Integration of isoelectric focusing with multi-channel gel electrophoresis by using microfluidic pseudo-valves

Abstract

Two-dimensional (2D) protein separation is achieved in a plastic microfluidic device by integrating isoelectric focusing (IEF) with multi-channel polyacrylamide gel electrophoresis (PAGE). IEF (the first dimension) is carried out in a 15 mm-long channel while PAGE (the second dimension) is in 29 parallel channels of 65 mm length that are orthogonal to the IEF channel. An array of microfluidic pseudo-valves is created for introducing different separation media, without cross-contamination, in both dimensions; it also allows transfer of proteins from the first to the second dimension. Fabrication of pseudo-valves is achieved by photo-initiated, in situ gel polymerization; acrylamide and methylenebisacrylamide monomers are polymerized only in the PAGE channels whereas polymerization does not take place in the IEF channel where a mask is placed to block the UV light. IEF separation medium, carrier ampholytes, can then be introduced into the IEF channel. The presence of gel pseudo-valves does not affect the performance of IEF or PAGE when they are investigated separately. Detection in the device is achieved by using a laser induced fluorescence imaging system. Four fluorescently-labeled proteins with either similar pI values or close molecular weight are well separated, demonstrating the potential of the 2D electrophoresis device. The total separation time is less than 10 minutes for IEF and PAGE, an improvement of 2 orders of magnitude over the conventional 2D slab gel electrophoresis.

Microfluidic 2-D PAGE using multifunctional in situ polyacrylamide gels and discontinuous buffers

“…16 Originally explored for capillary separations, 17,18 crosslinked PAAm has been widely used as a gel electrophoresis sieving medium in microfluidic platforms. 8,9,[19][20][21][22] The fabrication of long PAAm separation zones can result in voids along the gel-sidewall interface in silica capillaries due to shrinkage of the polymer chains during photopolymerization. [23][24][25] To eliminate bubble formation, a layer of linear polyacrylamide (LPA) may be formed by bonding pedant methacrylate groups within the LPA to acrylic groups grown on the silica surface via TPM.…”

“…In recent years, a number of efforts have targeted the development of microfluidic systems which mimic the functionality of 2-D PAGE in a miniaturized format, while promising significantly shorter analysis times and higher levels of automation. [5][6][7][8][9][10] A common feature of these systems is the combination of a single microchannel for performing a first-dimension IEF separation, and an array of discrete second-dimension microchannels for capillary gel electrophoresis (CGE), with the second-dimension array replacing the traditional slab gel format for gel electrophoresis. Although the peak capacities of chip-based IEF/SDS-CGE systems remain lower than traditional 2-D PAGE, the application of microfluidics technology has successfully reduced separation times by 2 orders of magnitude, with substantially lower sample loading requirements than slab gels.…”

“…Although the peak capacities of chip-based IEF/SDS-CGE systems remain lower than traditional 2-D PAGE, the application of microfluidics technology has successfully reduced separation times by 2 orders of magnitude, with substantially lower sample loading requirements than slab gels. 9,10 Furthermore, recent advances including the demonstration of on-chip differential gel electrophoresis 11 and separation repeatability comparable to slab gel analysis 10 suggest that further developments may allow microfluidic 2-D separations to emerge as a viable high-throughput alternative to slab-gel 2-D PAGE.…”

Microfluidics:A Boon for Biological Research

“…With the recent advances in microfluidic technology, protein targets have been characterized using microfluidic devices which can express, separate, label and quantify proteins to the level of single-cells. Researchers have developed microfluidic chips capable of separating the proteins through micellar electrokinetic chromatography (MEKC) 37 , capillary zone electrophoresis 37,38 and isoelectric focusing 39 in less than 20 min.…”

Two‐dimensional protein separation in microfluidic devices

“…A few shortcomings of the device in this pioneering effort were noted in later reports [45,46]. Firstly, bonding of different layers must be reversible in order to dissemble the four-layer IEF structure.…”