Journal
BIOSENSORS & BIOELECTRONICS
Volume 119, Issue -, Pages 42-47Publisher
ELSEVIER ADVANCED TECHNOLOGY
DOI: 10.1016/j.bios.2018.07.063
Keywords
Protein kinase; Protein phosphorylation; Peptide-templated nanoprobe; Copper nanoparticle; Electrochemical assay
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Funding
- National Natural Science Foundation of China [81671781, 21775101, 81401489]
- Shanghai Sailing Program [14YF1409000]
- Shanghai Pujiang Program [17PJ1402800]
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Protein kinases play a critical role in regulation of intracellular signal transduction, whose aberrant expression is closely associated with various dangerous human diseases. In this paper, we propose a feasible electrochemical assay of intracellular kinase by incorporating peptide nanoprobe-assisted signal labeling and signal amplification. Protein kinase A (PKA)-specific peptide P1 is self-assembled on the surface of a gold electrode, serine of which could be phosphorylated with catalysis of PKA in the presence of adenosine-5'-triphosphate (ATP). Another artificial peptide P2 contains a short template for preparation of copper nanoparticles-based nanoprobe (P2-CuNPs) and provides arginine residues for specific recognition of phosphorylation site. After PICA-catalyzed phosphorylation, phosphorylated P1 specially binds with P2-CuNPs through ultra-stable phosphate-guanidine interaction, and thus results in amplified electrochemical response from surface-attached CuNPs. Our method demonstrates a satisfactory sensitivity toward PICA detection with a detection limit of 0.0019 U/mL, which is also successfully applied in intracellular PKA assay and inhibitory study with high specificity comparable to ELISA. Therefore, the facile method suggests a promising potential use in kinase-related biochemical fundamental research, disease diagnosis and drug discovery in the future.
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