【作者】 吴波；

【导师】 曾钫；

【作者基本信息】 华南理工大学 ， 材料学， 2019， 硕士

【摘要】 不对称方酸染料在近红外区域(650-900 nm)具有优异的光学性质,但是由于它们的水溶性差,在水介质中表现出很强的聚集趋势,常常导致荧光淬灭,致使它们在生理环境下对酶的检测和成像受到限制。亮氨酸氨基肽酶(LAP)在多种病理过程中异常高水平地表达,因此实时准确地监测LAP活性和对LAP进行实时成像具有十分重要的意义。在本研究中,我们设计了一种乙二醇低聚体修饰的不对称方酸荧光探针USSQ-Leu,用来对内源性亮氨酸氨基肽酶的活性进行检测和成像。利用~1H NMR和HR-MS对USSQ-Leu的结构进行表征。通过溶解性测试考察了探针结构与溶解性之间的关系并证明了我们所设计的结构有效率地改善了探针的溶解性。通过测定探针的光谱性质和对酶的响应行为发现,USSQ-Leu具有良好的近红外吸收特性并在650 nm处有最大吸收峰,在生理条件下可与亮氨酸氨基肽酶发生反应发射出荧光并在710 nm有最大发射峰;探针对酶的响应具有时间依赖性和浓度依赖性,以及很好的检测专一性和检测灵敏度(0.61ng/mL)。利用HPLC和HR-MS验证了在LAP作用下USSQ-Leu上的L-亮氨酸基团脱落从而恢复荧光的检测机制。采用MTT法和流式细胞法研究并证实探针具有低细胞毒性和良好的细胞摄取能力。通过细胞荧光成像实验,考察并证实探针对内源性LAP的具有良好的检测能力;在异位移植瘤小鼠模型中研究了探针可以对小鼠体内的LAP的灵敏检测和成像。更多还原

【Abstract】 Unsymmetrical squaraine dyes have excellent optical properties in the near-infrared region(650-900nm).However,they exhibit a strong aggregation trend in the water medium,which often leads to fluorescence quenching due to their poor water solubility.As a result,their ability of detection and imaging of enzymes in the physiological environment are restricted.Leucine aminopeptidase(LAP)has abnormaliy high level of expression in various pathological processes.Thus,it is of great significance to accurately monitor the activity of LAP and conduct real-time imaging on LAP.In this study,we designed an ethylene glycol oligomer-modified asymmetric squaraine fluorescent probe to detect and image the activity of endogenous leucine aminopeptidase.The structure of USSQ-Leu has been confirmed by ~1H NMR and HR-MS.The relationship between probe structure and solubility was investigated by solubility test,and it was proved that the structure designed by us can effectively improved the solubility of the probe.By studying the spectral properties of the probe and the response behavior of the probe to the enzyme,we found that USSQ-Leu has good near infrared absorption properties and has maximum absorption peak at 650nm.The probe can react with leucine aminopeptidase to release fluorescence under physiological conditions and the largest emission peak is at 710nm.Besides,the response of the probe to the enzyme has time dependence,concentration dependence,good sensitivity and specificity(0.61ng/mL).HPLC and HR-MS were used to demonstrate the detection mechanism with L-leucine group being cleaved from USSQ-Leu to restore fluorescence under the reaction with LAP.MTT and flow cytometry showed that the probe had low toxicity and good cell uptake ability.Fluorescence imaging showed that the probe had good ability to detect endogenous LAP.In xenograft tumor mouse model,the probe was used for sensitive detection and imaging of LAP in mice.更多还原