Amperometric determination of the activity of protein kinase a using a glassy carbon electrode modified with IgG functionalized gold nanoparticles conjugated to horseradish peroxidase

The authors describe the fabrication of an electrochemical immunosensor for the determination of the activity of protein kinase A (PKA). The method involves (a) electrochemical deposition of gold nanoparticles (AuNPs) on a glassy carbon electrode, (b) PKA-induced catalytic phosphorylation of serine, and (c) the use of phosphoserine antibody and horseradish peroxidase conjugated to IgG on gold nanoparticles (HRP-IgG-AuNPs). The use of AuNPs and HRP-IgG-AuNPs results in large amplification so that the method, at a typical working potential as low as 0.08 V (vs. SCE), has a linear range that extends from 0.1 to 50 activity units per mL, and the detection limit is 0.026 units per mL (at an S/N ratio of 3). The assay is selective (not the least due to a rather low working potential) and well reproducible. It may also be applied to screening for PKA inhibitors and to quantify the PKA activity in human cell lysates. ᅟ