Single-molecule enzymology based on the principle of the Millikan oil drop experiment.
Single-molecule enzymology based on the principle of the Millikan oil drop experiment.
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基于密立根油滴实验原理的单分子酶学。
DOI：
10.1016/j.ab.2013.11.016
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发表时间：
2014
影响因子：
2.9
通讯作者：
Farinas,Javier
中科院分区：
文献类型：
--
作者：
Leiske,DanielleL;Chow,Andrea;Dettloff,Roger;Farinas,Javier
监测单分子酶反应进程的能力常常受到需要使用荧光底物的限制。开发了一种基于密立根油滴实验原理的方法，以监测与纳米颗粒结合的底物的电荷变化，并提供了一种无需荧光检测即可检测单酶反应的方法。作为监测导致底物电荷变化的反应的能力的原理证明，检测到单个DNA模板上的聚合。合成了一种定制的寡核苷酸，其允许单个DNA模板与具有单个聚合物系链的金纳米颗粒连接。然后将纳米颗粒拴系到微流体通道的表面，其中使用暗场显微镜监测经受振荡电场的纳米颗粒的位置。由于平均时间短，信噪比水平低到足以区分小于1.2%的电荷变化。长DNA模板的聚合证明了使用该系统监测单分子酶活性的能力。最后，用硫醇化部分修饰纳米颗粒表面以减少和/或屏蔽非生产性电荷的数量并允许提高灵敏度。
The ability to monitor the progress of single-molecule enzyme reactions is often limited by the need to use fluorogenic substrates. A method based on the principle of the Millikan oil drop experiment was developed to monitor the change in charge of substrates bound to a nanoparticle and offers a means of detecting single-enzyme reactions without fluorescence detection. As a proof of principle of the ability to monitor reactions that result in a change in substrate charge, polymerization on a single DNA template was detected. A custom oligonucleotide was synthesized that allowed for the attachment of single DNA templates to gold nanoparticles with a single polymer tether. The nanoparticles were then tethered to the surface of a microfluidic channel where the positions of the nanoparticles, subjected to an oscillating electric field, were monitored using dark field microscopy. With short averaging times, the signal-to-noise level was low enough to discriminate changes in charge of less than 1.2%. Polymerization of a long DNA template demonstrated the ability to use the system to monitor single-molecule enzymatic activity. Finally, nanoparticle surfaces were modified with thiolated moieties to reduce and/or shield the number of unproductive charges and allow for improved sensitivity.