Max Planck Institute for Solid State Research , D-70569 Stuttgat, Germany.
Department of Biology, University of Rome Tor Vergata , I-00133 Rome, Italy.
ACS Nano. 2015 Nov 24;9(11):11166-76. doi: 10.1021/acsnano.5b05709. Epub 2015 Oct 12.
Monolayer graphene field-effect sensors operating in liquid have been widely deployed for detecting a range of analyte species often under equilibrium conditions. Here we report on the real-time detection of the binding kinetics of the essential human enzyme, topoisomerase I interacting with substrate molecules (DNA probes) that are immobilized electrochemically on to monolayer graphene strips. By monitoring the field-effect characteristics of the graphene biosensor in real-time during the enzyme-substrate interactions, we are able to decipher the surface binding constant for the cleavage reaction step of topoisomerase I activity in a label-free manner. Moreover, an appropriate design of the capture probes allows us to distinctly follow the cleavage step of topoisomerase I functioning in real-time down to picomolar concentrations. The presented results are promising for future rapid screening of drugs that are being evaluated for regulating enzyme activity.
在液相中运行的单层石墨烯场效应传感器已被广泛用于检测各种分析物，通常在平衡条件下进行。在这里，我们报告了实时检测基本人类酶——拓扑异构酶 I 与固定在单层石墨烯条上的底物分子（DNA 探针）相互作用的结合动力学。通过在酶-底物相互作用过程中实时监测石墨烯生物传感器的场效应特性，我们能够以无标记的方式破译拓扑异构酶 I 活性的裂解反应步骤的表面结合常数。此外，通过适当设计捕获探针，我们能够实时实时跟踪拓扑异构酶 I 功能的裂解步骤，直至皮摩尔浓度。所呈现的结果对于未来快速筛选正在评估用于调节酶活性的药物具有重要意义。
