Department of Chemistry, University of Wyoming, Laramie, Wyoming 82071, USA.
Anal Chem. 2012 Aug 21;84(16):7029-36. doi: 10.1021/ac3011632. Epub 2012 Aug 9.
In this Article, we describe a microfluidic enzyme-linked immunosorbent assay (ELISA) method whose sensitivity can be substantially enhanced through preconcentration of the target analyte around a semipermeable membrane. The reported preconcentration has been accomplished in our current work via electrokinetic means allowing a significant increase in the amount of captured analyte relative to nonspecific binding in the trapping/detection zone. Upon introduction of an enzyme substrate into this region, the rate of generation of the ELISA reaction product (resorufin) was observed to increase by over a factor of 200 for the sample and 2 for the corresponding blank compared to similar assays without analyte trapping. Interestingly, in spite of nonuniformities in the amount of captured analyte along the surface of our analysis channel, the measured fluorescence signal in the preconcentration zone increased linearly with time over an enzyme reaction period of 30 min and at a rate that was proportional to the analyte concentration in the bulk sample. In our current study, the reported technique has been shown to reduce the smallest detectable concentration of the tumor marker CA 19-9 and Blue Tongue Viral antibody by over 2 orders of magnitude compared to immunoassays without analyte preconcentration. When compared to microwell based ELISAs, the reported microfluidic approach not only yielded a similar improvement in the smallest detectable analyte concentration but also reduced the sample consumption in the assay by a factor of 20 (5 μL versus 100 μL).
在本文中，我们描述了一种微流控酶联免疫吸附测定（ELISA）方法，通过将目标分析物在半透膜周围预浓缩，可以显著提高其灵敏度。在我们目前的工作中，通过电泳手段实现了这种预浓缩，与捕获/检测区域中的非特异性结合相比，可以显著增加捕获的分析物的量。在将酶底物引入该区域后，与没有分析物捕获的类似测定相比，观察到 ELISA 反应产物（resorufin）的生成速率在样品中增加了 200 多倍，在相应的空白中增加了 2 倍。有趣的是，尽管在我们的分析通道表面上捕获的分析物的量不均匀，但在 30 分钟的酶反应期间，预浓缩区域中的测量荧光信号随时间线性增加，并且增加速率与样品中分析物的浓度成正比。在我们目前的研究中，与没有分析物预浓缩的免疫测定相比，所报道的技术已被证明可以将肿瘤标志物 CA 19-9 和蓝舌病毒抗体的最小可检测浓度降低两个数量级以上。与微孔板 ELISA 相比，所报道的微流控方法不仅在最小可检测分析物浓度方面具有相似的改进，而且还将分析中的样品消耗减少了 20 倍（5 μL 对 100 μL）。
