School of Life Science, Beijing Institute of Technology, Beijing 100081, China; Center for Biomedical Engineering, University of Kentucky, Lexington, KY 40506, USA.
Talanta. 2010 Nov 15;83(1):156-61. doi: 10.1016/j.talanta.2010.08.055. Epub 2010 Sep 6.
Molecular imprinting is a method to fabricate a polymeric material (molecularly imprinted polymer or MIP) capable of selectively recognizing template molecules. Molecular imprinting of small molecules has been studied widely. Less common, however, is the imprinting of biological macromolecules, including proteins, among which lysozyme is an important molecule in the food, pharmaceutical, and diagnostic sciences. In this study, lysozyme MIP was fabricated in two steps. First, lysozyme, PEG600DMA, and methacrylic acid were used as the template molecule, cross-linking monomer, and the functional monomer, respectively, in a UV free-radical polymerization process to synthesize a polymeric gel. Second, lysozyme was removed by enzymatic digestion. Non-imprinted polymer (NIP) was synthesized without lysozyme addition. To evaluate the preferential binding capability of MIP, lysozyme, RNase A, or a 50:50 mixture of lysozyme and RNase A was added to MIP and NIP and then released by digestion. It was found that when more lysozyme was added to the reaction mixture, the quantity of protein released from the polymer increased, reflecting more potential binding sites. Tests of MIP with a competitive binding mixture of lysozyme and RNase A showed the MIP preferentially bound a greater amount of lysozyme, up to 20 times more than RNase A. NIP bound only small amounts of both proteins and did not show a preference for binding either lysozyme or RNase A. These results demonstrate that lysozyme was successfully imprinted into the MIP by UV free-radical polymerization, and the fabricated MIP was able to preferentially bind its template protein.
分子印迹是一种制备能够选择性识别模板分子的聚合物材料（分子印迹聚合物或 MIP）的方法。小分子的分子印迹已经得到了广泛的研究。然而，生物大分子的印迹，包括蛋白质，却不那么常见，其中溶菌酶是食品、制药和诊断科学中的重要分子。在这项研究中，溶菌酶 MIP 通过两步法制备。首先，将溶菌酶、PEG600DMA 和甲基丙烯酸用作模板分子、交联单体和功能单体，在紫外光自由基聚合过程中合成聚合物凝胶。其次，通过酶解去除溶菌酶。未印迹聚合物（NIP）是在没有添加溶菌酶的情况下合成的。为了评估 MIP 的优先结合能力，将溶菌酶、核糖核酸酶 A 或溶菌酶和核糖核酸酶 A 的 50:50 混合物添加到 MIP 和 NIP 中，然后通过酶解释放。结果发现，当向反应混合物中添加更多的溶菌酶时，从聚合物中释放的蛋白质量增加，这反映出更多的潜在结合位点。用溶菌酶和核糖核酸酶 A 的竞争性结合混合物对 MIP 进行测试表明，MIP 优先结合更多的溶菌酶，高达 20 倍于核糖核酸酶 A。NIP 仅结合少量的两种蛋白质，并且对结合溶菌酶或核糖核酸酶 A 没有偏好。这些结果表明，溶菌酶通过紫外光自由基聚合成功地印迹到 MIP 中，并且制备的 MIP 能够优先结合其模板蛋白。
