Efficient detection of bilirubin in human serum through a displacement approach

Abstract

The present study provides, for the first time, a method for the fluorescence “Turn ON” quantification of bilirubin (BR) in human serum through a displacement approach. The fluorescent probe THYQ exhibited a 55-fold increase in fluorescence intensity at 555 nm with HSA. The green fluorescent THYQ–HSA complex was highly selective towards bilirubin (BR) (subdomain IB) and exhibited >90% fluorescence quenching (log β = 4.83 ± 0.12) through the displacement of THYQ, whereas the subdomain IIA and subdomain IIIA selective drugs warfarin and ibuprofen did not displace THYQ. The non-fluorescent BR–HSA complex displayed a linear increase in fluorescence with THYQ (log β = 4.81 ± 0.13) to form THYQ–HSA complex. The slope of titration of BR–HSA with THYQ was linearly dependent on [bilirubin] in HSA, thus enabling the fluorescence-based detection of bilirubin below normal (0.7 mg dL⁻¹, 12 μM) to hyperbilirubinemia conditions (12.6 mg dL⁻¹, 216 μM), with the results in good agreement with the clinical findings. The LOD for the detection of BR was 0.004 mg dL⁻¹ (68 nM).