Fluorimetric detection of insulin in the presence of Eu(III)-{pyridine-2,6-dicarboxylate} tris complex

Bovine insulin solutions (pH=9.3) have maximum absorption at 278 +/- 2 nm a nd an intrinsic emission at 305 nm when excited at 282 nm. The relative flu orescence intensities show linear dependence on its concentration: 10 mug/m L< [Ins] < 200 mug/mL. When Eu(PDA)(3)(3-) tris complex is added to these s olutions, it has a hyperchromic effect at 278 nm absorption band of insulin , sensitizing the emission intensities of central Eu3+ metal ion of the com plex at 590 and 615 nm. and simultaneously quenching the emission intensity of hormone at 305 nm. Stern-Volmer plots show that a mechanism of bimolecu lar 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 i ntermolecular energy transfer is proposed. An apparent binding constant, lo g K-app=4.70 +/- 0.13, is calculated for Ins-[Eu(PDA)(3)(3-)](2) type produ ct, the presence of which may offer a new luminescence technique as a diagn ostic tool and art alternative to radio-iodinated (I-131 -) 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-o at la mbda (exc)/lambda (em)=282/615 nm and the difference, DeltaF after sensitiz ation when similar to 100 microliters of insulin sample is added. The coeff icient 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 +/- 0.2 mug/mL respectively.