Molecular Diagnostics using a Nanopore to Analyze Secretions from Single Cells

使用纳米孔分析单细胞分泌物的分子诊断

• 批准号: 10361196
• 负责人: Jun Li
• 金额: $ 40.46万
• 依托单位: UNIVERSITY OF NOTRE DAME
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-03-01 至 2025-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10361196
• 关键词: Address; BT 474; Binding Proteins; Biological Markers; Breast Cancer Cell; Cataloging; Catalogs; Categories; Cell Adhesion; Cell Communication; Cell Culture Techniques; Cell physiology; Cells; Charge; Collection; Complex; Data; Discriminant Analysis; Discrimination; Drug Targeting; Electrodes; Enzyme-Linked Immunosorbent Assay; Exhibits; Extracellular Fluid; Fibroblasts; Functional disorder; Gaussian model; Genes; Glass; Goals; Human; Immunoassay; Interstitial Collagenase; Knowledge; MCF7 cell; MDA MB 231; Mass Spectrum Analysis; Measurement; Measures; Mediating; Methods; Microfluidic Microchips; Microfluidics; Modeling; Molecular; Molecular Weight; Mus; PI3 gene; Peptides; Phenotype; Pilot Projects; Plasma; Positioning Attribute; Prognostic Factor; Property; Proteins; RANTES; Resistance; Serum; Signal Transduction; Speed; Statistical Study; Streptavidin; Surface; TIMP1 gene; Testing; Time; Tissue Engineering; Tissues; base; cell type; chemokine; drug discovery; electric field; embryonic stem cell; improved; induced pluripotent stem cell; integrated circuit; knowledge base; laser tweezer; malignant breast neoplasm; molecular diagnostics; nanofluidic; nanopore; notch protein; prevent; purge; response; single cell analysis; single molecule; specific biomarkers; technology research and development; time use; tool

Project Summary The proteins secreted from a cell constitute a complex subset of molecules referred to as the secretome. They are key factors mediating cell-cell communication. So, eavesdropping on the secretome informs molecular diagnostics, drug discovery and tissue engineering. The challenge then is to detect the proteins as they are secreted only in minute amounts, and diluted and/or contaminated in culture. Moreover, since tissue is heterogeneous, it is necessary to detect secretions from single cells, which is confounded by bulk-culture analysis. So, sensitivity is paramount. In response to the Focus Technology Research and Development solicitation, this proposal furnishes a plan to develop a tool that uses a nanopore to interrogate the secretome of single cells with extreme, single molecule sensitivity and high throughput. The blockades that develop in the ionic current through a nanopore, when a secreted, charged molecule is impelled through it by an electric field, measure the molecular volumes occluding the pore. A catalog of the blockades can be used to discriminate between different cellular phenotypes non-destructively, quickly, in real-time, and to interrogate the secretome for specific biomarkers. AIM #1: Single cell secretomics. As it reflects the different molecular constituencies comprising the secretome, the blockade current distributions should reveal distinctive aspects unique to the cell-type. To prove out this hypothesis, three categories of cells will be scrutinized: breast cancer cells; human induced pluripotent stem cells and their derivatives; and mouse embryonic stem cells and their derivatives. Single cells will be positioned with optical tweezers over a pore embedded in a microfluidic device and the resulting blockades will be classified by the Cramér’s distance, ?, and the expression of specific biomarkers will be tracked in real-time. AIM #2: Discriminant analysis of the single cell secretomes. To improve on ? for discriminating cell-types, a Gaussian-mixture-model (GMM) will be implemented that captures the profile of proteins in a secretome. This model will be fitted to the data with the number of components determined by a Bayesian Information Criterion and classifier will be developed to discriminate cell-types in real-time. The GMM will infer which proteins are up-/down-regulated in a cell, compared to the control, in an unbiased way. AIM #3: Micro/Nanofluidic integrated circuits for improved throughput. To boost throughput, arrays of eight nanopores will be fabricated and tested for concurrent single cell analysis. These arrays will be embedded in a microfluidic device incorporating integrated pneumatic valves to be used to convey cells to each pore, and each pore will be independently addressed by integrated electrodes for detecting blockades and producing di-electrophoretic forces for positioning the cell (instead of optical tweezers). To slash the down- time required to purge the microfluidic between measurements, fouling-resistant surfaces that relieve non- specific binding of protein and prevent cell adhesion to either glass and/or PDMS microfluidics will be tested.

项目摘要 从一个细胞分泌的蛋白质构成了称为分泌组的一个复杂的分子子集。 它们是介导细胞通信的关键因素。因此，窃取秘密的​​信息 分子诊断，药物发现和组织工程。那时的挑战是将蛋白质检测为 它们仅以微量的数量分泌，并在文化中被稀释和/或污染。而且，由于组织为 异质，有必要检测单个细胞的分泌 分析。因此，灵敏度至关重要。 为了响应重点技术研发的招标，该提案提供了 计划开发一种使用纳米孔来询问单个单元格的秘密对象的工具 分子灵敏度和高吞吐量。通过纳米孔在离子电流中形成的封锁， 当一个分泌的，带电的分子通过电场推动其促进，测量分子体积 阻塞孔。封锁的目录可用于区分不同的蜂窝 表型无损，快速，实时，并为特定的生物标志物询问分泌组。 目标＃1：单细胞秘密。因为它反映了完成的不同分子成分 分泌组，阻断电流分布应揭示细胞类型独特的独特方面。 从这个假设中，将仔细检查三类细胞：乳腺癌细胞；人类诱导的多能 干细胞及其衍生物；和小鼠胚胎干细胞及其衍生物。单细胞将是 在嵌入微流体设备中的孔上，用光镊子定位 由Cramér的距离进行分类，并且将实时跟踪特定生物标志物的表达。 目标＃2：单细胞秘密分析。改进？为了区分细胞类型， 将实施高斯混合模型（GMM），以捕获秘密组中蛋白质的剖面。 该模型将以贝叶斯信息确定的组件数量拟合到数据 标准和分类器将被开发以实时区分细胞类型。 GMM将推断哪个 与对照相比，蛋白质在细胞中以公正的方式向上/下调。 AIM＃3：微/纳米流体集成电路可改善吞吐量。为了增加吞吐量， 将制造八个纳米孔，并测试以进行并发单细胞分析。这些阵列将是 嵌入在编码集成气阀的微流体设备中，用于传达细胞到 每个孔，每个孔都会通过集成电极独立解决，以检测阻塞 并产生用于定位细胞（而不是光学镊子）的Di电动力量。削减下降 - 在测量，抗污染表面之间清除微流体所需的时间 将测试蛋白质的特异性结合，并防止细胞粘附于玻璃和/或PDMS微流体。