Zn2+-胆红素络合物的超快激发态动力学研究

胆红素(Bilirubin, BR)是脊椎动物分解代谢血红素的最终产物之一，具有抗氧化和消炎等作用。体内保持正常含量的胆红素对人类的健康起着非常重要的作用，被认为有利于预防癌症、中风、糖尿病和心血管等疾病的发生1-2]。然而胆红素过量则被认为是肝功能障碍的征兆，同时也是引起新生儿严重脑损伤的原因3]。因此，对人体中胆红素含量的快速精准检测具有十分重要的应用价值。目前为止，用于检测血清样品中胆红素含量的方法主要有重氮法、过氧化物酶法、光纤传感检测法和荧光光谱法等4]。其中，荧光光谱法具有检测迅速和操作简便的优势5]，吸引了越来越多研究人员的关注。但是，由于胆红素自身的荧光量子产率通常低至10-4量级6]，直接荧光测量难度很大，因此通常采用间接方式解决胆红素含量测量的难题。例如, Wabaidur等7]通过胆红素猝灭Ru(bopy)2+₃荧光的方式来测量胆红素的含量；Aparna等8]通过胆红素猝灭铜纳米团簇荧光的方式来测量胆红素的含量；Iwatani等9]通过UnaG蛋白结合胆红素增强荧光的方式来测量胆红素的含量。UnaG与胆红素具有极高的亲和力，且自身几乎不发射荧光，其与胆红素结合后，会将胆红素的荧光提高三个数量级10]，可以用作人体内的荧光传感器11]。然而，由于蛋白的制备和保存的成本十分高昂，人们更希望使用有机小分子或无机分子对胆红素的荧光进行增强。例如，Yang等12]发现Zn2+有助于显著增强胆红素的荧光，Kotal等13]提出了将Zn2+用于胆红素代谢物（尿胆素原）含量测量的方法。为了研究Zn2+增强胆红素荧光的机理并用于胆红素含量的测量，研究首先使用了稳态荧光光谱和紫外-可见吸收光谱表征了不同Zn2+浓度下胆红素的光学特性，并采用相对法测量了其量子产率。通过分析Zn2+-胆红素络合物在不同探测波长下的激发谱，提出了该络合物具有两个发光基团的设想。为了进一步验证关于发光基团的设想，使用了飞秒瞬态吸收光谱的手段研究分析了Zn2+-胆红素络合物的超快光动力学过程，分析结果与之前发表的对胆红素和Zn2+配位方式的结论有很好的吻合。根据瞬态吸收光谱和稳态荧光光谱的数据以及所提出的模型，得到了在有无Zn2+条件下胆红素的平均激发态寿命，进一步计算出了其辐射跃迁速率和非辐射跃迁速率的理论值。通过数据对比，得出了络合物相对单纯胆红素有更大的消光系数而具有更高的辐射跃迁速率的结论，同时发现胆红素与Zn2+的配位方式增加了胆红素以锥型交叉退激发态的效率。

Ultrafast Excited State Dynamicsof Zinc-Bilirubin Complex

Bilirubin (BR) is one of the final products of heme catabolism, which has functions of anti-oxidation and anti-inflammatory. Normal content of bilirubin in human body plays an indispensable role in human health, which has been confirmed to be protective against diseases such as cancer, stroke, diabetes and cardiovascular diseases etc.1-2]. However, excess bilirubin has been long recognized as a sign of liver dysfunction as well as a potential toxic factor causing severe brain damage in newborns3]. Thus, the analysis of bilirubin in body fluids is very important, speeding up the development of a cheap and accurate analytical method. Generally a lot of techniques have been used to detect bilirubin in serum samples, include diazo methods, peroxidase methods, fiber optic sensors, fluorometric methods etc.4]. Fluorometric methods have the advantages of quick response and simple operation5], attracting more and more researchers’ attention. However, the fluorescence quantum yield of bilirubin itself is quite low, which is on the order of 10-46], resulting in the difficulty in direct measurement of its content, so it is usually measured indirectly. Wabaidur et al. measured the content of bilirubin by using bilirubin to quench the fluorescence of Ru(bipy)2+₃7]; Aparna et al. measured that by using bilirubin to quench the fluorescence of copper nanocluster8]; Iwatani et al. measured that by using protein UnaG to conjugate bilirubin for its fluorescence enhancement9]. UnaG has a high affinity with bilirubin and UnaG itself is almost non-fluorescent. When combined with UnaG, the fluorescence of bilirubin would be increased by three orders of magnitude10] and thus it can be used as a fluorescent sensor in human body11]. However, the cost of protein production and preservation is pretty high, and researchers prefer to use organic small molecules or inorganic molecules to increase the fluorescence of bilirubin. For example, Yanget al. found that the zinc ions were helpful to significantly enhance the fluorescence of bilirubin12]. Kotal et al. proposed the use of zinc ions for the measurement of bilirubin metabolite (urobilinogen) content13]. Basing on using zinc ions to enhance the fluorescence of bilirubin for its content measurement, we applied the steady-state fluorescence and UV-visible absorption spectroscopy to characterize bilirubin with different concentration of zinc ions, and the fluorescence quantum yield was measured by using the relative method. By analyzing the excitation spectra of zinc-bilirubin complex at different emission wavelengths, we proposed a hypothesis that the complex has two fluorescence subunits. Besides, the femtosecond transient absorption spectroscopy was used to explore the photodynamic process of the complex and the hypothesis was confirmed. The experimental data was in a good agreement with previous studies of bilirubin and zinc ions complex. Moreover, combining together with the transient absorption spectra, steady-state spectra and the proposed hypothesis, we calculated the average excited-state lifetime of bilirubin with/without zinc ions, and obtained its theoretical value of radiative and non-radiative decay rate. Compared with bilirubin itself, the complex has a larger value of extinction coefficient, which caused a greater radiative decay rate. We believe the coordination of zinc ions and bilirubin has increased its deactivation efficiency byinternal conversion.