State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences , Changchun, Jilin 130022, China.
Department of Chemistry, University of Science and Technology of China , Hefei, Anhui 230026, China.
Anal Chem. 2016 Oct 4;88(19):9789-9795. doi: 10.1021/acs.analchem.6b02847. Epub 2016 Sep 22.
The emergence of fluorescent nanomaterials with excellent performances has triggered the development of fluorescence analysis technique, which possesses several advantages in the research and clinical applications. However, current strategies for fluorescence immunoassay usually involve the routine fluorophore-labeled antibody and/or awkward signal generation procedure that may not be available in conventional enzyme-linked immunosorbent assay (ELISA) systems. Herein, we circumvent this problem by imparting an exquisite signal generation mechanism to commercially available alkaline phosphatase (ALP)-based ELISA platform and putting forward a conceptual fluorescent ELISA system based on an original ALP-enabled in situ synthesis of fluorescent nanomaterials. After adding target antigen, the presence of ALP labeled on antibody catalyzes the transformation of the substrate ascorbic acid 2-phosphate into ascorbic acid. Then the resultant ascorbic acid (i.e., ascorbate) interacts with amine-containing silane molecules (no fluorescence) to produce intense cyan fluorescent silicon nanoparticles. For the proof-of-concept, alpha-fetoprotein and human immunoglobulin G are chosen as the model antigen targets, and our proposed immunoassay (designated as the nanoparticles generation-based fluorescent ELISA) enables the detection with either fluorescence spectroscopy or naked-eye readout under the ultraviolet lamp. The convincing recognition mechanism and assay performance ensure fluorescent ELISA to quantitatively evaluate the alpha-fetoprotein level in serologic test and potentially apply in the clinic diagnosis of hepatocellular carcinoma.
具有优异性能的荧光纳米材料的出现引发了荧光分析技术的发展，该技术在研究和临床应用中具有几个优势。然而，目前的荧光免疫分析策略通常涉及常规的荧光标记抗体和/或繁琐的信号产生过程，这在常规酶联免疫吸附测定（ELISA）系统中可能不可用。在此，我们通过为市售的碱性磷酸酶（ALP）-基于 ELISA 的平台赋予一种精密的信号产生机制，并提出了一种基于原始 ALP 实现的荧光 ELISA 系统的新概念，从而解决了这个问题。在加入目标抗原后，标记在抗体上的 ALP 催化将作为底物的抗坏血酸 2-磷酸转化为抗坏血酸。然后，所得的抗坏血酸（即抗坏血酸）与含胺的硅烷分子（无荧光）相互作用，产生强烈的青色荧光硅纳米粒子。为了验证概念，选择甲胎蛋白和人免疫球蛋白 G 作为模型抗原靶标，并且我们提出的免疫分析（被指定为基于纳米粒子生成的荧光 ELISA）能够通过荧光光谱或在紫外线灯下进行的肉眼读取进行检测。令人信服的识别机制和分析性能确保了荧光 ELISA 能够定量评估血清学测试中的甲胎蛋白水平，并有可能应用于肝细胞癌的临床诊断。
