Institute of Condensed Matter and Nanosciences , Université catholique de Louvain , Place Louis Pasteur, 1 bte L4.01.10 , B-1348 Louvain-la-Neuve , Belgium.
ACS Nano. 2018 Aug 28;12(8):8372-8381. doi: 10.1021/acsnano.8b03710. Epub 2018 Jul 6.
Layer-by-layer (LbL) assembly is an attractive method for protein immobilization at interfaces, a much wanted step for biotechnologies and biomedicine. Integrating proteins in LbL thin films is however very challenging due to their low conformational entropy, heterogeneous spatial distribution of charges, and polyampholyte nature. Protein-polyelectrolyte complexes (PPCs) are promising building blocks for LbL construction owing to their standardized charge and polyelectrolyte (PE) corona. In this work, lysozyme was complexed with poly(styrenesulfonate) (PSS) at different ionic strengths and pH values. The PPCs size and electrical properties were investigated, and the forces driving complexation were elucidated, in the light of computations of polyelectrolyte conformation, with a view to further unravel LbL construction mechanisms. Quartz crystal microbalance and atomic force microscopy were used to monitor the integration of PPCs compared to the one of bare protein molecules in LbL assemblies, and colorimetric assays were performed to determine the protein amount in the thin films. Layers built with PPCs show higher protein contents and hydration levels. Very importantly, the results also show that LbL construction with PPCs mainly relies on standard PE-PE interactions, independent of the charge state of the protein, in contrast to classical bare protein assembly with PEs. This considerably simplifies the incorporation of proteins in multilayers, which will be beneficial for biosensing, heterogeneous biocatalysis, biotechnologies, and medical applications that require active proteins at interfaces.
层层自组装（LbL）是一种在界面上固定蛋白质的有吸引力的方法，这是生物技术和生物医学非常需要的一步。然而，由于蛋白质的低构象熵、电荷的不均匀空间分布和两性电解质性质，将蛋白质整合到 LbL 薄膜中极具挑战性。蛋白质-聚电解质复合物（PPC）由于其标准化的电荷和聚电解质（PE）冠，是 LbL 构建的有前途的构建块。在这项工作中，溶菌酶在不同的离子强度和 pH 值下与聚苯乙烯磺酸盐（PSS）复合。研究了 PPC 的大小和电学性质，并根据聚电解质构象的计算阐明了驱动复合物形成的力，以期进一步揭示 LbL 构建机制。使用石英晶体微天平（QCM）和原子力显微镜（AFM）监测 PPC 与裸蛋白质分子在 LbL 组装中的整合情况，并进行比色测定以确定薄膜中的蛋白质含量。与 PPC 构建的层相比，PPC 构建的层显示出更高的蛋白质含量和更高的水合水平。非常重要的是，结果还表明，与经典的 bare protein 与聚电解质组装相比，PPC 的 LbL 构建主要依赖于标准的 PE-PE 相互作用，而与蛋白质的电荷状态无关。这大大简化了蛋白质在多层中的掺入，这将有益于需要界面处活性蛋白质的生物传感、多相生物催化、生物技术和医学应用。
