Two-step perpendicular free-solution isoelectric focusing in a microchamber array chip

Abstract

A free-solution microfluidic chip device was fabricated for small-scale protein fractionation based on the principle of two-step perpendicular isoelectric focusing (IEF) previously reported in a gel slab. The microchip was composed of two separate glass plates with a square array of 100 indentations etched on the facing side of each plate. By changing the relative position of the two plates, ten channels can be formed as ranges of the staggered indentations and can be switched to the perpendicular direction, or 100 chambers of 140 nL each can be produced by perfect overlapping of the indentations. The entire pH range of the carrier ampholyte was fractionated into 10 successive pH segments in the first IEF, and each of the segments was further fractionated into 10 fractions by the second IEF at a right angle to the first one. This chip enabled IEF fractionation at a lower voltage and in a shorter time, compared with a linear apparatus operated in one direction. Fluorescence-labeled peptides with a small isoelectric point (pI) difference (ΔpI = 0.3) were recovered in different chambers in a total separation time of 10 min at a maximum applied voltage of 500 V.