Application of plasma-polymerized films for isoelectric focusing of proteins in a capillary electrophoresis chip

Abstract

The first use of plasma polymerization technique to modify the surface of a glass chip for capillary isoelectric focusing (cIEF) of different proteins is reported. The electrophoresis separation channel was machined in Tempax^® glass chips with length 70 mm, 300 μm width and 100 μm depth. Acetonitrile and hexamethyldisiloxane monomers were used for plasma polymerization. In each case 100 nm plasma polymer films were coated onto the chip surface to reduce protein wall adsorption and minimize the electroosmotic flow. Applied voltages of 1000 V, 2000 V and 3000 V were used to separate mixtures of cytochrome c (pI 9.6), hemoglobin (pI 7.0) and phycocyanin (pI 4.65). Reproducible isoelectric focusing of each pI marker protein was observed in different coated capillaries at increasing concentration 2.22–5 μg μL⁻¹. Modification of the glass capillary with hydrophobic HMDS plasma polymerized films enabled rapid cIEF within 3 min. The separation efficiency of cytochrome c and phycocyanin in both acrylamide and HMDS coated capillaries corresponded to a plate number of 19 600 which compares favourably with capillary electrophoresis of neurotransmitters with amperometric detection.