Hacettepe University, Department of Biology, Ankara, Turkey.
Biosens Bioelectron. 2011 Oct 15;28(1):97-104. doi: 10.1016/j.bios.2011.07.004. Epub 2011 Jul 8.
F(ab) fragments imprinted surface plasmon resonance (SPR) chip was prepared for the real-time detection of human immunoglobulin G (IgG). In order to attach polymerization precursor on SPR chip, the SPR chip surface was modified with allyl mercaptan. F(ab) fragments of the IgG molecules were prepared by papain digestion procedure and collected by fast protein liquid chromatography (FPLC) system using Hi-Trap_r Protein A FF column. The collected F(ab) fragments were complexed with histidine containing specific monomer, N-methacryloyl-l-histidine methyl ester (MAH). Molecular imprinted polymeric nanofilm was prepared on SPR chip in the presence of ethylene glycol dimethacrylate and 2-hydroxyethylmethacrylate. The template molecules, F(ab) fragments, were removed from the polymeric nanofilm using 1M NaCl solution (pH: 7.4, phosphate buffer system). The molecular imprinted SPR chip was characterized by contact angle, atomic force microscopy and Fourier transform infrared spectroscopy. By the real-time IgG detection studies carried out using aqueous IgG solutions in different concentrations, the kinetics and isotherm parameters of the molecular imprinted SPR chip-IgG system were calculated. To show selectivity and specificity of the molecular imprinted SPR chip, competitive kinetic analyses were performed using bovine serum albumin (BSA), IgG, F(ab) and F(c) fragments in singular and competitive manner. As last step, IgG detection studies from human plasma were performed and the measured IgG concentrations were well matched with the results determined by enzyme-linked immunosorbent assay (ELISA). The results obtained with the molecular imprinted SPR chip were well fitted to Langmuir isotherm and the detection limit was found as 56 ng/mL. In the light of the results, we can conclude that the proposed molecular imprinted SPR chip can detect IgG molecules from both aqueous solutions and complex natural samples.
为了实时检测人免疫球蛋白 G（IgG），制备了 F（ab）片段印迹表面等离子体共振（SPR）芯片。为了将聚合前体附着在 SPR 芯片上，用烯丙基硫醇对 SPR 芯片表面进行了修饰。通过木瓜蛋白酶消化程序制备 IgG 分子的 F（ab）片段，并使用 Hi-Trap_r Protein A FF 柱通过快速蛋白液相色谱（FPLC）系统收集。收集的 F（ab）片段与含有组氨酸的特定单体 N-丙烯酰基-L-组氨酸甲酯（MAH）复合。在乙二醇二甲基丙烯酸酯和 2-羟乙基甲基丙烯酸酯存在的情况下，在 SPR 芯片上制备分子印迹聚合物纳米膜。使用 1M NaCl 溶液（pH：7.4，磷酸盐缓冲体系）从聚合物纳米膜中去除模板分子 F（ab）片段。通过接触角、原子力显微镜和傅里叶变换红外光谱对分子印迹 SPR 芯片进行了表征。通过使用不同浓度的水溶液 IgG 进行实时 IgG 检测研究，计算了分子印迹 SPR 芯片-IgG 系统的动力学和等温参数。为了展示分子印迹 SPR 芯片的选择性和特异性，使用牛血清白蛋白（BSA）、IgG、F（ab）和 F（c）片段以单一和竞争的方式进行了竞争动力学分析。最后，对来自人血浆的 IgG 检测研究进行了研究，并且用酶联免疫吸附测定（ELISA）测定的 IgG 浓度与测量的 IgG 浓度非常匹配。用分子印迹 SPR 芯片获得的结果与 Langmuir 等温线拟合良好，检测限为 56ng/mL。根据这些结果，我们可以得出结论，所提出的分子印迹 SPR 芯片可以从水溶液和复杂的天然样品中检测 IgG 分子。
