A multiplexed magnetic tweezer with precision particle tracking and bi-directional force control.
A multiplexed magnetic tweezer with precision particle tracking and bi-directional force control.
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DOI：
10.1186/s13036-017-0091-2
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发表时间：
2017
影响因子：
5.6
通讯作者：
Thomas WE
中科院分区：
文献类型：
--
作者：
Johnson KC;Clemmens E;Mahmoud H;Kirkpatrick R;Vizcarra JC;Thomas WE
在过去的二十年中，已经开发了测量单个生物分子力学性能的方法。其中一种方法，磁镊子，可以同时获取多个单分子的数据，但为了充分利用这种“多路复用”能力，有必要同时整合许多已经单独演示过的能力。我们定制的磁性镊子结合了高复用，精确的头跟踪和双向力控制，成为一个灵活稳定的平台，用于检测单分子行为。这是使用电磁铁完成的，它提供了高时间控制力，同时通过大的顺磁珠实现与永磁体相似的力水平。在这里，我们描述了该仪器及其同时对多达120个珠子施加2-260 pN力的能力，使用各种珠子尺寸和实验技术，最大空间精度为12 nm。我们还展示了一种利用密度分离和双向力相关相结合来提高非均质顺磁珠力估计精度的新方法，该方法将力的变化系数从27%降低到6%。然后，我们使用该仪器检查了大肠杆菌（E. coli）细菌的1型菌毛的开卷和反冲速度的力依赖性，并看到与先前研究相似的结果。该平台为高效采集单分子力谱测量数据提供了一种简单、有效、灵活的方法。
In the past two decades, methods have been developed to measure the mechanical properties of single biomolecules. One of these methods, Magnetic tweezers, is amenable to aquisition of data on many single molecules simultaneously, but to take full advantage of this "multiplexing" ability, it is necessary to simultaneously incorprorate many capabilities that ahve been only demonstrated separately. Our custom built magnetic tweezer combines high multiplexing, precision bead tracking, and bi-directional force control into a flexible and stable platform for examining single molecule behavior. This was accomplished using electromagnets, which provide high temporal control of force while achieving force levels similar to permanent magnets via large paramagnetic beads. Here we describe the instrument and its ability to apply 2–260 pN of force on up to 120 beads simultaneously, with a maximum spatial precision of 12 nm using a variety of bead sizes and experimental techniques. We also demonstrate a novel method for increasing the precision of force estimations on heterogeneous paramagnetic beads using a combination of density separation and bi-directional force correlation which reduces the coefficient of variation of force from 27% to 6%. We then use the instrument to examine the force dependence of uncoiling and recoiling velocity of type 1 fimbriae from Eschericia coli (E. coli) bacteria, and see similar results to previous studies. This platform provides a simple, effective, and flexible method for efficiently gathering single molecule force spectroscopy measurements.
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