Enrichment of putative stem cells from adipose tissue using dielectrophoretic field-flow fractionation.

We have applied the microfluidic cell separation method of dielectrophoretic field-flow fractionation (DEP-FFF) to the enrichment of a putative stem cell population from an enzyme-digested adipose tissue derived cell suspension. A DEP-FFF separator device was constructed using a novel microfluidic-microelectronic hybrid flex-circuit fabrication approach that is scaleable and anticipates future low-cost volume manufacturing. We report the separation of a nucleated cell fraction from cell debris and the bulk of the erythrocyte population, with the relatively rare (<2% starting concentration) NG2-positive cell population (pericytes and/or putative progenitor cells) being enriched up to 14-fold. This work demonstrates a potential clinical application for DEP-FFF and further establishes the utility of the method for achieving label-free fractionation of cell subpopulations.

A Continuous-Flow Polymerase Chain Reaction Microchip With Regional Velocity Control.

This paper presents a continuous-flow polymerase chain reaction (PCR) microchip with a serpentine microchannel of varying width for "regional velocity control." Varying the channel width by incorporating expanding and contracting conduits made it possible to control DNA sample velocities for the optimization of the exposure times of the sample to each temperature phase while minimizing the transitional periods during temperature transitions. A finite element analysis (FEA) and semi-analytical heat transfer model was used to determine the distances between the three heating assemblies that are responsible for creating the denaturation (96 degrees C), hybridization (60 degrees C), and extension (72 degrees C) temperature zones within the microchip. Predictions from the thermal FEA and semi-analytical model were compared with temperature measurements obtained from an infrared (IR) camera. Flow-field FEAs were also performed to predict the velocity distributions in the regions of the expanding and contracting conduits to study the effects of the microchannel geometry on flow recirculation and bubble nucleation. The flow fields were empirically studied using micro particle image velocimetry (mu-PIV) to validate the flow-field FEA's and to determine experimental velocities in each of the regions of different width. Successful amplification of a 90 base pair (bp) bacillus anthracis DNA fragment was achieved.

Dielectrophoresis-based programmable fluidic processors.

Droplet-based programmable processors promise to offer solutions to a wide range of applications in which chemical and biological analysis and/or small-scale synthesis are required, suggesting they will become the microfluidic equivalents of microprocessors by offering off-the-shelf solutions for almost any fluid based analysis or small scale synthesis problem. A general purpose droplet processor should be able to manipulate droplets of different compositions (including those that are electrically conductive or insulating and those of polar or non-polar nature), to control reagent titrations accurately, and to remain free of contamination and carry over on its reaction surfaces. In this article we discuss the application of dielectrophoresis to droplet based processors and demonstrate that it can provide the means for accurately titrating, moving and mixing polar or non-polar droplets whether they are electrically conductive or not. DEP does not require contact with control surfaces and several strategies for minimizing surface contact are presented. As an example of a DEP actuated general purpose droplet processor, we show an embodiment based on a scaleable CMOS architecture that uses DEP manipulation on a 32 x 32 electrode array having built-in control and switching circuitry. Lastly, we demonstrate the concept of a general-purpose programming environment that facilitates droplet software development for any type of droplet processor.

Dielectric characterization of complete mononuclear and polymorphonuclear blood cell subpopulations for label-free discrimination.

Dielectric spectroscopy is a powerful technique for the elucidation of a number of important cell biophysical properties, and it can provide information about cell morphology, physiological state, viability and identity. A high-impact application for dielectric cell analysis would be microfluidic flow-through impedance sensing to perform what is perhaps the most routinely ordered medical diagnostic assay, a complete blood count with white blood cell differential enumeration. To assess the biophysical feasibility of such an analysis, we obtained reference dielectric measurements of the complete complement of purified leukocyte subpopulations using the dielectrophoretic crossover frequency method. The sensitivity of this method can detect subtle changes in cell morphology and physiology, so we developed a leukocyte isolation protocol based on a suite of negative selection techniques to yield cell subpopulations that were minimally processed and in an as near native state as possible. This is the first reported study of the dielectric properties of all the major leukocyte subpopulations that includes separate analysis of the polymorphonuclear neutrophil, basophil and eosinophil cell types. We show that T-lymphocytes, B-lymphocytes, monocytes and granulocytes possess distinct membrane dielectric properties and that the morphologically similar granulocyte subpopulations can be identified via their dielectric and size properties. Finally, we discuss the application of our findings to label-free systems for the analysis of leukocytes.