National ESCA and Surface Analysis Center for Biomedical Problems, Department of Chemical Engineering, Box 351750, University of Washington, Seattle, Washington 98195-1750, USA.
Biointerphases. 2006 Jun;1(2):82-92. doi: 10.1116/1.2219110.
Reliable, direct "sample-to-answer" capture of nucleic acid targets from complex media would greatly improve existing capabilities of DNA microarrays and biosensors. This goal has proven elusive for many current nucleic acid detection technologies attempting to produce assay results directly from complex real-world samples, including food, tissue, and environmental materials. In this study, we have investigated mixed self-assembled thiolated single-strand DNA (ssDNA) monolayers containing a short thiolated oligo(ethylene glycol) (OEG) surface diluent on gold surfaces to improve the specific capture of DNA targets from complex media. Both surface composition and orientation of these mixed DNA monolayers were characterized with x-ray photoelectron spectroscopy (XPS) and near-edge x-ray absorption fine structure (NEXAFS). XPS results from sequentially adsorbed ssDNA/OEG monolayers on gold indicate that thiolated OEG diluent molecules first incorporate into the thiolated ssDNA monolayer and, upon longer OEG exposures, competitively displace adsorbed ssDNA molecules from the gold surface. NEXAFS polarization dependence results (followed by monitoring the N 1s-->pi(*) transition) indicate that adsorbed thiolated ssDNA nucleotide base-ring structures in the mixed ssDNA monolayers are oriented more parallel to the gold surface compared to DNA bases in pure ssDNA monolayers. This supports ssDNA oligomer reorientation towards a more upright position upon OEG mixed adlayer incorporation. DNA target hybridization on mixed ssDNA probe/OEG monolayers was monitored by surface plasmon resonance (SPR). Improvements in specific target capture for these ssDNA probe surfaces due to incorporation of the OEG diluent were demonstrated using two model biosensing assays, DNA target capture from complete bovine serum and from salmon genomic DNA mixtures. SPR results demonstrate that OEG incorporation into the ssDNA adlayer improves surface resistance to both nonspecific DNA and protein adsorption, facilitating detection of small DNA target sequences from these undiluted, unpurified complex biological mixtures unachievable with previously reported, analogous ssDNA/11-mercapto-1-undecanol monolayer surfaces [P. Gong, C.-Y. Lee, L. J. Gamble, D. G. Castner, and D. W. Grainger, Anal. Chem. 78, 3326 (2006)].
可靠的、直接的“从复杂介质中进行样本到答案的捕获”将极大地提高 DNA 微阵列和生物传感器的现有能力。对于许多当前试图直接从复杂的实际样本（包括食品、组织和环境材料）中产生检测结果的核酸检测技术来说，这一目标一直难以实现。在这项研究中，我们研究了混合自组装的巯基化单链 DNA（ssDNA）单层，其中包含短的巯基化聚（乙二醇）（OEG）表面稀释剂在金表面上，以提高从复杂介质中特异性捕获 DNA 靶标的能力。这些混合 DNA 单层的表面组成和取向都通过 X 射线光电子能谱（XPS）和近边 X 射线吸收精细结构（NEXAFS）进行了表征。金上依次吸附的 ssDNA/OEG 单层的 XPS 结果表明，巯基化 OEG 稀释剂分子首先掺入巯基化 ssDNA 单层中，并且在较长的 OEG 暴露后，从金表面上竞争取代吸附的 ssDNA 分子。NEXAFS 极化依赖性结果（随后监测 N 1s->pi(*)跃迁）表明，混合 ssDNA 单层中吸附的巯基化 ssDNA 核苷酸碱基环结构与纯 ssDNA 单层中的 DNA 碱基相比，更平行于金表面取向。这支持了 OEG 混合吸附层掺入后，ssDNA 低聚物向更垂直位置的重取向。通过表面等离子体共振（SPR）监测混合 ssDNA 探针/OEG 单层上的 DNA 靶标杂交。通过两种模型生物传感测定，从完整牛血清和鲑鱼基因组 DNA 混合物中捕获 DNA 靶标，证明了 OEG 掺入 ssDNA 吸附层中提高了对非特异性 DNA 和蛋白质吸附的特异性靶标捕获能力。SPR 结果表明，OEG 掺入 ssDNA 吸附层提高了表面对非特异性 DNA 和蛋白质吸附的抵抗力，从而可以从这些未经稀释、未经纯化的复杂生物混合物中检测到小的 DNA 靶序列，而这是以前报道的类似 ssDNA/11-巯基-1-十一醇单层表面无法实现的[P. Gong, C.-Y. Lee, L. J. Gamble, D. G. Castner, and D. W. Grainger, Anal. Chem. 78, 3326 (2006)]。
