双步激光质谱法对动物组织中药物分子的原位检测毕业论文.doc

双步激光质谱法对动物组织中药物分子的原位检测 双步激光质谱法对动物组织中药物分子的原位检测摘 要激光光电离作为一种独特的电离技术，已被广泛应用于质谱领域。基于单束激光的基质辅助激光解析(MALDI)质谱分析方法，已成为质谱分析生物大分子的标准方法之一。本文介绍的是另一种新的激光质谱分析方法：与MALDI相比，双步激光质谱法(L2MS)采用两束激光分别完成气化/解析和电离的任务。在双步激光系统中，解析阶段和电离阶段在空间和时间上是相互独立的，这样可以方便的对每一阶段进行单独优化。通过对实验仪器的改进，本论文详细探讨了应用双步激光质谱法对生物组织切片中药物分子的原位探测，并优化相关的实验条件。本文主要包括以下三个部分：1）综述了几种常用的生物质谱方法及其相关的“软”电离方法，重点描述了双步激光质谱法的独特优势和在分析领域的最新进展。2）实验仪器系统：在原有超声分子束光电离质谱装置的基础上，自主搭建一套基于双步激光质谱方法的实验装置。在双步激光质谱系统中较为关键的接口上，采用了进样杆模式。对于固体样品的进样，还自主设计安装了二维平移装置。整套装置包括一套可与质谱真空系统兼容的一维传动杆组件，并与二维平移台配套。组件的末端安装有样品承载平台。整套装置通过手动闸板阀和O型密封圈使真空系统与外界隔绝，可以在保持真空的状态下更换样品。3）应用双步激光质谱方法对生物组织中的药物分子进行了探测研究。实验中选用一种常见的光动力治疗药物亚甲基蓝（MB）作为模型分子，所选用的癌细胞为人体乳腺癌（Hela）细胞。实验结果表明，应用1064 nm的红外激光对纯肿瘤组织样品进行解析，同时用118 nm真空紫外激光对其进行单光子软电离，获得的质谱图背景简单，样品中组织的质谱信号主要来自于组织中的氨基酸或者短肽的碎片，质量数主要在100 Da以下。而预先在活体中注射有药物分子MB肿瘤组织的质谱图中，药物分子MB的质谱主峰为285 Da，出现在质谱图中质量数较高的区域。结果表明：该质谱峰避开了纯组织背景信号较复杂的区域，提高了检测信号的信噪比。在本实验过程中，对两束光之间的延迟进行了优化，结果发现最佳延迟大约在18-35 µs。本检测方法最大的特色是原位检测生物组织中的药物分子。经初步推测，其最低检测浓度可达10-6 mol/L以下。关键词：飞行时间质谱；单光子电离；生物组织；亚甲基蓝；原位检测IN SITU DETECTION OF DRUG MOLECULES IN THE ANIMAL TISSUE BY TWO-STEP LASER MASS SPECTROSCOPYABSTRACTAs a particular ionization technology, laser photoionization has been widely used in the field of the mass spectroscopy. MALDI (Matrix assisted laser desorption ionization), where only one laser was used, has become the standard method in the biological mass spectrometry. In this dissertation, a new mass spectrometry is introduced, i.e., Two-step laser desorption/laser ionization mass spectrometry (L2MS). Comparing this method with MALDI, no matrix is needed to add on the substrate and form a good cocrystallization with the sample. The signal can be optimized by changing the power and wavelength of two lasers independently. In present dissertation, we modify the home-made TOF-MS and optimize the L2MSexperimental condition. We also analyze drug molecules in situ in animal tissue by the introduced method. Detail contents for this dissertation are summarized as follows:(1). A couple of biological mass spectroscopy and their soft ionization methods are introduced. The unique advantages of L2MS are described and recent applications in analyzing field are reviewed.(2). Experiment instrument system: we build a two-step laser mass spectroscopic system on a home-made time of flight mass spectrometer which equips a supersonic jet source. The interface for L2MS method is an assembly with a linear transferred pole, which is compatible to the TOF mass spectrometer. An X-Y motion stage was designed to be compatible to the assembly. A sample holder is equipped in the end of the transferred pole. The vacuum is separated by a custom made gate-valve and two O-rings. So we can expediently change the sample while keeping the vacuum.(3). We analyze drug molecule in biological tissue by the use of L2MS. We choose one of the common photodynamic therapy drugs methylene blue (MB) and human Hela cells as the model systems. In present experiment, the tumor tissue without dosing MB is desorbed by an IR laser and soft ionized by 118 nm VUV radiation. The spectrum displays a variety of the fragments of amino acids and small other materials below m/z 100. For the tumor tissue with MB, the signal of MB m/z 285 is far away from the background signal of the tumor tissue. So it can reduce the background interference and improve the sensitivity of detection. The appropriate delay time of two lasers is optimized and is found to fall in the range of 18-35µs. For L2MS, the detection of in situ drugs in tissue is one of the most distinguishing features and the limit of detection (LOD) for this method is estimated to be lower than 10-6mol/L.KEY WORDS: TOF-MS; SPI; Tissue; Methylene Blue (MB)；In Situ目 录第一章 绪 论11.1 引 言11.2 几种常见的生物质谱方法11.3 软电离技术的发展及应用41.3.1电喷雾电离41.3.2 基质辅助激光解吸电离51.3.3 双步激光解析/激光电离7参考文献9第二章 实验系统的改进142.1 质谱系统142.1.1 束源室142.1.2 主腔体152.2 双步激光的进样系统172.3 光电离离子源182.3.1 1064nm激光光源182.3.2 118nm真空紫外激光光源182.4 时序控制系统19参考文献21第三章 生物组织中光动力药物MB的双步激光实验223.1 引 言223.2 双步激光实验和结果分析233.2.1 实验材料及实验方法233.2.2 实验条件的验证253.3 实验结果及讨论263.3.1 纯亚甲基蓝样品分析263.3.2 对两束光之间延迟的优化273.3.3 纯生物组织的质谱探测283.3.4 预先给药（MB）生物组织的质谱探测293.3.5 最低检测浓度的初步探究313.4 结 论32参考文献33第四章 全文总结及展望37致 谢39硕士期间发表论文和待发表论文情况4039第一章 绪 论1.1 引 言质谱仪是一种利用电场将不同大小荷质比的粒子区分开的一种检测仪器。世界上第一台质谱仪是于1910年由英国物理学家J. J. Thomson 研制成功，当时的质谱仪只是一种没有进行聚焦的抛物线质谱装置。随着离子光学理论的发展和完善，通过对离子运动轨迹的理论研究，发现适当的电场和磁场组合具有方向和速度聚焦的特性，于是质谱仪的质量分辨能力有了很大程度的提