Robert D. Chambers

Lymphocyte Electrotaxis In Vitro and In Vivo

Electric fields are generated in vivo in a variety of physiologic and pathologic settings, including penetrating injury to epithelial barriers. An applied electric field with strength within the physiologic range can induce directional cell migration (i.e., electrotaxis) of epithelial cells, endothelial cells, fibroblasts, and neutrophils suggesting a potential role in cell positioning during wound healing. In the present study, we investigated the ability of lymphocytes to respond to applied direct current (DC) electric fields. Using a modified Transwell assay and a simple microfluidic device, we show that human PBLs migrate toward the cathode in physiologically relevant DC electric fields. Additionally, electrical stimulation activates intracellular kinase signaling pathways shared with chemotactic stimuli. Finally, video microscopic tracing of GFP-tagged immunocytes in the skin of mouse ears reveals that motile cutaneous T cells actively migrate toward the cathode of an applied DC electric field. Lymphocyte positioning within tissues can thus be manipulated by externally applied electric fields, and may be influenced by endogenous electrical potential gradients as well.

Electrophoretic mobility measurements of fluorescent dyes using on-chip capillary electrophoresis

We present an experimental study of the effect of pH, ionic strength, and concentrations of the electroosmotic flow (EOF)‐suppressing polymer polyvinylpyrrolidone (PVP) on the electrophoretic mobilities of commonly used fluorescent dyes (fluorescein, Rhodamine 6G, and Alexa Fluor 488). We performed on‐chip capillary zone electrophoresis experiments to directly quantify the effective electrophoretic mobility. We use Rhodamine B as a fluorescent neutral marker (to quantify EOF) and CCD detection. We also report relevant acid dissociation constants and analyte diffusivities based on our absolute estimate (as per Nernst–Einstein diffusion). We perform well‐controlled experiments in a pH range of 3–11 and ionic strengths ranging from 30 to 90 mM. We account for the influence of ionic strength on the electrophoretic transport of sample analytes through the Onsager and Fuoss theory extended for finite radii ions to obtain the absolute mobility of the fluorophores. Lastly, we briefly explore the effect of PVP on adsorption–desorption dynamics of all three analytes, with particular attention to cationic R6G.

Imaging and Quantification of Isotachophoresis Zones Using Nonfocusing Fluorescent Tracers

We present a novel method for visualizing isotachophoresis (ITP) zones. We introduce negligibly small concentrations of a fluorophore that is not focused by isotachophoresis. This nonfocusing tracer (NFT) migrates through multiple isotachophoresis zones. As it enters each zone, the NFT concentration adapts to the local electric field in each zone. ITP zones can then be visualized with a point detector or camera. The method can be used to detect, identify, and quantify unknown analyte zones and can visualize complex and even transient electrophoresis processes. This visualization technique is particularly suited to microfluidic and laboratory-on-a-chip applications, as typical fluorescence microscopes and charge-coupled device (CCD) cameras can provide high-resolution spatiotemporal data. We present a theoretical description, a methodology for identifying analytes, and experimental validation. We also visualize and analyze a complex, transient DNA ITP preconcentration and separation.

Coupled isotachophoretic preconcentration and electrophoretic separation using bidirectional isotachophoresis.

We present a novel technique for coupling isotachophoretic preconcentration and electrophoretic separation using bidirectional isotachophoresis (ITP). Bidirectional ITP simultaneously sets up sharp ITP interfaces between relatively high- and low-mobility cations and high- and low-mobility anions. These two interfaces can migrate toward each other and be described as ion concentration shock waves. We here demonstrate a bidirectional ITP process in which we use the interaction of these anionic and cationic ITP shock waves to trigger a transformation from ITP preconcentration to electrophoretic separation. We use anionic ITP to focus anionic sample species prior to shock interaction. The interaction of the counter-propagating anionic and cationic ITP shocks then changes the local pH (and ionic strength) of the focused analyte zones. Under this new condition, the analytes no longer focus and begin to separate electrophoretically. The method provides faster and much less dispersive transition from ITP preconcentration to electrophoretic separation compared with traditional (unidirectional) transient ITP. It eliminates the need for intermediate steps between focusing and separation, such as manual buffer exchanges. We illustrate the technique with numerical simulations of species transport equations. We have validated our simulations with experimental visualization of bidirectional ITP zones. We then show the effectiveness of the technique by coupling ITP preconcentration and high-resolution separation of a 1 kbp DNA ladder via shock interaction in bidirectional ITP.

Effect of PVP on the electroosmotic mobility of wet-etched glass microchannels

We present an experimental study on the effect of polymer PVP on EOF mobility of microchannels wet etched into optical white soda lime glass, also known as Crown glass. We performed experiments to evaluate the effect of PVP concentration and pH on EOF mobility. We used on‐chip capillary zone electrophoresis and a neutral fluorescent dye as a passive marker to quantify the electroosmotic flow. We performed experiments under controlled conditions by varying pH from 5.2 and 10.3 and concentration of PVP from 0 to 2.0% w/w at constant ionic strength (30 mM). Our experiments show that PVP at concentrations of 1.0% or above very effectively suppress EOF at low pH (6.6). At high pH of 10.3, PVP has a much weaker suppressing effect on EOF and increasing its concentration above about 0.5% showed negligible effect on EOF mobility. Finally, we briefly discuss the effects of pH on using PVP as an adsorbed coating. Our experiments provide useful guidelines on choosing correct pH and concentration of PVP for effective EOF suppression in glass channels.