Fluorimetric Detection of Insulin in the Presence of Eu(III) - {Pyridine - 2,6 - Dicarboxylate} Tris Complex


Abstract: Bovine insulin solutions (pH=9.3) have maximum absorption at 278 \pm 2 nm and an intrinsic emission at 305 nm when excited at 282 nm. The relative fluorescence intensities show linear dependence on its concentration: 10 \mug/mL< [Ins] < 200 \mug/mL. When Eu(PDA)_{3}^{3-} tris complex is added to these solutions, it has a hyperchromic effect at 278 nm absorption band of insulin, sensitizing the emission intensities of central Eu^{3+} metal ion of the complex at 590 and 615 nm, and simultaneously quenching the emission intensity of hormone at 305 nm. Stern-Volmer plots show that a mechanism of bimolecular quenching at 305 nm and sensitization at 615 nm are valid up to a mole ratio, R = [Eu(PDA)_{3}^{3-}]/[Ins] < 2.0. An intramolecular rather than an intermolecular energy transfer is proposed. An apparent binding constant, log K_{app}=4.70 \pm 0.13, is calculated for Ins-[Eu(PDA)_{3}^{3-}]_{2} type product, the presence of which may offer a new luminescence technique as a diagnostic tool and an alternative to radio-iodinated (^{131}I -) insulin. A simple, rapid and accurate quantitation of insulin is proposed by using a fixed concentration of Eu(PDA)_{3}^{3-}, and measuring its initial F_{0} at \lambda_{exc}/ \lambda_{em}=282/615 nm and the difference, \DeltaF after sensitization when \sim 100 microliters of insulin sample is added. The coefficient of variation (CV), the relative error and minimum detectable amount of bovine insulin hormone are found to be 3.0%, 1.2% and 7.3 \pm 0.2 \mug /mL respectively.

Keywords: Insulin, Fluorescence Spectroscopy, Eu(III) - { Pyridine - 2,6 - Dicarboxylate } Tris Complex Stern-Volmer Plots, Apparent Binding Constant

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