Polyacrylamide gel plugs enabling 2‐D microfluidic protein separations via isoelectric focusing and multiplexed sodium dodecyl sulfate gel electrophoresis

Abstract

In situ photopolymerized polyacrylamide (PAAm) gel plugs are used as hydrodynamic flow control elements in a multidimensional microfluidic system combining IEF and parallel SDS gel electrophoresis for protein separations. The PAAm gel plugs offer a simple method to reduce undesirable bulk flow and limit reagent/sample crosstalk without placing unwanted constraints on the selection of separation media, and without hindering electrokinetic ion migration in the complex microchannel network. In addition to improving separation reproducibility, the discrete gel plugs integrated into critical regions of the chip enable the use of a simple pressure-driven sample injection method which avoids electrokinetic injection bias. The gel plugs also serve to greatly simplify operation of the spatially multiplexed system by eliminating the need for complex external fluidic interfaces. Using an FITC-labeled Escherichia coli cell lysate as a model system, the use of gel plugs is shown to significantly enhance separation reproducibility in a chip containing five parallel CGE channels, with an average variance in peak elution time of only 4.1%.

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

“…PAAm sieving gel While our previous efforts focused on using replaceable polyethylene oxide (PEO) as a sieving matrix in IEF/SDS-CGE chips, 6,10 there are several disadvantages to this approach. At lower gel concentrations ($2%), PEO can mobilize due to small on-chip pressure gradients, resulting in unstable interfaces between the first and second dimension channels, but filling the CGE separation channel array at higher PEO concentrations (>4%) is challenging due to the gel's high viscosity.…”

“…10 However, non-uniform injection of sample from the IEF channel to the SDS-CGE channel array remains problematic, resulting in significant broadening of the injected sample bands. Most critically, large electric field gradients across the width of a straight IEF channel can produce long sample tails during the injection process, preventing effective stacking following transfer to the second dimension and resulting in poor separation resolution.…”

“…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.…”

“…The gel plugs are also employed as on-chip SDS containers, allowing defined volumes of SDS to be injected and complexed with the IEF-focused proteins without the need for external intervention. Integrating SDS directly into the chip obviates the need for manual injection as required in previous studies, 6,10 improving automation of the system and providing photolithographic control over the amount of injected SDS.…”

Elastomer Application in Microsystem and Microfluidics

“…The design of elastomer microfluidics can be made based on these studies [71][72][73] . The electrokinetic injection of defined sample plugs within single dimension microfluidic systems has been extensively studied, with the most widely used injector configurations including the cross, double-T, and triple-T topologies 74,75 , and extended configurations employing continuous sample injection leveraging flow switching techniques.…”