Glazer Marc I, Fidanza Jacqueline A, McGall Glenn H, Trulson Mark O, Forman Jonathan E, Frank Curtis W
Stanford Department of Chemical Engineering, Stanford, CA 94305, USA.
Biophys J. 2007 Sep 1;93(5):1661-76. doi: 10.1529/biophysj.106.103275. Epub 2007 May 11.
We have investigated the kinetics of DNA hybridization to oligonucleotide arrays on high-capacity porous silica films that were deposited by two techniques. Films created by spin coating pure colloidal silica suspensions onto a substrate had pores of approximately 23 nm, relatively low porosity (35%), and a surface area of 17 times flat glass (for a 0.3-microm film). In the second method, latex particles were codeposited with the silica by spin coating and then pyrolyzed, which resulted in larger pores (36 nm), higher porosity (65%), and higher surface area (26 times flat glass for a 0.3-microm film). As a result of these favorable properties, the templated silica hybridized more quickly and reached a higher adsorbed target density (11 vs. 8 times flat glass at 22 degrees C) than the pure silica. Adsorption of DNA onto the high-capacity films is controlled by traditional adsorption and desorption coefficients, as well as by morphology factors and transient binding interactions between the target and the probes. To describe these effects, we have developed a model based on the analogy to diffusion of a reactant in a porous catalyst. Adsorption values (k(a), k(d), and K) measured on planar arrays for the same probe/target system provide the parameters for the model and also provide an internally consistent comparison for the stability of the transient complexes. The interpretation of the model takes into account factors not previously considered for hybridization in three-dimensional films, including the potential effects of heterogeneous probe populations, partial probe/target complexes during diffusion, and non-1:1 binding structures. The transient complexes are much less stable than full duplexes (binding constants for full duplexes higher by three orders of magnitude or more), which may be a result of the unique probe density and distribution that is characteristic of the photolithographically patterned arrays. The behavior at 22 degrees C is described well by the predictive equations for morphology, whereas the behavior at 45 degrees C deviates from expectations and suggests that more complex phenomena may be occurring in that temperature regime.
我们研究了通过两种技术沉积的高容量多孔二氧化硅薄膜上DNA与寡核苷酸阵列杂交的动力学。通过将纯胶体二氧化硅悬浮液旋涂到基板上形成的薄膜具有约23nm的孔、相对较低的孔隙率（35%）以及比平板玻璃大17倍的表面积（对于0.3μm的薄膜）。在第二种方法中，通过旋涂将乳胶颗粒与二氧化硅共沉积，然后进行热解，这导致形成更大的孔（36nm）、更高的孔隙率（65%）以及更高的表面积（对于0.3μm的薄膜，比平板玻璃大26倍）。由于这些有利特性，模板化二氧化硅比纯二氧化硅杂交更快，并且达到更高的吸附靶标密度（在22℃时为平板玻璃的11倍与8倍）。DNA在高容量薄膜上的吸附受传统吸附和解吸系数控制，同时也受形态因素以及靶标与探针之间的瞬时结合相互作用控制。为了描述这些效应，我们开发了一个基于反应物在多孔催化剂中扩散类比的模型。在平面阵列上针对相同探针/靶标系统测量的吸附值（k(a)、k(d)和K）为该模型提供参数，并且还为瞬时复合物的稳定性提供内部一致的比较。该模型的解释考虑了以前在三维薄膜杂交中未考虑的因素，包括异质探针群体的潜在影响、扩散过程中的部分探针/靶标复合物以及非1:1结合结构。瞬时复合物比完全双链体稳定性低得多（完全双链体的结合常数高三个数量级或更多），这可能是光刻图案化阵列特有的独特探针密度和分布的结果。22℃时的行为可以通过形态学预测方程很好地描述，而45℃时的行为偏离预期，这表明在该温度范围内可能发生更复杂的现象。
