydro-Cy3 in methanol (78 µm), various quantities of a hydrogen peroxide stock solution were added to generate hydrogen peroxide concentrations in the range 2–30 nm. Aqueous Fe2+ (1 µm) was then added to the hydro-Cy3/H2O2 solution to generate a 200 nm concentration. The resulting solution was kept at ambient temperature for 5 min, and the fluorescence intensity was measured (λex/λem = 535/560 nm) against a reagent blank at the same time. The sensitivity of DHE (λex/λem = 515/ 560 nm) and hydro-Cy7 (λex/λem = 735/760 nm) towards the hydroxyl radical was measured under similar experimental conditions.