Development and validation of a microfluidic immunoassay capable of multiplexing parallel samples in microliter volumes.
Development and validation of a microfluidic immunoassay capable of multiplexing parallel samples in microliter volumes.
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DOI：
10.1039/c5lc00398a
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发表时间：
2015-08-07
期刊：
影响因子：
6.1
通讯作者：
Yarmush ML
中科院分区：
文献类型：
--
作者：
Ghodbane M;Stucky EC;Maguire TJ;Schloss RS;Shreiber DI;Zahn JD;Yarmush ML
免疫测定由于其能够定量与生物研究和临床诊断相关的大量生物分子而被广泛使用。最近，通过开发同时测量单个样品中的多个分析物的多重测定，免疫测定能力已经得到改善。然而，这些测定受到试剂的高成本和相对大的样品需求的阻碍。例如，体外筛选系统目前将单独的威尔斯孔专用于每个感兴趣的时间点，并且当需要研究许多实验条件时，这种限制在筛选研究中被放大;导致用于分析的大体积、相应的高成本和有限的时间实验设计。为了克服这些缺点，已经开发了基于微流体的免疫测定。总之，先前的研究已经证明了具有大动态范围、高性能灵敏度和/或在单个芯片上并行处理样品的能力的芯片上测定。在这份报告中，我们开发了一种多重免疫测定拥有所有这些平行的特点，使用市售的试剂，它允许感兴趣的分析物很容易改变。该设备可以同时测量32个样品中的6种蛋白质，仅使用4.2微升的样品体积。为分析中包含的所有6种分析物生成高质量标准曲线，并在整个测定工作范围内对加标样品进行定量。此外，我们证明了使用我们的设备和从台式多重免疫测定获得的体外上清液测量之间的强相关性（R2=0.8999）。最后，我们描述了在体外炎症海马培养系统中的细胞因子分泌，建立了使用该平台作为体外筛选工具的能力的概念验证。本报告中描述的微型器件的低容量多路复用能力可以广泛应用于样品量和成本有限的许多情况。
Immunoassays are widely utilized due to their ability to quantify a vast assortment of biomolecules relevant to biological research and clinical diagnostics. Recently, immunoassay capabilities have been improved by the development of multiplex assays that simultaneously measure multiple analytes in a single sample. However, these assays are hindered by high costs of reagents and relatively large sample requirements. For example, in vitro screening systems currently dedicate individual wells to each time point of interest and this limitation is amplified in screening studies when the investigation of many experimental conditions is necessary; resulting in large volumes for analysis, a correspondingly high cost and a limited temporal experimental design. Microfluidics based immunoassays have been developed in order to overcome these drawbacks. Together, previous studies have demonstrated on-chip assays with either a large dynamic range, high performance sensitivity, and/or the ability to process samples in parallel on a single chip. In this report, we develop a multiplex immunoassay possessing all of these parallel characteristics using commercially available reagents, which allows the analytes of interest to be easily changed. The device presented can measure 6 proteins in 32 samples simultaneously using only 4.2 µL of sample volume. High quality standard curves are generated for all 6 analytes included in the analysis, and spiked samples are quantified throughout the working range of the assay. In addition, we demonstrate a strong correlation (R2=0.8999) between in vitro supernatant measurements using our device and those obtained from a bench-top multiplex immunoassay. Finally, we describe cytokine secretion in an in vitro inflammatory hippocampus culture system, establishing proof-of-concept of the ability to use this platform as an in vitro screening tool. The low-volume, multiplexing abilities of the microdevice described in this report could be broadly applied to numerous situations where sample volumes and costs are limiting.