Structure and DNA hybridization properties of mixed nucleic acid/maleimide-ethylene glycol monolayers
Structure and DNA hybridization properties of mixed nucleic acid/maleimide-ethylene glycol monolayers
复制标题
DOI：
10.1021/ac0703395
复制
发表时间：
2007-06-15
影响因子：
7.4
通讯作者：
Castner, David G.
中科院分区：
文献类型：
--
作者：
Lee, Chi-Ying;Nguyen, Phuong-Cac T.;Castner, David G.
研究了巯基化单链DNA（HS-ssDNA）与马来酰亚胺-乙二醇二硫化物（MEG）单分子膜的表面结构和DNA杂交性能。单层固定化化学和表面覆盖的反应ssDNA探针的X射线光电子能谱和飞行时间二次离子质谱法进行了研究。ssDNA探针的取向通过近边X射线吸收精细结构（NEXAFS）确定。通过表面等离子体共振（SPR）测量DNA-MEG探针表面上的靶DNA杂交，以证明这些探针表面用于检测来自纯化的靶DNA样品和复杂的生物混合物如血清的DNA靶的效用。来自互补技术的数据表明，固定化的ssDNA密度强烈依赖于点样的本体DNA浓度和缓冲液离子强度。固定化ssDNA密度的变化对DNA探针在表面的取向和随后的靶杂交效率有深远的影响。随着表面探针密度的增加，NEXAFS偏振依赖性结果（随后监测N 1 s-> pi* 跃迁）表明，固定化的ssDNA分子在MEG单层上朝向更直立的位置重新取向。从缓冲液和血清中的DNA靶的SPR测定表明，DNA杂交效率随着表面探针密度的降低而增加。然而，血清中的靶检测在“高密度”探针表面上比在“高效”探针表面上更好。由于非特异性血清蛋白吸附到传感表面上，两个ssDNA表面检测到的目标量在血清中比在纯化的DNA样品中差几个数量级。
The surface structure and DNA hybridization performance of thiolated single-strand DNA (HS-ssDNA) covalently attached to a maleimide-ethylene glycol disulfide (MEG) monolayer on gold have been investigated. Monolayer immobilization chemistry and surface coverage of reactive ssDNA probes were studied by X-ray photoelectron spectroscopy and time-of-flight secondary ion mass spectrometry. Orientation of the ssDNA probes was determined by near-edge X-ray absorption fine structure (NEXAFS). Target DNA hybridization on the DNA-MEG probe surfaces was measured by surface plasmon resonance (SPR) to demonstrate the utility of these probe surfaces for detection of DNA targets from both purified target DNA samples and complex biological mixtures such as blood serum. Data from complementary techniques showed that immobilized ssDNA density is strongly dependent on the spotted bulk DNA concentration and buffer ionic strength. Variation of the immobilized ssDNA density had a profound influence on the DNA probe orientation at the surface and subsequent target hybridization efficiency. With increasing surface probe density, NEXAFS polarization dependence results (followed by monitoring the N 1s -> pi* transition) indicate that the immobilized ssDNA molecules reorient toward a more upright position on the MEG monolayer. SPR assays of DNA targets from buffer and serum showed that DNA hybridization efficiency increased with decreasing surface probe density. However, target detection in serum was better on the "high-density" probe surface than on the "high-efficiency" probe surface. The amounts of target detected for both ssDNA surfaces were several orders of magnitude poorer in serum than in purified DNA samples due to nonspecific serum protein adsorption onto the sensing surface.