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.

项目概要 细胞分泌的蛋白质构成了一个复杂的分子子集，称为分泌组。 它们是介导细胞间通讯的关键因素，因此，窃听分泌蛋白组可以提供信息。 分子诊断、药物发现和组织工程面临的挑战是检测蛋白质。 它们仅分泌微量，并且在培养物中被稀释和/或污染。 异质性，有必要检测单细胞的分泌物，这会被大量培养所混淆 所以，敏感性至关重要。 为了响应焦点技术研究与开发征集，该提案提供了 计划开发一种工具，使用纳米孔以极端、单一的方式询问单细胞的分泌蛋白组 分子灵敏度和高通量通过纳米孔形成的离子电流， 当分泌的带电分子被电场推动通过时，测量分子体积 堵塞毛孔的目录可用于区分不同的细胞。 非破坏性、快速、实时地分析表型，并询问分泌蛋白组中的特定生物标志物。 目标#1：单细胞分泌组学，因为它反映了组成细胞的不同分子成分。 分泌组，封锁电流分布应揭示细胞类型独特的独特方面。 根据这一假设，将仔细检查三类细胞：乳腺癌细胞；人类诱导的多能细胞； 干细胞及其衍生物；以及小鼠胚胎干细胞及其衍生物。 用光镊将其定位在嵌入微流体装置中的孔上，由此产生的阻塞将 根据 Cramér 距离 ? 进行分类，并实时跟踪特定生物标志物的表达。 目标#2：单细胞分泌体的判别分析 为了改进 ? 判别细胞类型， 将实施高斯混合模型（GMM）来捕获分泌蛋白组中的蛋白质概况。 该模型将适合具有由贝叶斯信息确定的组件数量的数据 将开发标准和分类器来实时区分细胞类型，GMM 将推断出哪种细胞类型。 与对照相比，细胞中的蛋白质以无偏的方式上调/下调。 目标#3：用于提高吞吐量的微/纳米流体集成电路为了提高吞吐量，阵列。 将制造并测试八个纳米孔以用于并发单细胞分析。 嵌入微流体装置中，该装置包含集成气动阀，用于将细胞输送到 每个孔，每个孔将由集成电极独立寻址，用于检测堵塞 并产生介电泳力来定位细胞（而不是光镊）。 测量之间净化微流体所需的时间，防污表面可减轻非 将测试蛋白质的特异性结合并防止细胞粘附到玻璃和/或 PDMS 微流体。