On‐chip protein isoelectric focusing using a photoimmobilized pH gradient^†

Abstract

An immobilized pH gradient was directly constructed on the inner wall of a microfluidic chip channel by photoimmobilizing focused carrier ampholytes onto the wall. A mixture of carbonic anhydrase, myoglobin, and trypsin inhibitor was successfully isoelectric-focused and separated with good linearity between the pI values of proteins and the location of the focused bands. Furthermore, coating methods for the resistance of protein nonselective adsorption and simultaneously for pH gradient photocoupling were screened. The PEG-silane coating method was found to be better than the cross-linked polyacrylamide coating and aminosilane modification methods. Finally, based on the open tubular column mode of carrier ampholytes' immobilization and effective antiadsorption coating, the immobilized pH gradient was reused and the chip was recycled for the first time. By virtue of its remarkable features including simplicity, convenience, high efficiency of protein enrichment and separation, and potential for coupling site-selective IEF with other analytical or separation techniques, this novel method promises to be useful in several applications related with zwitterionic biomolecules.

Surface isoelectric focusing (sIEF) with carrier ampholyte pH gradient

“…Disadvantages of free‐flow μIEF include discontinuous pH gradients caused by local potential drops and undesired convective flows . Thus, hybrids of slab gel IEF and free‐flow μIEF were combined into polymer film coated μIEF devices , microchips with thin‐cut immobilized pH gradient (IPG) strips , microfluidic devices with polyacrylamide gels cast inside microchannels , followed by microchannel gels with photo‐immobilized pH gradients . These hybrid IEF techniques demonstrated stabilized pH gradients, sharp sample peak signals and that CA pH gradients effectively scaled down, while separation time and reproducibility improved as size decreased .…”

Analysis of proteins and peptides by electromigration methods in microchips

“…The focused narrow zones are then transported toward a detector by pressure or by electroelution (chemical mobilization). IEF was performed on a glass microchip using a photoimmobilized pH gradient [69]. From different channel surface coating procedures such as aminosilane modification, cross-linked polyacrylamide coating, and PEG-silane coating, the last one was found to be the most effective in avoiding protein adsorption.…”

“…In situ photo‐immobilized pH gradient IEF and ZE integrated on 2D microfluidic chip was employed for protein separation . As in the earlier work from this group (described in IEF section), the microchip channels were first coated by PEG‐silane, followed by photo‐immobilization of carrier ampholytes on the inner wall of the first channel of microchip mediated by benzophenone. Proteins (fluorescently labeled carbonic anhydrase, catalase, and myoglobin) focused during IEF in the first channel were further separated in the second channel in ZE mode; total separation time was 10 min.…”

“…With 2D photo‐immobilized pH gradient IEF × ZE chip, a better separation of proteins in native state than with free carrier ampholytes based on an IEF‐ZE 2D chip was achieved. Between the subsequent experiments, the second channel of the chip was washed with 1 M NaOH and Tween‐20 solution to remove nonspecifically adsorbed proteins, the first channel washing procedure was the same as in the previous study . Thus, the developed immobilized pH gradient based IEF × ZE 2D chip was reusable.…”

Outlook—Analysis of amino acids, peptides, and proteins: A virtual special issue

“…rapid determination of globin chains in red blood cells by CE , determination of genetic transferrin variants in human serum by CE , protein profiling of maize extracts by CE in bare or coated fused silica capillaries , investigation of sample pretreatment on protein stability by size‐exclusion chromatography , evaluation of complex formation between poliovirus and nanobodies by ACE , a comparison of three ACE modes for the study of drug‐protein interactions with emphasis on minimal consumption of proteins , and determination of metal ions content in superoxide dismutase enzymes by CE . Four methologically oriented papers present on‐chip protein CIEF using a photoimmobilized pH gradient , comparison of different mobilization strategies for CIEF of ovalbumin variants , optimization of free‐flow electrophoresis device for preparative separation of proteins , and study of adsorption and recovery issues of recombinant monoclonal antibodies in reversed‐phase LC .…”