A Frequency-multiplexed flow cytometer for high throughput screening and drug discovery

用于高通量筛选和药物发现的频率复用流式细胞仪

• 批准号: 8970595
• 负责人: Dino Di Carlo
• 金额: $ 19.25万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-08-01 至 2017-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8970595
• 关键词: Accounting; Address; Agreement; Air; Apoptosis; Area; Biological Assay; Calibration; Camptothecin; Cell Communication; Cells; Cellular biology; Collection; Color; Complex; Computer software; Coupled; Cytometry; Data; Detection; Disease; Flow Cytometry; Fluorescein-5-isothiocyanate; Fluorescence; Frequencies; Goals; Individual; Information Systems; Jurkat Cells; Laboratories; Lateral; Libraries; Light; Liquid substance; Literature; Marketing; Measurement; Measures; Microfluidics; Molecular Bank; Monitor; Nature; Noise; Optics; Patients; Performance; Pharmaceutical Preparations; Population; Positioning Attribute; Preclinical Drug Evaluation; Process; Propidium Diiodide; Protocols documentation; Pump; Reader; Running; Sampling; Scheme; Shapes; Side; Speed; Staging; Staurosporine; Stream; Syringes; System; Techniques; Technology; Testing; Time; Tube; United States National Institutes of Health; annexin A5; base; cost; cytotoxicity; design; detector; drug development; drug discovery; fluorescence imaging; high throughput screening; instrument; optical emission; particle; photomultiplier; photonics; public health relevance; radio frequency; radiofrequency; repository; response; screening; small molecule; success; time use

 DESCRIPTION: Drug discovery is an extremely lengthy and expensive process. On average, drugs today cost more than $5 billion to develop, and take more than a decade to reach the market. Most drugs today are discovered using high throughput screening, in which millions of trial compounds are assayed against cells to determine if they interact with a disease target. This is typically performed using well-level screening techniques, such as fluorescence, chemiluminescence, and optical absorbance. While these techniques are high throughput, they lack the ability to interrogate wells at the single-cell level, which provides a much more comprehensive picture of the drug-cell interaction than population-averaged measurements. Flow cytometry is a well established and widely used technique used in most areas of cell biology that provides multi-parameter, cell-level information by measuring optical scatter and fluorescence information from individual cells in flow at a high throughput (~10,000 cells/second). While this cellular throughput is high, the sample throughput of flow cytometers is relatively low, when compared to fluorescence plate readers, for example, due to the serial sample handling approach typically employed by modern flow cytometers. If the sample throughput could be increased to appropriate levels, it would enable multi-parameter drug screening assays using flow cytometry, which would obviate certain further downstream assays in the drug discovery workflow (e.g. cytotoxicity assays). This improvement would increase the efficiency of drug discovery by better elucidating the complex drug-cell interactions, reducing the overall cost and time-to-market of new drugs. We propose here to develop a parallel flow cytometer system using a combination of two technologies developed in the laboratories of the co-PI's. Fluorescence Imaging using Radiofrequency-tagged Emission (FIRE) is a high-speed optical technique that enables a single photomultiplier tube detector to measure fluorescence or scatter from multiple points on a sample using radiofrequency-domain multiplexing. We will use a modified FIRE optical system to probe fluorescence and scatter from cells flowing in multiple parallel intertially focused streams, created by an Inertial Microfluidic Parallel Stream (IMPS) microfluidic chip. IMPS chips create ordered streams of cells using inertial flow field shaping without the complexity of multiple sheath fluids to direct cells, allowing high numerical aperture optics to detect fluorescence from all streams simultaneously. Combining these techniques, we will develop an optical and fluidic system capable of measuring multi-color flow cytometry data from 10 samples of cells at the same time. This order of magnitude increase in the sample throughput will transform the utility of flow cytometry in drug discovery, ultimately enabling its widespread use in high throughput screening (>100,000 wells/day). We will characterize the system (precision, linearity, sensitivity) using standard protocols, and perform a basic two-color apoptosis time course assay and compare our results in this assay to those obtained using a conventional commercial flow cytometer.

 描述：当今的药物花费了超过50亿美元的开发，并且在市场上花费了更多的汉，而是针对细胞来确定它们是否与疾病靶标相互作用。单细胞水平的Nterrogate井，该井提供了流式相互作用平均相互作用的更加稳定图片。通过测量流量高的单个细胞的散射和荧光的水平信息。串行样品处理由现代流式细胞仪施加到适当的水平。减少 总的成本和新药的成本和时间。高速光学的光电层次能够使用射频式多域多路复用来测量荧光将鞘液塑造为导向细胞L流同时结合技术，我们将开发一种能够测量来自10个LS样本的多色流式细胞仪数据的光学和流体系统。牙齿将在药物发现中转化流式细胞仪的效用，最终使其在高含量筛选中广泛使用（> 100,000井/天）。 Tirse课程的课程将我们的刺激与使用转换通信流式赛仪获得的方法进行比较。