Sensitive detection of liver disease biomarkers can facilitate the diagnosis of primary hepatoma and other benign liver diseases, and the alpha fetoprotein (AFP) was selected as the model macromolecule in this work. Herein an enzyme cascade-amplified immunoassay (ECAIA) based on the nanobody-alkaline phosphatase fusion (Nb-ALP) and MnO₂ nanoflakes was developed for detecting AFP. The bifunctional biological macromolecule Nb-ALP serves as the detection antibody and the reporter molecule. The MnO₂ nanoflakes mimic the oxidase for catalyzing the 3,3′,5,5′-tetramethylbenzidine (TMB) into the blue oxidized TMB, which has a quantitative signal at the wavelength of 650 nm. Moreover, the Nb-ALP could dephosphorylate the ascorbic acid-2-phosphate (AAP) to form the ascorbic acid (AA) that can disintegrate the nanoflakes to reduce their oxidation capacity with the content decrease of the oxidized TMB. Using the constructed TMB-MnO₂ colorimetric sensing system for Nb-ALP and the optimized experimental parameters, the ECAIA has a limit of detection (LOD) of 0.148 ng/mL which is 18.7-fold lower than that of the p-nitrophenylphosphate (pNPP)-based method (LOD = 2.776 ng/mL). The ECAIA showed good selectivity for AFP with observed negligible cross-reactions with several common cancer biomarkers. The recovery rate for AFP spiked in human serum ranged from 94.8% to 113% with the relative standard deviation from 0.3% to 6.5%. For analysis of the actual human serum samples, a good linear correlation was found between the results tested by the ECAIA and the automatic chemiluminescence analyzer. Thus, the ECAIA was demonstrated to be a promising tool for highly sensitive and selective detection of AFP, providing a reference for analysis of other macromolecule biomarkers.

肝病生物标志物的灵敏检测可以促进原发性肝癌和其他良性肝病的诊断，本工作选择甲胎蛋白（AFP）作为模型大分子。在此，开发了基于纳米抗体-碱性磷酸酶融合 (Nb-ALP) 和 MnO ₂纳米薄片的酶级联放大免疫测定 (ECAIA)来检测 AFP。双功能生物大分子 Nb-ALP 作为检测抗体和报告分子。MnO ₂纳米薄片模拟氧化酶，用于将 3,3',5,5'-四甲基联苯胺 (TMB) 催化为蓝色氧化 TMB，其在 650 nm 波长处具有定量信号。此外，Nb-ALP 可以使 2-磷酸抗坏血酸 (AAP) 去磷酸化，形成抗坏血酸 (AA)，抗坏血酸 (AA) 可以分解纳米薄片，随着氧化 TMB 含量的降低，降低其氧化能力。使用构建的TMB-MnO ₂Nb-ALP 比色传感系统和优化的实验参数，ECAIA 的检测限 (LOD) 为 0.148 ng/mL，比基于对硝基苯磷酸盐 (pNPP) 的方法 (LOD = 2.776 纳克/毫升）。ECAIA 对 AFP 显示出良好的选择性，观察到与几种常见癌症生物标志物的交叉反应可忽略不计。人血清中加标 AFP 的回收率为 94.8% 至 113%，相对标准偏差为 0.3% 至 6.5%。对于实际人血清样品的分析，发现ECAIA和自动化学发光分析仪测试的结果之间存在良好的线性相关性。因此，ECAIA 被证明是一种具有高灵敏度和选择性检测 AFP 的有前途的工具，为其他大分子生物标志物的分析提供了参考。