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对单链DNA nanopatches的杂交效率测定的新方法

New approach for the determination of the hybridation efficiency of ssDNA nanopatches
课程网址: http://videolectures.net/slonano07_melli_nad/  
主讲教师: Mauro Melli
开课单位: TASC国家实验室
开课时间: 2008-02-12
课程语种: 英语
中文简介:
固定在表面的单链 dna (ssdna) 薄膜构成了许多重要的生物技术应用的基础, 包括 dna 微阵列和纳米阵列。然而, 关于薄膜结构如何影响杂交过程的问题在文献中没有得到适当解决。虽然人们普遍认为, 在广泛的自组装单层中, 杂交效率与表面探针的分子密度基本成反比, 但在单层时, 还没有达成同样的共识被限制在纳米结构中。利用纳米接枝 (一种在液体环境中使用 afm 尖端的纳米光刻技术), 我们能够制备出高度、侧向大小均定为明确的 ssdna 和纳米结构。杂交会导致高度的增加, 可以通过 afm 显微镜进行监测。令人惊讶的是, 我们观察到在半到饱和的密度下, ssdna 杂交的效率最高;此外, 在这种情况下, ssdna 杂化膜和纳米接枝双链 dna 膜的力学性能是等效的。 为了量化我们纳米结构的杂交效率, 更重要的是, 一般情况下, 只涉及少量分子的化学过程的效率, 分析化学技术已不再有效。相反, 物理方法提供了许多合适的解决方案。特别是微机械和纳米机械共振传感器在过去5年中取得了重大进展, 允许测量单个分子的枚举和10-21 克的质量灵敏度。我们制造了几种由5x2x15 m3 和5x2x15 组成的器件, 其尺寸分别为350和 580 pg 和谐振频率, 分别为10.3 和 3.7 mhz。顶部覆盖着一个超平坦的 au 层 (0.8 nm rms), 在上面形成了一个有机自组装的单层。采用纳米接枝法制备悬臂自由端的 ssdna 纳米结构。当 ssdna 杂交时, 悬臂的质量增加, 共振频率向较低的值移动。在真空条件下工作时, 我们得到的质量因子优于 104, 使我们获得 5x10-16 g 的质量灵敏度, 相当于只有1% 的3x3 m2 ssdna 纳米结构的杂交。
课程简介: Films of single-stranded DNA (ssDNA) immobilized on surfaces form the basis of a number of important biotechnology applications, including DNA micro- and nano-arrays. However, the question about how the film structure affects the hybridization process is not properly addressed in literature. While it is commonly accepted that the hybridization efficiency in extensive self assembled monolayers of ssDNA is basically inversely proportional to the molecular density of the probes on the surface, the same agreement has not been reached when the monolayers are confined in nanometric structures. Using nanografting (a nanolithographic technique performed in liquid environment with an AFM tip) we are able to fabricate ssDNA micro- and nanostructures with well defined height, lateral size. The hybridization causes an increase of height that can be monitored by AFM microscopy. Surprisingly, we observed the highest efficiency of ssDNA hybridization at densities halfway to saturation; moreover, in this regime the mechanical properties of the ssDNA hybridized film and of a nanografted double-stranded DNA film are equivalent. To quantify the hybridization efficiency on our nanostructures, and, more in general, the efficiency of chemical processes which involve only few molecules, the analytical chemistry techniques are no longer effective. On the contrary, a physical approach offers many suitable solution. In particular micro- and nano-mechanical resonating sensors experienced a significant progress in the last 5 years, allowing the measurement/enumeration of single molecules and a mass sensitivity of 10-21 g. We fabricate several devices consisting of silicon cantilevers 5x2x15m3 and 5x2x25m3 in size with mass of 350 and 580 pg and resonance frequency at 10.3 and 3.7 MHz respectively. The top side was covered with an ultraflat Au layer (0.8nm RMS) on which an organic self assembled monolayer was formed. ssDNA nanostructures on the free end of the cantilevers are fabricated by nanografting. When the ssDNA hybridizes the mass of the cantilever increases and the resonance frequency shifts toward lower values. Operating in vacuum conditions we obtained a quality factor better than 104 that allowed us to attain a mass sensitivity of 5x10-16 g, corresponding to the hybridization of only 1% of a 3x3m2 ssDNA nanostructure.
关 键 词: 单链DNA; 微纳米阵列; 结构
课程来源: 视频讲座网
最后编审: 2020-06-15：wuyq
阅读次数: 78

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