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：药物发现是一个非常漫长且昂贵的过程。当今的毒品平均成本超过50亿美元，并且需要十多年的时间才能到达市场。当今大多数药物都使用高吞吐量筛查发现，其中数百万个试验化合物针对细胞进行了确定是否与疾病靶标相互作用。通常使用良好的筛选技术（例如荧光，化学发光和光学吸收）进行。尽管这些技术是高通量的，但它们缺乏在单细胞水平上审问井的能力，这比人口平均测量值更全面地描绘了药物相互作用。流式细胞仪是一种在大多数细胞生物学领域中使用的良好建立且广泛使用的技术，可通过测量来自高吞吐量（约10,000个细胞/秒）的流量中的单个细胞的光散射和荧光信息，从而提供多参数，细胞级信息。尽管这种细胞吞吐量很高，但与荧光板读取器相比，流式细胞仪的样品吞吐量相对较低，例如，由于串行样品处理方法通常由现代流式细胞仪进行。如果样品吞吐量可以提高到适当的水平，则可以使用流式细胞仪进行多参数药物筛查测定法，这将消除药物发现工作流程中某些进一步的下游测定法（例如，细胞毒性测定）。这种改善将通过更好地阐明复杂的药物相互作用，从而提高药物发现的效率 总体成本和新药的时间。我们在这里建议使用在Co-Pi实验室中开发的两种技术的组合来开发平行的流式细胞仪系统。使用射频标签发射（FIR）的荧光成像是一种高速光学技术，它使单个光电倍增管检测器能够使用辐射频率域多路复用来测量样品上的多个点的荧光或散射。我们将使用改良的火光系统来探测荧光和散射，这些细胞在多个平行的侧面聚焦流中流动，这是由惯性微流体平行流（IMP）微流体芯片产生的。 IMPS芯片使用惯性流场的形状创建有序的细胞流，而无需多个鞘毛的复杂性直接引导细胞，从而使高数值光圈光学器件简单地从所有流中检测到荧光。结合了这些技术，我们将开发一个能够从10个细胞样本中测量多色流式细胞术数据的光学和流体系统。样品吞吐量的数量级增加将改变药物发现中流式细胞术的实用性，最终使其在高吞吐量筛选（> 100,000井/天）中的广泛使用。我们将使用标准方案来表征系统（精度，线性，灵敏度），并执行基本的两色凋亡时间课程测定法，并将我们的结果中的结果与使用常规商业流式细胞仪获得的结果进行比较。