A Phos-tag-based photoelectrochemical biosensor for assay of protein kinase activity and inhibitors

• A photoelectrochemical biosensor was fabricated for protein kinase A activity assay.
• Biotinylated Phos-tag was used to recognize phosphate group with high affinity.
• The activity of protein kinase A in HePG2 cell was higher than that in L-02 cell.
• The inhibition effect of ellagic acid and HA-1077 on PKA activity was evaluated.

A novel, sensitive and label-free photoelectrochemical assay for monitoring the activity and inhibition of protein kinase A (PKA) was presented based on the specific recognition ability of biotinylated Phos-tag for kinase-induced phosphopeptides. When the assembled substrate peptides were phosphorylated by PKA, they could bind specifically to the biotinylated Phos-tag in the presence of Zn2+. Then streptavidins could be further captured on the electrode surface through their specific interaction towards biotin, resulting in a significant decrease on the photocurrent of substrate Bi2S3 modified ITO electrode. The decreased photocurrent was proportional to the PKA concentration ranging from 0.05 to 100 unit/mL with a detection limit of 0.017 unit/mL (S/N = 3). Moreover, this photoelectrochemical method could be also used to quantitative analysis of kinase inhibition. In the light of the inhibitor concentration dependant photocurrent signal, the IC50 value for ellagic acid and HA-1077 was estimated to be 3.23 and 1.77 μM, respectively. With changing the sequence of substrate peptide, this method could also be applied to detect other kinase activity and inhibitors. Therefore, the developed protocol provides a new and promising platform for assay of kinase activity and its inhibitors with low cost and high sensitivity.

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