Microfluidic device having an immobilized pH gradient and PAGE gels for protein separation and analysis
Inventors
Sommer, Gregory J. • Hatch, Anson V. • Singh, Anup K. • Wang, Ying-Chih
Assignees
National Technology and Engineering Solutions of Sandia LLC • Sandia National Laboratories
Publication Number
US-8329016-B1
Publication Date
2012-12-11
Expiration Date
2028-07-30
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Abstract
Disclosed is a novel microfluidic device enabling on-chip implementation of a two-dimensional separation methodology. Previously disclosed microscale immobilized pH gradients (IPG) are combined with perpendicular polyacrylamide gel electrophoresis (PAGE) microchannels to achieve orthogonal separations of biological samples. Device modifications enable inclusion of sodium dodecyl sulfate (SDS) in the second dimension. The device can be fabricated to use either continuous IPG gels, or the microscale isoelectric fractionation membranes we have also previously disclosed, for the first dimension. The invention represents the first all-gel two-dimensional separation microdevice, with significantly higher resolution power over existing devices.
Core Innovation
The invention disclosed is a novel microfluidic device enabling on-chip implementation of a two-dimensional separation methodology combining previously disclosed microscale immobilized pH gradients (IPG) with perpendicular polyacrylamide gel electrophoresis (PAGE) microchannels to achieve orthogonal separations of biological samples. The device modifications enable inclusion of sodium dodecyl sulfate (SDS) in the second PAGE dimension and can be fabricated to use either continuous IPG gels or microscale isoelectric fractionation membranes for the first separation dimension. This device represents the first all-gel two-dimensional separation microdevice offering significantly higher resolution power than existing devices.
The problem being addressed arises from conventional two-dimensional (2-D) IEF-PAGE proteomics techniques that are reliable but labor-intensive, time-consuming, and involve bulky handling of gel strips, where the first dimension uses immobilized pH gradient strips transferred to a slab gel for a second dimension separation. Prior methods attempting to miniaturize or implement these separations in microfluidic formats were limited by difficulties in creating stable immobilized pH gradients on a microdevice, challenges in transferring focused bands into gels for secondary separation, complex instrumentation, lack of temporal stability, and bulk flow issues reducing resolution.
This invention addresses the long-felt need for a miniaturized, on-chip integrated 2-D IEF/PAGE platform with an immobilized pH gradient in the first dimension and orthogonal PAGE microchannels in the second dimension, allowing automated, rapid, high-resolution protein and molecular species separation in a continuous gel format. It overcomes limitations of free-solution IEF microdevices by covalently bonding gels to microchannel surfaces, allowing stable and reproducible immobilized pH gradients. The device also includes means for on-chip sodium dodecyl sulfate (SDS) incubation with focused bands to enhance resolution in the second dimension through SDS-PAGE.
Claims Coverage
The patent contains one primary independent claim, which establishes a comprehensive two-dimensional microchannel separation device, and further claims dependent features expanding on this device's components and functions. Below are the main inventive features claimed, extracted from the independent claim and substantiated by dependent claims.
Two-dimensional microchannel separation device with integrated IEF and orthogonal PAGE stages
A device having an isoelectric focusing (IEF) stage with a central microchannel including an acrylate-terminated coating covalently bonded to an immobilized pH gradient polymer gel that partially fills the microchannel, coupled to an orthogonal polyacrylamide gel electrophoresis (PAGE) separation stage comprising intersecting side microchannels for the second dimension.
Covalently bonded immobilized pH gradient polymer gel in the IEF stage
The immobilized pH gradient gel comprises a polymerized acrylamide/bisacrylamide solution containing multiple preselected acrylamido buffers with different pK values distributed to create a stable pH gradient along the central microchannel length.
Orthogonal PAGE separation stage with multiple intersecting side microchannels filled with polymerized acrylamide gel
One or more side microchannels intersect the central microchannel, each filled with a polymerized acrylamide gel covalently bonded to their surfaces, enabling orthogonal electrophoretic separation perpendicular to the IEF direction.
Incorporation of size exclusion membranes within PAGE microchannels for enhanced separation and SDS treatment
One or more size exclusion membranes span portions of the intersecting side microchannels, with secondary intersecting microchannels enabling stacking and in situ treatment of focused bands with sodium dodecyl sulfate (SDS) prior to PAGE separation.
Means for in situ treatment of focused bands with SDS prior to second dimension
Provision for introducing SDS in the presence of focused analyte bands in the IEF stage or at size exclusion membranes before PAGE to ensure SDS denaturation and uniform charge-to-mass ratio, improving resolution in the second dimension.
Alternative fabrication of the IEF stage using multiple porous membranes with specific pH values spaced along the microchannel
A variation where the central microchannel incorporates multiple porous membranes, each covalently bonded and having a distinct specific pH, enabling isoelectric fractionation by charge-based exclusion and more precise injection into the second dimension.
Single contiguous polymerized polyacrylamide gel combining gradients in pH and porosity for simultaneous 2-D separation
A single polymerized gel volume in the central micro-volume contains an immobilized pH gradient along one direction and an orthogonal gradient in porosity along the perpendicular direction, allowing simultaneous isoelectric focusing and pore-limit electrophoresis separation within the same gel.
The claims collectively cover a microfluidic device integrating an immobilized pH gradient isoelectric focusing stage covalently bonded in a central microchannel partially filled with polymer gel, coupled orthogonally to polyacrylamide gel electrophoresis side microchannels, with provisions for in situ SDS treatment, size exclusion membranes for improved separation, and variations including multiplexed porous membranes or combined gel gradients to achieve high-resolution two-dimensional molecular separations on-chip.
Stated Advantages
Reduced run times due to shorter separation length scales inherent in microfluidic formats.
Reduced sample volume requirements allowing analysis of small quantity samples.
On-chip automation resulting in decreased user interaction and simpler handling since no gel strips are physically transferred.
Significantly higher resolution power in 2-D separations over existing devices due to the all-gel microfluidic design minimizing diffusion and inter-diffusion effects.
Documented Applications
Proteomics and molecular species analysis by performing rapid, high-resolution two-dimensional isoelectric focusing and polyacrylamide gel electrophoresis separations on-chip.
On-chip biochemical and biological sample fractionation utilizing two orthogonal separation mechanisms in a continuous gel microdevice format.
Automated SDS-PAGE separations following isoelectric focusing through integrated SDS incubation in a microfluidic platform.
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