High volume high throughput affordable parallel acoustic flow cytometry

描述(由申请人提供)：流式细胞术的分析能力使其对于需要计数细胞群体和分析多细胞模型系统或生物体的众多生物医学应用具有无价的价值。然而，典型的流动细胞仪的样品分析流速被限制在250微升/分钟以下，分析速率被限制在70,000个/S，并且颗粒直径必须小于70微米。这些限制是由许多因素驱动的，这些因素包括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的颗粒的单点分析。因此，流式细胞术需要大量额外的样品制备步骤才能有效地分析非常稀有的细胞群，使用离线颗粒浓度来分析 大体积样品需要专用的大流量通道细胞仪，在宽通道中使用低线速度流体动力聚焦，以便在低分析率(200 S-1)下分析直径为70微米的颗粒。这些限制严重降低了它在许多关键应用中的有效性，包括检测稀有血细胞群、检测 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、细胞球体和一珠一化合物化学库)。为了向这些关键应用提供流式细胞仪的分析能力，我们必须大幅提高流式细胞仪的分析率、体积样品输送和可用颗粒大小。为此，我们开发了声流室，使用声学共振微制造通道和多节点声驻波产生多达300个聚焦的平行粒子流。这些流动池聚焦直径高达200微米的颗粒，体积输送速率高达25毫升/分钟。在这项计划中，我们将优化流动细胞的流体和光学特性，并将其与高度并行光学检测的新方法相结合，以创建经济实惠的并行声学流式细胞仪(APAfc)平台。为了满足广泛的应用需求，APAfc平台将分析直径从1到1000微米的细胞或颗粒，流速高达50毫升/分钟，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、分辨率、游离和结合探针的分辨率、相关多参数分析)，这些特性使其成为细胞和颗粒分析的首选技术。此外，APAfc平台的设计将使用负担得起的技术，以确保在转化为商业产品时，其成本与目前低端流式细胞仪的成本相当(约5万至10万美元)。我们将使用临床和研究分析的相关模型来演示APAfc平台的有效性，这些模型直接受到分析速率、容量吞吐量或颗粒大小的限制。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，以分析和体积传递速率提供高灵敏度和精确的多参数光学分析，足以提供成本效益 该解决方案可用于常规检测血液或环境样本中的稀有细胞，显著提高高温超导应用的样本处理速度，并显著加快多细胞粒子和模型生物的分析速度。我们预计，如果我们成功，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。

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.