Fluorometric detection of nitric oxide using 2,3-diaminonaphthalene incorporated in β-cyclodextrin

The aromatic diamino compound 2,3-diaminonaphthalene (DAN) has been used in numerous occasion to detect nitrite and nitric oxide in biological fluids; however, the sensitivity of this method relies on a strong alkalinisation of the sample which, in many cases, limits the general applicability of the method. In this work, we propose a new fluorescent method to quantify nitric oxide at physiological pH, based on the reaction of this radical with DAN, previously incorporated in β-cyclodextrin (β-CD). Changes in the fluorescence intensity of DAN were used to characterize the DAN/β-CD inclusion complexes and determine the association constant and stoichiometry of the process. Reactivity of the DAN/β-CD complex was then evaluated in presence of different concentrations of NO. Results show that the inclusion of DAN into the β-cyclodextrin does not prevent the formation of the fluorescent product 2,3-naphthotriazole (NATH), which remains into the cyclodextrin increasing considerably its fluorescence quantum yield, even at neutral pH. A plot between NO concentration and fluorescence intensity of NATH was built to determine the sensitivity of the DAN/β-CD complex to the radical. A linear relationship was found with a lower detection limit ∼20 nM.