Department of Chemistry, University of Wyoming, Laramie, WY 82071, USA.
Analyst. 2018 Feb 12;143(4):989-998. doi: 10.1039/c7an01671a.
The requirement for an 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 lower than that possible on commercial microwell plates primarily due to the better detectability of the rhodamine dye.
为了在酶底物的信号区域直接进行化学反应，从而使酶底物的可检测性发生显著变化，这对酶联免疫吸附测定（ELISA）的范围提出了重大限制。特别是，这种要求限制了可用于 ELISA 的酶标记-底物偶联物的种类，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较窄范围的测定条件/样品外，测定的检测限和多重检测能力受到限制。为了尝试解决其中的一些限制，本文介绍了一种微流控 ELISA 方法，该方法不需要酶标记在底物分子的信号区域周围起作用。合成了一种高可检测的基于若丹明的底物，以证明所报道的测定方法，该方法在酶标记物碱性磷酸酶切割后，从单阴离子转变为单阳离子，两者的荧光性质几乎相同。随后，这些物质基于其电荷差异使用集成设备中的毛细管区带电泳进行分离，从而为我们的样品中的分析物（人 TNF-α）提供了定量测量。令人印象深刻的是，该方法不仅允许使用新型酶底物进行 ELISA，而且还能够检测到人 TNF-α的浓度比商用微孔板低 54 倍以上，这主要是由于若丹明染料的可检测性更好。
