Molecular Diagnostics using a Nanopore to Analyze Secretions from Single Cells

项目摘要 从细胞分泌的蛋白质构成称为分泌组的分子的复杂子集。 它们是介导细胞间通讯的关键因素。所以，窃听秘密基因组 分子诊断、药物发现和组织工程。接下来的挑战是检测这些蛋白质， 它们仅以微量分泌，并且在培养物中被稀释和/或污染。此外，由于组织 由于细胞的异质性，有必要检测单个细胞的分泌物，这会被大量培养所混淆 分析.所以，敏感是最重要的。 为响应分众科技研发征集，本提案征集了 计划开发一种工具，使用纳米孔来询问单细胞的分泌组， 分子灵敏度和高通量。在离子电流通过纳米孔时产生的阻塞， 当一个分泌的带电分子被电场推动通过它时，测量分子体积 堵塞孔隙。一个封锁的目录可以用来区分不同的细胞 非破坏性地、快速地、实时地检测表型，并询问分泌组的特异性生物标志物。 目的#1：单细胞分泌组学。因为它反映了不同的分子组成， 在分泌蛋白质组中，阻断电流分布应该揭示细胞类型特有的独特方面。证明 根据这一假设，将仔细研究三类细胞：乳腺癌细胞;人类诱导多能细胞; 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 用光学镊子定位在嵌入微流体装置中的孔上， 按克拉美距离分类，并实时跟踪特定生物标志物的表达。 目的#2：单细胞分泌组的判别分析。为了改进？为了区分细胞类型， 将实施高斯混合模型（GMM），其捕获分泌组中蛋白质的分布。 该模型将拟合到具有由贝叶斯信息确定的组分数量的数据。 将开发标准和分类器以实时区分细胞类型。GMM将推断出 与对照相比，蛋白质在细胞中以无偏的方式上调/下调。 目标#3：微/纳米流体集成电路，用于提高吞吐量。为了提高吞吐量， 将制造八个纳米孔并测试用于同时进行的单细胞分析。这些数组将 包埋在微流体装置中，该微流体装置包括集成的气动阀，用于将细胞输送到 每个孔和每个孔将通过用于检测阻塞的集成电极独立地寻址 并产生用于定位细胞的介电泳力（代替光镊）。把下面的- 测量之间清洗微流体所需的时间，减轻非污染的抗污染表面， 将测试蛋白质特异性结合和防止细胞粘附到玻璃和/或PDMS微流体。

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.