A single-molecule enzymatic assay in a directly accessible femtoliter droplet array
A single-molecule enzymatic assay in a directly accessible femtoliter droplet array
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DOI：
10.1039/c0lc00062k
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发表时间：
2010-01-01
期刊：
影响因子：
6.1
通讯作者：
Noji, Hiroyuki
中科院分区：
文献类型：
--
作者：
Sakakihara, Shouichi;Araki, Suguru;Noji, Hiroyuki
飞升小室阵列中的酶分析是一种简单而有效的浓缩反应产物的方法，它极大地提高了检测灵敏度，低至单分子水平。然而，在以前的方法中，一旦封闭，很难控制每个反应室中反应的开始和结束。此外，酶和产物的回收也很困难。为了克服这些缺点，我们开发了一种飞升液滴阵列，其中单个液滴固定在衬底上，可以从外部直接接触到。液滴的制备使用了亲水亲水的微图案化表面。当表面的水溶液与油交换时，亲水表面保留了水溶液，可以同时制备10(6)多个可用于进一步分析的圆顶状液滴。液滴的曲率半径服从Young-Laplace方程，通过微吸管对液滴施加压力，可以精确控制液滴的体积。改变压力使单个液滴的水含量的添加、收集和交换成为可能。利用这些优点，我们成功地测量了单分子酶β-半乳糖苷酶和旋转马达蛋白F-1-ATPase在液滴中的动力学参数。
The enzyme assay in a femtoliter chamber array is a simple and efficient method for concentrating the reaction product; it greatly improves the detection sensitivity down to the single-molecule level. However, in previous methods, controlling the initiation and termination of the reaction in each chamber is difficult once enclosed. Furthermore, the recovery of the enzyme and product is also difficult. To overcome these drawbacks, we developed a femtoliter droplet array in which the individual droplets are fixed on the substrate and are directly accessible from outside. A hydrophilic-in-hydrophobic micropatterned surface was used for the preparation of the droplets. When the aqueous solution on the surface is exchanged with oil, the hydrophilic surface retains the aqueous solution, and more than 10(6) dome-shaped droplets that are usable for further assay can be prepared simultaneously. The curvature radius of the droplet obeys the Young-Laplace equation, and the volume can be precisely controlled by the micropipette, which applies pressure into the droplet. Changing the pressure makes the addition, collection, and exchange of the aqueous content for individual droplets possible. Using these advantages, we successfully measured the kinetic parameters of the single-molecule enzyme beta-galactosidase and rotary motor protein F-1-ATPase enclosed in a droplet.