Halliwell C M, Cass A E
Department of Biochemistry, Imperial College of Science, Technology and Medicine, University of London, UK.
Anal Chem. 2001 Jun 1;73(11):2476-83. doi: 10.1021/ac0010633.
The modification of glass surfaces with (3-mercaptopropyl)trimethoxysilane and the application of this to DNA chip technology are described. A range of factors influencing the silanization method, and hence the number of surface-bound, chemically active thiol groups, were investigated using a design of experiment approach based on analysis of variance. The number of thiol groups introduced on glass substrates were measured directly using a specific radiolabel, [14C]cysteamine hydrochloride. For liquid-phase silanization, the number of surface-bound thiol groups was found to be dependent on both postsilanization thermal curing and silanization time and relatively independent of silane concentration, reaction temperature, and sample pretreatment. Depending on the conditions used in liquid-phase silanization, (1.3-9.0) x 10(12) thiol groups/cm2 on the glass samples were bound. The reliability and repeatability of liquid- and vacuum-phase silanization were also investigated. Eighteen-base oligonucleotide probes were covalently attached to the modified surfaces via a 3'-amino modification on the DNA and subsequent reaction with the cross-linking reagent N-(gamma-maleimidobutyryloxy) succinimide ester (GMBS). The resulting probe levels were determined and found to be stoichiometric with that of the introduced thiol groups. These results demonstrate that silanization of glass surfaces under specific conditions, prior to probe attachment, is of great importance in the development of DNA chips that use the simple concept of the covalent attachment of presynthesized oligonucleotides to silicon oxide surfaces.
描述了用（3-巯基丙基）三甲氧基硅烷对玻璃表面进行改性及其在DNA芯片技术中的应用。采用基于方差分析的实验设计方法，研究了一系列影响硅烷化方法以及表面结合的化学活性硫醇基团数量的因素。使用特定的放射性标记物[14C]盐酸半胱胺直接测量玻璃基板上引入的硫醇基团数量。对于液相硅烷化，发现表面结合的硫醇基团数量既取决于硅烷化后的热固化和硅烷化时间，又相对独立于硅烷浓度、反应温度和样品预处理。根据液相硅烷化所使用的条件，玻璃样品上结合了（1.3 - 9.0）×10（12）个硫醇基团/cm2。还研究了液相和真空相硅烷化的可靠性和可重复性。通过对DNA进行3'-氨基修饰并随后与交联试剂N-（γ-马来酰亚胺丁酰氧基）琥珀酰亚胺酯（GMBS）反应，将18碱基的寡核苷酸探针共价连接到改性表面。测定了所得探针水平，发现其与引入的硫醇基团化学计量相关。这些结果表明，在探针连接之前，在特定条件下对玻璃表面进行硅烷化对于开发利用预合成寡核苷酸共价连接到氧化硅表面这一简单概念的DNA芯片至关重要。
