糖芯片制备的关键技术:基于氨基改性表面的糖分子固定方法及其表面表征

Preparation and characterization of APTES-modified surfaces immobilized with glycans

糖芯片已经成为了研究糖生物学和发展相关生物医学应用的重要工具。糖芯片制备的关键技术是如何在芯片基质表面固定糖分子。虽然氨基改性表面已被用于蛋白质、核酸和小分子芯片的制备,然而利用该表面制备糖芯片的策略鲜有报道。该文以乳糖为例,提出了在氨基改性表面采用二乙烯基砜为偶联试剂共价键固定糖分子的策略。利用天然糖以及衍生化物,设计了三种固定乳糖的方法,构筑了基于氨基改性表面的糖芯片样品。通过酶联凝集素分析技术(enzyme-linked lectin assay,ELLA)初步评价了上述样品,并确定最优路线为乙烯基砜衍生化的脱氧氨基乳糖(化合物1)与氨基改性表面反应。在此基础上,采用生物膜干涉实验定量研究了固定的乳糖与血凝素蛋白质之间多价态作用;通过X-射线光电子能谱(XPS)考察了乳糖固定前后样品表面上元素和碳化学物种的变化。该文发展的以二乙烯基砜为偶联试剂的糖分子固定策略,为氨基改性表面在糖芯片制备方面的应用提供了有效的方法和评价手段,也适用于多种含糖生物表界面的制备和表征。

Glycan arrays have becoming increasingly important for both glycobiology and glycan-involved bio- medical applications. A key step for glycan array fabrication is the immobilization of glycan molecules on the surface of an array substrate. Many strategies based on APTES-modified surfaces have been developed to fabricate protein, DNA and small molecules arrays, though few were documented for glycan arrays. To make this type of readily available surfaces of value for glycan arrays, we employed divinyl sulfone （DVS） as a crosslinker to enable covalent immobilization of lactose, an example of the glycan family, onto such surfaces. Three routes were investigated, differed by the type of lactose used, i.e., derivative with amine group, or vinyl sulfone （VS） group, or without any functionalization, respectively. By studying protein binding activity on these lactose-immobilized surfaces via enzyme linked lectin assay （ELLA）, the most optimal route was revealed to be the one using VS-bearing lactose （compound 1）. Furthermore, multivalent interactions between lectin and the bound lactose were quantitatively studied using Biolayer interferometry （BLI）. Changes in the surface chemical compositions before and after glycan immobilization were also characterized using X-ray photoelectron spectroscopy （XPS）. The DVS-based immobilization strategy reported herein opens up the potential application of APTES-modified surfaces for glycan array preparation. This study also signifies the role of surface analysis techniques for characterization of various biointerfaces presenting glycan molecules.