微纳颗粒在细胞中的摄入、分布差异与细胞状态改变密切相关，因此，研究其与单细胞的相互作用具有重要意义。由于微纳颗粒和细胞内生物分子的理化性质差异很大，实现两者的同时检测对分析方法提出了很高的要求。飞行时间二次离子质谱（TOF-SIMS）是一种具有高空间分辨能力的质谱成像技术，不仅能实现微纳颗粒在单细胞中的原位鉴定和成像，还能获得细胞代谢物的指纹图谱信息，反映细胞状态改变。本论文旨在发展用于微纳颗粒与单细胞相互作用的TOF-SIMS分析方法，主要内容如下：1．建立了基于TOF-SIMS的单细胞中磁性纳米颗粒原位成像分析方法，用于单细胞水平铁磁纳米颗粒摄入、分布与细胞代谢物变化的同时分析。研究了铁磁纳米颗粒导致的细胞膜和胞内代谢物的变化，分析了铁磁纳米颗粒摄入量与细胞代谢状态的关系，结果表明两者具有非线性相关性，当铁磁纳米颗粒摄入到达一定量时，胞内代谢状态会发生快速改变。2．建立了基于TOF-SIMS的单细胞中小尺寸微塑料的原位鉴定与成像分析方法，对十余种常见的微塑料建立指纹图谱库和特征离子库，实现多种微塑料的快速、准确鉴定和成像分析。该方法用于单细胞生物草履虫中多种微塑料的原位检测，避免了微塑料的提取纯化等前处理过程，揭示了微塑料在生物体内的聚集状态。发展了基于像素点指纹图谱的数据处理算法，实现草履虫亚细胞结构和微塑料的可视化分析和共定位成像。3．建立了基于TOF-SIMS的单颗粒微塑料吸附物的原位成像分析方法，探究了吸附卤代污染物的微塑料对细胞代谢的影响。通过与冷冻切片技术结合实现了单颗粒中污染物的表面吸附和内部分布的原位成像，探究了吸附物在不同种类单颗粒微塑料上的空间分布。细胞实验表明吸附全氟化合物的微塑料与细胞相互作用会导致细胞脂肪酸代谢异常。4．针对TOF-SIMS质谱难以鉴定代谢物种类和无法准确定量的问题，我们研究了基于电喷雾质谱的单细胞代谢物定量分析方法。通过微阵列与液滴微萃取质谱结合，实现了nL-pL级内标溶液的精准引入，对单细胞中67 种代谢物进行了指认，实现了单细胞中代谢物的准确定量。我们将该定量方法用于单细胞中药物代谢的异质性研究。
The uptake and distribution of micro-nano particles in cells is closely related to the changes of cell state. Hence，the study of the interaction between micro-nano particles and single cells is of great significance. Due to the huge difference in physical and chemical properties of biomolecules and micro-nano particles, it is challenging for the simultaneous detection of them. Time-of-flight secondary ion mass spectrometry (TOF-SIMS) is a mass spectrometry imaging technology with high spatial resolution, which can not only allow in-situ identification and imaging of micro-nano particles in single cells, but also acquire fingerprints of cellular metabolites. Herein, we develop TOF-SIMS based analytical methods for studying the interaction between micro-nano particles and single cells. The main contents of the present thesis include:1. We developed a method for in-situ mapping of magnetic nanoparticles in single cells by TOF-SIMS and applied it to study the interaction between the uptake and distribution of magnetic nanoparticles and cellular metabolites. The changes of component of cell membrane and intracellular metabolites caused by magnetic nanoparticles was explored. We analyzed the relationship between the intakes of magnetic nanoparticles and the changes of metabolites in single cells and the results showed that they had a non-liner correlation that once the intakes reached a certain amount, some metabolites would change rapidly.2. We developed a method for in situ identification and mapping of microlpastics in single cells by TOF-SIMS. Eleven different common microplastics were identified by their characteristic ions and fingerprints. In situ mass imaging of multiple microplastics in Paramecium was achieved, avoiding complex pretreatments such as extraction and purification of microplastics and revealing the origin state of microplastics in Paramecium. And the cellular structure and microplastics were visualized and colocalized through the home-made algorithm that interpretating data and grouping pixels from the mass spectrometry images. 3. We developed a method for the detection of adsorbed pollutants in single microplastic particle by TOF-SIMS, exploring the combined effects of microplastics and halogenated pollutants on cell metabolites. The in-situ mass imaging of adsorbed pollutant in single particles was realized through combination with cryosection technology and the heterogeneous spatial distributions of adsorbents on different types of single microplastic particle were investigated. The result indicated that the microplastics that adsorbed perfluorinated compound could jointly cause abnormal fatty acid metabolism.4. We developed a quantitative method for metabolites in single cell based on electrospray mass spectrometry in view of the difficulties in identification and accurate quantification of metabolites by TOF-SIMS. Through microwell based nanoliter droplet microextraction, the precise introduction of nL-pL internal standard solution was realized. Sixty-seven metabolites in single cells were identified and the accurate quantification of metabolite in single cells was realized. We applied this method to study drug metabolism at single-cell level, revealing the heterogeneity of drug metabolism.
