TY - JOUR
T1 - Probing kinase activities by electrochemistry, contact-angle measurements, and molecular-force interactions
AU - Wilner, Ofer I.
AU - Guidotti, Claudio
AU - Wieckowska, Agnieszka
AU - Gill, Ron
AU - Willner, Itamar
PY - 2008/9/8
Y1 - 2008/9/8
N2 - Three different methods to investigate the activity of a protein kinase (casein kinase, CK2) are described. The phosphorylation of the sequence-specific peptide (1) by CK2 was monitored by electrochemical impedance spectroscopy (EIS). Phosphorylation of the peptide monolayer assembled on a Au electrode yields a negatively charged surface that electrostatically repels the negatively charged redox label [Fe(CN)6]3-/4-, thus increasing the interfacial electron-transfer resistance. The phosphorylation process by CK2 is further amplified by the association of the anti-phosphorylated peptide antibody to the monolayer. Binding of the antibody insulates the electrode surface, thus increasing the interfacial electron-transfer resistancein the presence of the redox label. This method enabled the quantitative analysis of the concentration of CK2 with a detection limit of ten units. The second method employed involved contact-angle measurements. Although the peptide 1-functionalized electrode revealed a contact angle of 67.5°, phosphorylation of the peptide yielded a surface with enhanced hydrophilicity, 36.8°. The biocatalyzed cleavage of the phosphate units with alkaline phosphatase regenerates the hydrophobic peptide monolayer, contact angle 55.3°. The third method to characterize the system involved chemical force measurements between the phosphorylated peptide monolayer associated with the Au surface and a Au tip functionalized with the anti-phosphorylated peptide antibody. Although no significant rupture forces existed between the modified tip and the 1-functionalized surface (6±2pN), significant rupture forces (multiples of 120±20 pN) were observed between the phosphorylated monolayer-modified surface and the antibody-functionalized tip. This rupture force is attributed to the dissociation of a simple binding event between the phosphorylated peptide and the fluorescent antibody (Fab) binding region. ω 2008 Wiley-VCH Verlag GmbH& Co. KGaA.
AB - Three different methods to investigate the activity of a protein kinase (casein kinase, CK2) are described. The phosphorylation of the sequence-specific peptide (1) by CK2 was monitored by electrochemical impedance spectroscopy (EIS). Phosphorylation of the peptide monolayer assembled on a Au electrode yields a negatively charged surface that electrostatically repels the negatively charged redox label [Fe(CN)6]3-/4-, thus increasing the interfacial electron-transfer resistance. The phosphorylation process by CK2 is further amplified by the association of the anti-phosphorylated peptide antibody to the monolayer. Binding of the antibody insulates the electrode surface, thus increasing the interfacial electron-transfer resistancein the presence of the redox label. This method enabled the quantitative analysis of the concentration of CK2 with a detection limit of ten units. The second method employed involved contact-angle measurements. Although the peptide 1-functionalized electrode revealed a contact angle of 67.5°, phosphorylation of the peptide yielded a surface with enhanced hydrophilicity, 36.8°. The biocatalyzed cleavage of the phosphate units with alkaline phosphatase regenerates the hydrophobic peptide monolayer, contact angle 55.3°. The third method to characterize the system involved chemical force measurements between the phosphorylated peptide monolayer associated with the Au surface and a Au tip functionalized with the anti-phosphorylated peptide antibody. Although no significant rupture forces existed between the modified tip and the 1-functionalized surface (6±2pN), significant rupture forces (multiples of 120±20 pN) were observed between the phosphorylated monolayer-modified surface and the antibody-functionalized tip. This rupture force is attributed to the dissociation of a simple binding event between the phosphorylated peptide and the fluorescent antibody (Fab) binding region. ω 2008 Wiley-VCH Verlag GmbH& Co. KGaA.
KW - Antibodies
KW - Biosensors
KW - Contact angles
KW - Electrochemistry
KW - Protein kinases
UR - http://www.scopus.com/inward/record.url?scp=53849147610&partnerID=8YFLogxK
U2 - 10.1002/chem.200800765
DO - 10.1002/chem.200800765
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C2 - 18698570
AN - SCOPUS:53849147610
SN - 0947-6539
VL - 14
SP - 7774
EP - 7781
JO - Chemistry - A European Journal
JF - Chemistry - A European Journal
IS - 26
ER -