Department of Chemistry, University of Illinois at Urbana-Champaign, 600 S. Matthews Ave., Urbana, IL 61801, USA.
Phys Chem Chem Phys. 2010 May 7;12(17):4301-8. doi: 10.1039/b920713a. Epub 2009 Dec 4.
We describe a modified bifunctional analogue of polyacrylamide that spontaneously forms self-assembled polymeric thin films on Au surfaces. The film is engineered to specifically bind histidine tagged proteins (6His), while simultaneously remaining inherently resistant to the non-specific adsorption of proteins in solution. The backbone of a polyacrylamide-co-n-acryloxysuccinimide copolymer is functionalized via tandem active ester (NHS) couplings with 3-(methylthio)propylamine (MTP) and nitrilotriacetic acid (NTA). The resulting functionalized polymers form stable and exceptionally hydrophilic thin films that are approximately 2-5 nm thick, a mass coverage that varies with the MTP graft density. These films are characterized using a variety of techniques (X-ray photoelectron spectroscopy (XPS), reflection absorption infrared spectroscopy (RAIRS), ellipsometry, surface plasmon resonance (SPR), and matrix assisted laser desorption ionization (MALDI)) to establish their structure and function. The protein resistance of the films, as demonstrated by their exposure to solutions of bovine serum albumin (BSA), can be modulated by the amount of MTP grafted to the polymer, which in turn, affects their mass coverage. We show that it is possible to specifically capture hexahistidine tagged proteins with low incidences of nonspecific adsorption using these materials, a discrimination quantified using surface plasmon resonance (SPR) at concentrations down to approximately 20 nM. These polymers also bind strongly to the surfaces of Au nanoparticles, stabilizing them against aggregation, providing them with a similar capacity to selectively bind 6His tagged proteins that can then be speciated using MALDI.
我们描述了一种聚丙酰胺的改良双功能类似物，它能在 Au 表面自发形成自组装聚合物薄膜。该薄膜经过设计，可特异性结合组氨酸标记蛋白（6His），同时对溶液中蛋白质的非特异性吸附具有固有抗性。聚丙烯酰胺-co-n-丙烯酰氧基琥珀酰亚胺共聚物的骨架通过与 3-（甲硫基）丙胺（MTP）和氮三乙酸（NTA）的串联活性酯（NHS）偶联进行功能化。所得功能化聚合物形成稳定且非常亲水的薄膜，厚度约为 2-5nm，其质量覆盖率随 MTP 接枝密度而变化。使用各种技术（X 射线光电子能谱（XPS）、反射吸收红外光谱（RAIRS）、椭圆光度法、表面等离子体共振（SPR）和基质辅助激光解吸电离（MALDI））对这些薄膜进行表征，以确定其结构和功能。这些薄膜对蛋白质的抗性，如通过它们在牛血清白蛋白（BSA）溶液中的暴露来证明，可以通过接枝到聚合物上的 MTP 量来调节，这反过来又影响它们的质量覆盖率。我们表明，使用这些材料可以特异性地捕获六组氨酸标记的蛋白质，而不会发生非特异性吸附，这种选择性使用表面等离子体共振（SPR）在大约 20nM 的浓度下进行量化。这些聚合物还能强烈结合 Au 纳米粒子的表面，防止它们聚集，为它们提供了类似的能力来特异性结合 6His 标记的蛋白质，然后可以使用 MALDI 对其进行特异性分析。
