Label-free assay of protein kinase A activity and inhibition in cancer cell using electrochemically-prepared AuNP/rGO nanohybrid electrode modified with C-Kemptide.

A simple, label-free and sensitive electrochemical assay is described for the detection of protein kinase A (PKA) activity and inhibition in cancer cell by measuring the change in electrochemical impedance upon phosphorylation. The assay utilized gold nanoparticle (AuNP) and reduced graphene oxide (rGO) nanohybrid which was synthesized and deposited on the electrode from GO and Au3+ precursors by one-pot electrochemical synthesis. As-prepared AuNP/rGO electrode was employed to immobilize C-Kemptide peptide substrates which was phosphorylated in the presence of PKA and ATP. The resulting assay allowed effective, selective and sensitive monitoring of PKA activity according to the impedimetric change in the range of 0.1-500 U/mL, and the detection limit (LOD) is 53 mU/mL. It could also be used for the screening of protein kinase inhibitors. Furthermore, the assay could be applied for the evaluation of PKA activity and inhibition in HeLa cell samples. Therefore, the proposed assay provides one promising tool for PKA activity detection and inhibitor screening with excellent performance.