Microfluidic ELISA employing an enzyme substrate and product species with similar detection properties.
Microfluidic ELISA employing an enzyme substrate and product species with similar detection properties.
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DOI：
10.1039/c7an01671a
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发表时间：
2018-02-12
期刊：
影响因子：
--
通讯作者：
Dutta D
中科院分区：
文献类型：
--
作者：
Giri B ;Liu Y ;Nchocho FN ;Corcoran RC ;Dutta D
酶标签必须直接在酶底物的信号区进行化学反应，以使其可检测性发生较大变化，这一要求极大地限制了酶联免疫吸附测定（elisa）的范围。特别是，这一要求限制了酶标记-底物对在elisa中使用的种类，并阻止了它们在酶反应和酶反应底物/产物的可检测性方面的独立优化。因此，除了使该技术适用于狭窄范围的分析条件/样品外，该分析的检测限和多路复用能力也受到限制。为了解决其中的一些限制，本文描述了一种微流体ELISA方法，该方法不需要酶标签在底物分子的信号区域周围起作用。合成了一种高度可检测的罗丹明基底物，以证明所报道的测定方法，该底物经酶标记碱性磷酸酶裂解，从单阴离子转化为单阳离子，两者具有几乎相同的荧光特性。这些物种后来根据它们的电荷差异在一个集成装置中使用毛细管区带电泳分离，产生了我们样品中分析物（人TNF-α）的定量测量。令人印象深刻的是，该方法不仅可以使用一种新的酶底物进行elisa，而且由于罗丹明染料的可检测性更好，因此可以在比商业微孔板低54倍的浓度下检测人TNF-α。
The requirement for the enzyme label to carry out a chemical reaction directly at the signaling region of the enzyme substrate in order to produce a large change in its detectability places a significant constraint on the scope of enzyme-linked immunosorbent assays (ELISAs). In particular, this requirement limits the kinds of enzyme label-substrate couples employable in ELISAs and prevents their independent optimization with respect to the enzyme reaction and the detectability of the enzyme reaction substrate/product. The detection limit and multiplexing capabilities of the assay are consequently restricted in addition to rendering the technique applicable to a narrow range of assay conditions/samples. Attempting to address some of these limitations, the current article describes a microfluidic ELISA method that does not require the enzyme label to act around the signaling region of the substrate molecule. A highly detectable rhodamine based substrate was synthesized to demonstrate the reported assay which upon cleavage by the enzyme label, alkaline phosphatase, transformed from a monoanionic to a monocationic species both of which had nearly identical fluorescence properties. These species were later separated based on their charge difference using capillary zone electrophoresis in an integrated device yielding a quantitative measure for the analyte (human TNF-α) in our sample. Impressively, the noted approach not only enabled the use of a new kind of enzyme substrate for ELISAs but also allowed the detection of human TNF-α at concentrations over 54-fold smaller than that possible on commercial microwell plates primarily due to the better detectability of the rhodamine dye.
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