Selective and sensitive homogenous assay of serum albumin with 1-anilinonaphthalene-8-sulphonate as a biosensor

Homogenous selective assay of albumin (ALB) in clinical sera was tested with 1-anilinonaphthalene-8-sulphonate (ANS) as Förster-resonance-energy-transfer (FRET) acceptor of tryptophan residues and biosensor of ALB. Between the excitation at 280 and 350   nm, the ratio of the fluorescence at 470   nm of free ANS in ethanol was about 1.9 while that of the complexes of ALB and ANS was about 3.9, supporting FRET in complexes of ANS and ALB. ANS below 1.0   mM saturated one site of ALB with K d of about 0.13   μM in 20   mM sodium phosphate buffer at pH 7.0. For selective assay of ALB, 0.30   μM ANS was used to quantify fluorescence of the complexes at 470   nm under the excitation at 280   nm. ALB from 1.8 to 25   nM was quantified, whose lower limit was below 1% than that by bromocresol green assay while one-third than that by immunoturbidimetric assay. Globular proteins at comparable levels gave negligible signals. This new method showed reasonable resistance to other interfering substances in clinical sera. Quantities of ALB in clinical sera by this method were consistent with those by bromocresol green assay and immunoturbidimetric assay. Hence, homogenous assay of ALB with ANS as FRET biosensor was effective.