A Novel Colorimetric Immunoassay of Ultrasensitive Alpha-Fetoprotein Sensing in Magnetic Bead-Based Mimic Enzyme-Chromogenic Substrate System

This work described a simple and feasible colorimetric immunoassay. Using hemin (a horseradish peroxidase (HRP) mimic enzyme) and the mimic enzyme-chromogenic substrate system, it can qualitatively and quantitatively detect a-fetoprotein (AFP) at an ultralow concentration. The glucose oxidase (GOx) catalyzed oxidation of glucose leads to the formation of gluconic acid and hydrogen peroxide (H2O2). The latter can oxidize 4-aminoatipyrine (4-AAP) to chromogenic products, and the reaction was catalyzed by hemin. With the increase of H2O2, the absorbance increased, and the color of the solution changed from colorless to pink. On the basis of the system, monitored by recording the color or absorbance (λ = 505 nm), a new immunoassay protocol with GOx-labeled anti-AFP detection antibody was designed. A wide linear dependence was obtained in the range from 0.075 to 280 ng mL-1 with a low detection limit (LOD) of 0.0247 ng mL-1 (S/N = 3).

本研究报道了一种简便可行的比色免疫分析法（colorimetric immunoassay）。该方法利用血红素（hemin，一种辣根过氧化物酶（horseradish peroxidase，HRP）模拟酶）及模拟酶-显色底物体系，可实现超低浓度甲胎蛋白（α-fetoprotein，AFP）的定性与定量检测。葡萄糖氧化酶（glucose oxidase，GOx）可催化葡萄糖氧化生成葡萄糖酸与过氧化氢（hydrogen peroxide，H₂O₂）；后者可在血红素的催化作用下，将4-氨基安替比林（4-aminoatipyrine，4-AAP）氧化为显色产物。随着过氧化氢浓度升高，体系吸光度上升，溶液颜色由无色逐渐变为粉红色。基于该体系，通过记录溶液颜色或吸光度（λ=505 nm）进行监测，本研究设计了一种采用GOx标记抗AFP检测抗体的新型免疫分析方案。该方案的线性检测范围为0.075~280 ng·mL⁻¹，检测限（detection limit，LOD）低至0.0247 ng·mL⁻¹（信噪比（signal-to-noise ratio，S/N）=3）。