Characterization and application of controllable local chemical changes produced by reagent delivery from a nanopipet
Characterization and application of controllable local chemical changes produced by reagent delivery from a nanopipet
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DOI：
10.1021/ja8022253
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发表时间：
2008-08-06
影响因子：
15
通讯作者：
Klenerman, David
中科院分区：
文献类型：
--
作者：
Piper, Joe D.;Li, Chao;Klenerman, David
我们介绍了一种多功能的方法，该方法允许在离子溶液中从纳米移液管局部和可重复地递送（或耗尽）任何水溶性试剂，以在表面处进行试剂浓度的局部控制变化。在这项工作中，Na+或OH-离子从移液管使用脉冲电压驱动的输送。来自用于Na+离子的浴中的CoroNa绿色染料或用于pH的浴中的荧光素的全内反射荧光定量了局部表面浓度的所得变化。这些变化的时间响应短至10 ms，半径为1-30 μ m，并取决于所用移液管的直径、施加的电压和移液管-表面分离。在详细表征移液管给药后，然后对单细胞和单分子进行两次概念验证实验。我们证明了本地控制的钠敏感的鞭毛电机在单一的大肠杆菌嵌合体的时间尺度为1秒，通过给药钠和监测旋转的1 μ m直径的珠固定到鞭毛。我们还证明了触发的单分子解折叠，通过从移液管中给药酸以局部熔化双链体DNA的单个分子，如使用荧光共振能量转移所观察到的。
We introduce a versatile method that allows local and repeatable delivery (or depletion) of any water-soluble reagent from a nanopipet in ionic solution to make localized controlled changes in reagent concentration at a surface. In this work, Na+ or OH- ions were dosed from the pipet using pulsed voltage-driven delivery. Total internal reflection fluorescence from CoroNa Green dye in the bath for Na+ ions or fluorescein in the bath for pH quantified the resulting changes in local surface concentration. These changes had a time response as short as 10 ms and a radius of 1-30 mu m and depended on the diameter of the pipet used, the applied voltage, and the pipet-surface separation. After the pipet dosing was characterized in detail, two proof-of-concept experiments on single cells and single molecules were then performed. We demonstrated local control of the sodium-sensitive flagellar motor in single Escherichia coli chimera on the time scale of 1 s by dosing sodium and monitoring the rotation of a 1 mu m diameter bead fixed to the flagellum. We also demonstrated triggered single-molecule unfolding by dosing acid from the pipet to locally melt individual molecules of duplex DNA, as observed using fluorescent resonance energy transfer.