State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry , Chinese Academy of Sciences , Changchun , Jilin 130022 , China.
University of Chinese Academy of Sciences , Beijing 100049 , China.
Anal Chem. 2018 Jun 19;90(12):7754-7760. doi: 10.1021/acs.analchem.8b01845. Epub 2018 Jun 7.
An enzyme cascade-triggered reaction with novel signal generation mechanism is beneficial for the development and insight of the enzyme cascade, which is extensively used for signal transduction in potential applications. Inspired by the fluorogenic and chromogenic reaction between dopamine and resorcinol, and the specific catalytic properties of alkaline phosphatase (ALP) and tyrosinase, we designed and synthesized an unconventional substrate of ALP, named p-aminoethyl-phenyl phosphate disodium salt (PAPP). As expected, the ALP and tyrosinase-incubated PAPP solution exhibited pale yellow with intense blue fluorescence upon addition of resorcinol, owing to the ALP-catalyzed transformation of PAPP into an intermediate tyramine, and the tyrosinase-catalyzed hydroxylation of tyramine to dopamine, as well as the specific reaction between dopamine and resorcinol. Therefore, an enzyme cascade system has been developed herein based on the ALP and tyrosinase coupled enzymes-triggered fluorogenic and chromogenic reaction. According to the direct relationship between the activity of ALP/tyrosinase and absorbance/fluorescence intensity of the resultant solution, the proposed enzyme cascade-triggered reaction was utilized for assaying ALP and tyrosinase activity with fluorometric and colorimetric dual-readout signals. Furthermore, such enzyme cascade catalysis process was integrated into the ALP-based cascade enzyme-linked immunosorbent assay with dual-readout signals, resulting in the sensitive detection of cardiac troponin I in diluted serum.
一种具有新型信号产生机制的酶级联触发反应有利于酶级联的发展和深入研究，该反应在潜在应用中的信号转导中得到了广泛应用。受多巴胺和间苯二酚之间的荧光和显色反应以及碱性磷酸酶 (ALP) 和酪氨酸酶的特定催化特性的启发，我们设计并合成了一种非传统的 ALP 底物，命名为对氨基乙基苯膦酸二钠盐 (PAPP)。正如预期的那样，加入间苯二酚后，孵育有 ALP 和酪氨酸酶的 PAPP 溶液呈现出淡黄色，并带有强烈的蓝色荧光，这是由于 ALP 催化 PAPP 转化为中间产物酪胺，以及酪氨酸酶催化酪胺羟化为多巴胺，以及多巴胺和间苯二酚之间的特异性反应。因此，本文基于 ALP 和酪氨酸酶偶联酶触发的荧光和显色反应，开发了一种酶级联系统。根据所得溶液的 ALP/tyrosinase 活性与吸光度/荧光强度之间的直接关系，提出的酶级联触发反应可用于测定 ALP 和酪氨酸酶活性，具有荧光和比色双重读数信号。此外，该酶级联催化过程被整合到基于 ALP 的级联酶联免疫吸附测定中，具有双重读数信号，从而实现了对稀释血清中心肌肌钙蛋白 I 的灵敏检测。
