Department of Chemistry, Buffalo State, State University of New York, 1300 Elmwood Avenue, Buffalo, New York 14222, USA.
Langmuir. 2010 Feb 16;26(4):2599-608. doi: 10.1021/la904027p.
Fourier transform infrared spectroscopy by grazing-angle attenuated total reflection (FTIR-GATR), ellipsometry, atomic force microscopy (AFM), UV-visible spectroscopy, and fluorescence microscopy were employed to investigate chemical modifications of amino-terminated organic thin films on silicon substrates, protein immobilization, and the biological activity and hydrolytic stability of immobilized proteins. Amino-terminated organic films were prepared on silicon wafers by self-assembling 3-aminopropyltriethoxysilane (APTES) in anhydrous toluene. Surface amino groups were derivatized into three different linkers: N-hydroxysuccinimide (NHS) ester, hydrazide, and maleimide ester groups. UV-visible absorption measurements and fluorescence microscopy revealed that more than 40% of surface amino groups were chemically modified. Protein immobilization was carried out on modified APTES films containing these linkers via coupling with primary amines (-NH(2)) in intact monoclonal rabbit immunoglobulin G (IgG), the aldehyde (-CHO) of an oxidized carbohydrate residue in IgG, or the sulfhydryl (-SH) of fragmented half-IgG, respectively. FTIR spectra contain vibrational signatures of these functional groups present in modified APTES films and immobilized IgGs. Changes in the APTES film thickness after chemical modifications and protein immobilization were also observed by ellipsometric measurements. The biological activity and long-term hydrolytic stability of immobilized IgGs on modified APTES films were estimated by fluorescence measurements of an adsorbed antigen, fluorescein isothiocyanate (FITC)-labeled goat anti-rabbit IgG (FITC-Ab). Our results indicate that the FITC-Ab binding capacity of half-IgG immobilized via maleimide groups is greater than that of the oxidized IgG and the intact IgG immobilized via hydrazide and NHS ester groups, respectively. In addition, IgGs immobilized using all coupling chemistries were hydrolytically stable in phosphate-buffered saline (PBS).
采用掠角衰减全反射傅里叶变换红外光谱（FTIR-GATR）、椭圆光度法、原子力显微镜（AFM）、紫外-可见光谱和荧光显微镜研究了氨基末端有机薄膜在硅衬底上的化学修饰、蛋白质固定化以及固定化蛋白质的生物活性和水解稳定性。在无水甲苯中，通过自组装 3-氨丙基三乙氧基硅烷（APTES）在硅晶片上制备氨基末端有机膜。表面氨基被衍生为三种不同的连接体：N-羟基琥珀酰亚胺（NHS）酯、酰肼和马来酰亚胺酯。紫外-可见吸收测量和荧光显微镜表明，超过 40%的表面氨基被化学修饰。通过与完整的单克隆兔免疫球蛋白 G（IgG）中的伯胺（-NH2）、IgG 中氧化碳水化合物残基的醛基（-CHO）或片段化的半 IgG 中的巯基（-SH）分别偶联，将蛋白质固定在含有这些连接体的修饰的 APTES 膜上。FTIR 光谱包含存在于修饰的 APTES 膜和固定化 IgGs 中的这些官能团的振动特征。通过椭圆光度测量还观察到化学修饰和蛋白质固定化后 APTES 膜厚度的变化。通过吸附抗原荧光测量，即异硫氰酸荧光素（FITC）标记的山羊抗兔 IgG（FITC-Ab），评估修饰的 APTES 膜上固定化 IgG 的生物活性和长期水解稳定性。我们的结果表明，通过马来酰亚胺基团固定的半 IgG 的 FITC-Ab 结合能力大于通过酰肼和 NHS 酯基团固定的氧化 IgG 和完整 IgG。此外，使用所有偶联化学固定的 IgG 在磷酸盐缓冲盐水（PBS）中水解稳定。
