Optical films for protamine detection with lipophilic dichlorofluorescein derivatives

Lipophilic 2′,7′-dichlorofluorescein esters have been synthesized and incorporated into thin plasticized polymeric membrane films as chromoionophores for the optical sensing and detection of polycationic protamine. The membrane response is based on the extraction of protamine into the film due to the interaction between the anionic fluorescein and protamine polycation which results in a cation exchange between the protamine and proton and thus, induces an absorbance spectra change of the polymeric film. The response speed is controlled by protamine diffusion through the stagnant diffusion layer adjacent to the film surface as well as within the bulk of the polymer film. When limited exposure time (3 min) and non-stirring detection modes are used in a buffer solution of pH 7.4, absorbance (at 536 nm) of a film (thickness: 2 μm) changes as a function of the protamine concentration in the range of 2 to 60 μg m −1 (0.44–13.3 μM). The sensing film showed good selectivity over most of the common small cations, and, therefore, it can be used in the determination of protamine in diluted serum. No response was observed when a protamine complexing reagent such as heparin is present, thus it can be used as an indicator for the protamine-heparin titration. The operating principle and applications of the present sensing membrane are discussed.