Microfluidic preparative free-flow isoelectric focusing in a triangular channel: system development and characterization.

A preparative scale free-flow IEF device is developed and characterized with the aim of addressing needs of molecular biologists working with protein samples on the milligrams and milliliters scale. A triangular-shape separation channel facilitates the establishment of the pH gradient with a corresponding increase in separation efficiency and decrease in focusing time compared with that in a regular rectangular channel. Functionalized, ion-permeable poly(acrylamide) gel membranes are sandwiched between PDMS and glass layers to both isolate the electrode buffers from the central separation channel and also to selectively adjust the voltage efficiency across the separation channel to achieve high electric field separation. The 50 x 70 mm device is fabricated by soft lithography and has 24 outlets evenly spaced across a pH gradient between pH 4 and 10. This preparative free-flow IEF system is investigated and optimized for both aqueous and denaturing conditions with respect to the electric field and potential efficiency and with consideration of Joule-heating removal. Energy distribution across the functionalized polyacrylamide gel is investigated and controlled to adjust the potential efficiency between 15 and 80% across the triangular separation channel. The device is able to achieve constant electric fields high as 370+/-20 V/cm through the entire triangular channel given the separation voltage of 1800 V, enabling separation of five fluorescent pI markers as a demonstration example.

Cascaded free-flow isoelectric focusing for improved focusing speed and resolution.

This work presents the first implementation of cascaded stages for a microfabricated free-flow isoelectric focusing (FF-IEF) device. Both analytical and computational models for IEF suggest device performance will be improved by utilizing multiple stages to reduce device residence time. These models are shown to be applicable by using focusing of small IEF markers as a demonstration. We also show focusing of fluorescently tagged proteins under different channel geometries, with the most efficient focusing occurring in the cascaded design, as predicted by theory. An additional aim of this work is to demonstrate the compatibility of cascaded FF-IEF with common bioanalytical tools. As an example, outlet fractions from cascaded FF-IEF were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Processing of whole cell lysate followed by immunoblotting for cell signaling markers demonstrates the reduction of albumin from samples, as well as the enrichment of apoptotic markers.

Micro free-flow IEF enhanced by active cooling and functionalized gels.

Rapid free-flow IEF is achieved in a microfluidic device by separating the electrodes from the focusing region with porous buffer regions. Moving the electrodes outside enables the use of large electric fields without the detrimental effects of bubble formation in the active region. The anode and cathode porous buffer regions, which are formed by acrylamide functionalized with immobilized pH groups, allow ion transport while providing buffering capacity. Thermoelectric cooling mitigates the effects of Joule heating on sample focusing at high field strengths (approximately 500 V/cm). This localized cooling was observed to increase device performance. Rapid focusing of low-molecular-weight p/ markers and Protein G-mouse IgG complexes demonstrate the versatility of the technique. Simulations provide insight into and predict device performance based on a well-defined sample composition.