title: Fluorescence sensor for the quantification of unbound bilirubin concentrations.
abstract: 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.
authors.affiliation: FFA Sciences LLC, San Diego, CA, USA.
authors.forename: Andrew H
authors.initials: AH
authors.lastname: Huber
authors.forename: Baolong
authors.initials: B
authors.lastname: Zhu
authors.forename: Thomas
authors.initials: T
authors.lastname: Kwan
authors.forename: J Patrick
authors.initials: JP
authors.lastname: Kampf
authors.forename: Thomas
authors.initials: T
authors.lastname: Hegyi
authors.forename: Alan M
authors.initials: AM
authors.lastname: Kleinfeld
mesh_terms: D015081:2-Naphthylamine / Q000031:analogs & derivatives; D000328:Adult; D000818:Animals; D001663:Bilirubin / Q000097:blood*; D015374:Biosensing Techniques; D002138:Calibration; D050556:Fatty Acid-Binding Proteins / Q000235:genetics; D005456:Fluorescent Dyes; D006801:Humans; D007231:Infant, Newborn; D009154:Mutation; D011485:Protein Binding; D051381:Rats; D012235:Rhodamines; D012709:Serum Albumin / Q000378:metabolism
chemical_list: D050556:Fatty Acid-Binding Proteins; D005456:Fluorescent Dyes; D012235:Rhodamines; D012709:Serum Albumin; C038518:acrylodan; D015081:2-Naphthylamine; C029773:rhodamine B; D001663:Bilirubin