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A bead-based immunogold-silver staining assay on capillary-driven microfluidics

机译：基于微珠的毛细管驱动微流控免疫金银染色法

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摘要

Point-of-care (POC) diagnostics are critically needed for the detection of infectious diseases, particularly in remote settings where accurate and appropriate diagnosis can save lives. However, it is difficult to implement immunoassays, and specifically immunoassays relying on signal amplification using silver staining, into POC diagnostic devices. Effective immobilization of antibodies in such devices is another challenge. Here, we present strategies for immobilizing capture antibodies (cAbs) in capillary-driven microfluidic chips and implementing a gold-catalyzed silver staining reaction. We illustrate these strategies using a species/anti-species immunoassay and the capillary assembly of fluorescent microbeads functionalized with cAbs in "bead lanes", which are engraved in microfluidic chips. The microfluidic chips are fabricated in silicon (Si) and sealed with a dry film resist. Rabbit IgG antibodies in samples are captured on the beads and bound by detection antibodies (dAbs) conjugated to gold nanoparticles. The gold nanoparticles catalyze the formation of a metallic film of silver, which attenuates fluorescence from the beads in an analyte-concentration dependent manner. The performance of these immunoassays was found comparable to that of assays performed in 96 well microtiter plates using "classical" enzyme-linked immunosorbent assay (ELISA). The proof-of-concept method developed here can detect 24.6 ng mL(-1) of rabbit IgG antibodies in PBS within 20 min, in comparison to 17.1 ng mL(-1) of the same antibodies using a similar to 140-min-long ELISA protocol. Furthermore, the concept presented here is flexible and necessitate volumes of samples and reagents in the range of just a few microliters.
机译：即时诊断（POC）诊断对于检测传染病至关重要，特别是在偏远地区，准确而适当的诊断可以挽救生命。然而，难以在POC诊断装置中实施免疫测定，特别是依赖于使用银染的信号放大的免疫测定。将抗体有效地固定在此类装置中是另一个挑战。在这里，我们提出了在毛细管驱动的微流控芯片中固定捕获抗体（cAb）并实施金催化的银染反应的策略。我们举例说明了使用物种/反物种免疫测定和在“珠子通道”中用cAbs功能化的荧光微珠的毛细管装配，这些微雕被刻在微流控芯片中。微流体芯片用硅（Si）制成，并用干膜抗蚀剂密封。样品中的兔IgG抗体被捕获在珠子上，并与与金纳米颗粒偶联的检测抗体（dAb）结合。金纳米颗粒催化银金属膜的形成，该银金属膜以分析物浓度依赖性的方式减弱了来自珠子的荧光。发现这些免疫测定的性能与使用“经典”酶联免疫吸附测定（ELISA）在96孔微量滴定板中进行的测定相当。本文开发的概念验证方法可在20分钟内检测到PBS中的24.6 ng mL（-1）兔IgG抗体，而使用相同的140-min方法可检测到17.1 ng mL（-1）的相同抗体。长ELISA方案。此外，这里提出的概念很灵活，样品和试剂的体积必须在几微升的范围内。

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  • 来源
    《Biomedical Microdevices》 |2018年第2期|41.1-41.9|共9页
  • 作者

    Pham Ngoc M.; Rusch Sebastian; Temiz Yuksel; Lovchik Robert D.; Beck Hans-Peter; Karlen Walter; Delamarche Emmanuel;

  • 作者单位

    ETH, Mobile Hlth Syst Lab, Inst Robot & Intelligent Syst, Dept Hlth Sci & Technol,BAA, Lengghalde 5, CH-8092 Zurich, Switzerland;

    Swiss Trop & Publ Hlth Inst, Socinstr 57, CH-4051 Basel, Switzerland;

    IBM Res Zurich, Saumerstr 4, CH-8803 Ruschlikon, Switzerland;

    IBM Res Zurich, Saumerstr 4, CH-8803 Ruschlikon, Switzerland;

    Swiss Trop & Publ Hlth Inst, Socinstr 57, CH-4051 Basel, Switzerland;

    ETH, Mobile Hlth Syst Lab, Inst Robot & Intelligent Syst, Dept Hlth Sci & Technol,BAA, Lengghalde 5, CH-8092 Zurich, Switzerland;

    IBM Res Zurich, Saumerstr 4, CH-8803 Ruschlikon, Switzerland;

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  • 正文语种 eng
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  • 关键词

    Microfluidics; Silver staining; Immunoassays; Microbeads;

    机译：微流控;银染;免疫分析;微珠;

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