A new approach for developing protein-protein interactions based kinase biosensors

Abnormal phosphorylation often leads to uncontrolled cell proliferation and cancer. Kinases are biomarkers of various diseases, and therefore biosensors are being developed for sensing kinases. These include radiometric, spectroscopic and electrochemical biosensors. All these require labeling, which increases the complexity of the process.

The kinase ERK2 is responsible for the phosphorylation and activation of many substrates involved in cell proliferation and cancer. Our labs previously developed an electrochemical kinase biosensor for the detection of ERK2, which is comprised of a peptide monolayer on a gold electrode. The peptide is derived from the phosphorylation site of HDGF, which is overphosphorylated by ERK2 in lung cancer. Using electrochemical impedance spectroscopy (EIS), the changes in the dielectric properties of the peptide monolayer after exposure to ERK2 were monitored. Phosphorylation of the peptide monolayer by ERK2 resulted in decrease in R_CT values, suggesting conformational changes in the peptide monolayer due to the phosphorylation. Here we show that using this assay, we were able to sense ERK2 at concentrations of 10 nM – 1 µM, which correlate with its celluar concentration, 50 nM – 2 μM. Similar results were observed even in the absence of ATP, which led us to suggest that the ERK2 – HDGF peptide interaction itself may cause the conformational changes in the peptide monolayer. Indeed, ellipsometry and IR measurements suggested conformational changes of the peptides in the layer, from compact to linear conformation, due to the interaction with ERK2. BSA caused no significant changes in the properties of the monolayer, indicating that specific interactions are required for sensing. In summary, our results show a new approach for developing kinase biosensors, which are based also on the interactions between the kinase and its substrate and not exclusively on the enzymatic phosphorylation reaction.