【作者】 李霄；

【导师】 邬建敏；

【作者基本信息】 浙江大学 ， 化学， 2015， 硕士

【摘要】 血清中的低分子量物质中含有大量可用于疾病诊断的生物标志物。这些生物标志物主要为肽类标志物。目前对人类血清中肽类生物标志物的分析鉴定已经成为医学研究的热门领域。然而由于血清中肽段的含量很低,且有大量共存的高丰度大蛋白,因此很难通过质谱技术直接检测血清获得高质量的肽指纹图谱。因而,去除高分子量蛋白的干扰并提高样品分子在基质辅助激光解吸离子化飞行时间质谱(MALDI-TOF-MS)中的解吸离子化效率,成为当前肽组学研究中急需的技术。多孔硅具有大的比表面积和良好的尺寸排阻效应,可以有效地富集血清中的低分子量物质。此外,当其与贵金属纳米颗粒复合在一起时,构成了贵金属-半导体接触界面,该复合材料不仅具有局域表面等离子共振效应,同时在光照时能提高二者之间的电荷转移能力,有利于MALDI的解吸离子化。基于贵金属-多孔硅半导体材料独特的物理、化学性质,本文初步探索了其对MALDI离子化效率的提高效果以及其在血清低分子物质分析中的应用。(1)综述了肽组学研究的重要意义,以及生物质谱技术在肽组学研究中的应用。介绍了介孔材料在蛋白质富集方面的研究现状以及影响MALDI离子化效率的因素。综述了贵金属纳米颗粒以及半导体复合材料的光电性质及其应用。(2)本文基于多孔硅的尺寸排阻效应及其半导体特性,结合了贵金属纳米颗粒独特的物理和化学性质,研发了一种新型的MALDI样品靶——金纳米颗粒修饰的多孔硅芯片。通过胰岛素模型样本探索了该芯片对检测信号的增强效果并用辣根过氧化物酶(HRP)的胰酶酶解液和牛血清白蛋白(BSA)的混合样本对人体的血液环境进行了模拟,验证了该芯片材料对大分子量蛋白的排阻效应以及对低丰度小分子物质的检测效果。此外,还将此技术应用于结直肠癌生物标志物的临床分析检测中。结果表明,点在此芯片上的血清样品可以通过MALDI-TOF-MS技术直接检测,并获得较高保真度的肽指纹图谱,联用统计学软件聚类分析得到正常人和癌症病人的区分度为80%,体现了该芯片材料在肽组学研究中的潜在应用。(3)本文基于贵金属-半导体材料的独特性质,制备了钯纳米颗粒修饰的多孔硅芯片,研究了该样品靶对血清中肽段检测性能的提高效果,并系统探索了其对MALDI检测信号的增强机理。研究发现多孔硅具有很好的尺寸排除作用,可有效地抑制大蛋白的干扰,对小分子物质进行富集,并且多孔硅基底提高了表面粗糙度,有利于基质的分散并形成均匀的结晶,提高了MALDI的检测性能和重复性；钯纳米颗粒的局域表面等离子共振效应可以提高其对激光能量的吸收能力,并传递给样品,促进样品分子的解吸离子化；钯纳米颗粒与多孔硅半导体之间产生电荷转移现象,钯纳米颗粒中的电子传递给了半导体,并与多孔硅中的空穴复合,使得大量的正电荷积累在钯纳米颗粒的表面,对正离子检测模式下低分子量物质的解吸具有促进作用。更多还原

【Abstract】 Low molecular weight peptides (LMWPs) in serum are potential biomarkers for the diagnosis of diseases. The identification of peptide biomarkers in human serum has become an area of high interest in medical research. However, the direct analysis of peptides in serum samples using mass spectrometry is challenging and it is difficult to obtain high quality peptide fingerprint profile due to the low concentration of LMWPs and the significant signal suppression caused by the high concentration of high molecular weight proteins (HMWPs). Therefore, the methods for improving laser desorption/ionization efficiency of analytes in matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) and excluding the interference of HMWPs are urgently needed. Porous silicon (PSi) has high surface area and can effectively enrich the LMWPs in serum sample due to its size exclusion effect. When the PSi is combined with the noble metal nanoparticles, the new metal-semiconductor hybrid structure has the localized surface plasmon resonance (LSPR) effect and the charge transfer capability has been improved. Both of these characteristics are benefit to the ionization process in MALDI detection. In this thesis, the ionization efficiency of MALDI based on the special properties of noble metal-PSi semiconductor material has been explored and its application in serum LMWPs analysis has also been studied.(1) It is summarized the significance of the peptidomics research, and the application of bio-mass spectrometry in peptidomics research. The mesoporous materials’ application in peptide enrichment and the influence factors of ionization efficiency in MALDI have also been introduced. In addition, the photoelectric properties and applications of the noble metal nanoparticles-semiconductor hybrid material are also reviewed.(2) This thesis reports a novel sample plate of MALDI, which is obtained by integration of porous silicon with Au nanoparticles. The new chip combines the size exclusion effect and semiconductor property of the PSi with the special physical and chemical properties of noble metal nanoparticles. The enhancement effect of signal intensity by this chip is proved with insulin as the model sample. To confirm whether the hybrid nanostructure can selectively capture the small peptides and exclude the HMWPs, a model sample consisting of horseradish peroxidase digest and an excess of bovine serum albumin has been tested. Besides, its application to capture low molecular weight peptides from serum sample of colorectal cancer patients and normal subjects in clinical testing are studied. The results show that the new plate could be used for the direct detection of serum sample and obtaining serum peptide fingerprints with high fidelity, and successful discrimination of colorectal cancer patients based on peptide fingerprints is demonstrated. Its accuracy is as high as80%and the chip shows potential application in peptidomics research.(3) The palladium nanoparticles (PdNPs) modified porous silicon (Pd-PSi) chip has been prepared, and its improvement of serum peptides detection has been explored. The mechanisms of MALDI enhancement in this chip have also been studied. With the help of size exclusion effect of PSi, the interference of HMWPs can be efficiently reduced and small molecules in serum would be enriched, leading to higher detection sensitivity of peptides in the low molecular weight region. Besides, the rough surface of PSi is benefit to form uniform crystallization, which can improve the repeatability of the MALDI detection. Due to the localized surface plasmon resonance (LSPR) effect of palladium nanoparticles, the Pd-PSi chip also has a plasmon resonance in the UV region and can improve the laser energy absorption capability, which contributes to the desorption of analytes. The charge transfer property between palladium nanoparticles and porous silicon semiconductor would help the accumulation of positive charge on the surface of the palladium nanoparticles, which can also promote the desorption and ionization process of small analytes under positive liner detection mode.更多还原