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.

抽象的 该提案旨在开发一种高通量的基于液滴的数字二次采样工具，该工具能够 从单个活细胞中连续提取细胞内分子（>1000个细胞/秒）并实现数字化 单分子检测。单细胞分析的技术进步解决了细胞异质性 并使得稀有细胞亚群的发现成为可能。他们开辟了检测微妙现象的新机会 生物系统中存在变异性时分子会发生变化。由于个体细胞的独特性 在其组成、功能和结构方面，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。为了准确地 为了分析细胞动力学和行为，纵向监测细胞变化和反应至关重要 随着时间的推移。然而，从同一细胞获取时间分子信息一直具有挑战性 种群，因为需要在观察过程中保持它们的生存，同时尽量减少它们的数量 扰动。 最近，纳米技术方法（例如纳米线、纳米活检、纳米吸管）已被开发用于 纵向细胞监测，其中纳米级尺寸用于穿透细胞并采样细胞内 分子，同时提供最小的细胞毒性。他们成功实现了纵向细胞二次采样 和分析，但仍然很难解决单个细胞及其内容物固有的异质性 由于通量和灵敏度较低。在这些研究中，细胞被放置在由以下物质组成的基质上： 不同的纳米结构和少量的分子被提取出来，限制了实现高水平的能力 吞吐量采样和全面的下游分析。构建强大的纵向细胞内 提取工具，需要以高通量对单个细胞进行空间条形码和分析并测量 具有超高灵敏度的少量分子，可在提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二次采样技术，该技术将细胞膜穿孔器和 使用液滴微流体进行数字检测，以实现 i) 通过流体动力学拉伸进行活细胞二次采样 单个活细胞，ii) 对从单个细胞采样的单个分子进行超灵敏数字分析，以及 iii) 监测表型和基因型营养传感途径相关分子以进行早期诊断 阿尔茨海默病的研究和新疗法的开发。我们将把这个平台建设得更好 了解细胞生物学并将其应用于神经科学、免疫学和癌症生物学等不同领域。