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题名: 低压光电离质谱对挥发性生物标志物的检测
作者: 李震1
学位类别: 博士
答辩日期: 2017-04
授予单位: 中国科学院大学
授予地点: 北京
导师: 束继年
关键词: VOC,生物标志物,低压光电离质谱 ; VOC, biomarkers, LPPI-MS
其他题名: Detection of Volatile Biomarkers by Low-Pressure Photoionization Mass Spectrometry
学位专业: 环境科学
中文摘要: 呼吸组学是生物科技的一个新兴领域。依靠分析人类呼吸中挥发性有机物 (VOC)的组成模式可以诊断一系列疾病。呼吸诊断疾病的方法有着安全无创、 简单便捷、患者接受度高的特点,有望用于癌症筛查与辅助诊断。为癌症早发现、 早治疗、提高治愈率、降低死亡率提供新的检查手段。然而这些 VOCs通常是 ppbv或 pptv量级,且大部分疾病相关 VOCs的代谢来源还没有被阐述。所以发 展呼出 VOCs高灵敏实时检测技术有着重要意义。本论文介绍了对自主研发的低 压光电离质谱(LPPI-MS)的性能研究,并以此质谱仪搭建分析诊断平台,对癌 细胞代谢 VOCs、人体呼出 VOCs、细胞与药物作用产生 VOCs进行高灵敏在线 检测。主要研究成果和创新性如下: (1)仪器性能研究表明,该低压光电离质谱仪取得了超越其他竞争技术的 检测限。此仪器的核心是一个自制的低压光电离源,射频放电氪灯以每秒 5×1014 的光通量放出 10.0 eV (~80%)和 10.6 eV (~20%)的光子能,这些光子在一个带挡 板的电离器中对分析物进行“软”电离。从电离器推出的离子被离子透镜的静电 场聚焦,随后被引入 V-型反射式飞行时间质量分析器。通过调整电压、气压等参 数,飞行时间质量分析器与低压光电离源得到了更好的协调匹配,不仅保持了较 高分辨率,更减少了离子的额外损失。此外,该低压光电离质谱有着 95%-105% 的准确度,3%-30%的精确度,以及 12小时内标准差仅为 0.025 ppbv的稳定度 (检测 1ppbv BTE)。低压光电离质谱对几种苯系物的灵敏度达到了 4-7 counts/pptv,检测限约为 0.5-0.8 pptv(S/N=2)。空气中的水分是最主要的干扰因 素,且会导致背景值的显著增高。在相对湿度 90%时,仪器对 BTE的检测限依 然能够达到 1-2pptv。说明这台高灵敏且可靠的低压光电离质谱仪可以投入到有 着较高工作湿度的呼出气体检测中。 (2)利用低压光电离质谱仪顶空实时检测了四种癌细胞和一种非癌细胞 (HeLa, A549, HepG-2, MGC-803和 293T)呼出的 VOCs组成模式。通过比较 20个 VOC标志物峰,可以显著区分癌细胞和正常细胞。继而使用低压光电离质 谱分别对乳腺癌、肺癌、肝癌的三个可能的 VOC标志物(即:二甲基三硫醚、 甲硫醚、2-丁酮)在上述不同种类癌细胞中的代谢情况进行了评估。低压光电离 质谱对这三种 VOCs的检测限分别达到了 3.1, 3.9和 23.2 pptv; 2-丁酮作为肺癌 VOC标志物之一,在肺癌细胞 A549中的释放最多;甲硫醚作为肝功能障碍的 VOC标志物之一,在肝癌细胞 HepG-2的检测中最多。 (3)应用 CH2Cl2对极性 VOCs的质子化增强效应,对呼吸中的四种极性肺 癌 VOC标志物(即:正丙醇、正戊醛、丙酮、乙酸丁酯)进行了检测。在烟雾 箱模拟环境下,低压光电离质谱对上述四种 VOCs的信号响应可以分别提升了 53, 18, 16,和 43倍。CH2Cl2的加入不仅可以增强母离子峰,更可以对碎片峰起 到抑制或降低的作用。在 20%的相对湿度以及 582 ppmv CH2Cl2增强下,低压光 电离质谱对正丙醇、正戊醛、丙酮、乙酸丁酯的灵敏度达到了 116 – 452 counts/ppbv。 然而过多的水分(50%,90%相对湿度)会作为干扰因素抑制质子转移反应和光 电离效率。在对人体呼气和标记呼气样本的实验中,正丙醇、正戊醛、丙酮、 乙酸丁酯的信号有效的提升了约 8倍。 (4)二甲基亚砜不仅在生物实验中有着广泛地应用,而且在临床医学上也 被证明有着重要价值,但是二甲基亚砜被生物代谢产生的臭味气体(二甲基硫醚) 却限制了其作为药物的广泛应用。该部分研究利用低压光电离质谱实时检测了人 体细胞(HeLa, 293T)在不同浓度紫杉醇和二甲基亚砜条件下,鉴别了痕量级别 VOC的代谢情况。实验说明,DMSO与低浓度(5-10 nM)紫杉醇联用可在维持 较高细胞活性的前提下显著降低 DMS的产生;然而更高浓度紫杉醇(> 50nM) 的胁迫则会诱发广泛的细胞凋亡,伴随蛋氨酸的分解,更多的二甲基硫醚将被产 生。 以上低压光电离质谱的初步研究工作,说明这台仪器有着高灵敏、稳定可靠 的特点,并可以应用于人体和细胞呼吸检测。凭借该仪器的优越性能,有望通过 实时检测发现更多的疾病相关 VOC标志物。 关键词:VOC,生物标志物,低压光电离质谱
英文摘要: Breathomics is an emerging field of biotechnology. A series of diseases could be diagnosed by analysing the composition models of volatile organic compound (VOC) in human breath. Breath-based diagnostic method is characterized by noninvasive, convenient, as well as high degree of acceptance among patients. Therefore, it is anticipated to be used for the cancer screening and supplementary diagnosis, which would provide a new method for the early detection and treatment of cancers, improving the cure rate, and reducing the mortality. However, these VOCs generally exist at ppbv or pptv (part per billion by volume, part per trillion by volume) levels, and the metabolic origin of most disease-related VOCs have not been elucidated. Therefore, it is significant to develop the ultrasensitive real-time detecting technology for exhaled VOCs. In this paper, we introduce the performance study of our newly developed low pressure photoionization mass spectrometry (LPPI-MS). Based on the LPPI-MS, an analytic and diagnostic platform was constructed, then employed in the ultrasensitive real-time detection of cancer cell emitted VOCs, human exhaled VOCs, and cell emitted VOCs under drug treatment. The main research contents and innovations are listed as the following: (1) In the performance study of instrument, the LPPI-MS obtained the limit of detection beyond other competing technologies. The core part of the instrument is a laboratory-assembled krypton lamp, which emits approximately 5×10 14 photon with energies of 10.0 eV (~80%) and 10.6 eV (~20%). Analyte chould be “soft”ionized by the photons in a photoionizer with an optical baffle. The ions expelled from the photoionizer are focused by the electrostatic fields of the ion lenses, followed by injection into the V-shaped TOF mass analyzer. By adjust the parameters of voltage and pressure, mass analyzer and LPPI-source could be coordinated matched. Not only maitain a high resolution, but also reduce the additional loss of ions. In addition, the measurement accuracy is 95% - 105%, and the corresponding precision ranges from 3% to 30%. The stability of LPPI-MS for a 1 ppbv BTE mixture is less than 0.025 (>12 h). The detection sensitivity toward BTE is 4-7 counts/pptv and the limit of detection (LOD) is 0.5-0.8 pptv (S/N=2). Water in ambient air is the most significant interfering factor, leading to the increased background, and inferior LODs of 1-2 pptv for BTE under an RH of ~90% is observed. Experimental results indicated that LPPI-MS is reliable for the detection of exhaled breath with highly moisture. (2) LPPI-MS was applied to the heaspace detection of four cancerous and 1 non- malignant cell lines (i.e. HeLa, A549, HepG-2, MGC-803, and 293T). By comparing the mass peaks of 20 VOC biomarkers, cancer cell lines and non-malignant one could be significantly distinguished. Then, the metabolism of three VOC biomarker candidates (dimethyl trisulfide, dimethyl sulfide, and 2-butanone) for breast, liver, and lung cancers were evaluated in different cancer cell lines. The limits of detection (LODs) for DMTS, DMS, and 2-butanone were 3.1, 3.9, and 23.2 pptv, respectively. Moreover, relatively high concentrations of 2-butanone and DMS were respectively observed in the A549 lung cancer cell line and the HepG2 liver cancer cell line. (3) According to the protonation enhancement effect of dichloromethane (CH2Cl2) for polar VOCs, four lung cancer-related polar VOCs (n-propanol, n-pentanal, acetone, and butyl acetate) in breath were measured. Compared with conventional LPPI-MS, CH2Cl2 doping-assisted LPPI-MS specifically boosted the peak intensities of the four VOCs respectively 53, 18, 16, and 43 times in chamber. The signal intensities of their daughter ions were inhibited or reduced. At relative humidity (RH) of 20%, the sensitivities of n-propanol, n-pentanal, acetone, and butyl acetate detection ranged from 116 to 452 counts/ppbv. Water at a high concentration (RH levels of 50% and 90%.) competitively inhibits the proton transfer reaction assisted by CH2Cl2 and reduces the photoionization efficiency of polar VOCs. The signal intensities for four polar VOCs were effectively enhanced by ~8 times in volunteers’ breath and spiked breath. (4) Methylsulfinylmethane (dimethyl sulfoxide; DMSO) is not only widely used in bioresearch, but also has been proved to be of great value in clinical treatment. However, the principal side effect is an odd odor (dimethyl sulfide; DMS), which put off many drug companies and hindered its clinical application. Here, we report the characterization of trace-level volatile organic compounds (VOCs) produced following DMSO and paclitaxel (PTX) treatment of cultured human cells using LPPI-MS. Our experimental results demonstrate that by co-administering 5 to 10 nM PTX with DMSO, it is possible to moderate the production of DMS considerably. However, at higher doses of PTX (> 50nM), increased apoptosis was observed that likely contributed to higher DMS production by cells. As illustrated in the abovementioned study on LPPI-MS, this instrument is characterized as ultrasensitive, stable and reliable, and can be used for the detection of exhaled breath and cell lines. Rely on the excellent performance of LPPI-MS, it is expected to be able to discover more disease-related VOC biomarkers via real-time detection.
内容类型: 学位论文
URI标识: http://ir.rcees.ac.cn/handle/311016/38657
Appears in Collections:大气环境研究室_学位论文

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作者单位: 1.中国科学院生态环境研究中心

Recommended Citation:
李震. 低压光电离质谱对挥发性生物标志物的检测[D]. 北京. 中国科学院大学. 2017.
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