Functionalized surfaces of mixed alkanethiols on gold as a platform for oligonucleotide microarrays
Functionalized surfaces of mixed alkanethiols on gold as a platform for oligonucleotide microarrays
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DOI：
10.1021/la011732n
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发表时间：
2002-09-03
期刊：
影响因子：
3.9
通讯作者：
Ortigao, F
中科院分区：
文献类型：
--
作者：
Riepl, M;Enander, K;Ortigao, F
在金表面制备了生物素化和乙二醇端长链烷硫醇的混合自组装单层，试图建立一种可靠的固定链霉亲和素的方案。采用广泛的表面分析技术，包括椭偏、原子力显微镜、红外、荧光和x射线光电子能谱，对链霉亲和素固定前后的SAMs进行了表征。第一部分的工作重点是寻找导致链霉亲和素的最佳结合能力的混合条件。由含有75-95%生物素化烷硫醇的负载溶液制备的混合SAMs导致了高固定化水平的功能性链霉亲和素。链亲和素的薄层随后可用于固定化广泛的生物素化生物分子（例如寡核苷酸，cDNA，肽，蛋白质，抗体和碳水化合物），因此为制造用于生物传感器和筛选应用的芯片/阵列提供了一个极好的平台。通过使用基于荧光的DNA微阵列检测系统监测生物素化的24-mer捕获寡核苷酸和标记的89-mer目标DNA之间的杂交，成功地证明了这一点。此外，dna微阵列实验还显示(i)互补寡核苷酸与随机捕获的24-mer寡核苷酸的反应相比具有良好的选择性，（ii）与链霉亲和素表面的非特异性结合水平低。
Mixed self-assembled monolayers of biotinylated- and ethylene glycol-terminated long-chain alkanethiols were prepared on gold surfaces in an attempt to develop a reliable protocol for immobilization of streptavidin. A broad range of surface analytical techniques including ellipsometry, atomic force microscopy, and infrared, fluorescence, and X-ray photoelectron spectroscopy were used to characterize the SAMs before and after immobilization of streptavidin. The first part of the work was focused on finding the mixing conditions that lead to optimum binding capacity of streptavidin. Mixed SAMs prepared from loading solutions containing 75-95% of the biotinylated alkanethiol resulted in high immobilization levels of functional streptavidin. The thin layers of streptavidin subsequently can be used for the immobilization of a broad spectrum of biotinylated biomolecules (e.g. oligonucleotides, cDNA, peptides, proteins, antibodies, and carbohydrates) and provides therefore an excellent platform for the fabrication of chips/arrays for biosensor and screening applications. This is successfully demonstrated by monitoring the hybridization between a biotinylated 24-mer capturing oligonucleotide and a labeled target 89-mer DNA using a fluorescence-based DNA-microarray detection system. Moreover, the DNA-microarray experiments also revealed (i) good selectivity when comparing the response of the complementary oligonucleotide with that of a random 24-mer capturing oligonucleotide and (ii) low levels of nonspecific binding to the streptavidin surface.