Department of Chemical and Biological Engineering , University of Colorado Boulder , Boulder , Colorado 80309 , United States.
ACS Nano. 2019 Jul 23;13(7):7850-7859. doi: 10.1021/acsnano.9b02157. Epub 2019 Jun 25.
Adsorption of soluble DNA to surfaces decorated with complementary DNA plays an important role in many bionanotechnology applications, and previous studies have reported complex dependencies of the surface density of immobilized DNA on hybridization. While these effects have been speculatively ascribed to steric or electrostatic effects, the influence of surface-mediated molecular transport (i.e., intermittent "hopping diffusion") has not been fully appreciated. Here, single-molecule tracking and Förster resonance energy transfer (FRET) were employed to characterize the mobility and the hybridization efficiency of adsorbed ssDNA oligonucleotides ("target") at solid-liquid interfaces exhibiting surface-immobilized ssDNA ("probe") over a wide range of surface grafting densities. Two distinct regimes were observed, with qualitatively different transport and hybridization behaviors. At dilute grafting density, only 1-3% of target molecules were observed to associate with probes (i.e., to hybridize). Adsorbing target molecules often searched unsuccessfully and "flew", via desorption-mediated diffusion, to secondary locations before hybridizing. In contrast, at high probe grafting density, approximately 20% of target DNA hybridized to immobilized probes, and almost always in the vicinity of initial adsorption. Moreover, following a dehybridization event, target molecules rehybridized at high probe density, but rehybridization was infrequent in the dilute density regime. Interestingly, the intermittent interfacial transport of mobile target molecules was suppressed by the presence of immobilized probe DNA, presumably due to an increased probability of readsorption following each "hop". Together, these findings suggested that many salient effects of grafting density on surface-mediated DNA hybridization can be directly related to the mechanisms of surface-mediated intermittent diffusion.
表面修饰互补 DNA 可吸附可溶性 DNA，这在许多生物纳米技术应用中发挥着重要作用，先前的研究报告称固定化 DNA 的表面密度与杂交之间存在复杂的依赖性。虽然这些效应被推测归因于空间或静电效应，但表面介导的分子传输（即间歇性“跳跃扩散”）的影响尚未得到充分认识。在这里，通过单分子跟踪和Förster 共振能量转移（FRET），我们在广泛的表面接枝密度范围内，对在具有表面固定化 ssDNA（“探针”）的固液界面上吸附的 ssDNA 寡核苷酸（“靶标”）的流动性和杂交效率进行了表征。观察到两种不同的状态，具有定性不同的传输和杂交行为。在稀释的接枝密度下，只有 1-3%的靶分子观察到与探针结合（即杂交）。吸附的靶分子通常会不成功地搜索并通过解吸介导的扩散“飞行”到杂交之前的次要位置。相比之下，在高探针接枝密度下，大约 20%的靶 DNA 与固定化探针杂交，而且几乎总是在初始吸附的附近。此外，在解杂交事件之后，在高探针密度下靶分子重新杂交，但在稀释密度下重新杂交的频率较低。有趣的是，固定化探针 DNA 的存在抑制了可动靶分子的间歇性界面传输，这可能是由于每次“跳跃”后重新吸附的可能性增加所致。总之，这些发现表明，接枝密度对表面介导 DNA 杂交的许多显著影响可以直接与表面介导的间歇性扩散机制相关。
