M. Smoluchowski Institute of Physics , Jagiellonian University , Łojasiewicza 11 , 30-348 Kraków , Poland.
Institute of Nuclear & Radiological Sciences & Technology, Energy & Safety , NCSR Demokritos , 15310 Agia Paraskevi , Greece.
Langmuir. 2019 Feb 26;35(8):3058-3066. doi: 10.1021/acs.langmuir.8b03509. Epub 2019 Feb 14.
The orientation of biotin-binding sites of streptavidin adsorbed to thin films of three polythiophenes (PTs), namely, regioregular poly(3-hexylthiophene) (RP3HT), regiorandom poly(3-butylthiophene) (P3BT), and poly(3,3‴-didodecylquaterthiophene) (PQT12), has been investigated. Polymer films were examined prior to and after protein adsorption with atomic force microscopy and time-of-flight secondary ion mass spectrometry (ToF-SIMS). Principal component analysis (PCA) applied to ToF-SIMS data revealed subtle changes in surface chemistry of polymer films and orientation of adsorbed streptavidin. PCA resolved the surface alignment of alkyl side chains and differentiated the ToF-SIMS data for PQT12, RP3HT, and P3BT, verifying an amorphous morphology for P3BT and a semicrystalline one for PQT12 and RP3HT. After the characterization of the polymeric films, streptavidin adsorption from solutions with different protein concentrations (up to 300 μg/mL) has been conducted. The PCA results distinguished between amino acids characteristic for external regions of streptavidin molecules adsorbed to different PTs suggest that streptavidin adsorbed to PQT12 exposes molecular regions rich in tryptophan and tyrosine, which are components of the biotin-binding sites. The latter results were confirmed using biotin-labeled horse radish peroxidase to estimate the exposed binding sites of streptavidin adsorbed onto the different PT films. The analysis of streptavidin structure suggests that interaction between polythiophene film and dipole moment of streptavidin subunit is responsible for orientation of biotin-binding sites.
已研究了吸附在三种聚噻吩（PT）薄膜上的链霉亲和素的生物素结合位的取向，这三种聚噻吩分别是：（1）具有规整形（regioregular）的聚（3-己基噻吩）（RP3HT），（2）具有规乱型（regiorandom）的聚（3-丁基噻吩）（P3BT），和（3）聚（3,3‴-二十二烷基-四噻吩）（PQT12）。在吸附蛋白质前后，利用原子力显微镜和飞行时间二次离子质谱法（ToF-SIMS）对聚合物薄膜进行了检查。主成分分析（PCA）应用于 ToF-SIMS 数据，揭示了聚合物薄膜表面化学和吸附链霉亲和素的取向的细微变化。PCA 解析了烷基侧链的表面排列，并区分了 PQT12、RP3HT 和 P3BT 的 ToF-SIMS 数据，验证了 P3BT 的无定形形态和 PQT12 和 RP3HT 的半晶形态。在对聚合物薄膜进行了表征之后，从不同蛋白质浓度（高达 300μg/mL）的溶液中进行了链霉亲和素的吸附。PCA 结果区分了吸附在不同 PT 上的链霉亲和素分子的外部区域的特征氨基酸，表明吸附在 PQT12 上的链霉亲和素暴露了富含色氨酸和酪氨酸的分子区域，这些区域是生物素结合位的组成部分。后者的结果使用生物素标记的辣根过氧化物酶进行了确认，以估计吸附在不同 PT 薄膜上的链霉亲和素暴露的结合位。链霉亲和素结构的分析表明，聚噻吩薄膜与链霉亲和素亚基的偶极矩之间的相互作用负责生物素结合位的取向。
