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. 2019 Jun 18;91(12):7828-7834. doi: 10.1021/acs.analchem.9b01553. Epub 2019 May 31.
Alkaline phosphatase (ALP) usually acts as a signal transmitter in enzyme-linked immunosorbent assay (ELISA); therefore, developing an attractive ALP activity assay, especially using a preferable substrate, would help improve the efficiency and convenience of ELISA in practical applications. Herein we have first prepared an original and creative substrate, named m-hydroxyphenyl phosphate sodium salt ( m-HPP), with a desirable dephosphorylation site for ALP. On the basis of the ALP-catalyzed hydrolysis of m-HPP to resorcinol and its subsequent specific nucleophilic reaction with dopamine, we have exploited a fluorometric and colorimetric dual-readout ALP activity assay and ALP-based ELISA system. Under the employed experimental conditions, highly sensitive and specific assay of ALP and cardiac troponin I (cTnI) has been accomplished in a straightforward way. Furthermore, the commendable sensing performance of our proposed ELISA in the determination of the cTnI level in diluted human serum unambiguously illustrates great potential in the early diagnosis of acute myocardial infarction.
碱性磷酸酶（ALP）通常在酶联免疫吸附测定（ELISA）中充当信号转导物；因此，开发一种有吸引力的 ALP 活性测定方法，特别是使用更优选的底物，将有助于提高 ELISA 在实际应用中的效率和便利性。在此，我们首次制备了一种原始且有创意的底物，命名为 m-对羟苯基磷酸钠盐（m-HPP），它具有 ALP 理想的去磷酸化位点。基于 ALP 催化 m-HPP 水解生成间苯二酚及其随后与多巴胺的特异性亲核反应，我们开发了一种荧光和比色双读数 ALP 活性测定法和基于 ALP 的 ELISA 系统。在采用的实验条件下，以简单的方式实现了对 ALP 和心肌肌钙蛋白 I（cTnI）的高灵敏和特异性测定。此外，我们提出的 ELISA 在测定稀释人血清中 cTnI 水平方面令人赞赏的传感性能清楚地表明了其在急性心肌梗死早期诊断中的巨大潜力。
