【作者】 张源；

【导师】 胡斌；

【作者基本信息】 武汉大学 ， 分析化学， 2015， 博士

【摘要】 癌症是世界范围内主要的致死疾病之一。外周血液中循环肿瘤细胞(CTCs)的数目是重要的标志物,检测CTCs可实现癌症的早期诊断。近年来,基于元素标记ICP-MS检测的新方法逐渐在细胞分析中发挥作用,该方法通过元素标签与细胞标志物特异性识别而标记目标细胞,ICP-MS测定标记元素,间接得到细胞信息。在该方法中,选择合适的元素标签和标记方法是关键因素。纳米粒子生物相容性好,在ICP-MS检测时可供选择的原子数目多,检测灵敏度高,检出限低,是理想的元素标签。尽管ICP-MS检测具有动态范围宽、检出限低、灵敏度高以及受基体干扰小等优点,但由于细胞基质复杂且胞内金属元素含量低,使得单细胞内金属元素的定量分析十分困难。时间分辨ICP-MS技术(SP-ICP-MS)是单颗粒检测的良好方式,灵敏度高,可分析单细胞,完成质谱细胞计数和定量分析。实际血液样品中CTCs数量极少,且复杂的生物基质也会影响基于元素标记ICP-MS检测方法的准确性。因此在ICP-MS检测前,寻求合适的细胞前处理方法,对目标细胞进行富集,实现细胞与基质分离,是细胞分析的一个重要内容。此外,与传统的荧光分析不同,ICP-MS作为细胞分析新技术,可实现细胞计数,但是不能对细胞进行分选；另外ICP-MS检测方式是有损分析,无法给出直观可视化成像结果。因此设计合成多功能标记探针,将多种检测方式相结合,对获得更准确和全面的癌症诊断信息具有重要意义。本论文的研究目的是：建立磁免疫分析策略与纳米粒子标记ICP-MS检测联用的分析新方法用于癌细胞分析；设计合成新型的多功能探针,将荧光细胞成像、稀土上转换发光材料标记细胞成像以及ICP-MS检测三种方法联用分析癌细胞；建立时间分辨ICP-MS检测方法用于单细胞中金属元素的分析。本论文的主要研究内容包括：(1)建立了硫化铅纳米粒子PbS NPs标记ICP-MS检测的免疫新方法用于HepG2细胞的分析。羧基功能化的PbS NPs经活化后共价偶联anti-EpCAM抗体作为标记探针,特异性识别HepG2细胞表面过表达的EpCAM抗原。以不表达EpCAM抗原的HeLa细胞作为对照考察了方法的特异性。考察了影响免疫实验中的各项影响因素,包括探针与细胞的孵育时间、探针的用量和洗脱条件等。在最优的实验条件下,评价了方法的分析性能。方法对HepG2细胞的检出限LOD为282个,线性范围为800-40000个HepG2细胞,相对标准偏差RSD为5.0%(采用3000个HepG2细胞平行测定7次)。(2)探索了磁免疫分析策略用于癌细胞的分离和富集,建立了细胞磁免疫样品前处理技术与纳米金标记ICP-MS检测相结合的免疫分析新方法用于Jurkat T细胞的测定。由于Jurkat T细胞表面特异性表达CD2和CD3抗原,以修饰anti-CD3抗体的免疫磁珠作为捕获探针,从混合细胞中特异性捕获了Jurkat T细胞。在外加磁场作用下,捕获细胞的磁珠与样品基质可实现快速分离。以纳米金Au NPs偶联anti-CD2抗体作为标记探针,标记在Jurkat T细胞表面,经酸解吸后引入ICP-MS进行后续测定,通过检测Au的信号可得到细胞信息。对实验中的影响因素进行了详细的考察,包括磁珠捕获细胞的时间和温度、纳米金标记探针与细胞作用的时间和用量以及洗脱条件等。以不表达CD2和CD3抗原的两种细胞作为阴性对照,考察了方法的特异性和交叉反应性。在最优的实验条件下,考察了方法的分析性能,方法的检出限为86个Jurkat T细胞,线性范围为300-30000个Jurkat T细胞,相对标准偏差RSD为5.2%(采用3000个Jurkat T细胞平行测定7次)。(3)设计合成了集元素标记、近红外标记和荧光标记于一体的三功能标记探针,将多种检测方式相结合用于CTCs的分析,以得到更为全面、准确的检测结果。在实验中,将羧基改性的稀土上转换发光材料UCNPs与荧光染料Cy3标记的二抗化学偶联作为标记探针识别目标细胞,实现了近红外激发下细胞成像、荧光染料标记细胞成像和ICP-MS检测三种方式同时对HepG2细胞进行分析。采用水热法合成了UCNPs材料,并使用多种表征手段对其偶联抗体后合成的新型三功能探针进行了表征,表明探针的制备成功。将所制备的三功能探针标记HepG2细胞,在561 nm激发波长下可观察Cy3荧光细胞成像,在980 nm激发波长下可观察UCNPs标记细胞成像,ICP-MS对UCNPs材料中掺杂的稀土元素进行检测。以Vero细胞为阴性对照组,考察了方法的特异性。(4)建立了时间分辨ICP-DRC-MS检测方法用于Jurkat T单细胞中Mg元素的测定。时间分辨ICP-MS技术可实现单颗粒检测,具体操作是将单分散的JurkatT细胞悬浮液直接引入DS-5微流全耗雾化器中,经ICP-MS测定后得到24Mg信号-时间谱图,其中24Mg跳峰个数与细胞个数正相关,可实现质谱的细胞计数；跳峰强度代表单细胞中元素Mg的含量。以92% He作为碰撞气体和8% H2作为反应气体,采用碰撞/反应池DRC技术可有效消除测定24Mg时存在的多原子离子干扰,对实验中影响ICP-DRC-MS检测的因素进行了优化。实验结果表明跳峰数目与细胞浓度成正比。在固定细胞浓度下,对跳峰强度进行了统计学分析,跳峰个数与强度符合对数-常数分布。在最优的实验条件下,检测了20个单细胞中Mg元素含量为0.09到0.29fgcell-1不等,体现出细胞的异质性。这20个细胞中Mg的平均含量为0.18 fg cell-1。对大量细胞(细胞浓度为1.7 ×106 cell mL-*1)分别经酸消解和细胞破膜处理,检测了Jurkat T细胞中Mg元素含量的平均值分别为0.94 fg cell-1和0.48 fg cell-1。对比三种检测方式,实验结果在一个数量级上,验证了所建立方法的准确性。(5)设计并构建了微流控芯片以获得单行排列的细胞流的方法。设计了对称的十字交叉型芯片,利用芯片上的流体力学聚焦原理,控制聚焦流的宽度与细胞的尺寸在一个数量级上,以获得单细胞流。通过在芯片两侧引入鞘流,进口处引入细胞样品流,鞘流液体对样品流进行挤压获得聚焦流,优化了鞘流液体种类、微流控芯片通道宽度以及鞘流和细胞流的流速比值,对聚焦流的方向和宽度进行了控制。在优化的实验条件下,选择了十二烷醇作为鞘流液体,芯片通道宽度为100μm,可满足聚焦流宽度与Jurkat T细胞(10μm)大小在一个数量级上。更多还原

【Abstract】 Cancer is one of the most serious diseases that cause death worldwide. The presence of CTCs in the peripheral blood has been regarded as an important cancer biomarker. The detection of CTCs especially at low concentration has a drastic effect on the accurate early diagnosis of cancer. Recently, element-tagged ICP-MS-based immunoassay techniques have gained much attention for cancer cell detection. ICP-MS is one of the most sensitive techniques providing a wide dynamic range, low detection limits, low matrix effects and element specificity. Recently, ICP-MS technique has become one of the most versatile and sensitive complementary tools in the emerging field of life-science related research. To take full advantages of element-tagged ICP-MS-based immunoassay techniques for cell analysis, considerable attention has been paid to the labeling tags. Inorganic nanoparticles are employed to improve the tag signal intensity of cancer cells detection, high sensitivity can be easily obtained because of large quantities of detectable atoms in each nanoparticle tag. It is well known that individual cells differ from each other in many aspects even under identical environmental condition. In consideration of average data can mask the underlying information, except for the bulk analysis, it is necessary to study individual cell for better understanding of cellular heterogeneity. Time-resolved ICP-MS technique emerges as a powerful tool for single cell analysis. The spike signal obtained for each element is corresponding to the presence of a cell and the spike signal intensity can be used for the determination of the metal quality of the cell.Disseminated cells circulate in the blood at extremely low concentrations, making the detection of low-frequency cancer cells difficult. To achieve acceptable detection sensitivity, appropriate sample preparation prior to ICP-MS detection is required for the specific identification, isolation and enrichment of target cells from the heterogeneous suspension. Currently, immunomagnetic separation is routinely used for target cells separation from a blood sample.Unlike conventional fluorescence-based cytometry, mass cytometry is a destructive technique. It can replace fluorescence cytometry in multiplex cell analysis, but can not perform cell sorting.The design and synthesis of novel multifunctional probes, in which cellular biomarkers label with fluorescent agents and element tags, are greatly expected to perform a more comprehensive multiplex assay of cancer cells, achieving more accurate early diagnosis of cancers.The aim of this dissertation is to develop immunomagnetic separation technique combined with element-tagged ICP-MS-based immunoassay method for cancer cells analysis using nanoparticles as a label probe; to design a trifunctional probe (IgG-Cy3-conjugated UCNPs) for two ways of visualization of CTCs and combination with ICP-MS determination; to explore time-resolved ICP-MS technique for single cell analysis. The major contents of this dissertation are described as follows:(1) A new method for detecting tumor cells at low levels based on coupling of element-tagged immunoassay with ICP-MS detection has been developed In this method, PbS NPs are functionalized to bond covalently with the anti-EpCAM antibody, which is specific to the highly expressed EpCAM antigen on the surface of HepG2 cells, but not on HeLa cells. The interference of HeLa cells on detection of HepG2 cells was investigated. A series of conditions influencing the immunoassay were carefully optimized, including the incubation time, the concentration of the labeling probe and the elution conditions. Under the optimized conditions, the linear range of 800-40000 and the limit of detection of 282 HepG2 cells were obtained, and the relative standard deviation for seven replicate determinations of HepG2 cells was 5.0%(3000 HepG2 cells).(2) An efficient, specific and sensitive immunoassay protocol for detection of tumor cells by using inductively coupled plasma mass spectrometry (ICP-MS) with two probes has been developed. Magnetic nanobeads modified with anti-CD3 were used as capture probes for efficient and fast magnetic separation of Jurkat T cells from a mixture of cells, and gold nanoparticles (Au NPs) conjugated with anti-CD2 were used as detection probes for ICP-MS measurement.The interference of A549 and 97L cells (CD2/CD3-negative cells) on the capturing and detection of Jurkat T cells was investigated. The conditions for this immunoassay were carefully optimized, including the incubation time and temperature, the concentration of the labeling probe, and the elution conditions. Under the optimized conditions, the linear range of 300-30000 and the limit of detection of 86 Jurkat T cells were obtained, and the relative standard deviation for seven replicate detections of Jurkat T cells was 5.2% (3000 Jurkat T cells).(3) A new method of UCNPs-Ab2-Cy3 nanoprobe for two ways of visualization of tumors and combined with ICP-MS for the detection of cancer cells has been developed. This concept of the all-in-one probe facilitates the selective labeling of targeted biomarkers and thus a subsequent multi technology for cancer cells using various detection techniques, offering more comprehensive information over a single assay. In this work, EpC AM-positive HepG2 cells were used as a model and EpCAM-negative Vero cells as the control group. The tirfunctional probe consisted of Ab2 unit to catch cancer cells via targeting anti-EpCAM which has reacted with EpCAM antigen overexpressed on cellular surface, Cy3 moiety for fluorescence imaging, UCNPs for upconversion luminescence imaging under near-infrared irradiation and subsequent ICP-MS determination of Yb in UCNPs(NaYF4:Yb3+, Er3+). Under the optimized conditions, Cy3 confocal images excited at 561 nm and upconversion luminescence images excited at 980 nm were observed, and ICP-MS determination of Yb was obtained.(4) A new method of time-resolved ICP-DRC-MS for the determination of Mg contents in single Jurkat T cell for the first time has been developed. Time-resolved ICP-MS technique for single cell analysis can be considered as a special case of single-particle analysis in which intact biological cells are introduced directly into ICP for measurement. Cells are directly sprayed as single-cell aerosols into torch to produce a plume of metal atoms through the way of vaporizing and ionization of its atomic constituents, and then quantified by mass detector. The spike signal obtained is corresponding to the presence of a cell, and the spike signal intensity is the metal quality in a single cell. DRC was used to reduce spectral interferences to improve the analytical performance.92% He as collision gas and 8% H2 as reaction gas were selected to reduce spectral interferences. The factors were optimized in detail. The distribution of Mg spike intensity of Jurkat T cell was approximately log-normal. The number of spikes was proportional to the cell number density. Under the optimized conditions, Mg quality in 20 individual cells is ranging from 0.09 to 0.29 fg cell-1. Thus, the average Mg content for these 20 cells was 0.18 fg cell-1. Because of the cellular heterogeneity, Mg contents in the individual Jurkat T cell were not equal even though they were the same cell population. The average Mg content was 0.94 fg cell-1 and 0.48 fg cell-1 respectively determined by acid digestion and cell rupture of Jurkat T cell suspensions (number density was 1.7 × 106 cell mL-1).(5) A new method to provide a single cell flow has been developed by using a symmetric cross-over microfluidic chip based on the symmetric hydrodynamic focusing effect. This is achieved by controlling the width of focused stream to the same order magnitude of the size of cell on a cross-over microchannel device. The hydrodynamic focusing experiments were accomplished on a cross-over microchannel device using sheath flows from the two side channels and cell suspensions for the sample flow from the inlet channel. Optimization of the experimental conditions, including sheath flow reagent, the microchannel width in a symmetric cross-over chip and the velocity ratio of sheath flow to sample flow to make sure the width of the focused stream reduced to the same order of magnitude as the cell size. Under the optimized conditions, a single cell flow could be obtained.更多还原