title: Electrochemical detection of tyrosine derivatives and protein tyrosine kinase activity using boron-doped diamond electrodes.
abstract: In this report, we determined protein tyrosine kinase (PTKs) activity in human epidermoid carcinoma cells (A431) by employing a novel electrochemical method using boron-doped diamond (BDD) electrodes that enables the electrochemical oxidation of tyrosine (Tyr), phosphorylated Tyr (Tyr-P) and sulfated Tyr (Tyr-S) in water-based solutions. Cyclic voltammetry for Tyr, Tyr-P and Tyr-S showed well-defined oxidation peaks at 0.8 V for Tyr, 1.4V for Tyr-P and 1.7 V for Tyr-S, respectively. Very little work has been reported previously on the detection of Tyr-P and Tyr-S, probably due to their high oxidation potentials. We utilized electrochemical methods for the detection of kinase activity in connection with poly(Glu-Tyr) modified magnetic beads. Linear-sweep voltammograms for the electrochemical detection of PTKs activity were carried out using BDD electrodes consisting of peptide-modified magnetic beads. Without phosphorylation of the peptide-modified magnetic beads using PTKs, we observed clear oxidation peaks for Tyr oxidation and no significant electrochemical responses for Tyr-P oxidation at 1.4V for the background. On the other hand, with phosphorylation of the beads using PTKs, the peak oxidation current at 1.4V clearly increased, while the peak oxidation current for Tyr oxidation decreased. This indicates that PTKs activity could be successfully detected by using electrochemical methods employing BDD electrodes. This method was utilized for the in vitro kinase activity detection of human cell lysate, and the electrochemical measurements were compatible with the Enzyme-Linked ImmunoSorbent Assay based method. Our results indicate that the electrochemical method can be applied to real samples such as cell lysate.
authors.affiliation: Department of Chemistry, Faculty of Science and Technology, Keio University, Yokohama 223-8522, Japan.
authors.forename: Masanobu
authors.initials: M
authors.lastname: Chiku
authors.forename: Kenichi
authors.initials: K
authors.lastname: Horisawa
authors.forename: Nobuhide
authors.initials: N
authors.lastname: Doi
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Yanagawa
authors.forename: Yasuaki
authors.initials: Y
authors.lastname: Einaga
mesh_terms: D015374:Biosensing Techniques / Q000295:instrumentation*; D001895:Boron / Q000737:chemistry*; D003217:Conductometry / Q000295:instrumentation*; D018130:Diamond / Q000737:chemistry*; D004566:Electrodes; D004789:Enzyme Activation; D004800:Enzymes, Immobilized / Q000737:chemistry; D004867:Equipment Design; D019544:Equipment Failure Analysis; D011505:Protein-Tyrosine Kinases / Q000737:chemistry*; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000032:analysis*
chemical_list: D004800:Enzymes, Immobilized; D014443:Tyrosine; D018130:Diamond; D011505:Protein-Tyrosine Kinases; D001895:Boron