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Field-free remobilization of proteins after isoelectric focusing in packed capillaries.

ABSTRACT: Pressure-driven remobilization without an applied electric field is shown to be possible with capillary isoelectric focusing using packed capillaries. The capillary dimensions are 100 ?m i.d. and 2 cm in length, and the packing is made of 0.9 ?m nonporous silica particles that are chemically modified with a brush layer of polyacrylamide. Both reversible and irreversible adsorption are shown to be negligible. The packed capillaries eliminate the problem of unwanted hydrodynamic flow between reservoirs. Three proteins are focused: trypsin inhibitor, carbonic anhydrase II, and myoglobin. The time required for focusing in the packed capillaries is increased by only a factor of 2 compared to the open capillary, giving complete focusing in less than 15 min at 200 V/cm. The packed capillaries allow the use of higher electric fields, with resolution continually increasing up to at least 1500 V/cm. The packing obstructs diffusional broadening after the field is turned off: for trypsin inhibitor, D = 6.1(±0.3) × 10(-8) cm(2)/s for the packed capillary vs D = 28.8(±0.3) × 10(-8) cm(2)/s for the open capillary. The broadening contributed by the packing during remobilization is from eddy diffusion, and it is described by its plate height, H, which is the variance per unit length: H = ?(2)/L = 0.64 ?m. This limits the resolution to 0.1 pH units for the 2 cm capillary having a pH range of 3-10, giving a theoretical peak capacity of 47.

SUBMITTER: Hua Y

PROVIDER: S-EPMC3042039 | biostudies-literature | 2010 Nov

REPOSITORIES: biostudies-literature

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Field-free remobilization of proteins after isoelectric focusing in packed capillaries.

Hua Yimin Y Koshel Brooke M BM Wirth Mary J MJ

Analytical chemistry 20101008 21

Pressure-driven remobilization without an applied electric field is shown to be possible with capillary isoelectric focusing using packed capillaries. The capillary dimensions are 100 μm i.d. and 2 cm in length, and the packing is made of 0.9 μm nonporous silica particles that are chemically modified with a brush layer of polyacrylamide. Both reversible and irreversible adsorption are shown to be negligible. The packed capillaries eliminate the problem of unwanted hydrodynamic flow between reser ...[more]

PMID: 20931968

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Project description:Particle concentration is a key unit operation in biochemical assays. Although there are many techniques for particle concentration in continuous-phase microfluidics, relatively few are available in multiphase (plug-based) microfluidics. Existing approaches generally require external electric or magnetic fields together with charged or magnetized particles. This paper reports a passive technique for particle concentration in water-in-oil plugs which relies on the interaction between particle sedimentation and the recirculating vortices inherent to plug flow in a cylindrical capillary. This interaction can be quantified using the Shields parameter ([Formula: see text]), a dimensionless ratio of a particle's drag force to its gravitational force, which scales with plug velocity. Three regimes of particle behavior are identified. When [Formula: see text] is less than the movement threshold (region I), particles sediment to the bottom of the plug where the internal vortices subsequently concentrate the particles at the rear of the plug. We demonstrate highly efficient concentration (∼100%) of 38 μm glass beads in 500 μm diameter plugs traveling at velocities up to 5 mm/s. As [Formula: see text] is increased beyond the movement threshold (region II), particles are suspended in well-defined circulation zones which begin at the rear of the plug. The length of the zone scales linearly with plug velocity, and at sufficiently large [Formula: see text], it spans the length of the plug (region III). A second effect, attributed to the co-rotating vortices at the rear cap, causes particle aggregation in the cap, regardless of flow velocity. Region I is useful for concentrating/collecting particles, while the latter two are useful for mixing the beads with the solution. Therefore, the two key steps of a bead-based assay, concentration and resuspension, can be achieved simply by changing the plug velocity. By exploiting an interaction of sedimentation and recirculation unique to multiphase flow, this simple technique achieves particle concentration without on-chip components, and could therefore be applied to a range of heterogeneous screening assays in discrete nl plugs.

Dataset Information

Field-free remobilization of proteins after isoelectric focusing in packed capillaries.

Publications

Field-free remobilization of proteins after isoelectric focusing in packed capillaries.

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