State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University , Nanjing, Jiangsu 210093, China.
Anal Chem. 2014 Jan 7;86(1):959-66. doi: 10.1021/ac403736y. Epub 2013 Dec 24.
Molecularly imprinted polymers (MIPs), as inexpensive and stable substitutes of antibodies, have shown great promise in immunoassays. Glycoproteins are of significant diagnostic value. To facilitate the application of MIPs in clinical diagnostics, a general and facile imprinting method toward glycoproteins oriented for an enzyme-linked immunosorbent assay (ELISA) in the form of a 96-well microplate is essential but has not been fully explored yet. In this study, a new method called boronate affinity-based oriented surface imprinting was proposed for facile preparation of glycoprotein-imprinted microplates. A template glycoprotein was first immobilized by a boronic acid-modified microplate through boronate affinity binding, and then, a thin layer of polyaniline was formed to cover the microplate surface via in-water self-copolymerization. After the template was removed by an acidic solution, 3D cavities that can rebind the template were fabricated on the microplate surface. Using horseradish peroxidase (HRP) as a model target, the effects of imprinting conditions as well as the properties and performance of the prepared MIPs were investigated. α-Fetoprotein (AFP)-imprinted microplate was then prepared, and thereby, a MIP-based ELISA method was established. The prepared MIPs exhibited several highly favorable features, including excellent specificity, widely applicable binding pH, superb tolerance for interference, high binding strength, fast equilibrium kinetics, and reusability. The MIP-based ELISA method was finally applied to the analysis of AFP in human serum. The result was in good agreement with that by radioimmunoassay, showing a promising prospect of the proposed method in clinical diagnostics.
分子印迹聚合物（MIPs）作为抗体的廉价且稳定替代品，在免疫分析中显示出巨大的应用前景。糖蛋白具有重要的诊断价值。为了促进 MIP 在临床诊断中的应用，需要开发一种通用且简便的糖蛋白印迹方法，用于以 96 孔微孔板形式进行酶联免疫吸附测定（ELISA），但目前尚未得到充分探索。在这项研究中，提出了一种称为硼酸亲和定向表面印迹的新方法，用于简便制备糖蛋白印迹微孔板。首先通过硼酸改性微孔板通过硼酸亲和结合将模板糖蛋白固定化，然后通过在水中自聚合形成薄的聚苯胺层覆盖微孔板表面。通过酸性溶液去除模板后，在微孔板表面上制造出可以重新结合模板的 3D 腔。使用辣根过氧化物酶（HRP）作为模型靶标，研究了印迹条件以及制备的 MIP 的性质和性能的影响。然后制备了α-胎蛋白（AFP）印迹微板，并建立了基于 MIP 的 ELISA 方法。所制备的 MIPs 具有几个非常有利的特征，包括优异的特异性、广泛适用的结合 pH 值、对干扰的出色耐受性、高结合强度、快速平衡动力学和可重复使用性。最后，将基于 MIP 的 ELISA 方法应用于人血清中 AFP 的分析。结果与放射免疫测定法吻合良好，表明该方法在临床诊断中有很好的应用前景。
