A microfluidics-based technique for automated and rapid labeling of cells for flow cytometry
A microfluidics-based technique for automated and rapid labeling of cells for flow cytometry
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DOI：
10.1088/0960-1317/24/3/034002
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发表时间：
2014-03-01
影响因子：
2.3
通讯作者：
Sethu, Palaniappan
中科院分区：
文献类型：
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作者：
Patibandla, Phani K.;Estrada, Rosendo;Sethu, Palaniappan
流式细胞术是一种强大的技术，能够同时对异质细胞群体进行多参数分析，用于研究和临床应用。近年来，流式细胞仪已经小型化，并使其便于在临床和资源有限的环境中应用。样品制备程序，即用与荧光标记相结合的抗体标记细胞，是一项耗时(类似于45分钟)和劳动密集型程序。微流控技术提供了实现快速和自动化样品制备的技术。使用由标签和洗涤模块组成的集成微流控设备，我们展示了一种新的方案，它可以省去样品处理，并以快速自动化的方式完成样品和试剂的计量、高效混合、标签和洗涤。标记模块由一个带有集成混沌混合器的长微流控通道组成。样品和试剂精确地计量到该装置中，实现快速高效的混合。混合的样品和试剂被收集在一个持有注射器中，并保持长达8分钟，然后将混合物引入惯性洗涤模块，以获得用于分析的样品。洗涤模块由能够将细胞聚焦到接近通道壁的平衡位置的高纵横比通道组成。通过在两侧两侧的洗涤缓冲液两侧的通道中心处的狭窄溪流中引入细胞和标记试剂，实现了细胞从游离抗体洗脱到洗涤缓冲液中的洗脱。在初步校准实验以确定允许抗体结合的适当“保持时间”之后，两个模块被联合使用来同时用三种不同的抗体标记MOLT-3细胞(T淋巴母细胞系)。结果证实，在常规程序(45分钟)和我们的微流控方法(12分钟)之间，所有三种抗体标记(p<；0.01)的平均荧光强度值没有显著差异。
Flow cytometry is a powerful technique capable of simultaneous multi-parametric analysis of heterogeneous cell populations for research and clinical applications. In recent years, the flow cytometer has been miniaturized and made portable for application in clinical-and resource-limited settings. The sample preparation procedure, i.e. labeling of cells with antibodies conjugated to fluorescent labels, is a time consuming (similar to 45 min) and labor-intensive procedure. Microfluidics provides enabling technologies to accomplish rapid and automated sample preparation. Using an integrated microfluidic device consisting of a labeling and washing module, we demonstrate a new protocol that can eliminate sample handling and accomplish sample and reagent metering, high-efficiency mixing, labeling and washing in rapid automated fashion. The labeling module consists of a long microfluidic channel with an integrated chaotic mixer. Samples and reagents are precisely metered into this device to accomplish rapid and high-efficiency mixing. The mixed sample and reagents are collected in a holding syringe and held for up to 8 min following which the mixture is introduced into an inertial washing module to obtain 'analysis-ready' samples. The washing module consists of a high aspect ratio channel capable of focusing cells to equilibrium positions close to the channel walls. By introducing the cells and labeling reagents in a narrow stream at the center of the channel flanked on both sides by a wash buffer, the elution of cells into the wash buffer away from the free unbound antibodies is accomplished. After initial calibration experiments to determine appropriate 'holding time' to allow antibody binding, both modules were used in conjunction to label MOLT-3 cells (T lymphoblast cell line) with three different antibodies simultaneously. Results confirm no significant difference in mean fluorescence intensity values for all three antibodies labels (p < 0.01) between the conventional procedure (45 min) and our microfluidic approach (12 min).