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.

Real-time reliable determination of binding kinetics of DNA hybridization using a multi-channel graphene biosensor

“…By far, graphene has been employed in various biosensing platforms827 for detection of glucose28, nitric oxide29, DNA303132, biomarkers33, bacteria34, pathogens27 and nervous system35. Such a large number of studies successfully demonstrated a wide-range application of 2D biosensors with low detection limit, and a few further illustrated their capabilities in quantifying kinetics and affinity of protein-ligand binding363738. However, the quantitative determination of DNA–DNA or DNA–RNA hybridization kinetics have not yet been achieved due to controllability and reproducibility across different devices and fabrication batches.…”

Bioelectronics and Interfaces Using Monolayer Graphene

“…At a total ionic strength of around 0.1 M, the EDL thickness is around 1 nm, and hence it is believed that it is not possible to detect larger biomolecules such as antibody or enzyme interactions. Despite this belief, numerous examples of sensitive detection of biomolecules have been reported not only using graphene, but also with nanowires, nanotubes and organic FETs ,,. The Debye screening hypothesis assumes that the surface of the nanostructure in the sensor is rather flat and an ideal EDL capacitor is taken as a prerequisite.…”

“…The modulation of charge at the graphene‐liquid interface is possible by the attachment of a broad range of organic or inorganic chemical functionalities to the graphene surface . This allows the realization of sensors for targets like DNA, peptides, and proteins ,. As in carbon electrodes, the interfacial charge of graphene is modulated by the presence of chemical moieties covalently or non‐covalently attached to the surface.…”

Chemical Sensors and Biosensors in Italy: A Review of the 2015 Literature

“…This is a relevant result for future rapid screening of drugs, being evaluated for regulating enzyme activity. It is worth to observe that this is one of the very few examples of a sensor device fabricated using a CVD monolayer graphene substrate [74]. …”