High throughput and accuracy single cell dielectric analysis and dielectric dyes

高通量和高精度的单细胞介电分析和介电染料

• 批准号: RGPIN-2020-05340
• 负责人: Thomson, Douglas
• 金额: $ 2.04万
• 依托单位: University of Manitoba
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2020
• 资助国家: 加拿大
• 起止时间: 2020-01-01 至 2021-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=717601
• 关键词: throughput; accuracy; single; cell; dielectric

Measuring the dielectric properties of cells has important applications in research, biomedical and industrial sectors. For example, in all its manufacturing the biopharma company Biogen maximizes drug productivity using bulk dielectric analysis to control feeding of mammalian cell cultures. Bulk dielectric analysis has limitations due to the inhomogeneous dielectric nature of cells and cell populations. Single cell dielectric analysis is far superior for determining the true dielectric properties of cells in suspension. My program will focus on: 1) Development of an optical transit dielectrophoresis (DEP) cytometer with a highly beneficial combination of throughput, accuracy, analysis time and frequency range. The optical transit DEP cytometer analyzes signals from transit light curves from cells, similar to planets passing in front of a star, to measure particle velocity and infer dielectric properties. Accuracy will be improved using differential signals approaches. Electrodes will be designed to allow analysis frequencies up to 500 MHz, to glean information on cell nuclei and organelles. The channel and detector geometry will be designed to dramatically increase throughput and reduce analysis time. Throughputs of greater than 1000 cells per and the ability to capture mutli-frequency spectra in 10's of seconds. 2) Development of unique dielectric dyes of cellular physiological status. A dielectric dye is a compound that produces a measurable dielectric contrast compared to untreated cells that is dependent on a particular cell property (e.g. biochemical process). For example, exposing a cell to Oligamycin inhibits mitochondrial ATP production. Lower ATP slows membrane ion pumps and causes a drop in cytoplasm conductivity that can be used to estimate the level of mitochondrial ATP production. I propose to test some of the most promising candidates using temporal fits to double shell dielectric models. This unique research will result in new modalities of single cell dielectric analysis. This research will provide a new single cell dielectric analysis tool with a combination of properties, including simple components, that make it a very promising candidate to become one of the preferred methods for single cell dielectric analysis. The dielectric dyes have the potential to expand the types of assays that can be carried out using single cell dielectric analysis. For example, having a simple assay for mitochondrial ATP would have application in biopharma control and for cancer detection where the lack of mitochondrial ATP is considered a hallmark of cancer. The optical transit cytometer single cell dielectric analysis will benefit the Canadian biopharma industries such as Emergent, Therapure, Sanofi by maximizing the productivity of the mammalian cell cultures. With the HQP and technology from this program microfluidics instrumentation companies such as Micralyne and Dalsa will be able to offer new products and markets.

测量细胞的介电特性在研究、生物医学和工业领域具有重要的应用。例如，生物制药公司Biogen在其所有生产中使用体介电分析来控制哺乳动物细胞培养物的进料，从而最大限度地提高药物生产率。由于细胞和细胞群的不均匀介电性质，体介电分析具有局限性。单细胞介电分析对于确定悬浮液中细胞的真实介电性质是优越得多的上级。我的计划将侧重于： 1)开发了一种光学透射介电电泳（DEP）细胞仪，该细胞仪具有通量、准确度、分析时间和频率范围的高度有益组合。光学透射DEP细胞仪分析来自细胞的透射光变曲线的信号，类似于行星经过星星前面，以测量粒子速度并推断介电特性。使用差分信号方法将提高精度。电极将被设计为允许分析频率高达500 MHz，以收集细胞核和细胞器的信息。通道和检测器的几何形状将被设计为显著增加通量并减少分析时间。每个细胞的数量超过1000个，并且能够在10秒内捕获多个频谱。 2)细胞生理状态的独特介电染料的开发。介电染料是与未处理的细胞相比产生可测量的介电对比度的化合物，其取决于特定的细胞性质（例如生化过程）。 例如，将细胞暴露于Oligamycin会抑制线粒体ATP的产生。较低的ATP减慢膜离子泵，并导致细胞质电导率下降，可用于估计线粒体ATP产生的水平。我建议测试一些最有前途的候选人使用时间适合双壳电介质模型。这项独特的研究将导致单细胞介电分析的新模式。 这项研究将提供一种新的单细胞介电分析工具，具有多种特性，包括简单的组件，使其成为非常有前途的候选者，成为单细胞介电分析的首选方法之一。介电染料具有扩展可以使用单细胞介电分析进行的测定类型的潜力。例如，对线粒体ATP进行简单的测定将应用于生物制药控制和癌症检测，其中缺乏线粒体ATP被认为是癌症的标志。光学透射式细胞仪单细胞介电分析将通过最大限度地提高哺乳动物细胞培养物的生产率而使加拿大生物制药行业（如Emergent、Therapure、Sanofi）受益。通过HQP和该计划的技术，Micralyne和Dalsa等微流体仪器公司将能够提供新的产品和市场。