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.