Molecular Diagnostics using a Nanopore to Analyze Secretions from Single Cells

项目摘要 细胞分泌的蛋白质构成一个复杂的分子子集，称为分泌体。 它们是调节细胞间通讯的关键因素。所以，窃听秘密告诉我们 分子诊断学、药物发现和组织工程。接下来的挑战是将蛋白质检测为 它们仅以微量分泌，并在培养中稀释和/或被污染。此外，由于组织是 异质性，需要检测单个细胞的分泌物，这一点被批量培养搞混了。 分析。因此，敏感性是至高无上的。 作为对Focus Technology Research and Development征集的回应，本提案提供了一个 计划开发一种工具，使用纳米孔来询问具有极端、单一 分子灵敏度和高通量。在通过纳米孔的离子流中形成的阻塞， 当一个分泌的带电分子在电场的推动下穿过它时，测量分子体积。 堵塞毛孔。封锁的目录可以用来区分不同的细胞 非破坏性、快速、实时的表型，并询问分泌组的特定生物标记物。 目标1：单细胞分泌组学。因为它反映了不同的分子组成 分泌体，阻断电流的分布应该显示细胞类型特有的独特特征。为了证明 在这一假设之外，将仔细研究三类细胞：乳腺癌细胞；人类诱导的多能性 干细胞及其衍生物；以及小鼠胚胎干细胞及其衍生物。单个单元格将是 用光学镊子定位在嵌入微流控设备中的孔上，由此产生的阻塞将 根据Cramér距离？进行分类，将实时跟踪特定生物标记物的表达。 目的#2：单细胞分泌体的判别分析。来改进吗？为了区分细胞类型， 将实现一个高斯混合模型(GMM)来捕获分泌组中蛋白质的轮廓。 该模型将与由贝叶斯信息确定的分量数量的数据进行拟合 将开发标准和分类器来实时区分细胞类型。GMM会推断出 与对照组相比，细胞中的蛋白质以一种公正的方式上调/下调。 目标#3：用于提高产量的微/纳米流体集成电路。为提高吞吐量， 将制造8个纳米孔并进行测试，以便同时进行单细胞分析。这些阵列将是 嵌入集成气动阀门的微流控装置中，用于将细胞输送到 每个孔和每个孔将由集成电极独立寻址，以检测堵塞 并产生用于定位细胞的双电泳力(而不是光学镊子)。为了削减开支- 在两次测量之间清洗微流体所需的时间，防污表面释放非 将测试蛋白质的特定结合和防止细胞与玻璃和/或PDMS微流体的粘连。

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.