阳离子铝酞菁-黏多糖缔合物荧光探针测定生物样品中的溶菌酶  

Novel Method for Determination of Lysozyme Based on Fluorescence Recovery of a Cationic Aluminum Phthalocyanine-Mucopolysaccharides Association Complex Used as a Red Emitting Fluorogenic Substrate

作　　者：陈林[1] 黄萍[1] 杨惠卿 邓雅斌[1] 李东辉[1] 

机构地区：[1]厦门大学医学院抗癌研究中心,厦门361102

出　　处：《分析化学》2014年第7期962-967,共6页Chinese Journal of Analytical Chemistry

基　　金：国家自然科学基金重大研究计划项目(No.90206016);福建省科技重点项目(No.2012Y0081);福建省属公益类科研院所基本科研专项(No.2010R1039-3)~~

摘　　要：在弱酸性介质中,具有红区发射特性的强荧光化合物阳离子铝酞菁（ TTMAAlPc）在带磺酸基团的低浓度阴离子黏多糖（肝素,HP）的存在下,发生诱导聚集,导致酞菁荧光几乎完全猝灭。此聚集缔合物可作为溶菌酶的荧光底物,在溶菌酶的水解作用下,HP降解为小分子片段,破坏了TTMAAlPc的诱导聚集行为而使其释放,体系荧光显著恢复。据此现象,建立了测定溶菌酶的新方法。结合荧光光谱与荧光各向异性技术对反应机理进行了探讨。确定了最佳反应条件（醋酸缓冲体系,pH 4.0、反应温度70°C、反应时间30 min）,考察了共存物质的影响。在最佳条件下,方法的线性回归方程为y=-30.12121＋214.65772x, r=0.99871,线性范围为0.2~2 mg/L,检出限0.015 mg/L。本研究操作简便且有较好的选择性和灵敏度。本方法用于溶菌酶实际样品的测定,并与常规的比浊法进行了比较,结果符合良好。本研究将阳离子金属酞菁荧光探针用于酶分析,开拓了酞菁荧光探针的应用范围。We developed a novel method for the rapid determination of lysozyme using a new fluorogenic substrate that consists of a cationic aluminum phthalocyanine （ tetra （ trimethylammonio ） aluminum phthalocyanine, TTMAAlPc ） , and an anionic mucopolysaccharide （ heparin, HP ） . We found that fluorescence from the cationic aluminum phthalocyanine, a red-region fluorescence probe, was quenched significantly in acidic media in the presence of low concentrations of anionic mucopolysaccharide heparin （ HP） bearing anionic sulfonic acid groups, because of induced aggregation. The practically non-fluorescent substrate degraded into small molecular fragments upon the hydrolysis of lysozyme, and thus the phthalocyanine molecules aggregated in HP were released, resulting in significant fluorescence recovery in the reaction system. This phenomenon forms the foundation of the proposed method. The reaction mechanism was determined using fluorescence spectroscopy and fluorescence anisotropy techniques. Factors that affected the determination were investigated. Under optimal conditions, the linear range was 0. 2-2 mg/L, and the detection limit was 0. 015 mg/L. The developed method is easy to operate and has good selectivity and sensitivity. This method was used in the analysis of practical samples of lysozyme, and the results were in agreement with those determined by a conventional turbidimetric method.

关 键 词：溶菌酶 酞菁 荧光 测定 

分 类 号：O657.3[理学—分析化学] O629.8[理学—化学]