Molecular tags such as fluorophores are increasingly being replaced with nanoparticles thanks to their superior optical properties, substantial chemical stability, and stability against photobleaching. Herein, we innovatively constructed a new ratiometric fluorescence enzyme-linked immunosorbent assay (RF-ELISA) for the screening of alpha-fetoprotein (AFP) in early hepatocellular carcinoma in vitro diagnostics using carbon dots@SiO₂@CdTe quantum dots (CDs@SiO₂@CdTe QDs). Carbon dots with blue fluorescence were initially encapsulated into SiO₂ nanospheres through the typical Stöber method. Thereafter, CdTe QDs with red fluorescence were modified onto the surface of CDs@SiO₂ nanospheres. Dual-emission nanotags with blue and red fluorescent signals were utilized to design a RF-ELISA method for the determination of AFP on the anti-AFP capture antibody-coated microplate using glucose oxidase (GOx)-labeled anti-AFP secondary antibody. After the formation of the sandwiched immunocomplex, GOx catalyzed glucose to generate hydrogen peroxide (H₂O₂), which could quench the red fluorescence of CdTe QDs on the surface of nanotags. Meanwhile, the encapsulated carbon dots in the nanotags could still maintain the initial blue fluorescence intensity. The ratio between red fluorescence intensity and blue-emission intensity could be used for the quantitative monitoring of AFP concentration under optimum conditions. The experimental results indicated that CDs@SiO₂@CdTe QDs-based RF-ELISA could exhibit a good fluorescence signal with a dynamic linear range of 0.05–60 ng mL⁻¹ at a low detection limit of 8.7 pg mL⁻¹. Moreover, the fluorescence color of the solution including CDs@SiO₂@CdTe QDs changed from pink to purple to blue with the increasing AFP level when viewed by the naked eye. Good reproducibility, high specificity, and acceptable stability were achieved for the analysis of target AFP. Importantly, the accuracy of ratiometric fluorescence immunoassay was evaluated to determine human serum samples, giving well-matched results relative to commercially usable human AFP ELISA method.

中文翻译：

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甲胎蛋白双功能碳点@SiO2@CdTe量子点的比率荧光酶联免疫吸附测定

荧光团等分子标签正越来越多地被纳米颗粒取代，因为它们具有出色的光学特性、显着的化学稳定性和抗光漂白的稳定性。在此，我们创新构建了一种新的比率荧光酶联免疫吸附试验（RF-ELISA），用于使用碳点@SiO ₂ @CdTe量子点（CDs@SiO _{2 个}@CdTe 量子点）。具有蓝色荧光的碳点最初通过典型的 Stöber 方法封装到 SiO _{2纳米球中。}此后，将具有红色荧光的 CdTe QDs 修饰到 CDs@SiO _{2的表面上}纳米球。利用具有蓝色和红色荧光信号的双发射纳米标签设计了一种 RF-ELISA 方法，用于使用葡萄糖氧化酶 (GOx) 标记的抗 AFP 二抗测定涂有抗 AFP 捕获抗体的微孔板上的 AFP。夹层免疫复合物形成后，GOx 催化葡萄糖生成过氧化氢（H ₂ O ₂），可以淬灭纳米标签表面CdTe量子点的红色荧光。同时，纳米标签中封装的碳点仍能保持初始的蓝色荧光强度。红色荧光强度与蓝色发射强度的比值可用于定量监测最佳条件下的AFP浓度。实验结果表明，CDs@SiO₂基于@CdTe QDs 的RF-ELISA 可以表现出良好的荧光信号，动态线性范围为0.05-60 ng mL ^-1，检测限低至8.7 pg mL ^-1。此外，当肉眼观察时，包括CDs@SiO _{2 @CdTe QDs的溶液的荧光颜色随着AFP水平的增加而从粉红色变为紫色变为蓝色。}目标 AFP 的分析具有良好的重现性、高特异性和可接受的稳定性。重要的是，对比率荧光免疫测定法的准确性进行了评估，以确定人血清样本，相对于商业上可用的人 AFP ELISA 方法给出了良好匹配的结果。