Tercero Napoleon, Wang Kang, Gong Ping, Levicky Rastislav
Department of Chemical & Biological Engineering, Polytechnic Institute of New York University, Brooklyn, New York 11201, USA.
J Am Chem Soc. 2009 Apr 8;131(13):4953-61. doi: 10.1021/ja810051q.
Surface hybridization, a reaction in which nucleic acid molecules in solution react with nucleic acid partners immobilized on a surface, is widely practiced in life science research. In these applications the immobilized partner, or "probe", is typically single-stranded DNA. Because DNA is strongly charged, high salt conditions are required to enable binding between analyte nucleic acids ("targets") in solution and the DNA probes. High salt, however, compromises prospects for label-free monitoring or control of the hybridization reaction through surface electric fields; it also stabilizes secondary structure in target species that can interfere with probe-target recognition. In this work, initial steps toward addressing these challenges are taken by introducing morpholinos, a class of uncharged DNA analogues, for surface-hybridization applications. Monolayers of morpholino probes on gold supports can be fabricated with methods similar to those employed with DNA and are shown to hybridize efficiently and sequence-specifically with target strands. Hybridization-induced changes in the interfacial charge organization are analyzed with electrochemical methods and compared for morpholino and DNA probe monolayers. Molecular mechanisms connecting surface hybridization state to the interfacial capacitance are identified and interpreted through comparison to numerical Poisson-Boltzmann calculations. Interestingly, positive as well as negative capacitive responses (contrast inversion) to hybridization are possible, depending on surface populations of mobile ions as controlled by the applied potential. Quantitative comparison of surface capacitance with target coverage (targets/area) reveals a nearly linear relationship and demonstrates sensitivities (limits of quantification) in the picogram per square millimeter range.
表面杂交是一种溶液中的核酸分子与固定在表面的核酸配体发生反应的过程，在生命科学研究中广泛应用。在这些应用中，固定的配体，即“探针”，通常是单链DNA。由于DNA带有很强的电荷，因此需要高盐条件才能使溶液中的分析物核酸（“靶标”）与DNA探针结合。然而，高盐会影响通过表面电场对杂交反应进行无标记监测或控制的前景；它还会稳定靶标物种中可能干扰探针-靶标识别的二级结构。在这项工作中，通过引入吗啉代寡核苷酸（一类不带电荷的DNA类似物）用于表面杂交应用，朝着应对这些挑战迈出了初步步骤。金载体上的吗啉代探针单层可以用与DNA类似的方法制备，并显示出能与靶链高效且序列特异性地杂交。用电化学方法分析杂交引起的界面电荷组织变化，并对吗啉代和DNA探针单层进行比较。通过与数值泊松-玻尔兹曼计算进行比较，确定并解释了将表面杂交状态与界面电容联系起来的分子机制。有趣的是，根据施加电位控制的移动离子的表面数量，杂交可能产生正电容响应和负电容响应（对比度反转）。表面电容与靶标覆盖率（靶标数/面积）的定量比较显示出近乎线性的关系，并证明了皮克每平方毫米范围内的灵敏度（定量限）。
