Spectrofluorimetric determination of heparin using doxycycline-europium probe

A new spectrofluorimetric method was developed for the determination of the trace amount of heparin (Hep). Using doxycycline (DC)-europium ion (Eu3+) as a fluorescent probe, in the buffer solution of pH=8.9, Hep can remarkably enhance the fluorescence intensity of the DC-Eu3+ complex at λ=612 nm and the enhanced fluorescence intensity of Eu3+ ion is in proportion to the concentration of Hep. Optimum conditions for the determination of Hep were also investigated. The linear range and detection limit for the determination of Hep are 0.04-0.8 μg/mL and 19.7 ng/mL, respectively. This method is simple, practical, and relatively free of interference from coexisting substances and can be successfully applied to assess Hep in biological samples. By the Rosenthal graphic method, the association constant and binding numbers of Hep with the probe are 6.60×104 L/mol and 33.9. Moreover, the enhancement mechanism of the fluorescence intensity in the DC-Eu3+ system and the DC-Eu3+-Hep-CTMAB system have been also discussed.