Michael W. L. Watson

Technique for Real-Time Measurements of Endothelial Permeability in a Microfluidic Membrane Chip Using Laser-Induced Fluorescence Detection

Characterizing permeability of the endothelium that lines blood vessels and heart valves provides fundamental physiological information and is required to evaluate uptake of drugs and other biomolecules. However, current techniques used to measure permeability, such as Transwell insert assays, do not account for the recognized effects of fluid flow-induced shear stress on endothelial permeability or are inherently low-throughput. Here we report a novel on-chip technique in a two-layer membrane-based microfluidic platform to measure real-time permeability of endothelial cell monolayers on porous membranes. Bovine serum albumin (a model protein) conjugated with fluorescein isothiocyanate was delivered to an upper microchannel by pressure-driven flow and was forced to permeate a poly(ethylene terephthalate) membrane into a lower microchannel, where it was detected by laser-induced fluorescence. The concentration of the permeate at the point of detection varied with channel flow rates in agreement to less than 1% with theoretical analyses using a pore flow model. On the basis of the model, a sequential flow rate stepping scheme was developed and applied to obtain the permeability of cell-free and cell-bound membrane layers. This technique is a highly sensitive, novel microfluidic approach for measuring endothelial permeability in vitro, and the use of micrometer-sized channels offers the potential for parallelization and increased throughput compared to conventional shear-based permeability measurement methods.

Gradient elution in microchannel electrochromatography.

There is great interest in using microfluidic channels packed with a stationary phase for chemical separations of complex mixtures. A key advantage of such techniques is the use of electroosmotic flow (EOF), controlled simply by applying electrical potentials between reservoirs. A disadvantage for this technique, however, is a lack of compatibility with gradient elution separations. This limitation arises from the dependence of EOF velocity on run buffer content (including the concentration of organic modifier). Here, we introduce a method for implementing gradient elution in electrochromatography in which multiple run buffers are velocity-matched, such that the elution profile resembles that found in conventional HPLC. This method is driven entirely with EOF, meaning that pumps, valves, and pressure fittings are not required. The method was validated by application to separations of peptide standards and protein digests. These results suggest that microfluidic electrochromatography may be compatible with a wide range of applications that have previously been unexplored.