High volume high throughput affordable parallel acoustic flow cytometry

高容量、高通量、经济实惠的并行声学流式细胞仪

• 批准号: 8575382
• 负责人: STEVEN W GRAVES
• 金额: $ 17.68万
• 依托单位: UNIVERSITY OF NEW MEXICO
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-01 至 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8575382
• 关键词: Acoustics; Address; Animal Model; Area; Bacteria; Binding; Biological Assay; Biological Models; Biomedical Research; Blood; Blood Cells; Blood specimen; Caliber; Calibration; Cells; Cellular Spheroids; Clinical; Clinical Research; Detection; Development; Diagnostic; Effectiveness; Ensure; Flow Cytometry; Generations; Goals; Image; Lasers; Light; Liquid substance; Microspheres; Modeling; Optics; Particle Size; Pathogen detection; Population; Population Analysis; Preparation; Process; Property; Resolution; Sampling; Solutions; Source; Speed; Stream; System; Techniques; Technology; Translating; Ursidae Family; Whole Blood; clinical application; cost; cost effective; data acquisition; design; detector; fluid flow; high throughput analysis; high throughput screening; instrument; meter; novel strategies; particle; pressure; prototype; public health relevance; small molecule libraries; success; tumor

DESCRIPTION (provided by applicant): The analytical power of flow cytometry makes it invaluable for numerous biomedical applications that require the enumeration of cell populations and the analysis of multicellular model systems or organisms. However, sample analysis flow rates of typical flow cytometers are limited to less than 250 uL/min, analytical rates are limited o 70,000 cells/s, and particle diameters must be less than 70 um. These limitations are driven by a number of factors that include pressure induced by high linear velocity fluid flows, turbulence in wide channels, and the single point analysis of stochastically arriving particles. Therefore, flow cytometry requires significant additional sample preparation steps to be effective in the analysis of very rare cell populations, uses offline particle concentration to analyze particles in large volume samples, and requires special purpose large flow channel cytometers using low linear velocity hydrodynamic focusing in wide channels to analyze particles that are >70 um in diameter at low analysis rates (200 s-1). Such limitations severely reduce its effectiveness in many critical applications including the detection of rare blood cell populations, the detection of pathogens in liquid samples, and the high throughput analysis model systems (e.g. multicellular model organisms, cellular spheroids, and one-bead-one-compound chemical libraries) that use large particles. To provide the analytical power of flow cytometry to these critical applications, we must dramatically increase the analytical rate, volumetric sample delivery, and the useable particle size of flow cytometers. To this end, we have developed acoustic flow cells that generate up to 300 focused parallel streams of particles using both acoustically resonant micro fabricated channels and multi-node acoustic standing waves. These flow cells focus particles up to 200 um in diameter at volumetric delivery rates as high as 25 mL/min. In this proposal, we will optimize the fluidics and optical properties of our flow cells and couple them with new approaches for highly parallel optical detection to create an affordable parallel acoustic flow cytometer (APAfc) platform. To address the broad set of unmet application needs, the APAfc platform will analyze cells or particles, ranging from 1 to 1000 um in diameter, at flow rates up t 50 mL/min, and at rates up to 1 x 106 particles/s. Importantly, the APAfc will achieve these specifications while retaining the analytical properties of flow cytometry (sensitivity, resolutionof free vs. bound probes, correlated multipara meter analysis) that make it the technology of choice for cell and particle analysis. Furthermore, the APAfc platform will be designed using affordable technologies to ensure that when translated into a commercial product, it will cost about what a low- end flow cytometer does today (~$50 to $100K). We will demonstrate the effectiveness of the APAfc platform using relevant models of clinical and research assays that are directly limited by analytical rates, volumetric throughput, or particle size. Development of the APAfc will have significant impact on both biomedical research and clinical diagnostics. It will provide a prototype instrument that provides highly sensitive and precise multipara meter optical analysis at analytical and volumetric delivery rates sufficient to provide a cost effective solution to routine detection of rare cells in blood or environmental samples, dramatically increase sample processing rates for HTS applications, and dramatically speed the analysis of multicellular particles and model organisms. We anticipate that if we are successful, our approaches to large volume high throughput flow cytometry will bring powerful analytical techniques to bear on a new spectrum of clinical and research problems.

描述（由申请人提供）：流式细胞术的分析能力使其对于需要细胞群计数和多细胞模型系统或生物体分析的许多生物医学应用非常宝贵。然而，典型流式细胞仪的样品分析流速被限制为小于250 μ L/min，分析速率被限制为70，000个细胞/s，并且颗粒直径必须小于70 μ m。这些限制是由许多因素驱动的，这些因素包括由高线速度流体流动引起的压力、宽通道中的湍流以及随机到达颗粒的单点分析。因此，流式细胞术需要显著的额外样品制备步骤以在非常罕见的细胞群体的分析中有效，使用离线颗粒浓度来分析流式细胞术中的颗粒。 大体积样品，并且需要在宽通道中使用低线速度流体动力学聚焦的专用大流动通道细胞仪，以低分析速率（200 s-1）分析直径>70 μ m的颗粒。这种限制严重降低了其在许多关键应用中的有效性，包括检测稀有血细胞群体、检测血细胞中的细胞毒性、以及检测血细胞中的细胞毒性。 液体样品中的病原体，以及使用大颗粒的高通量分析模型系统（例如，多细胞模型生物、细胞球状体和一珠一化合物化学文库）。为了向这些关键应用提供流式细胞仪的分析能力，我们必须显著提高流式细胞仪的分析速率、体积样品递送和可用的颗粒尺寸。为此，我们开发了声学流动池，其使用声学谐振微制造通道和多节点声学驻波产生多达300个聚焦的平行颗粒流。这些流动池集中颗粒直径高达200微米，体积输送速率高达25毫升/分钟。在这个建议中，我们将优化我们的流动池的流体和光学特性，并将它们与高度并行光学检测的新方法相结合，以创建一个负担得起的并行声学流式细胞仪（APAfc）平台。为了解决广泛的未满足的应用需求，APAfc平台将分析直径从1到1000 μ m的细胞或颗粒，流速高达50 mL/min，速率高达1 x 106个颗粒/s。重要的是，APAfc将实现这些规格，同时保留流式细胞术的分析特性（灵敏度、游离探针与结合探针的分辨率、相关多参数分析），使其成为细胞和颗粒分析的首选技术。此外，APAfc平台将使用负担得起的技术进行设计，以确保当转化为商业产品时，其成本约为今天低端流式细胞仪的成本（约50至10万美元）。我们将使用直接受分析速率、体积通量或粒度限制的临床和研究测定的相关模型来证明APAfc平台的有效性。APAfc的发展将对生物医学研究和临床诊断产生重大影响。它将提供一个原型仪器，提供高灵敏度和精确的多参数仪光学分析，分析和体积输送率足以提供一个成本效益 这是血液或环境样品中稀有细胞常规检测的解决方案，显著提高了HTS应用的样品处理速率，并显著加快了多细胞颗粒和模式生物的分析。我们预计，如果我们成功的话，我们的大容量高通量流式细胞术方法将带来强大的分析技术，以应对一系列新的临床和研究问题。