Analysis of Biomolecular Interactions Using Fast Electrokinetic Microfluidic Chip

Abstract

The analysis of biomolecular interactions is a key factor in ultimately regulating biological phenomena, such as metabolism, immune response, cell development, and homeostasis, in the cell. The conventional methods of analysis of biomolecular interactions have required long assay time, expensive equipment, and a large amount of samples. Therefore, for overcoming the limitations of conventional methods, this study addresses analysis of biomolecular interactions using electrokinetic phenomena, such as isoelectric focusing (IEF) and ion concentration polarization (ICP), on a PDMS-based microfluidic chip. First, this study presented a simple method for analyzing the degree of phosphorylation by protein kinases using fluorescent peptide substrates and microfluidic IEF (μIEF). When a dye-labeled peptide substrate was sequentially phosphorylated by two consecutive protein kinases (mitogen-activated protein kinase (MAPK) and glycogen synthase kinase 3 (GSK3)), its differently phosphorylated forms were easily separated and visualized by fluorescent focusing zones in the IEF channel based on a change in the isoelectric point (pI) by phosphorylation. As a result, ratiometric and quantitative analysis of the fluorescent focusing regions shifted by phosphorylation enabled the analysis of phosphorylation efficiency and the relevant inhibition of protein kinases (MAPK and GSK3) with high simplicity and selectivity. Furthermore, the GSK3 activity in the cell lysates was elucidated by IEF in combination with immunoprecipitation. These results suggest that this method has great potential for analyzing the phosphorylation degree by multiple protein kinases that are implicated in cellular signaling pathways. In addition, this study reported an easy method for analyzing the DNA-protein interactions using ICP and the DNA labeled with fluorescence. The DNA-protein complex is formed by binding organic hydroperoxide resistance (OhrR), a type of repressor protein, and dsDNA labeled with fluorescence. When the complex substrate was dissociated by organic hydroperoxide or low molecule weight thiols containing organic hydroperoxide, the undissociated complex and the dissociated dsDNA was easily separated in the upstream of depletion zone based on electrophoretic mobility differences and visualized by fluorescence in dsDNA. As a result, ratiometric analysis of the focused bands enabled the quantitative analysis of dissociation percentage. Furthermore, using ICP, the dissociation percentage through the reaction with the low molecule weight thiols in mouse serum was quantitatively measured. Finally, the dissociation percentage through the reaction with the spiking mouse serum was also measured successfully on a microfluidic chip using ICP. These results suggest that the ICP method has a great possibility for analyzing the various DNA-protein interactions.