Direct On-Paper Inkjet Printing of Kinase-to-Kinase Phosphorylation Cascade Reactions

Abstract

In this study, we described a novel method to detect the kinase-to-kinase phosphorylation cascade reaction using a commercially available inkjet printing machine. It is very difficult to accomplish the kinase phosphorylation cascade reaction with inkjet printing because of the complex optimization of various reaction conditions inherent to transient protein-protein interactions. To demonstrate the proposed approach, the c-Jun N-terminal kinase (JNK1/2) and mitogen-activated protein kinase (MAPK)-mediated phosphorylation cascade reaction was chosen as a model. After printing, the resolution and biofunctional activity of the kinase enzyme was determined, and the reproducibility was evaluated. Our results showed a negligible loss in phosphorylation activity, which confirms the success of the phosphorylation cascade reaction on the printed substrate. Considering the success of the cascade reaction on paper, the assay was extended to determine JNK1/2 inhibition activity by SP600125. It reveals that the method was able to assess the percentage of phosphorylation and inhibition activity by utilizing a relatively small amount of the enzyme; data analysis does not require specialized instrumentation. Thus, the inkjet printing technology-based method will be a suitable platform to screen kinase inhibitors. This is the first report to validate the kinase-kinase phosphorylation cascade reaction using an inkjet printing method.