Protein
kinase activity correlates closely with that of many human
diseases. However, the existing methods for quantifying protein kinase
activity often suffer from limitations such as low sensitivity, harmful
radioactive labels, high cost, and sophisticated detection procedures,
underscoring the urgent need for sensitive and rapid detection methods.
Herein, we present a simple and sensitive approach for the homogeneous
detection of protein kinase activity based on nanoimpact electrochemistry
to probe the degree of aggregation of silver nanoparticles (AgNPs)
before and after phosphorylation. Phosphorylation, catalyzed by protein
kinases, introduces two negative charges into the substrate peptide,
leading to alterations in electrostatic interactions between the phosphorylated
peptide and the negatively charged AgNPs, which, in turn, affects
the aggregation status of AgNPs. Via direct electro-oxidation of AgNPs
in nanoimpact electrochemistry experiments, protein kinase activity
can be quantified by assessing the impact frequency. The present sensor
demonstrates a broad detection range and a low detection limit for
protein kinase A (PKA), along with remarkable selectivity. Additionally,
it enables monitoring of PKA-catalyzed phosphorylation processes.
In contrast to conventional electrochemical sensing methods, this
approach avoids the requirement of complex labeling and washing procedures.