Electrical cell manipulation in microfluidic systems

This dissertation reports on the development of devices and concepts for electrical and microfluidic cell manipulation. In the present context, the term cell manipulation stands for both cell handling and cell modification. The combination of microfluidic channels with micropatterned electrodes allows for the definition of highly localised chemical and electrical environments with spatial resolution comparable to the size of a cell. The devices fabricated in the frame of this thesis employ dielectrophoretic particle handling schemes such as deflection and trapping in pressure-controlled laminar flows to bring cells to – or immobilise them at – locations where cell altering electric fields or chemicals are present. The two concepts of dielectrophoretic cell dipping and cell immersion are introduced and experimentally shown for erythrocytes dipped into Rhodamine in flow, and for individually immobilised Jurkat cells immersed by Trypan Blue. Also, in-situ membrane breakdown in high intensity AC electric fields is optically assessed by efflux of haemoglobin (haemolysis) and by influx of nucleic stains or fluorescence-enhancing ions. The most advanced experiments are on-chip medium exchange followed immediately by electropermeablisation or electrodeformation. The majority of assays presented in this thesis are carried out in microfabricated glass-polymer-glass chips featuring top-bottom electrodes. The devices are fluidically controlled by external gas pressure bridging circuits. Experimental evidence of the unmatched precision of pressure bridging is given in the case of micrometric xy positioning of cells at the intersection of two perpendicular microfluidic channels. Further shown in this document are two methods of optical in-situ temperature measurements, important for bioinstrument characterisation. The two concepts of thermoquenching of a fluorescent dye and the original thermoprecipitation of "smart polymers" are used. The last part of this work deals with the innovative, conceptual engineering tool Liquid Electrode. The general concept and its advantages over solid-state electrodes are given, followed by numerical particle tracking in the case of the novel lateral nDEP particle deflection. The chapter on liquid electrodes concludes with preliminary experimental results of buffer swapping of cells in flow and of AC electropermeabilisation of erythrocytes at frequencies far below the cut-off frequency of corresponding solid-state microelectrodes

Impaired neutrophil extracellular trap formation : a novel defect in the innate immune system of aged individuals

Neutrophil extracellular traps (NETs) are a recently discovered addition to the defensive armamentarium of neutrophils, assisting in the immune response against rapidly dividing bacteria. Although older adults are more susceptible to such infections, no study has examined whether aging in humans influences NET formation. We report that TNF-α-primed neutrophils generate significantly more NETs than unprimed neutrophils and that lipopolysaccharide (LPS)- and interleukin-8 (IL-8)-induced NET formation exhibits a significant age-related decline. NET formation requires generation of reactive oxygen species (ROS), and this was also reduced in neutrophils from older donors identifying a mechanism for reduced NET formation. Expression of IL-8 receptors (CXCR1 and CXCR2) and the LPS receptor TLR4 was similar on neutrophils from young and old subjects, and neutrophils challenged with phorbol-12-myristate-13-acetate (PMA) showed no age-associated differences in ROS or NET production. Taken together, these data suggest a defect in proximal signalling underlies the age-related decline in NET and ROS generation. TNF-α priming involves signalling through p38 MAP kinase, but activation kinetics were comparable in neutrophils from young and old donors. In a clinical setting, we assessed the capacity of neutrophils from young and older patients with chronic periodontitis to generate NETs in response to PMA and hypochlorous acid (HOCL). Neutrophil extracellular trap generation to HOCL, but not PMA, was lower in older periodontitis patients but not in comparison with age-matched controls. Impaired NET formation is thus a novel defect of innate immunity in older adults but does not appear to contribute to the increased incidence of periodontitis in older adults

Analytical expression for electric filed between two facing strip electrodes in microchannels

An analytical solution is presented for the electric field between two facing strip electrodes situated in a microchannel, as frequently used in impedance cytometry applications. The measured change in resistance as induced by a 5 mm bead moving through the microchannel is in good agreement with the model. It is also demonstrated that the centre sensitivity is maximal for an electrode width equal to ~56% of the channel height

Dielectric spectroscopy in a micromachined flow cytometer: theoretical and practical considerations

We propose a model to determine the influence of different cell properties, such as size, membrane capacitance and cytoplasm conductivity, on the impedance spectrum as measured in a microfabricated cytometer. A dielectric sphere of equivalent complex permittivity is used as a simplified model to describe a biological cell. The measurement takes place between a pair of facing microelectrodes in a microchannel filled with a saline solution. The model incorporates various cell parameters, such as dielectric properties, size and position in the channel. A 3D finite element model is used to evaluate the magnitude of the electric field in the channel and the resultant changes in charge densities at the measurement electrode boundaries as a cell flows past. The charge density is integrated on the electrode surface to determine the displacement current and the channel impedance for the computed frequency range. The complete impedance model combines the finite element model, the electrode-electrolyte interface impedance and stray impedance, which are measured from a real device. The modeled dielectric complex spectra for various cell parameters are discussed and a measurement strategy for cell discrimination with such a system is proposed. We finally discuss the amount of noise and measurement fluctuations of the sensor

Temperature measurements in microfluidic systems: Heat dissipation of negative dielectrophoresis barriers

The manipulation of living biological cells in microfluidic channels by a combination of negative dielectrophoretic barriers and pressure-driven flows is widely employed in lab-on-a-chip systems. However, electric fields in conducting media induce Joule heating. This study investigates if the local temperatures reached under typical experimental conditions in miniaturized systems cause a potential risk for hyperthermic stress or cell damage. Two methods of optical in situ temperature detection have been tested and compared: (i) the exposure of the thermo-dependent fluorescent dye Rhodamine B to heat sources situated in microfluidic channels, and (ii) the use of thermoprecipitating N-alkyl-substituted acrylamide polymers as temperature threshold probes. Two-dimensional images of temperature distributions in the vicinity of active negative dielectrophoresis (nDEP)- barriers have been obtained and local temperature variations of more than 20 degrees C have been observed at the electrode edges. Heat propagation via both buffer and channel walls lead to significant temperature increases within a perimeter of 100 mu m and more. These data indicate that power dissipation has to be taken into account when experiments at physiological temperatures are planned