Spectrofluorimetric determination of human serum albumin using a doxycycline-europium probe

A new spectrofluorimetric method is proposed for determination of human serum albumin (HSA) with the limit of detection at ng levels. Using doxycycline (DC)-europium (Eu³⁺) as a fluorescent probe, in a buffer solution of pH 10.2, HSA can remarkably enhance the fluorescence intensity of the DC-Eu³⁺ complex at 612 nm and the enhanced fluorescence intensity of Eu³⁺ is proportional to the concentration of HSA. Optimum conditions for the determination of HSA are also investigated. The linear ranges for HSA are 0-9.2 and 9.2-34.5 μg ml⁻¹ with limits of detection of 64 and 115 ng ml⁻¹, respectively. This method is simple, practical and relatively free of interference from coexisting substances, as well as much more sensitive than most of the existing assays. The determination results for human serum and urine samples are identical to those by the AOAO method, with relative standard deviations of five determinations of 1.1-3.6%. By the Rosenthal graphic method, the binding number and association constant of human serum albumin with the probe are 1.8 and 3.71×10⁵ l mol⁻¹, respectively.