Abstract：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 newborns[3]. 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 operation[5], attracting more and more researchers’ attention. However, the fluorescence quantum yield of bilirubin itself is quite low, which is on the order of 10-4[6], 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+3[7]; Aparna et al. measured that by using bilirubin to quench the fluorescence of copper nanocluster[8]; Iwatani et al. measured that by using protein UnaG to conjugate bilirubin for its fluorescence enhancement[9]. 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 magnitude[10] and thus it can be used as a fluorescent sensor in human body[11]. 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 bilirubin[12]. Kotal et al. proposed the use of zinc ions for the measurement of bilirubin metabolite (urobilinogen) content[13]. 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.
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