A gold nanoparticles colorimetric assay for label-free detection of protein kinase activity based on phosphorylation protection against exopeptidase cleavage

• A novel colorimetric protein kinase activity assay has been developed based on the unmodified gold nanoparticles (AuNPs)/peptide platform.
• The recognition of kinase phosphorylation relies on the phosphorylation-induced suppression of carboxypeptidase Y (CPY) cleavage.
• Our assay is simple and homogenous without multistep washing process, sophistical peptide labeling, and the modification of AuNPs.

Protein kinases are significant regulators in the cell signaling pathways, and it is still greatly desirable to achieve simple and quick kinase detection. Herein, we present a novel colorimetric gold nanoparticles (AuNPs)/peptide platform for probing the activity and inhibition of protein kinases based on phosphorylation-induced suppression of carboxypeptidase Y (CPY) cleavage. This AuNPs/peptide platform can easily monitor the kinase activity by a UV–vis spectrometer or even by the naked eye. The feasibility of the method has been demonstrated by sensitive measurement of the cAMP-dependent protein kinase (PKA) activity with a low detection limit of 0.232 mU/µL and assessment of kinase inhibition by H-89 with an IC50 value of 18.13 nM. The assay was also successfully put into practice for the detection of kinase activity in cell lysate. Because of its label-free, homogenous and colorimetric merits, the proposed assay presents great potential in high-throughput screening for kinase-targeted drug discovery.