Droplet-based Spatially Encoded Live Cell Digital Extraction

基于液滴的空间编码活细胞数字提取

• 批准号: 10687620
• 负责人: JINA KO
• 金额: $ 136.88万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2026-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10687620
• 关键词: Alzheimer' s disease diagnosis; Bar Codes; Behavior; Biological Process; Cancer Biology; Cell membrane; Cells; Cellular biology; Detection; Development; Diagnostic; Dimensions; Early Diagnosis; Genotype; Heterogeneity; Immunology; Individual; Measures; Methods; Microfluidics; Molecular; Molecular Analysis; Monitor; Nanostructures; Nanotechnology; Neurosciences; Pathway interactions; Penetration; Phenotype; Population; Sampling; Stretching; Structure; Technology; Therapeutic; Time; biological systems; cytotoxicity; detection of nutrient; digital; individual variation; nanoscale; nanowire; new technology; novel therapeutics; response; single cell analysis; single molecule; tool

Abstract This proposal aims to develop a high throughput droplet-based digital subsampling tool that enables continuous extraction of intracellular molecules (>1000 cells/sec) from individual living cells and achieve digital single molecule detection. Technological advances in single cell analysis have resolved cellular heterogeneity and enabled discovery of rare cell subpopulations. They have opened up new opportunities to detect subtle molecular changes in the presence of variability in biological systems. Due to the uniqueness of individual cells in their composition, functionality, and structures, molecular analyses at the single-cell level are critical for understanding the complexity of biological processes and cellular responses to perturbations. To accurately profile cellular dynamics and behaviors, it is essential to longitudinally monitor cellular changes and responses over time. However, it has been challenging to acquire temporal molecular information from the same cell populations due to the need of keeping them alive during the course of observation while minimizing their perturbations. Recently, nanotechnology methods (e.g. nanowire, nanobiopsy, nanostraw) have been developed for longitudinal cell monitoring where nanoscale dimensions are used to penetrate cells and sample intracellular molecules while providing minimal cytotoxicity. They have successfully achieved longitudinal cell subsampling and analysis, but it has still been difficult to resolve inherent heterogeneity of individual cells and their contents due to low throughput and sensitivity. In these studies, cells were placed on a substrate that consists of different nanostructures and a scarce amount of molecules were extracted, limiting the ability to achieve high throughput sampling and comprehensive downstream analysis. To build a robust longitudinal intracellular extraction tool, there is a need to spatially barcode and profile individual cells at high throughput and measure scant molecules with ultra-high sensitivity to maintain minimal perturbations during extraction. Here, we propose a live cell digital subsampling technology that will combine a cell membrane perforator and digital detection using droplet microfluidics to achieve i) live cell subsampling via hydrodynamic stretching of individual living cells, ii) ultrasensitive digital profiling of individual molecules sampled from single cells, and iii) monitoring of phenotypic and genotypic nutrient sensing pathway associated molecules for the early diagnosis of Alzheimer's disease and the development of new therapeutics. We will advance this platform to better understand cell biology and apply it to diverse fields including neuroscience, immunology, and cancer biology.

摘要 该建议旨在开发一种基于液滴的高通量数字亚采样工具，以实现 从单个活细胞中连续提取细胞内分子(&gt；1000个/秒)并实现数字化 单分子检测。单细胞分析技术的进步解决了细胞的异质性 使稀有细胞亚群的发现成为可能。他们开辟了新的机会来发现细微之处 生物系统中存在可变性时的分子变化。由于单个细胞的独特性 在它们的组成、功能和结构上，单细胞水平的分子分析对于 了解生物过程的复杂性和细胞对扰动的反应。准确地说 Profile细胞动力学和行为，必须纵向监控细胞的变化和响应 随着时间的推移。然而，从同一细胞中获取临时分子信息一直是一项挑战 由于需要在观察过程中保持它们的生命，同时尽量减少它们的 微扰。 最近，纳米技术方法(例如纳米线、纳米生物体、纳米稻草)已经被开发用于 纵向细胞监测，使用纳米尺度穿透细胞并采样细胞内 分子，同时提供最小的细胞毒性。他们成功地实现了纵向细胞亚采样 和分析，但仍然很难解决单个细胞及其内容物的固有异质性 由于吞吐量和灵敏度较低。在这些研究中，细胞被放置在由以下物质组成的基质上 不同的纳米结构和稀有的分子被提取出来，限制了实现高 吞吐量采样和全面的下游分析。要建立一个强大的纵向细胞内 提取工具，需要以高通量和测量对单个细胞进行空间条形码和fi编码 稀有的分子具有超高的灵敏度，在萃取过程中保持最小的扰动。 在这里，我们提出了一种活细胞数字亚采样技术，它将细胞膜穿孔器和 使用液滴微fl指南实现数字检测，以实现i)通过流体动力拉伸进行活细胞亚采样 单个活细胞，ii)从单个细胞采样的单个分子的超灵敏数字fi，以及iii) 监测表型和基因型营养传感通路相关分子用于早期诊断 阿尔茨海默氏症和新疗法的发展。我们将把这个平台推进得更好 了解细胞生物学，并将其应用于不同的fi领域，包括神经科学、免疫学和癌症生物学。