【正文】 雙步激光質(zhì)譜法對動物組織中藥物分子的原位檢測 I 雙步激光質(zhì)譜法對動物組織中藥物分子的原位檢測 專業(yè)名稱： 光學(xué) 申請者姓名：王紅磊 導(dǎo)師姓名： 胡勇軍 摘 要 激光光電離作為一種獨特的電離技術(shù)，已被廣泛應(yīng)用于質(zhì)譜領(lǐng)域?；趩问す獾幕|(zhì)輔助激光解析 (MALDI)質(zhì)譜分析方法，已成為質(zhì)譜分析生物大分子的標(biāo)準(zhǔn)方法之一。本文介紹的是另一種新的激光質(zhì)譜分析方法：與 MALDI相比，雙步激光質(zhì)譜法 (L2MS)采用兩束激光分別完成氣化 /解析和電離的任務(wù)。在雙步激光系統(tǒng)中，解析階段和電離階段在空間和時間上是相互獨立的，這樣可以方便的對每一 階段進(jìn)行單獨優(yōu)化。 通過對實驗儀器的改進(jìn)，本論文詳細(xì)探討了應(yīng)用雙步激光質(zhì)譜法對生物組織切片中藥物分子的原位探測，并優(yōu)化相關(guān)的實驗條件。本文 主要包括以下三個部分： 1）綜述了幾種常用的生物質(zhì)譜方法及其相關(guān)的 “軟 ”電離方法，重點描述了雙步激光質(zhì)譜法的獨特優(yōu)勢和在分析領(lǐng)域的最新進(jìn)展。 2）實驗儀器系統(tǒng)：在原有超聲分子束光電離質(zhì)譜裝置的基礎(chǔ)上，自主搭建一套基于雙步激光質(zhì)譜方法的實驗裝置。在雙步激光質(zhì)譜系統(tǒng)中較為關(guān)鍵的接口上，采用了進(jìn)樣桿模式。對于固體樣品的進(jìn)樣，還自主設(shè)計安裝了二維平移裝置。整套裝置包括一套可與質(zhì)譜真 空系統(tǒng)兼容的一維傳動桿組件，并與二維平移臺配套。組件的末端安裝有樣品承載平臺。整套裝置通過手動閘板閥和 O型密封圈使真空系統(tǒng)與外界隔絕，可以在保持真空的狀態(tài)下更換樣品。 雙步激光質(zhì)譜法對動物組織中藥物分子的原位檢測 II 3）應(yīng)用雙步激光質(zhì)譜方法對生物組織中的藥物分子進(jìn)行了探測研究。實驗中選用一種常見的光動力治療藥物亞甲基藍(lán)（ MB）作為模型 分子 ，所選用的癌細(xì)胞為人體乳腺癌（ Hela）細(xì)胞。實驗結(jié)果表明，應(yīng)用 1064 nm的紅外激光對純腫瘤組織樣品進(jìn)行解析，同時用 118 nm真空紫外激光對其進(jìn)行單光子軟電離，獲得的質(zhì)譜圖背景簡單，樣品中組織的質(zhì)譜信號主要來自于組 織中的氨基酸或者短肽的碎片，質(zhì)量數(shù)主要在 100 Da以下。而預(yù)先在活體中注射有藥物分子 MB腫瘤組織的質(zhì)譜圖中，藥物分子 MB的質(zhì)譜主峰為 285 Da，出現(xiàn)在質(zhì)譜圖中質(zhì)量數(shù)較高的區(qū)域。結(jié)果表明：該質(zhì)譜峰避開了純組織背景信號較復(fù)雜的區(qū)域，提高了檢測信號的信噪比。在本實驗過程中，對兩束光之間的延遲進(jìn)行了優(yōu)化，結(jié)果發(fā)現(xiàn)最佳延遲大約在 1835 181。s。本檢測方法最大的特色是原位檢測生物組織中的藥物分子。經(jīng)初步推測，其最低檢測濃度可達(dá) 106 mol/L以下。 關(guān)鍵詞 ：飛行時間質(zhì)譜；單光子電離；生物組織；亞甲基藍(lán)；原位 檢測 雙步激光質(zhì)譜法對動物組織中藥物分子的原位檢測 III IN SITU DETECTION OF DRUG MOLECULES IN THE ANIMAL TISSUE BY TWOSTEP LASER MASS SPECTROSCOPY Major: Optics Name: Honglei Wang Supervisor: Yongjun Hu ABSTRACT As 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 bee the standard method in the biological mass spectrometry. In this dissertation, a new mass spectrometry is introduced, ., Twostep 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 homemade TOFMS and optimize the L2MS experimental 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 雙步激光質(zhì)譜法對動物組織中藥物分子的原位檢測 IV in analyzing field are reviewed. (2). Experiment instrument system: we build a twostep laser mass spectroscopic system on a homemade 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 patible to the TOF mass spectrometer. An XY motion stage was designed to be patible to the assembly. A sample holder is equipped in the end of the transferred pole. The vacuum is separated by a custom made gatevalve and two Orings. 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 mon 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 1835181。s. 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 106mol/L. KEY WORDS: TOFMS。 SPI。 Tissue。 Methylene Blue (MB)； In Situ 雙步激光質(zhì)譜法對動物組織中藥物分子的原位檢測 V 目 錄 第一章 緒 論 ........................................................................................................ 1 引 言 ........................................................................................................... 1 幾種常見的生物質(zhì)譜方法 .............................................................. 1 軟電離技術(shù)的發(fā)展及應(yīng)用 .............................................................. 4 電噴霧電離 ........................................................................................ 4 基質(zhì)輔助激光解吸電離 ............................................................... 5 雙步激光解析 /激光電離 .............................................................. 7 參考文獻(xiàn) .................................................................................................................. 9 第二章 實驗系統(tǒng)的改進(jìn) .............................................................................. 14 質(zhì)譜系統(tǒng) .................................................................................................. 14 束源室 .............................................................................................. 14 主腔體 .............................................................................................. 15 雙步激光的進(jìn)樣系統(tǒng) ...................................................................... 17 光電離離子源 ...............................................