Sigolaeva Larisa V, Mergel Olga, Evtushenko Evgeniy G, Gladyr Snezhana Yu, Gelissen Arjan P H, Pergushov Dmitry V, Kurochkin Ilya N, Plamper Felix A, Richtering Walter
Department of Chemistry, M.V. Lomonosov Moscow State University , 119991 Moscow, Russia.
Institute of Physical Chemistry II, RWTH Aachen University , 52056 Aachen, Germany.
Langmuir. 2015 Dec 1;31(47):13029-39. doi: 10.1021/acs.langmuir.5b03497. Epub 2015 Nov 17.
This work examines the adsorption regime and the properties of microgel/enzyme thin films deposited onto conductive graphite-based substrates. The films were formed via two-step sequential adsorption. A temperature- and pH-sensitive poly(N-isopropylacrylamide)-co-(3-(N,N-dimethylamino)propylmethacrylamide) microgel (poly(NIPAM-co-DMAPMA microgel) was adsorbed first, followed by its interaction with the enzymes, choline oxidase (ChO), butyrylcholinesterase (BChE), or mixtures thereof. By temperature-induced stimulating both (i) poly(NIPAM-co-DMAPMA) microgel adsorption at T > VPTT followed by short washing and drying and then (ii) enzyme loading at T < VPTT, we can effectively control the amount of the microgel adsorbed on a hydrophobic interface as well as the amount and the spatial localization of the enzyme interacted with the microgel film. Depending on the biomolecule size, enzyme molecules can (in the case for ChO) or cannot (in the case for BChE) penetrate into the microgel interior and be localized inside/outside the microgel particles. Different spatial localization, however, does not affect the specific enzymatic responses of ChO or BChE and does not prevent cascade enzymatic reaction involving both BChE and ChO as well. This was shown by the methods of electrochemical impedance spectroscopy (EIS), atomic force microscopy (AFM), and amperometric analysis of enzymatic responses of immobilized enzymes. Thus, a novel simple and fast strategy for physical entrapment of biomolecules by the polymeric matrix was proposed, which can be used for engineering systems with spatially separated enzymes of different types.
本工作研究了沉积在导电石墨基衬底上的微凝胶/酶薄膜的吸附机制及其性质。这些薄膜通过两步顺序吸附形成。首先吸附温度和pH敏感的聚（N-异丙基丙烯酰胺）-共-（3-（N，N-二甲基氨基）丙基甲基丙烯酰胺）微凝胶（聚（NIPAM-共-DMAPMA微凝胶）），然后使其与胆碱氧化酶（ChO）、丁酰胆碱酯酶（BChE）或它们的混合物相互作用。通过温度诱导刺激（i）在T > VPTT时吸附聚（NIPAM-共-DMAPMA）微凝胶，随后进行短时间洗涤和干燥，然后（ii）在T < VPTT时加载酶，我们可以有效地控制吸附在疏水界面上的微凝胶量以及与微凝胶薄膜相互作用的酶的量和空间定位。根据生物分子大小，酶分子（对于ChO的情况）可以或（对于BChE的情况）不能渗透到微凝胶内部并定位在微凝胶颗粒的内部/外部。然而，不同的空间定位不会影响ChO或BChE的特定酶促反应，也不会阻止涉及BChE和ChO的级联酶促反应。这通过电化学阻抗谱（EIS）、原子力显微镜（AFM）以及固定化酶的酶促反应的安培分析方法得以证明。因此，提出了一种通过聚合物基质物理捕获生物分子的新颖简单且快速的策略，该策略可用于构建具有空间分离的不同类型酶的工程系统。
