Laser-induced fluorometric determination of albumin using longwave absorbing molecular probes

A novel fluorescence assay for HSA is described. It is based on longwave-absorbing probes that selectively bind to HSA to form fluorescent complexes. The two probes reported here (Albumin Blue 633 and Albumin Blue 670) are tailored to match the lines of the 633-nm HeNe laser and the 670-nm diode laser, respectively. In both cases, the strong laser-induced fluorescence of the HSA/probe complexes makes the assay sensitive to HSA at trace levels. Detection limits of 0.2 mg/liter were obtained. In addition to its sensitivity, the assay is highly selective for HSA in that the response to other serum proteins is weaker by a factor of at least 100. It also works well on urine, and no significant interferences could be located. Potential interferents are bovine serum albumin and some detergents. Parameters of the probe--HSA interaction were obtained from a Scatchard evaluation. The high specificity and sensitivity of the assay are discussed in terms of molecular geometry of the probes. A photophysical mechanism that leads to the effect is proposed. The assay presents a promising alternative to immunological determinations of HSA since it is less expensive and much more quickly performed.