NanoSMS: single molecule secretome analysis for non-destructive cellular fingerprinting

项目摘要/摘要 在接下来的5年里，NanoSMS将被开发成一种提供第一眼的技术 进入单细胞分泌体；具有单分子敏感性。这一新技术的发展 技术是基于单细胞封装技术与纳米孔的集成- 基于单分子传感。释放到细胞外的分子的集合 单细胞的环境，称为单细胞分泌组，提供了一个独特的一瞥 细胞生命和细胞间通讯的复杂性。这项技术的主要特点 将在项目期结束时开发的方法包括：(1)大规模并行方式 附着在玻璃片上并包含单个细胞的形状液滴，以及(2)基于纳米孔的 测量源自单个细胞的分泌分子的方法。一旦纳米孔和 开发了相关方法，我们的目标是回答围绕重要的四个关键问题 生物医学问题。首先，纳米孔将量化起源于 从不同疾病状态的患者身上分离出的人类浆细胞 包括自身免疫性疾病。第二，B细胞和T细胞将共同孵化和刺激 用单一的抗原来诱导免疫反应。开始产生抗体的时间延迟 以及影响免疫反应时间的因素将被研究。第三, 嗜酸性粒细胞与单个癌细胞(或癌细胞衍生的小泡)共同孵育的研究将被研究。 以提供对癌症早期识别机制的洞察。最后，单细胞分泌体 指纹将被用来获得区分干细胞谱系的能力 非破坏性的方式。干细胞的特性对于细胞治疗很重要(在细胞治疗中 作用机制是分泌分子)以及评估再生的细胞状态 医药。不用说，分泌体在这两种正常情况下都发挥着功能但却不明显的作用 以及病态的细胞状态。纳米颗粒的应用(一种基于纳米孔的方法) 分泌组分析)将为单细胞鉴定提供一种独特的新工具。

Project Summary/Abstract Over the next 5 years, NanoSMS will be developed into a technology which offers the first look into the single cell secretome; with single molecule sensitivity. The development of this new technology is based on the integration of single cell encapsulation technologies with nanopore- based single molecule sensing. The collection of molecules released into the extracellular environment by single cells, termed the single cell secretome, offers a unique glimpse into the complexities of cellular life and cell-to-cell communication. The key features of the technology which will be developed at the end of the project period include (1) a massively parallel way to form droplets adhered onto a glass slide and containing a single cell, and (2) a nanopore-based approach for measuring secreted molecule originating from a single cell. Once the nanopores and related methodologies are developed, we aim to answer four key questions surrounding important biomedical issues. First, the nanopore will quantify single cell antibody production originating from human plasma cells which have been isolated from patients in various disease states including auto-immune disorders. Second, B cells and T cells will be co-incubated and stimulated with a single antigen to induce an immune response. The time lag to start generating antibodies as well as the factors which influence the timing of the immune response will be studied. Third, eosinophils co-incubated with a single cancer cell (or cancer-derived vesicles) will be investigated to provide insight into early cancer recognition mechanisms. Lastly, single cell secretome fingerprinting will be used to access the ability to discriminate between stem cell lineages in a non-destructive manner. Stem cell characterization is important for cell therapy (in which the mechanism of action is secreted molecules) as well as assessing cell state for regenerative medicine. Needless to say, the secretome plays a functional and yet obscured role in both normal and diseased cellular states. The application of NanoSMS (a nanopore-based approach towards secretome analysis) will provide a unique and new tool for single cell characterization.