Free-flow electrophoresis (FFE) is a technique that performs an electrophoretic separation on a continuous stream of analyte as it flows through a planar flow channel. The electric field is applied perpendicularly to the flow to deflect analytes laterally according to their mobility as they flow through the separation channel. Miniaturization of FFE (μFFE) over the past 15 years has allowed analytical and preparative separation of small volume samples. Advances in chip design have improved separations by reducing interference from bubbles generated by electrolysis. Mechanisms of band broadening have been examined theoretically and experimentally to improve resolution in μFFE. Separations using various modes such as zone electrophoresis, isoelectric focusing, isotachophoresis, and field-step electrophoresis have been demonstrated.
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 minutes. This is a significant improvement in speed and precision over affinity capillary electrophoresis (ACE) assays. The dissociation constant of the aptamer-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:HIVRT), 2:1 and 1:1 stoichiometries.
Synthetic silica preforms with an inverse opal or three-dimensionally ordered macroporous (3DOM) structure were converted to 3DOM TiOF 2 and subsequently to 3DOM TiO 2 by solidgas pseudomorphic transformation reactions, reactions which maintain the shape and structural features of the original material. 3DOM SiO 2 preforms with periodic arrays of macropores and hierarchical feature sizes (e.g., macropore separation 334 nm, average wall thickness 59 nm) were prepared by colloidal crystal templating. They were reacted with TiF 4 in sealed steel pipes at 190, 235, and 300 °C. At 190 °C no conversion took place, while at 300 °C the material was converted mostly to crystalline TiOF 2 with an irregular structure. However, at 235 °C the periodic macroporous structure of the preform was maintained with little change in average pore separation. In these samples, the initially smooth wall structure of 3DOM SiO 2 was largely replaced by interconnected TiOF 2 cubes with edge lengths of 133 nm. The X-ray diffraction (XRD) pattern showed sharp lines of TiOF 2 . The product exhibited opalescence similar to that of the preform, giving a visual confirmation of the success of the pseudomorphic transformation on an extended length scale. An analogous transformation was also investigated with spherical silica preforms. Different stages of transformation were observed by scanning electron microscopy, permitting a discussion of critical parameters in these conversions. The macroporous TiOF 2 product was subsequently converted to TiO 2 (anatase) by reaction with moist air at 300 °C. In this reaction, pseudomorphism was observed on the scale of tens of micrometers, on the submicrometer macropore scale, and on the scale of the cubic particles forming the wall skeleton. The sample was still composed of interconnected cubes with similar edge lengths, and the pore spacing was nearly maintained. XRD showed only TiO 2 anatase reflections. The synthetic paradigms demonstrated for the silica to anatase conversion may be transferable to other 2D or 3D material shapes within the applicable range of feature sizes.
Microfluidic free-flow electrophoresis (μFFE) is a separation technique that separates continuous streams of analytes as they travel through an electric field in a planar flow channel. The continuous nature of the μFFE separation suggests that approaches more commonly applied in spectroscopy and imaging may be effective in improving sensitivity. The current paper describes the S/N improvements that can be achieved by simply averaging multiple images of a μFFE separation; 20-24-fold improvements in S/N were observed by averaging the signal from 500 images recorded for over 2 min. Up to an 80-fold improvement in S/N was observed by averaging 6500 images. Detection limits as low as 14 pM were achieved for fluorescein, which is impressive considering the non-ideal optical set-up used in these experiments. The limitation to this signal averaging approach was the stability of the μFFE separation. At separation times longer than 20 min bubbles began to form at the electrodes, which disrupted the flow profile through the device, giving rise to erratic peak positions.
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