Gong Ping, Lee Chi-Ying, Gamble Lara J, Castner David G, Grainger David W
Department of Chemistry, Colorado State University, Fort Collins, CO 80523-1872, USA.
Anal Chem. 2006 May 15;78(10):3326-34. doi: 10.1021/ac052138b.
Nucleic acid assay from a complex biological milieu is attractive but currently difficult and far from routine. In this study, DNA hybridization from serum dilutions into mixed DNA/mercaptoundecanol (MCU) adlayers on gold was monitored by surface plasmon resonance (SPR). Immobilized DNA probe and hybridized target densities on these surfaces were quantified using 32P-radiometric assays as a function of MCU diluent exposure. SPR surface capture results correlated with radiometric analysis for hybridization performance, demonstrating a maximum DNA hybridization on DNA/MCU mixed adlayers. The maximum target surface capture produced by MCU addition to the DNA probe layer correlates with structural and conformational data on identical mixed DNA/MCU adlayers on gold derived from XPS, NEXAFS, and fluorescence intensity measurements reported in a related study (Lee, C.-Y.; Gong, P.; Harbers, G. M.; Grainger, D. W.; Castner, D. G.; Gamble, L. J. Anal. Chem. 2006, 78, 3316-3325.). MCU addition into the DNA adlayer on gold also improved surface resistance to both nonspecific DNA and serum protein adsorption. Target DNA hybridization from serum dilutions was monitored with SPR on the optimally mixed DNA/MCU adlayers. Both hybridization kinetics and efficiency were strongly affected by nonspecific protein adsorption from a complex milieu even at a minimal serum concentration (e.g., 1%). No target hybridization was detected in SPR assays from serum concentrations above 30%, indicating nonspecific protein adsorption interference of DNA capture and hybridization from complex milieu. Removal of nonsignal proteins from nucleic acid targets prior to assay represents a significant issue for direct sample-to-assay nucleic acid diagnostics from food, blood, tissue, PCR mixtures, and many other biologically complex sample formats.
从复杂生物环境中进行核酸检测很有吸引力，但目前难度较大且远未成为常规操作。在本研究中，通过表面等离子体共振（SPR）监测了血清稀释液与金表面混合的DNA/巯基十一醇（MCU）吸附层之间的DNA杂交。使用³²P放射性测定法对这些表面上固定的DNA探针和杂交靶标的密度进行了定量，作为MCU稀释剂暴露的函数。SPR表面捕获结果与杂交性能的放射性分析相关，表明在DNA/MCU混合吸附层上DNA杂交达到最大值。向DNA探针层中添加MCU所产生的最大靶标表面捕获与相关研究（Lee, C.-Y.; Gong, P.; Harbers, G. M.; Grainger, D. W.; Castner, D. G.; Gamble, L. J. Anal. Chem. 2006, 78, 3316 - 3325.）报道的源自XPS、NEXAFS和荧光强度测量的金表面相同混合DNA/MCU吸附层的结构和构象数据相关。向金表面的DNA吸附层中添加MCU还提高了表面对非特异性DNA和血清蛋白吸附的抗性。在最佳混合的DNA/MCU吸附层上，用SPR监测了血清稀释液中靶标DNA的杂交。即使在最低血清浓度（例如1%）下，复杂环境中的非特异性蛋白质吸附也会强烈影响杂交动力学和效率。在血清浓度高于30%的SPR检测中未检测到靶标杂交，表明复杂环境中DNA捕获和杂交受到非特异性蛋白质吸附的干扰。在检测前从核酸靶标中去除非信号蛋白是食品、血液、组织、PCR混合物和许多其他生物复杂样品形式的直接样品到检测核酸诊断中的一个重要问题。
