High density single-molecule-bead arrays for parallel single molecule force spectroscopy.
High density single-molecule-bead arrays for parallel single molecule force spectroscopy.
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DOI：
10.1021/ac3001622
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发表时间：
2012-06-05
影响因子：
7.4
通讯作者：
Vezenov, Dmitri
中科院分区：
文献类型：
--
作者：
Barrett, Michael J.;Oliver, Piercen M.;Cheng, Peng;Cetin, Deniz;Vezenov, Dmitri
高度并行力谱工具的组装需要在基板上仔细放置单分子目标，并精心操纵多个力探针。由于探针必须接近目标生物分子进行共价附着，同时避免不可逆地粘附到基底上，因此使用聚合物微球作为力探针来创建系留珠阵列会带来问题。因此，力探针和表面之间的相互作用在非常短的距离（< 5 nm）处必须是排斥的，而在长距离处必须是吸引的。为了实现这种平衡，必须调整基材、力探针和溶液的化学成分以控制探针与表面的相互作用。除了适当设计的化学物质之外，还必须控制目标分子的表面密度，以确保单个力探针仅询问一个分子。我们使用金硫醇化学来控制基质的表面化学和所研究分子的间距，通过硫醇封端的 DNA 和形成阻挡层的惰性硫醇的竞争性结合。对于我们的单分子阵列，我们使用 DLVO 理论对探针和基底之间的力进行建模，并使用胶体探针显微镜测量它们的大小和方向。使用探针结合测定来测试每个系统的实用性，以评估施力后仍粘附在表面上的珠子的比例。我们已经将我们系统的具体结果转化为制备系留珠阵列的一般指导原则，并证明了该系统能够在微孔基板中产生高产量的主动力光谱探针。这项研究概述了创建这种力谱阵列所需的化学特性。
The assembly of a highly-parallel force spectroscopy tool requires careful placement of single-molecule targets on the substrate and the deliberate manipulation of a multitude of force probes. Since the probe must approach the target biomolecule for covalent attachment, while avoiding irreversible adhesion to the substrate, the use of the polymer microsphere as force probes to create the tethered bead array poses a problem. Therefore, the interactions between the force probe and the surface must be repulsive at very short distances (< 5 nm) and attractive at long distances. To achieve this balance, the chemistry of the substrate, force probe, and solution must be tailored to control the probe-surface interactions. In addition to an appropriately designed chemistry, it is necessary to control the surface density of the target molecule in order to ensure that only one molecule is interrogated by a single force probe. We used gold-thiol chemistry to control both the substrate’s surface chemistry and the spacing of the studied molecules, through a competitive binding of the thiol-terminated DNA and an inert thiol forming a blocking layer. For our single molecule array, we modeled the forces between the probe and the substrate using DLVO theory and measured their magnitude and direction with colloidal probe microscopy. The practicality of each system was tested using a probe binding assay to evaluate the proportion of the beads remaining adhered to the surface after application of force. We have translated the results specific for our system to general guiding principles for preparation of tethered bead arrays and demonstrated the ability of this system to produce a high yield of active force spectroscopy probes in a microwell substrate. This study outlines the characteristics of the chemistry needed to create such a force spectroscopy array.
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