Simulation and experiment of asymmetric electrode placement for electrophoretic exclusion in a microdevice.

Electrophoretic exclusion (EE) is a counterflow gradient technique that exploits hydrodynamic flow and electrophoretic forces to exclude, enrich, and separate analytes. Resolution for this technique has been theoretically examined and the smallest difference in electrophoretic mobilities that can be completely separated is estimated to be 10-13  cm2 /Vs. Traditional and mesoscale systems have been used, whereas microfluidics offers a greater range of geometries and configurations towards approaching this theoretical limit. To begin to understand the impact of seemingly subtle changes to the entrance flow and the electric field configurations, three closely related microfluidic interfaces were modeled, fabricated, and tested. These interfaces consisted of systematically varying placement of an asymmetric electrode relative to a channel entrance: leading electrode placed outside the channel entrance, leading electrode aligned with the channel, and leading electrode placed within the channel. A charged fluorescent dye is used as a sensitive and accurate probe for the model and to test the concentration variation at these interfaces. Models and experiments focused on visualizing the concentration profile in areas of high temporal dynamics, thus providing a severe test of the models. Experimental data and simulation results showed strong qualitative agreement. The complexity of the electric and flow fields about this interface and the agreement between models and testing suggests the theoretical assessment capabilities can be used to faithfully design novel and more efficient interfaces.

Electrophoretic exclusion microscale sample preparation for cryo-EM structural determination of proteins.

Transmission electron microscopy (TEM) of biological samples has a long history and has provided many important insights into fundamental processes and diseases. While great strides have been made in EM data collection and data processing, sample preparation is still performed using decades-old techniques. Those sample preparation methods rely on extensive macroscale purification and concentration to achieve homogeneity suitable for high-resolution analyses. Noting that relatively few bioparticles are needed to generate high-quality protein structures, this work uses microfluidics that can accurately and precisely manipulate and deliver bioparticles to grids for imaging. The use of microfluidics enables isolation, purification, and concentration of specific target proteins at these small scales and does so in a relatively short period of time (minutes). These capabilities enable imaging of more dilute solutions and obtaining pure protein images from mixtures. In this system, spatially isolated, purified, and concentrated proteins are transferred directly onto electron microscopy grids for imaging. The processing enables imaging of more dilute solutions, as low as 5 × 10-6 g/ml, with small total amounts of protein (<400 pg, 900 amol). These levels may be achieved with mixtures and, as proof-of-principle, imaging of one protein from a mixture of two proteins is demonstrated.