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

Abstract

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 Fe 2+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 Fe 3+ ions with bilirubin produces Fe 2+ 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 (R 2 = 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.

Efficient detection of bilirubin in human serum through a displacement approach

“…Moreover, BR is detected through spiking methods and does not represent a clinical detection. In the case of "Turn ON" fluorescent organic molecular systems 16,17 (Table S3, † ref.…”

“…Recently reported coumarin-based N -oxide–Fe 3+ mixtures have revealed “Turn ON” fluorescence in the presence of bilirubin owing to the deoxygenation process by the in situ generated Fe 2+ (ref. 16 and 17) and have been tested and even used for the detection of bilirubin from human serum. In another approach, in non-fluorescent resorcinol-sucrose CDs and Cu 2+ complex, the competitive binding of Cu 2+ with bilirubin released the fluorescence of the CDs.…”

Conjugated Polymer Nanoparticles as a Fluorescence Probe for Amplified Detection of Human Serum Bilirubin

“…Bilirubin (BR) is a neurotoxic, yellow-orange degradation product of heme catabolism whose turnover is greatly associated with jaundice. , Under a typical physiological state, BR is enzymatically converted to mono- and di-glucuronide conjugates in the liver and gets rid of the body along with the body fluids . The human serum contains <25 μM (<1.2 mg/dL) BR, which increases up to 50 μM during the jaundice condition. , Such a medical condition is fatal as it can accumulate in body organs; thereby, its toxicity leads to mental disorder, hepatitis, Gilbert syndrome, brain damage, anemia, and even death to neonatals due to the inhibition of mitochondrial functions. − Several reports have found that a low serum BR level is also connected with several diseases like coronary artery disease and atherosclerosis. , Thus, monitoring the abnormal concentration of BR, a key biomarker for various physiological processes in our body, precisely and early in low concentrations is of immense importance to ensure early treatment.…”

“…3 The human serum contains <25 μM (<1.2 mg/dL) BR, which increases up to 50 μM during the jaundice condition. 4,5 Such a medical condition is fatal as it can accumulate in body organs; thereby, its toxicity leads to mental disorder, hepatitis, Gilbert syndrome, brain damage, anemia, and even death to neonatals due to the inhibition of mitochondrial functions. 6−11 Several reports have found that a low serum BR level is also connected with several diseases like coronary artery disease and atherosclerosis.…”

Using Blue Mini-LEDs as a Light Source Designed a Miniaturized Optomechanical Device for the Detection of Direct Bilirubin

“…The fluorescence method detected purification bilirubin has a high linear correlation, but the detection method is dependent on chemical reactions. In addition, for biological samples with a complex composition, such as urine and blood, the anti-interference and linear correlation of fluorescence-based methods do not reach the expected level of accuracy [ 38 – 40 ]. Besides the methods above, the excess bilirubin in the blood will lead to the skin becoming yellow, and then, the method using diffuse reflectance spectroscopy to quantify bilirubin from skin reflectance spectra has been proposed [ 41 ].…”