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.

中文翻译：

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脱氧反应开关触发荧光探针对人血清中胆红素的定量分析

成功设计和合成了一种基于香豆素的荧光化合物胆红素荧光探针N-氧化物 (BFPNox)，用于高选择性和灵敏地检测游离胆红素，响应时间短。探针的荧光“开启”响应是基于原位产生的 Fe ^{2+介导的来自探针二乙基芳胺基团的}N-氧化物的脱氧反应，其中连接到香豆素 π 共轭系统的基团负责对于探针的荧光猝灭状态，BFPNox。在这里，添加的 Fe ³⁺离子与胆红素反应生成 Fe ²⁺离子在水性缓冲液中原位。当在 25 °C 在 400 nm 激发下将双当量胆红素溶液添加到 pH 7.2 的反应缓冲液（50 mM HEPES，5% DMSO）中时，BFPNox 的荧光增强超过 12 倍。它在水系统中检测出低至 76 nM 的游离胆红素，不受金属离子、阴离子和其他重要生物分子的干扰，线性浓度范围为 0-10 μM ( R ² = 0.991)。该探针还用于估计人血清样本中的游离胆红素，以验证该探针的功效。有了这些，表明该探针是一个很好的候选者，可用作评估游离胆红素的强大诊断工具，具有显着的准确性和可靠性。