High-Efficient and Controllable Purification of Protein by Cyclic Injection in an Orthogonal Micro-Free-Flow Electrophoresis.

High-abundance proteins (HAPs) in plasma significantly interfere with the purification of low-abundance proteins (LAPs). This study proposed a unique orthogonality microfluidic free-flow electrophoresis (OMFFE) platform and then set up a self-made device that can perform cyclic injection for protein purification. The OMFFE device consists of a focusing channel, an orthogonal separation channel, and two sets of voltage-controlled electrodes. In the scan voltage mode, the voltage and deflection angle are positively correlated for proteins, allowing the determination of the separation voltage for the target protein. In the selective voltage mode, proteins with a specific mass-to-charge ratio could be deflected into a specific channel, enabling selective separation of proteins. When the 75 V voltage was applied, the optimal resolution of the device between HAPs (human serum albumin, HSA, and immunoglobulin IgG) and LAP (green fluorescent protein, GFP) was achieved with Rs = 0.37. The OMFFE device achieved a removal rate of 94.7 ± 4.1% for HSA and IgG and a recovery rate of 95.3 ± 3.7% for GFP. The purity of GFP has increased by approximately 32 times. Furthermore, in human plasma, the removal rates of HAPs (HSA and IgG) and the recovery rates of LAP (GFP) are as high as 83.2 ± 3.9 and 76.3 ± 7.5%, respectively. The OMFFE device can achieve the removal and purification of proteins, hence posing promising potential in proteomics, protein function analysis, and medical diagnostics.