Shen Gang, Anand Maria Francis G, Levicky Rastislav
Columbia University, 500 West 120th Street Room 801, New York, NY 10027, USA.
Nucleic Acids Res. 2004 Nov 10;32(20):5973-80. doi: 10.1093/nar/gkh932. Print 2004.
Surface-tethered nucleic acids are widely applied in solid-phase assays in which complementary strands must be detected against a complex mixture of other sequences. In response to such needs, numerous methods have been developed for immobilizing nucleic acids on solid supports. Often, detailed analysis of associated chemical transformations and of potential side reactions is difficult to obtain. Combined use of planar and high surface area powder supports allows characterization using surface as well as bulk diagnostic techniques. This approach is followed in the present study in which X-ray photoelectron spectroscopy (XPS), transmission infrared spectroscopy (FTIR) and reactivity titrations are used to investigate siliceous supports modified with an aminosilane precursor followed by a maleimide-bearing crosslinker for attachment of nucleic acids. The supports retain maleimide activity for approximately a day when stored under buffer, but deactivation is accelerated under basic conditions or by incomplete conversion of the precursor aminosilane monolayer. Reactions involving the olefinic bond of the imide as well as its carbonyl groups are observed and analyzed. Attachment of sulfhydryl-terminated oligodeoxyribonucleotides is highly site specific, and immobilized strands exhibit excellent hybridization activity. Quantitative use of XPS for label-free determination of DNA coverage based on calibration against reference materials is also described.
表面 tethered 核酸广泛应用于固相分析中，在这种分析中必须针对其他序列的复杂混合物检测互补链。为满足此类需求，已开发出多种将核酸固定在固体支持物上的方法。通常，很难获得相关化学转化及潜在副反应的详细分析。平面和高表面积粉末支持物的联合使用允许使用表面以及体相诊断技术进行表征。本研究采用了这种方法，其中使用 X 射线光电子能谱（XPS）、透射红外光谱（FTIR）和反应性滴定来研究用氨基硅烷前体修饰，随后用带有马来酰亚胺的交联剂修饰以连接核酸的硅质支持物。当在缓冲液中储存时，支持物的马来酰亚胺活性可保持约一天，但在碱性条件下或前体氨基硅烷单层未完全转化时，失活会加速。观察并分析了涉及酰亚胺的烯烃键及其羰基的反应。巯基末端的寡脱氧核糖核苷酸的连接具有高度的位点特异性，并且固定化的链表现出优异的杂交活性。还描述了基于与参考材料校准的 XPS 用于无标记测定 DNA 覆盖率的定量方法。
