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Gradient micro free flow electrophoresis (μFFE) was used to observe the equilibria of DNA aptamers with their targets (IgE or HIVRT) across a range of ligand concentrations. A continuous stream of aptamer was mixed online with an increasing concentration of target and introduced into the μFFE device, which separated ligand-aptamer complexes from the unbound aptamer. The continuous nature of μFFE allowed the equilibrium distribution of aptamer and complex to be measured at 300 discrete target concentrations within 5 min. This is a significant improvement in speed and precision over affinity capillary electrophoresis (ACE) assays. The dissociation constant of the aptainer-IgE complex was estimated to be 48 ± 3 nM. The high coverage across the range of ligand concentrations allowed complex stoichiometries of the aptamer-HIVRT complexes to be observed. Nearly continuous observation of the equilibrium distribution from 0 to 500 nM HIVRT revealed the presence of complexes with 3:1 (aptamer/HlVRT), 2:1, and 1:1 stoichiometries. [ABSTRACT FROM AUTHOR]

Fast, continuous separation of mitochondria from rat myoblasts using micro free-flow electrophoresis (μFFE) with online laser-induced fluorescence detection (HF) is reported. Mitochondrial electrophoretic proffles were acquired in less than 30 s. In comparison to macroscale FFE instnnments, μFFE devices consumed approximately 100-fold less sample, used 10-fold less buffer, and required a 15-fold lower electric field. Mitochondrial electrophoretic mobility distributions measured using μFFE were compared to those measured with a capillary electrophoresis instrument with laser-induced fluorescence detection (CE-HF). There was high similarity between the two distributions with CE-HF distribution being offset by 1.8 x 10-4 cm2 V-1 s-1 with respect to the μFFE distribution. We hypothesize that this offset results from the differences in electric field strength used in the techniques. In comparison to CE-HF, analysis of mitochondria using μFFE greatly decreased separation time and required less separation voltage, while maintaining low sample (125nL) and buffer (250 μL) volumes. These features together with the potential for collecting separated organelle fractions for further characterization make μFFE a very attractive tool for the high-throughput analysis of organelle sub-populations as well as investigating the fundamentals of the electrophoretic mobility of biological particles. [ABSTRACT FROM AUTHOR]

The broadening mechanisms for micro-free flow electrophoresis (μ-FFE) have been investigated using a van Deemter analysis. Separation power, the product of electric field and residence time, is presented as a parameter for predicting the position of sample streams and for comparing separations under different conditions. Band broadening in μ-FFE is governed by diffusion at lower linear velocities and a migration distance-dependent mechanism at higher linear velocities. At higher linear velocities, the parabolic flow profile is elongated, generating a distribution of analyte residence times in the separation channel. This distribution of residence times gives rise to a distribution of migration distances in the lateral direction since analytes spend different amounts of time in the electric field. Equations were derived to predict the effect of electric field and buffer flow rate on broadening. Experimental data were collected to determine whether the derived equations were useful in explaining broadening caused by diffusion and hydrodynamic flow at different linear velocities and electric fields. Overall there was an excellent correlation between the predicted and experimentally observed values allowing linear velocity and electric field to be optimized. Suppression of electroosmotic flow is proposed as a means of reducing μ-FFE band broadening due to hydrodynamic effects and maximizing resolution and peak capacity. [ABSTRACT FROM AUTHOR]

A multiple-depth micro free-flow electrophoresis chip (μ-FFE) has been fabricated with a 20-μm-deep separation channel and 78-μm-deep electrode channels. Due to the difference in channel heights, the linear velocity of buffer in the electrode channels is ∼15 times that of the buffer in the separation channel. Previous μ-FFE devices have been limited by electrolysis product formation at the electrodes. These electrolysis products, manifested as bubbles, decreased the electric field and disrupted the buffer flow profile, limiting performance and preventing continuous operation. Using channel depth to control buffer flow over the electrodes and in the separation channel effectively removes electrolysis products, allowing continuous operation. The linear velocities in the channels were confirmed using particle velocimetry and compared well with values predicted using lubrication theory. A separation potential of 645 V could be applied before significant Joule heating was observed. This corresponded to an electric field of 586 V/cm in the separation channel, a 4-fold increase over our previous design. A separation of fluorescent standards was demonstrated using the new μ-FFE device. Resolution increased by a factor of 1.3 over our previous design, even when operated under similar conditions, suggesting that effective removal of electrolysis products is more important than originally thought. [ABSTRACT FROM AUTHOR]

A micro-free-flow electrophoresis chip has been fabricated into a glass wafer etched with 20-μm-deep channels. Wafers were bonded anodically using an intermediate amorphous silicon film. Electric fields as high as 283 V/cm were applied across the separation channel to obtain baseline resolution of fluorescent standards in 4.8 s. The effect of electric fields ranging from 0 to 283 V/cm on the separations and resulting resolutions were examined. Resolution was shown to increase linearly with the applied electric field. Joule heating was not significant under the conditions tested. Instead, the generation of electrolysis products at higher currents proved to be the limiting factor preventing higher separation potentials from being used. [ABSTRACT FROM AUTHOR]