二硫双琥珀酰亚胺基丙酸酯 (dithiobis-succinimidyl propionate, DTSP)是一种重要的同源双功能偶联分子, 广泛地应用于蛋白质在载体表面的共价固定。 研究利用表面增强红外光谱分析了DTSP分子在真空蒸镀金岛膜表面的吸附特性。 首先通过密度泛函理论对吸附于金表面的DTSP分子进行了结构优化, 计算了该分子的振动模式和红外强度。 表面增强红外吸收和变角偏振反射吸收测量结果表明TSP分子在金表面有序排列, 其五元杂环平面与金表面法线成65°左右的二面角。 此外, 表面增强红外光谱还成功地监测到了TSP分子和Nα′, Nα″二(羧甲基)-L-赖氨酸在金表面的实时组装过程。

Dithiobis-succinimidyl propionate (DTSP), an important homobifunctional crosslinker, has been widely used for the covalent immobilization of proteins onto solid supports by amine coupling. In the present study, adsorption of DTSP on vacuum-deposited gold island films was analyzed by means of surface-enhanced infrared absorption spectroscopy (SEIRAS). For the sake of a reliable assignment of the vibrational spectra, IR intensity of the adsorption model of TSP on one gold surface was calculated using density functional theory (DFT) at the Beck’s three-parameter Lee-Yang-Parr (B3LYP) level with the LANL2DZ basis set. SEIRAS and multiple-angle-of-incidence polarization infrared reflection-absorption spectroscopy indicated that TSP is arranged orderly in a tilted fashion with a dihedral angle of 65 degrees between the plane of succinimidyl ring and the gold surface. The binding kinetics revealed that that the time constant of self-assembly of the TSP layer is 220 sec. Furthermore, the coupling process of amino-nitrilotriacetic acid (ANTA) with surface-bound TSP monolayer was monitored in situ by SEIRAS. Three negative bands observed at 1 807, 1 776, and 1 728 cm-1 respectively provided direct evidence for the reaction of the succinimidyl ester. The appearance of one intense band at 1 566 cm-1 gave a clear support for the presence of the cross-link between ANTA and TSP. We hope that the results in current investigation will contribute to the better understanding of properties of DTSP and related reactions at the molecular level.