School of Materials Science and Engineering , University of Jinan , Jinan , Shandong 250022 , China.
State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry , Chinese Academy of Sciences , Changchun , Jilin 130022 , China.
Anal Chem. 2018 Mar 6;90(5):3505-3511. doi: 10.1021/acs.analchem.7b05325. Epub 2018 Feb 9.
A simple and cost-effective fluorescence immunoassay for the sensitive quantitation of disease biomarker α-fetoprotein (AFP) has been developed based on the phosphate-triggered fluorescence turn-on detection of alkaline phosphatase (ALP), with the reversible binding between calcein and Ce as a signaling element. In this immunoassay, fluorescent calcein is readily quenched by Ce via a coordination process. The ALP-catalyzed hydrolysis of p-nitrophenyl phosphate leads to the formation of p-nitrophenol and inorganic orthophosphate, and the newly formed orthophosphate could potently combine with Ce due to the higher affinity, thus, recovering the fluorescence of calcein. The corresponding fluorescence signal triggered by phosphate is related to ALP activities labeled on antibody, and thus could be applied to detect target antigen in an enzyme-linked immunosorbent assay (ELISA) platform. The fluorescence intensity correlated well to the AFP concentration ranges of 0.2-1.0 and 1.0-4.0 ng/mL, with a detection limit of 0.041 ng/mL. The proposed fluorescence ELISA possesses convincing recognition mechanism and exhibits excellent assay performance in the evaluation of the AFP level in serologic test, which unambiguously reveals great application potential in the clinic diagnosis of disease biomarkers.
基于碱性磷酸酶（ALP）的磷酸触发荧光开启检测，利用钙黄绿素和铈之间的可逆结合作为信号元件，开发了一种用于灵敏定量疾病生物标志物甲胎蛋白（AFP）的简单且经济高效的荧光免疫分析方法。在该免疫分析中，荧光钙黄绿素可通过配位过程被铈迅速猝灭。ALP 催化对硝基苯磷酸酯的水解生成对硝基苯酚和无机正磷酸盐，新形成的正磷酸盐由于更高的亲和力而与铈强力结合，从而恢复钙黄绿素的荧光。由磷酸盐触发的相应荧光信号与抗体上标记的 ALP 活性相关，因此可应用于酶联免疫吸附测定（ELISA）平台中检测靶抗原。荧光强度与 AFP 浓度范围 0.2-1.0 和 1.0-4.0ng/mL 相关，检测限为 0.041ng/mL。所提出的荧光 ELISA 具有令人信服的识别机制，并在血清学测试中 AFP 水平的评估中表现出优异的分析性能，明确显示了其在疾病生物标志物临床诊断中的巨大应用潜力。
