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Label-free electrochemical impedance detection of kinase and phosphatase activities using carbon nanofiber nanoelectrode arrays.

ABSTRACT: We demonstrate the feasibility of a label-free electrochemical method to detect the kinetics of phosphorylation and dephosphorylation of surface-attached peptides catalyzed by kinase and phosphatase, respectively. The peptides with a sequence specific to c-Src tyrosine kinase and protein tyrosine phosphatase 1B (PTP1B) were first validated with ELISA-based protein tyrosine kinase assay and then functionalized on vertically aligned carbon nanofiber (VACNF) nanoelectrode arrays (NEAs). Real-time electrochemical impedance spectroscopy (REIS) measurements showed reversible impedance changes upon the addition of c-Src kinase and PTP1B phosphatase. Only a small and unreliable impedance variation was observed during the peptide phosphorylation, but a large and fast impedance decrease was observed during the peptide dephosphorylation at different PTP1B concentrations. The REIS data of dephosphorylation displayed a well-defined exponential decay following the Michaelis-Menten heterogeneous enzymatic model with a specific constant, k(cat)/K(m), of (2.1±0.1)×10(7) M(-1)s(-1). Consistent values of the specific constant was measured at PTP1B concentration varying from 1.2 to 2.4 nM with the corresponding electrochemical signal decay constant varying from 38.5 to 19.1s. This electrochemical method can be potentially used as a label-free method for profiling enzyme activities in fast reactions.

SUBMITTER: Li Y

PROVIDER: S-EPMC3432243 | biostudies-literature | 2012 Sep

REPOSITORIES: biostudies-literature

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Label-free electrochemical impedance detection of kinase and phosphatase activities using carbon nanofiber nanoelectrode arrays.

Li Yifen Y Syed Lateef L Liu Jianwei J Hua Duy H DH Li Jun J

Analytica chimica acta 20120724

We demonstrate the feasibility of a label-free electrochemical method to detect the kinetics of phosphorylation and dephosphorylation of surface-attached peptides catalyzed by kinase and phosphatase, respectively. The peptides with a sequence specific to c-Src tyrosine kinase and protein tyrosine phosphatase 1B (PTP1B) were first validated with ELISA-based protein tyrosine kinase assay and then functionalized on vertically aligned carbon nanofiber (VACNF) nanoelectrode arrays (NEAs). Real-time e ...[more]

PMID: 22935373

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Project description:In the last decade, researchers have been searching for innovative platforms, methods, and techniques able to address recurring problems with the current cancer detection methods. Early disease detection, fast results, point-of-care sensing, and cost are among the most prevalent issues that need further exploration in this field. Herein, studies are focused on overcoming these problems by developing an electrochemical device able to detect telomerase as a cancer biomarker. Electrochemical platforms and techniques are more appealing for cancer detection, offering lower costs than the established cancer detection methods, high sensitivity inherent to the technique, rapid signal processing, and their capacity of being miniaturized. Therefore, Au interdigital electrodes and electrochemical impedance spectroscopy were used to detect telomerase activity in acute T cell leukemia. Different cancer cell concentrations were evaluated, and a detection limit of 1.9 × 105 cells/mL was obtained. X-ray photoelectron spectroscopy was used to characterize the telomerase substrate (TS) DNA probe self-assembled monolayer on gold electrode surfaces. Atomic force microscopy displayed three-dimensional images of the surface to establish a height difference of 9.0 nm between the bare electrode and TS-modified Au electrodes. The TS probe is rich in guanines, thus forming secondary structures known as G-quadruplex that can be triggered with a fluorescence probe. Confocal microscopy fluorescence images showed the formation of DNA G-quadruplex because of TS elongation by telomerase on the Au electrode surface. Moreover, electrodes exposed to telomerase containing 2',3'-dideoxyguanosine-5'-triphosphate (ddGTP) did not exhibit high fluorescence, as ddGTP is a telomerase inhibitor, thus making this device suitable for telomerase inhibitors capacity studies. The electrochemical method and Au microchip device may be developed as a biosensor for a point-of-care medical device.

Dataset Information

Label-free electrochemical impedance detection of kinase and phosphatase activities using carbon nanofiber nanoelectrode arrays.

Publications

Label-free electrochemical impedance detection of kinase and phosphatase activities using carbon nanofiber nanoelectrode arrays.

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