High-speed Analysis of Proteins by Microchip Isoelectric Focusing with Linear-imaging UV Detection

Abstract

High-speed analyses of proteins by microchip isoelectric focusing (MCIEF) were investigated on straight channel chips. By employing a commercially available microchip electrophoresis instrument equipped with a linear-imaging UV detector, sample proteins focused in a separation channel could be detected without a mobilization step. Typically, standard proteins were focused within 210 s at different positions in a separation channel with its total length of 38.8 mm on the basis of their pI values. The linear relationship between the focused position of each protein and its pI value was observed in the pH range from 6 to 10. The formation process of a pH gradient in the microchannel and the effects of the applied voltage and the channel length on the MCIEF separation were also investigated.

A Simple and Easy-to-Use Capillary Isoelectric Focusing Technique Using Reagent-Release Hydrogels

“…In conventional CIEF, however, the focused analytes must be transported toward a detection window, which can sometimes decrease the resolution due to a band broadening during transportation [5]. Recently, many IEF techniques using microfluidic devices (microfluidic IEF, MIEF) have been developed to overcome this drawback [6][7][8][9][10][11][12]. MIEF has many advantages compared to CIEF, including a short analysis time (within 5 min) due to a short separation channel (several cm) and the requirement of a minimal amount of reagents and samples (several L).…”

“…MIEF has many advantages compared to CIEF, including a short analysis time (within 5 min) due to a short separation channel (several cm) and the requirement of a minimal amount of reagents and samples (several L). In particular, a short separation length in MIEF makes whole-column imaging possible, which enables real-time observation of an IEF process over the whole length of the microchannel without the requirement of any of the transportation processes necessary for conventional CIEF [9][10][11][12]. More recently, a microfluidic device for two-dimensional electrophoresis was independently developed by Kim and Moon and Yang et al to obtain a higher resolution for analyzing a complicated mixture of biogenic samples [13,14].…”

Label-free real-time imaging in microchip free-flow electrophoresis applying high speed deep UV fluorescence scanning

“…Although a fascinating technology, commercially it was not very successful, possibly because the achieved sensitivities were only moderate as evident from various literature applications. [31][32][33][34][35][36] In capillary electrophoresis (CE), microchip electrophoresis (MCE) and gel electrophoresis (GE) label-free analyte detection and imaging can be achieved by excitation of the native fluorescence of many small molecules and proteins in the deep UV spectral range (<300 nm). [37][38][39][40][41][42][43][44][45][46] Although less general than UV absorbance detection it can lead to higher sensitivities for natively fluorescent compounds.…”

Recent Progress of On-line Sample Preconcentration Techniques in Microchip Electrophoresis

“…Besides this, some novel sample preconcentration techniques utilizing specific characteristics of microchips have appeared in MCE. 8 The on-line sample preconcentration techniques for MCE have four classifications: preconcentration by analyte velocity change in two or three discontinuous solutions system, focusing, [9][10][11][12][13][14][15][16][17][18][19][20] solid phase extraction, [21][22][23] and electrokinetic trapping (filtering). [24][25][26][27] Among them, novel focusing and electrokinetic trapping techniques have been exhaustively investigated in the recent decade.…”