Fluorescence Sensor for the Quantification of Unbound Bilirubin Concentrations

BACKGROUND: Hyperbilirubinemia in jaundiced neonates is routinely assessed by use of total serum bilirubin. However, the unbound or free form (B-f), not total bilirubin, crosses the blood brain barrier and can be neurotoxic. Although the peroxidase-mediated oxidation of bilirubin can be used to measure plasma concentrations of B-f, this measurement is relatively complex and the assay is not routinely used. We describe a fluorescence sensor for quantifying B-f in plasma. METHODS: Our method uses a mutated fatty acid binding protein labeled with the fluorescent molecule acrylodan (BL22P1B11), whose fluorescence is quenched upon binding bilirubin. Another configuration (BL22P1B11-Rh) was developed that uses BL22P1B11 together with the fluorophore rhodamine B, which responds by a change in the ratio of its fluorescence. RESULTS: The B-f probes were calibrated with aqueous solutions of bilirubin and yielded similar bilirubin dissociation constants [K-d = 16 (1.5) nmol/L]. We used the probes to determine B-f concentrations in equilibrium with human serum albumin (HSA) and in human plasma samples supplemented with bilirubin. We obtained equivalent B-f values in both systems, and the B-f probe results were in agreement with the peroxidase assay. B-f measurements revealed that bilirubin HSA binding was well described by 2 sites with K-d values of 15.4 (1) nmol/L and 748 (14) nmol/L. We measured B-f concentrations in the range expected in jaundiced neonates with a mean CV of approximately 3%. CONCLUSIONS: The BL22P1B11-Rh probe provides accurate plasma sample B-f concentrations with a single measurement, in 1 min with either a handheld B-f meter or a laboratory fluorometer. (C) 2012 American Association for Clinical Chemistry