Rapid microchip-based electrophoretic immunoassays for the detection of swine influenza virus
Rapid microchip-based electrophoretic immunoassays for the detection of swine influenza virus
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DOI：
10.1039/b801396a
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发表时间：
2008-08-01
期刊：
影响因子：
6.1
通讯作者：
Singh, Anup K.
中科院分区：
文献类型：
--
作者：
Reichmuth, David S.;Wang, Serena K.;Singh, Anup K.
为了开发快速、便携式的诊断方法来检测人畜共患疾病，我们开发了基于微芯片的电泳免疫测定法，用于灵敏、快速地检测病毒。开发了两种基于微芯片的电泳免疫测定法。最初的检测使用开放通道电泳和激光诱导荧光检测以及标记抗体来检测流感病毒。然而，该测定方法没有足够的灵敏度来检测用于诊断应用的相关浓度的病毒。因此，开发了一种新颖的检测方法，可以使用带有集成纳米孔膜的微流控芯片同时浓缩和检测病毒。利用原位聚合聚丙烯酰胺膜的尺寸排阻特性来同时浓缩病毒颗粒并将病毒/荧光抗体复合物与未结合的抗体分离。该测定通过两个简单的步骤进行——向样品中添加荧光标记的抗体，然后将抗体-病毒复合物浓缩在多孔膜上。通过膜电泳去除多余的抗体，然后在膜下游检测复合物。这种新检测方法检测到的灭活猪流感病毒浓度比开通道电泳检测方法低四倍。总检测时间（包括设备再生）为六分钟，需要
Towards developing rapid and portable diagnostics for detecting zoonotic diseases, we have developed microchip-based electrophoretic immunoassays for sensitive and rapid detection of viruses. Two types of microchip-based electrophoretic immunoassays were developed. The initial assay used open channel electrophoresis and laser-induced fluorescence detection with a labeled antibody to detect influenza virus. However, this assay did not have adequate sensitivity to detect viruses at relevant concentrations for diagnostic applications. Hence, a novel assay was developed that allows Simultaneous concentration and detection of viruses using a microfluidic chip with an integrated nanoporous membrane. The size-exclusion properties of the in situ polymerized polyacrylamide membrane are exploited to simultaneously concentrate viral particles and separate the virus/fluorescent antibody complex from the unbound antibody. The assay is performed in two simple steps-addition of fluorescently labeled antibodies to the sample, followed by concentration of antibody-virus complexes on a porous membrane. Excess antibodies are removed by electrophoresis through the membrane and the complex is then detected downstream of the membrane. This new assay detected inactivated swine influenza virus at a concentration four times lower than that of the open-channel electrophoresis assay. The total assay time, including device regeneration, is six minutes and requires