A single-molecule digital enzyme assay using alkaline phosphatase with a cumarin-based fluorogenic substrate
A single-molecule digital enzyme assay using alkaline phosphatase with a cumarin-based fluorogenic substrate
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DOI：
10.1039/c5an00714c
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发表时间：
2015-01-01
期刊：
影响因子：
4.2
通讯作者：
Noji, Hiroyuki
中科院分区：
文献类型：
--
作者：
Obayashi, Yusuke;Iino, Ryota;Noji, Hiroyuki
通过使用飞升室阵列技术进行荧光酶测定的数字化是实现具有单分子灵敏度的高度定量生物测定的新兴方法。然而，仅报道了少数数字荧光酶测定，并且数字酶测定的变化基本上限于基于荧光素和试卤灵的荧光测定。这种限制阻碍了多重数字酶测定（例如数字酶联免疫吸附测定（ELISA））的实现。在本研究中，在优化缓冲液条件后，我们使用基于香豆素的荧光底物 4-甲基unbelliferyl 磷酸盐 (4-MUP) 实现了单分子数字酶碱性磷酸酶 (ALP) 测定。当 ALP 分子以每个室少于 1 个分子的低比率封装在 44 飞升室阵列中时，每个室以全或无的方式显示离散荧光信号，从而可以对活性酶分子的数量进行数字计数。荧光室的分数随着酶浓度线性下降，符合预期的泊松分布。我们还展示了使用 ALP/4-MUP 和 β-半乳糖苷酶 (β-gal)/试卤灵-β-D-吡喃半乳糖苷组合的双色数字酶测定。同时清楚地检测到单个 ALP 和 β-gal 分子的活性。本研究中开发的方法将使我们能够进行并行、多重数字 ELISA。
Digitalization of fluorogenic enzymatic assays through the use of femtoliter chamber array technology is an emerging approach to realizing highly quantitative bioassays with single-molecule sensitivity. However, only a few digital fluorogenic enzyme assays have been reported, and the variations of the digital enzyme assays are basically limited to fluorescein- and resorufin-based fluorogenic assays. This limitation hampers the realization of a multiplex digital enzyme assay such as a digital enzyme-linked immunosorbent assay (ELISA). In this study, after optimization of buffer conditions, we achieved a single-molecule digital enzyme alkaline phosphatase (ALP) assay with a cumarin-based fluorogenic substrate, 4-methylunbelliferyl phosphate (4-MUP). When ALP molecules were encapsulated in a 44-femtoliter chamber array at a low ratio of less than 1 molecule per chamber, each chamber showed a discrete fluorescence signal in an all-or-none manner, allowing the digital counting of the number of active enzyme molecules. The fraction of fluorescent chambers linearly decreased with the enzyme concentration, obeying the Poisson distribution as expected. We also demonstrated a dual-color digital enzyme assay with a ALP/4-MUP and beta-galactosidase (beta-gal)/resorufin-beta-D-galactopyranoside combination. The activities of single ALP and beta-gal molecules were clearly detected simultaneously. The method developed in this study will enable us to carry out a parallelized, multiplex digital ELISA.