title: Real-Time Label-Free Direct Electronic Monitoring of Topoisomerase Enzyme Binding Kinetics on Graphene.
abstract: 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.
authors.affiliation: Max Planck Institute for Solid State Research , D-70569 Stuttgat, Germany.|Department of Biology, University of Rome Tor Vergata , I-00133 Rome, Italy.
authors.forename: Laura
authors.initials: L
authors.lastname: Zuccaro
authors.affiliation: Department of Biology, University of Rome Tor Vergata , I-00133 Rome, Italy.|Department of Molecular Biology & Genetics, Aarhus University , DK-8000 Aarhus, Denmark.
authors.forename: Cinzia
authors.initials: C
authors.lastname: Tesauro
authors.affiliation: Max Planck Institute for Solid State Research , D-70569 Stuttgat, Germany.
authors.forename: Tetiana
authors.initials: T
authors.lastname: Kurkina
authors.affiliation: Department of Biology, University of Rome Tor Vergata , I-00133 Rome, Italy.|Institute of Translational Pharmacology , National Research Council CNR, I-00133 Rome, Italy.
authors.forename: Paola
authors.initials: P
authors.lastname: Fiorani
authors.affiliation: Max Planck Institute for Biophysical Chemistry , 37077 Göttingen, Germany.
authors.forename: Hak Ki
authors.initials: HK
authors.lastname: Yu
authors.affiliation: Department of Molecular Biology & Genetics, Aarhus University , DK-8000 Aarhus, Denmark.|Interdisciplinary Nanoscience Center (iNANO), Aarhus University , DK-8000 Aarhus, Denmark.
authors.forename: Birgitta R
authors.initials: BR
authors.lastname: Knudsen
authors.affiliation: Max Planck Institute for Solid State Research , D-70569 Stuttgat, Germany.|Institut de Physique de la Matière Condensée, École Polytechnique Fédérale de Lausanne , CH-1015 Lausanne, Switzerland.
authors.forename: Klaus
authors.initials: K
authors.lastname: Kern
authors.affiliation: Department of Biology, University of Rome Tor Vergata , I-00133 Rome, Italy.
authors.forename: Alessandro
authors.initials: A
authors.lastname: Desideri
authors.affiliation: Max Planck Institute for Solid State Research , D-70569 Stuttgat, Germany.
authors.forename: Kannan
authors.initials: K
authors.lastname: Balasubramanian
mesh_terms: D001483:Base Sequence; D055162:Biocatalysis; D003199:Computer Systems; D004264:DNA Topoisomerases, Type I / Q000378:metabolism*; D004581:Electronics / Q000379:methods*; D006108:Graphite / Q000737:chemistry*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D011485:Protein Binding; D013194:Staining and Labeling
chemical_list: D006108:Graphite; D004264:DNA Topoisomerases, Type I; C528909:TOP1 protein, human
keywords: biosensor; enzyme activity; field effect; human topoisomerase; label-free biosensing