Simple and rapid detection of disease-related bio-markers are significant for early clinical diagnosis and can potentially improve the survival rate. However, establishing a high-specificity colorimetric detection method for bio-markers are still challenges due to their inevitable natural antibody used or enzymatic labeling. Herein, a cost-efficient and easy-to-use approach, which called dual molecular imprinting immunosandwich colorimetric strategy (DMI-ICS) was constructed for detection alpha-2-macroglobulin (α2MG) by janus imprinted nanoparticles. The unique detection principle was contained with two mimic antibody parts, the first part was α2MG glass slides molecularly imprinted material (GS-MIP) as a “Separation antibody”, which can specifically rapid separate the protein in the complex sample; Another part was asymmetrically modified janus molecularly imprinted gold nanoparticles nanozyme (J-GNPs-MIP) as a “Detection antibody”, which has the properties of specific recognition and catalytic substrate color performance at the same time. The concentration of α2MG can be determined by the substrate color changes and observed with naked eyes. Under the optimized conditions, the DMI-ICS had a great performance and offering lower relative standard deviation (RSD, 7.69%), good linear range (0.297–130 μg/mL, R² = 0.994), high imprinting factor (IF: 3.74) with lower detection limit (0.089 μg/mL). This strategy provides an easy operation and low cost signal readout method for direct detection and separation of α2MG in human serum samples, which is a versatile tool for point-of-care diagnosis, while also offering a new perspective on antibody simulation technology, multifunctional antibody preparation and contribute to detection of disease-related bio-marker in nonspecialized laboratory infrastructure.

简单快速地检测疾病相关的生物标志物对于早期临床诊断具有重要意义，并可能提高生存率。然而，由于不可避免地使用天然抗体或酶标记，为生物标志物建立高特异性比色检测方法仍然是一个挑战。本文构建了一种经济高效且易于使用的方法，称为双分子印迹免疫夹心比色策略 (DMI-ICS)，用于通过 Janus 印迹纳米粒子检测 α-2-巨球蛋白 (α2MG)。独特的检测原理包含两个模拟抗体部分，第一个部分是α2MG载玻片分子印迹材料（GS-MIP）作为“分离抗体”，可以特异性快速分离复杂样品中的蛋白质；另一部分是不对称修饰的janus分子印迹金纳米粒子纳米酶（J-GNPs-MIP）作为“检测抗体”，它同时具有特异性识别和催化底物显色性能。α2MG的浓度可以通过底物颜色的变化来确定，并用肉眼观察。在优化条件下，DMI-ICS 具有出色的性能，并提供较低的相对标准偏差（RSD，7.69%），良好的线性范围（0.297–130 μg/mL，R²  = 0.994)，印记因子高 (IF: 3.74)，检测限低 (0.089 μg/mL)。该策略为直接检测和分离人血清样品中的α2MG提供了一种操作简单、成本低的信号读出方法，是一种用于即时诊断的多功能工具，同时也为抗体模拟技术、多功能抗体提供了新的视角准备并有助于在非专业实验室基础设施中检测疾病相关的生物标志物。