High-sensitivity miniaturized immunoassays for tumor necrosis factor a using microfluidic systems
High-sensitivity miniaturized immunoassays for tumor necrosis factor a using microfluidic systems
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DOI：
10.1039/b408964b
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发表时间：
2004-01-01
期刊：
影响因子：
6.1
通讯作者：
Delamarche, E
中科院分区：
文献类型：
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作者：
Cesaro-Tadic, S;Dernick, G;Delamarche, E
我们使用微流控芯片，使用亚微升体积的样品和试剂，以皮摩尔灵敏度检测生物学上重要的细胞因子肿瘤坏死因子 α (TNF-α)。该芯片由多个独立的毛细管系统（CS）组成，每个毛细管系统由一个填充口、一个附加的微通道和一个毛细管泵组成。每个 CS 通过毛细管力自发填充，并包含一个自我调节机制，可防止微通道的意外排水。因此，通过两个可以独立加热和冷却的珀耳帖元件对所有 CS 中的蒸发进行集体控制，可以轻松实现每个 CS 中流量的交互式控制。长孵育时间对于高灵敏度测定至关重要，并且可以通过调整蒸发速率以具有类似于 30 nL min(-1) 的低流速来方便地获得。该测定是一种夹心荧光免疫测定，在放置在微通道上的聚二甲基硅氧烷 (PDMS) 板的表面上进行。我们用捕获抗体 (Abs) 预涂 PDMS，使用芯片定位分析物分子的捕获，然后使用第二个芯片将捕获的分析物分子与荧光标记的检测 Abs 结合。该测定结果在 PDMS 表面上产生荧光信号马赛克，并使用荧光扫描仪进行测量。我们证明，如果使用具有长激发波长荧光团（大于或等于 580 nm）的检测抗体，PDMS 是高灵敏度荧光测定的兼容材料。芯片设计、长孵育时间、荧光团的正确选择以及检测抗体浓度的优化相结合，可实现高灵敏度检测。一个实验证明了这一点，该实验有 170 个测定点，在 PDMS 上占据约 0.6 mm(2) 的面积，用于检测 600 nL 树突状细胞 (DC) 培养基中的 TNF-α，灵敏度约为 20 pg mL(-1) (1.14 pM)。
We use microfluidic chips to detect the biologically important cytokine tumor necrosis factor alpha (TNF-alpha) with picomolar sensitivity using sub-microliter volumes of samples and reagents. The chips comprise a number of independent capillary systems (CSs), each of which is composed of a filling port, an appended microchannel, and a capillary pump. Each CS fills spontaneously by capillary forces and includes a self-regulating mechanism that prevents adventitious drainage of the microchannels. Thus, interactive control of the flow in each CS is easily achieved via collective control of the evaporation in all CSs by means of two Peltier elements that can independently heat and cool. Long incubation times are crucial for high sensitivity assays and can be conveniently obtained by adjusting the evaporation rate to have low flow rates of similar to30 nL min(-1). The assay is a sandwich fluorescence immunoassay and takes place on the surface of a poly( dimethylsiloxane) ( PDMS) slab placed across the microchannels. We precoat PDMS with capture antibodies (Abs), localize the capture of analyte molecules using a chip, then bind the captured analyte molecules with fluorescently-tagged detection Abs using a second chip. The assay results in a mosaic of fluorescence signals on the PDMS surface which are measured using a fluorescence scanner. We show that PDMS is a compatible material for high sensitivity fluorescence assays, provided that detection antibodies with long excitation wavelength fluorophores (greater than or equal to580 nm) are employed. The chip design, long incubation times, proper choice of fluorophores, and optimization of the detection Ab concentration all combine to achieve high-sensitivity assays. This is exemplified by an experiment with 170 assay sites, occupying an area of similar to0.6 mm(2) on PDMS to detect TNF-alpha in 600 nL of a dendritic cell (DC) culture medium with a sensitivity of similar to20 pg mL(-1) (1.14 pM).