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 (Bf), 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 Bf, this measurement is relatively complex and the assay is not routinely used. We describe a fluorescence sensor for quantifying Bf 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 “Bf 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 Bf concentrations in equilibrium with human serum albumin (HSA) and in human plasma samples supplemented with bilirubin. We obtained equivalent Bf values in both systems, and the Bf probe results were in agreement with the peroxidase assay. Bf 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 Bf concentrations in the range expected in jaundiced neonates with a mean CV of approximately 3%. CONCLUSIONS: The BL22P1B11-Rh probe provides accurate plasma sample Bf concentrations with a single measurement, in 1 min with either a handheld Bf meter or a laboratory fluorometer.