Simultaneous electrokinetic concentration and separation of proteins on a paper-based analytical device

In this report, we introduced an electrokinetic strategy for simultaneous concentration and separation of charged analytes in an open and free standing paper fluidic channel. This was achieved by taking advantage of the field amplified sample stacking (FASS) effect. An electric field gradient was developed in the channel due to the conductivity difference between the background electrolyte (BGE) and sample solution. Through adjustment of the electroosmotic flow level and BGE concentration, charged analytes were not only stacked but also separated in the gradient based on their difference in electrophoretic mobility. Proof of concept was demonstrated firstly by two food dyes, brilliant blue and amaranth, and each dye was about 100-fold concentrated within 300 s. Simultaneous stacking and separation of two colored proteins, bovine hemoglobin and cytochrome c, was also demonstrated visually with the proposed method. Sample consumption was reduced to 5 mu L by directly loading the sample onto the open channel, and the separation was completed within 30 s. This method is simple, rapid and easy to operate, which is particularly beneficial to the development of sensitive and multifunctional paper-based analytical devices (PADs) for field testing.