In this work, we demonstrate a novel molecular imprinting method by unfolding and refolding an auxiliary protein in the presence of a small-molecular template, followed by fixation of the resultant protein/template assembly via polymerization of additional functional monomers. Employing doxorubicin (DOX) as a model template and bovine serum albumin (BSA) as an auxiliary binding protein, we optimized the conformation of BSA through thermal denaturation and renaturation process in the presence of template DOX. Then, in-situ polymerization of isopropylacrylamide, N-[3-(dimethylamino)propyl]methacrylamide and acrylamide was carried out to form imprinted hydrogels of the dBSA&DOX complex. The conformation of BSA in the resultant polymer was further fixed via formaldehyde cross-linking with the hydrogel network. The obtained imprinted hydrogel showed much higher affinity and selectivity to DOX than both native BSA and the control hydrogel prepared by using native BSA instead of unfolded/refolded BSA. The method may be further extended to prepare water compatible imprinted hydrogel against other small molecules by using easily accessible proteins as auxiliary ligands.

在这项工作中，我们展示了一种新的分子印迹方法，通过在小分子模板存在的情况下展开和重新折叠辅助蛋白质，然后通过附加功能单体的聚合固定所得蛋白质/模板组件。使用阿霉素（DOX）作为模型模板，牛血清白蛋白（BSA）作为辅助结合蛋白，我们在模板 DOX 存在下通过热变性和复性过程优化了 BSA 的构象。然后，进行异丙基丙烯酰胺、N-[3-(二甲氨基)丙基]甲基丙烯酰胺和丙烯酰胺的原位聚合以形成dBSA&DOX复合物的印迹水凝胶。所得聚合物中 BSA 的构象通过甲醛与水凝胶网络的交联进一步固定。与天然 BSA 和使用天然 BSA 代替未折叠/重折叠 BSA 制备的对照水凝胶相比，获得的印迹水凝胶对 DOX 显示出更高的亲和力和选择性。该方法可以进一步扩展到通过使用容易获得的蛋白质作为辅助配体来制备针对其他小分子的水相容印迹水凝胶。