A DNA-based electrochemical strategy for label-free monitoring the activity and inhibition of protein kinasew

Protein phosphorylation by kinase plays a significant regulatory role in many vital biological processes involving metabolic pathways, cellular signal communications, neural activities, and so on. Aberrant protein phosphorylation states and kinase activity are associated with many human diseases. The identification of kinase activities and their potential inhibitors is not only necessary for understanding of many fundamental biochemical events, but also valuable for protein–kinase-targeted drug discovery. The general kinase activity assays rely on radioactive, electroactive, fluorescent or biotin labeling techniques and phosphorylation-specific recognition protein (e.g. antibody or SH2 domain) based methods. Such assays are effective but require labor-intensive labeling procedures or sophisticated preparation of recognition protein. Therefore, developing a simple, sensitive and labelfree kinase activity assay is still challenging. Recent reports described a few intriguing kinase assays based on electrochemical oxidation of tyrosine, volatammetric determination of Ag ion associated with the phosphorylated site, and TiO2-assisted silver enhanced protocol. These works proved that the electrochemical sensing system is a promising way to achieve label-free kinase activity measurement for its high sensitivity, low cost and simplicity in microarray fabrication. Herein, we developed a novel label-free electrochemical sensing strategy for profiling kinase activity based on zirconium cation (Zr) mediated signal transition. Since Zr has been proven as an effective capture agent for selective enrichment of phosphorylated peptides, Zr presents a potential phosphorylation-recognition element. Furthermore, the multicoordinative interactions between Zr ions and phosphates indicate that Zr could be used as a linkage between the phosphorylated site of peptide and the ‘‘signal reporter’’ containing phosphate groups. DNA functionalized gold nanoparticles (DNA–AuNPs) are chosen as the ‘‘signal reporter’’ by virtue of the intrinsic 50-phosphate end of DNA and the versatile properties of DNA–AuNPs in electrochemical signal generation and amplification. Taking advantage of these principles, we demonstrated that using Zr ion as a phosphorylation indentifier, DNA-based methods could be employed for facile and label-free monitoring the activity of protein kinase. The schematic diagram of this label-free kinase activity assay is shown in Scheme 1 and the electrode modification process was confirmed by electrochemical impedance spectroscopy (EIS, the details are provided in ESI,w Fig. S1). cAMP–dependent protein kinase (PKA), a typical serine/ threonine protein kinase, was used as the model kinase. The PKA-specific peptide substrate (LRRASLGGGGC) was self-assembled onto the surface of the gold electrode through its cysteine end. The surface coverage of the peptide on the Au electrode was estimated to be 4.8 10 10 mol cm 2 by quartz crystal microbalance measurements (see ESIw). After the peptide phosphorylation catalyzed by PKA in the presence of ATP, the functional groups on the phosphorylated peptides were recorded by FT-IR. Two additional peaks at 920.56 cm 1 and 1080.62 cm 1 are attributed to the presence of phosphate groups (ESI,w Fig. S2). This implies that phosphate groups with negative charge were incorporated into the serine residue of the peptides. Then, the Zr ions were attached to the phosphorylated peptide (P-peptide). After a careful washing process to remove the excess and nonspecifically adsorbed Zr ions, the DNA–AuNPs were added and linked with the P-peptide through the steady interaction between Zr and phosphate groups (as in the schematic illustration in Scheme 1B). The scanning electron microscopy (SEM) image of the resulting DNA–AuNPs bound to the phosphorylated electrode is shown in Scheme 1C, I. It is noted that plenty of the DNA–AuNPs with diameters of 10–15 nm were uniformly distributed on the surface of the electrode, and no aggregation of the DNA–AuNPs was found. However, only a few DNA–AuNPs were observed on the surface of the control electrode without PKA treatment (Scheme 1C, II), which is probably due to nonspecific absorption of DNA–AuNPs. The SEM results indicated that the peptide phosphorylation by kinase can significantly increase the Zr mediated binding of the DNA–AuNPs to the electrode surface, which implies that the proposed protocol for monitoring the activity of protein a State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha, 410082, P. R. China. E-mail: [email protected], [email protected]; Fax: +86-731-88821848 Key Laboratory of Chemical Biology and Traditional Chinese Medicine Research (Ministry of Education of China), College of Chemistry and Chemical Engineering, Hunan Normal University, Changsha, 410081, P. R. China w Electronic supplementary information (ESI) available: Details description of the experimental section and additional figures. See DOI: 10.1039/b913943e