Cavic B A, McGovern M E, Nisman R, Thompson M
Department of Chemistry, University of Toronto, 80 St. George Street, Toronto, Ontario, Canada M5S 3H6.
Analyst. 2001 Apr;126(4):485-90. doi: 10.1039/b008945l.
Oligonucleotide (11-mer) molecules are immobilized on silicon in high surface population using either a permanent thioether bond or a chemo-selectively reversible disulfide bond to the surface thiol functionality. Substrate hydroxy groups are first silanized with an 11 carbon trichlorosilane containing a terminal, protected thiol moiety. Oligonucleotide modified with a tether possessing a terminal thiol group is further derivatized with a water-soluble, halobenzylic bifunctional reagent, which allows the complete conjugate to be attached to the surface through a permanent thioether bond. Alternatively, the oligonucleotide-tether complex can be combined with a pyridyldisulfide compound, which, in turn, facilitates the formation of a reversible disulfide bond with surface thiol. The amount of immobilized oligonucleotide was determined by radiochemical labeling with 32P. Additional verification of surface amounts was obtained from X-ray photoelectron spectroscopic analysis of substrates. The results of the immobilization protocols are compared with the oligonucleotide surface population achieved through the conventional silanizing agent, mercaptopropyltrimethoxysilane. Finally, a preliminary confirmation of duplex formation of a TTU-attached 25-mer with its complementary strand is outlined.
使用与表面硫醇官能团的永久性硫醚键或化学选择性可逆二硫键，将寡核苷酸（11聚体）分子以高表面密度固定在硅上。首先用含有末端保护硫醇部分的11碳三氯硅烷对底物羟基进行硅烷化。用具有末端硫醇基团的连接子修饰的寡核苷酸进一步用一种水溶性卤代苄基双功能试剂进行衍生化，这使得完整的缀合物能够通过永久性硫醚键连接到表面。或者，寡核苷酸-连接子复合物可以与吡啶二硫化物化合物结合，这反过来又促进了与表面硫醇形成可逆二硫键。通过用32P进行放射化学标记来确定固定化寡核苷酸的量。从底物的X射线光电子能谱分析中获得了表面量的额外验证。将固定化方案的结果与通过传统硅烷化剂巯基丙基三甲氧基硅烷实现的寡核苷酸表面密度进行了比较。最后，概述了与互补链形成TTU连接的25聚体双链体的初步确认。
