University of Washington Engineered Biomaterials, Department of Bioengineering, University of Washington, Seattle, Washington 98195-5061, USA.
Biointerphases. 2006 Mar;1(1):61. doi: 10.1116/1.2187980.
Thorough studies of protein interactions with stimulus responsive polymers are necessary to provide a better understanding of their applications in biosensors and biomaterials. In this study, protein behavior on a thermoresponsive polymer surface, plasma polymerized N-isopropyl acrylamide (ppNIPAM), is investigated using multiple characterization techniques above and below its lower critical solution temperature (LCST). Protein adsorption and binding affinity are probed using radiolabeled proteins. Protein activity is estimated by measuring the immunological activity of an antibody adsorbed onto ppNIPAM using surface plasmon resonance. Conformation/orientation of the proteins is probed by time-of-flight secondary ion mass spectrometry (TOF-SIMS) and principal component analysis (PCA) of the TOF-SIMS data. In this work, we find that at low protein solution concentrations, ppNIPAM-treated surfaces are low fouling below the LCST, but protein retentive above it. The protein adsorption isotherms demonstrate that apparent affinity between soluble protein molecules and the ppNIPAM surface are an order of magnitude lower at room temperature than at 37 degrees C. Although direct protein desorption is not observed in our study when the surface temperature drops below the LCST, the binding affinity of surface adsorbed protein with ppNIPAM is reduced, as judged by a detergent elution test. Furthermore, we demonstrated that proteins adsorbed onto ppNIPAM are functionally active, but the activity is better preserved at room temperature than 37 degrees C. The temperature dependent difference in protein activity as well as TOF-SIMS and PCA study suggest that proteins take different conformations/orientations after adsorption on ppNIPAM above and below the LCST.
深入研究蛋白质与刺激响应聚合物的相互作用对于更好地理解它们在生物传感器和生物材料中的应用是必要的。在这项研究中，使用多种表征技术研究了蛋白质在热响应聚合物表面（等离子体聚合的 N-异丙基丙烯酰胺（ppNIPAM））上的行为，高于和低于其低临界溶液温度（LCST）。使用放射性标记的蛋白质探测蛋白质的吸附和结合亲和力。通过表面等离子体共振测量吸附在 ppNIPAM 上的抗体的免疫活性来估计蛋白质的活性。通过飞行时间二次离子质谱（TOF-SIMS）和 TOF-SIMS 数据的主成分分析（PCA）探测蛋白质的构象/取向。在这项工作中，我们发现，在低蛋白质溶液浓度下，ppNIPAM 处理的表面在 LCST 以下是低污染的，但在 LCST 以上是保留蛋白质的。蛋白质吸附等温线表明，在室温下，可溶性蛋白质分子与 ppNIPAM 表面之间的表观亲和力比在 37°C 时低一个数量级。尽管当表面温度低于 LCST 时，我们的研究中没有观察到蛋白质的直接解吸，但通过去污剂洗脱试验判断，表面吸附的蛋白质与 ppNIPAM 的结合亲和力降低。此外，我们证明吸附到 ppNIPAM 上的蛋白质具有功能活性，但在室温下比在 37°C 时保存得更好。蛋白质活性的温度依赖性差异以及 TOF-SIMS 和 PCA 研究表明，蛋白质在 LCST 以上和以下吸附到 ppNIPAM 上时会采取不同的构象/取向。
