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二次采样工具， 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 并使稀有细胞亚群的发现成为可能它们为探测细微的 生物系统中存在变异性时的分子变化。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。准确 为了了解细胞动力学和行为，必须纵向监测细胞变化和反应 随着时间然而，从同一细胞中获取时间分子信息一直具有挑战性 由于需要在观察过程中保持它们的存活，同时最大限度地减少它们的数量， 扰动 最近，已经开发了纳米技术方法（例如，纳米线、纳米活体解剖、纳米管）用于 纵向细胞监测，其中纳米尺度用于穿透细胞并在细胞内取样 分子，同时提供最小的细胞毒性。他们成功实现了纵向细胞亚采样 但它仍然很难解决单个细胞及其内容物的固有异质性 这是由于低吞吐量和灵敏度。在这些研究中，将细胞置于由以下组成的基底上： 不同的纳米结构和稀缺量的分子被提取，限制了实现高性能的能力。 吞吐量采样和全面的下游分析。为了建立一个强大的纵向细胞内 作为一种提取工具，需要以高通量对单个细胞进行空间条形码化和轮廓化，并测量 具有超高灵敏度的少量分子，以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，该技术将联合收割机结合细胞膜穿孔器和 使用液滴微流控的数字检测，以实现i）通过液滴微流控的流体动力学拉伸的活细胞二次取样， 单个活细胞，ii）从单个细胞取样的单个分子的超灵敏数字分析，以及iii） 用于早期诊断的表型和基因型营养传感途径相关分子的监测 阿尔茨海默氏症和新疗法的发展。我们将把这个平台发展得更好。 了解细胞生物学，并将其应用于不同领域，包括神经科学，免疫学和癌症生物学。