Bilirubin Quantification in Human Blood Serum by Deoxygenation Reaction Switch-Triggered Fluorescent Probe

A coumarin-based fluorescent compound, bilirubin fluorescent probe N-oxide (BFPNox), was successfully designed and synthesized for highly selective and sensitive detection of free bilirubin with short response time. The fluorescence "turn-on" response of the probe is based on the in situ generated Fe²⁺-mediated deoxygenation reaction of N-oxide from the diethylarylamine group of the probe, where the group attached to the coumarin π-conjugated system is responsible for the fluorescence quenching state of the probe, BFPNox. Here, the reaction of the added Fe³⁺ ions with bilirubin produces Fe²⁺ ions in situ in aqueous buffer. Fluorescence enhancement of BFPNox was achieved by more than 12-fold when a double equivalent of bilirubin solution was added in reaction buffer at pH 7.2 (50 mM HEPES, 5% DMSO) at 25 °C under excitation at 400 nm. It detected free bilirubin as low as 76 nM in an aqueous system without any interference of metal ions, anions, and other important biomolecules with a linear concentration range of 0-10 μM (R² = 0.991). The probe was also employed in the estimation of free bilirubin in human serum specimens to verify the efficacy of this probe. With these, it is revealed that this probe is a good candidate to be used as a powerful diagnostic tool for the assessment of free bilirubin with significant accuracy and reliability.