摘要： 肝素是一种聚阴离子生物活性多糖，具有重要的临床价值，简便快速、特异性高的分析方法的研发一直受到重视。基于分子缔合作用原理，构建了酞菁离子缔合物荧光探针，并籍此建立了肝素高特异、高灵敏的荧光分析新方法，分析浓度可达纳克级。发射强红色荧光的四磺基铝酞菁（AlS4Pc）是一种带负电的金属酞菁荧光化合物，研究发现，母体结构相同、荷电相反的阳离子铜酞菁化合物阿利新蓝（Alcian blue 8 GX）对AlS4Pc具有高效荧光猝灭作用。由于二者均具有大平面的酞菁母体，但电性相反，分子结构有着高度的匹配性，易于通过静电作用、平面疏水作用等分子间力而形成强的缔合作用，生成近乎无荧光的缔合物。据此现象构建了AlS4Pc-Alcian blue 8 GX缔合物荧光探针。进一步的糖类物质考察、筛选实验显示，具有强负电性的阴离子生物多糖可以使AlS4Pc-Alcian blue 8 GX缔合体系的荧光发生不同程度的恢复，重新发出AlS4Pc的荧光，其中尤以肝素存在下的荧光恢复行为最为显著，且恢复程度与肝素的浓度呈正相关。这是因为肝素带有大量磺酸根阴离子，对AlS4Pc构成强的竞争结合（Alcian blue 8GX）的作用，导致AlS4Pc从缔合体系中游离出来，体系荧光得以恢复。基于这一发现，研究建立了一种高灵敏、高特异性测定肝素的荧光增强分析法。考察了体系的分子光谱（荧光光谱和吸收光谱）行为以探讨缔合物形成和反应体系荧光恢复的作用机理；对反应参数（包括pH、反应温度、反应时间、AlS4Pc以及Alcian blue 8 GX的用量等）进行了优化；在最佳条件下，方法的线性回归方程y=1.08x+58.62，工作曲线线性区间为6.0～600.0 ng·mL-1，检测限为5.7 ng·mL-1；提出了简便实用的样品前处理方法（极性有机溶剂沉淀分离法）以解决实际样品测定时存在偏差的问题；此外，较为全面地考察了多种类物质（常见阴、阳离子、小分子、表面活性剂及生物大分子）对肝素测定的干扰行为；本法应用于实际样品（肝素钠注射液）的测定，取得了较为满意的结果。开拓了酞菁类化合物作为分子光谱探针在分析科学中的应用。
关键词：酞菁；荧光；肝素；探针
Abstract：Heparin, a polyanionic bio-polysaccharide, has important clinical values. The development of simple, rapid and specific method for the detection of heparin has been being an attractive topic in the last decades. According to the principle of molecular association, a phthalocyanine-based ion-associate fluorescent probe was prepared, followed by the establishment of a novel fluorimetry for the determination of heparin with high specificity at nanogram level. Tetrasulfonated aluminum phthalocyanine (AlS4Pc), which emits strong red fluorescence is negatively charged, was found that its fluorescence could be dramatically quenched by a structure-matched cationic copper phthalocyanie (Alcian blue 8 GX) bearing a same parent structure and oppositely charged. Because of their highly matched molecular structures the two phthalynines, strong association is easy to occur through intermolecular force, leading to the formation of none-fluorescent associate. Based on this founding, a fluorescent probe of AlS4Pc-Alcian blue 8 GX associate was developed in this study. Screening experiments on carbohydrates showed that the fluorescence of AlS4Pc-Alcian blue 8 GX recovered when polyanionic bio-polysaccharide existed. The fluorescence recovery is particularly significant in the presence of heparin. We believe that this phenomenon could be attributed to the presence of a large number of sulfonate anion on the skeleton of heparin. The polyanionic structure leads to strong competitive binding to Alcian blue 8GX with AlS4Pc, resulting in the dissociation of AlS4Pc from the associate, and the fluorescence of the system restored. Based on the above findings, a highly sensitive and specific method for the determination of heparin by fluorescence enhancement was established. The molecular spectra (fluorescence and absorption spectra) of the system were investigated to deduce the mechanism of the formation of associate and the fluorescence recovery. The reaction parameters (including pH, reaction temperature, reaction time, usage of AlS4Pc and Alcian blue 8 GX) were optimized. Under the optimal conditions, the linear range of calibration curve is 6.0～600.0 ng·mL-1, and the detection limit is 5.7 ng·mL-1. A facile pretreatment method employing polar organic solvent as precipitant was developed to avoid the deviation in the detection of practical samples. In addition, the interference behavior of foreign substances on the determination of heparin was investigated comprehensively, which has made up for the deficiency in literatures. The proposed method has been applied to the determination of practical sample (heparin sodium injection) with satisfactory results. This study expends the application of fluorescent phthalocyanines as molecular optical probes in analytical sciences.
周 涛，杜光心，郑 霄，张 艳，黄 萍，邓雅斌，李东辉. 结构匹配酞菁缔合物红区荧光探针在纳克级肝素测定中的应用[J]. 光谱学与光谱分析, 2019, 39(04): 1109-1113.
ZHOU Tao, DU Guang-xin, ZHENG Xiao, ZHANG Yan, HUANG Ping, DENG Ya-bin, LI Dong-hui. Application of Structure-Matched Phthalocyanines Associate as a Red-Emitting Fluorescent Probe in the Determination of Heparin at Nano-Gram Level. SPECTROSCOPY AND SPECTRAL ANALYSIS, 2019, 39(04): 1109-1113.
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