Electrochemical assay of protein kinase activity based on the Fe3O4@PNE-Ti4+ functionalized PDMS microchip

Abstract

A new strategy for the sensitive assay of protein kinase activity has been constructed using Fe3O4@PNE-Ti4+ nanocomposite as stationary phase in microchip electrophoresis. Through the self-polymerization of norepinephrine in weak alkaline solution, a thin poly(norepinephrine) (PNE) film can be spontaneously formed on the surface of Fe3O4 nanoparticles (NPs) to produce Fe3O4@PNE NPs. Due to the good magnetic property, Fe3O4@PNE NPs can be easily fixed in poly(dimethylsiloxane) (PDMS) channel under external magnetic field. On the other hand, the catechol hydroxyl groups in PNE can chelate Ti4+ to form Fe3O4@PNE-Ti4+ in PDMS channel. When peptides are phosphorylated by protein kinase A (PKA) in the presence of adenosine 5′-triphosphate (ATP), the Fe3O4@PNE-Ti4+ packed in PDMS channels can specifically coordinate with the phosphorylated sites of phosphopeptides, but the non-phosphorylated peptides cannot bind with Ti4+. Thus, the retention time of the phosphorylated peptides in the Fe3O4@PNE-Ti4+ packed PDMS channel was increased, and phosphorylated peptides and non-phosphorylated peptides can be separated successfully. Due to the high loading of Ti4+ on the Fe3O4@PNE, this strategy can be used for the enrichment of phosphorylated peptides and electrochemical assay of PKA activity. This method can also be used for quantitative analysis of PKA activity in cell lysate, indicating its potential application in clinical diagnosis.