Lee Chi-Ying, Gong Ping, Harbers Gregory M, Grainger David W, Castner David G, Gamble Lara J
National ESCA and Surface Analysis Center for Biomedical Problems, University of Washington, Seattle, WA 98195-1750, USA.
Anal Chem. 2006 May 15;78(10):3316-25. doi: 10.1021/ac052137j.
Self-assembly of thiol-terminated single-stranded DNA (HS-ssDNA) on gold has served as an important model system for DNA immobilization at surfaces. Here, we report a detailed study of the surface composition and structure of mixed self-assembled DNA monolayers containing a short alkylthiol surface diluent [11-mercapto-1-undecanol (MCU)] on gold supports. These mixed DNA monolayers were studied with X-ray photoelectron spectroscopy (XPS), near-edge X-ray absorption fine structure spectroscopy (NEXAFS), and fluorescence intensity measurements. XPS results on sequentially adsorbed DNA/MCU monolayers on gold indicated that adsorbed MCU molecules first incorporate into the HS-ssDNA monolayer and, upon longer MCU exposures, displace adsorbed HS-ssDNA molecules from the surface. Thus, HS-ssDNA surface coverage steadily decreased with MCU exposure time. Polarization-dependent NEXAFS and fluorescence results both show changes in signals consistent with changes in DNA orientation after only 30 min of MCU exposure. NEXAFS polarization dependence (followed by monitoring the N 1s --> pi* transition) of the mixed DNA monolayers indicated that the DNA nucleotide base ring structures are oriented more parallel to the gold surface compared to DNA bases in pure HS-ssDNA monolayers. This indicates that HS-ssDNA oligomers reorient toward a more-upright position upon MCU incorporation. Fluorescence intensity results using end-labeled DNA probes on gold show little observable fluorescence on pure HS-ssDNA monolayers, likely due to substrate quenching effects between the fluorophore and the gold. MCU diluent incorporation into HS-ssDNA monolayers initially increases DNA fluorescence signal by densifying the chemisorbed monolayer, prompting an upright orientation of the DNA, and moving the terminal fluorophore away from the substrate. Immobilized DNA probe density and DNA target hybridization in these mixed DNA monolayers, as well as effects of MCU diluent on DNA hybridization in complex milieu (i.e., serum) were characterized by surface plasmon resonance (SPR) and (32)P-radiometric assays and reported in a related study (Gong, P.; Lee, C.-Y.; Gamble, L. J.; Castner, D. G.; Grainger, D. W. Anal. Chem. 2006, 78, 3326-3334.).
硫醇封端的单链DNA（HS-ssDNA）在金表面的自组装已成为表面固定DNA的重要模型系统。在此，我们报告了一项关于在金载体上含有短链烷基硫醇表面稀释剂[11-巯基-1-十一醇（MCU）]的混合自组装DNA单分子层的表面组成和结构的详细研究。这些混合DNA单分子层通过X射线光电子能谱（XPS）、近边X射线吸收精细结构光谱（NEXAFS）和荧光强度测量进行研究。对金表面依次吸附的DNA/MCU单分子层的XPS结果表明，吸附的MCU分子首先掺入HS-ssDNA单分子层，并且随着MCU暴露时间延长，从表面取代吸附的HS-ssDNA分子。因此，HS-ssDNA表面覆盖率随着MCU暴露时间稳步下降。偏振相关的NEXAFS和荧光结果均显示，仅在MCU暴露了30分钟后，信号变化与DNA取向变化一致。混合DNA单分子层的NEXAFS偏振依赖性（通过监测N 1s→π*跃迁）表明，与纯HS-ssDNA单分子层中的DNA碱基相比，DNA核苷酸碱基环结构更平行于金表面取向。这表明HS-ssDNA寡聚物在掺入MCU后重新取向至更直立的位置。在金上使用末端标记的DNA探针的荧光强度结果显示，纯HS-ssDNA单分子层上几乎没有可观察到的荧光，这可能是由于荧光团与金之间的底物猝灭效应。将MCU稀释剂掺入HS-ssDNA单分子层最初通过使化学吸附单分子层致密化来增加DNA荧光信号，促使DNA直立取向，并使末端荧光团远离底物。这些混合DNA单分子层中固定的DNA探针密度和DNA靶标杂交，以及MCU稀释剂在复杂环境（即血清）中对DNA杂交的影响，通过表面等离子体共振（SPR）和（32）P放射性测定进行表征，并在相关研究中报道（龚，P.；李，C.-Y.；甘布尔，L. J.；卡斯特纳，D. G.；格兰杰，D. W.《分析化学》, 2006, 78, 3326 - 3334.）。
