INTEGRATED ISOLATION & MOLECULAR ANALYSIS OF TUMOR CELLS

集成隔离

• 批准号: 6514761
• 负责人: PETER Russell Charles GASCOYNE
• 金额: $ 52.19万
• 依托单位: UNIVERSITY OF TX MD ANDERSON CAN CTR
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-07-23 至 2004-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6514761
• 关键词: biomedical equipment development; breast neoplasms; dielectric property; flow cytometry; human tissue; immunomagnetic separation; neoplastic cell; surface antigens

Our goal for the combined R21 and R33 phases of this proposal is to make an integrated fluidic device that is capable of the automatic isolation of target cells from a cell mixture and their analysis for molecular markers for cancer detection and diagnosis. The final integrated device will be comprised of the following stages: (1) a cell separator able to process 2x106 cells/minute and to trap suspect tumor cells from them; (2) a cell fractionator that will separate suspect cells according to their dielectric and surface marker properties; (3) a microfluidic isolator stage that will collect and concentrate cell subpopulations emerging from the fractionator, combine them with a mixture of beads carrying multiple molecular probes, and burst them in the presence of the beads; and (4) an impedance sensor stage able to identify beads according to their dielectric fingerprints and thereby index them to the probes carried on their surfaces. Fluorescence of molecular probes on each bead will also be measured at this stage. By using a mixture of multiple bead types, several molecular assays will be run simultaneously. To achieve this goal we will develop and integrate fluidic and microfluidic technologies to automate the central problems of molecular diagnosis, namely the enrichment and isolation of a suspect cell subpopulation from a mixture of cells in a biopsy sample and the quantitative analysis of that subpopulation for molecular markers including proteins and mRNA's. The key technologies to be developed are: 1) A prefilter able to trap suspect tumor cells from large numbers of blood or lymph node cells; 2) A force balance method that exploits the dielectric and density properties, and, optionally, the immunomagnetic labeling properties, of target cells to enable the isolation; and 3) A dielectric indexing and manipulation method for carrier beads that, when combined with established molecular assays, will allow the simultaneous quantification of multiple molecular markers in parallel assays. The sample preparation aspects represent an extension of our successful dielectrophoretic trapping and dielectrophoretic field-flow-fractionation (DEP-FFF) methods that have been demonstrated for sorting and isolating different cell types from a cell mixture. The dielectric bead methods represent a novel approach to identifying individual molecular tests in a parallel molecular marker analysis scheme. Dielectric indexing of carrier beads will be used instead of the spatial indexing used on a gene chip. This will allow different subpopulations of beads, each carrying a different marker probe, to be identified and differentially manipulated by exploiting their dielectric signatures. The need to immobilize molecular probes in a tightly specified location on a fixed substrate, as demanded by spatial indexing is thereby eliminated and customized mixtures of probes, each carried on a separately-indexed bead type, may be identified as they emerge in a mixture from a reusable assay system. The study will be divided into a one year R21 phase and a three year R33 phase. During the R21 phase, proofs-of-concept will be developed for scaled-up dielectrophoretic trapping of suspect cells from large cell populations, for combined dielectrophoretic-magnetophoretic field flow fractionation, and for dielectric indexing of beads in mixed molecular assays. During the R33 phase, these technologies will be further developed and integrated in order to accomplish all steps of sample preparation and molecular analysis. The technologies will then be tested and refined using clinically relevant samples.

我们对该提案合并的R21和R33阶段的目标是制造一种集成的流体装置，该设备能够从细胞混合物中自动隔离靶细胞，并分析分子标记物以进行癌症检测和诊断。 最终的集成装置将由以下阶段组成：（1）能够处理2x106单元/分钟的细胞分离器并从中捕获可疑肿瘤细胞； （2）一个细胞分级器，该细胞分级器将根据其介电和表面标记性能分离可疑细胞； （3）一个微流体隔离器阶段，它将收集和浓缩从分级器中出现的细胞亚群，将它们与携带多个分子探针的珠子混合在一起，并在珠子存在下破裂。 （4）一个阻抗传感器阶段，能够根据其介电指纹识别珠子，从而将它们索引到其表面上的探针。 在此阶段，也将测量每个珠子上分子探针的荧光。 通过使用多种珠类型的混合物，将同时运行几种分子测定。为了实现这一目标，我们将开发和整合流体和微流体技术，以使分子诊断的核心问题自动化，即从活检样品中的细胞混合物中的可疑细胞亚群的富集和隔离以及对包括蛋白质和mRNA在内的分子标记的该亚群的定量分析中的定量分析。 要开发的关键技术是：1）能够从大量血液或淋巴结细胞中捕获可疑肿瘤细胞的前滤器； 2）一种利用靶细胞的介电和密度特性的力平衡法，以及选择的靶细胞的免疫磁性标记特性，以实现分离； 3）载体珠的介电索引和操纵方法，当与已建立的分子测定时，将允许在平行测定中同时定量多个分子标记。样品制备方面代表了我们成功的介电摄取诱捕和介电练习场磁场 - 流动分级（DEP-FFF）方法的扩展，这些方法已被证明用于从细胞混合物中分类和分离不同的细胞类型。 介电珠方法代表了一种在平行分子标记分析方案中识别单个分子测试的新方法。 将使用载体珠的介电索引，而不是基因芯片上使用的空间索引。 这将允许通过利用其介电特征来识别并差异化的珠子的不同亚群，每个珠子，每个珠子，每个珠子，均带有不同的标记探针。 从空间索引要求的情况下，可以将分子探针固定在固定基板上的紧密指定位置中，从而消除并定制了探针的定制混合物，每种探针都可以在分别索引的珠子类型上携带，可以识别，因为它们从可重用的测定系统中出现在混合物中。该研究将分为一年的R21阶段和三年R33阶段。 在R21阶段，将开发概念证明，用于从大细胞群体中缩放的可疑细胞的介电术诱捕，用于混合介摄取 - 磁性遗传学场流量分馏，以及用于混合分子鉴定珠的介电指数。在R33阶段，将进一步开发和集成这些技术，以完成样品制备和分子分析的所有步骤。 然后，将使用临床相关样品对技术进行测试和完善。