Low-cost, high-throughput and rapid-prototyped 3D-integrated dielectrophoretic channels for continuous cell enrichment and separation.

Microfluidic devices for dielectrophoretic cell separation are typically designed and constructed using microfabrication methods in a clean room, requiring time and expense. In this paper, we describe a novel alternative approach to microfluidic device manufacture, using chips cut from conductor-insulator laminates using a cutter plotter. This allows the manufacture of microchannel devices with micron-scale electrodes along every wall. Fabrication uses a conventional desktop cutter plotter, and requires no chemicals, masks or clean-room access; functional fluidic devices can be designed and constructed within a couple of hours at negligible cost. As an example, we demonstrate the construction of a continuous dielectrophoretic cell separator capable of enriching yeast cells to 80% purity at 10 000 cells/s.

Rhythmic potassium transport regulates the circadian clock in human red blood cells.

Circadian rhythms organize many aspects of cell biology and physiology to a daily temporal program that depends on clock gene expression cycles in most mammalian cell types. However, circadian rhythms are also observed in isolated mammalian red blood cells (RBCs), which lack nuclei, suggesting the existence of post-translational cellular clock mechanisms in these cells. Here we show using electrophysiological and pharmacological approaches that human RBCs display circadian regulation of membrane conductance and cytoplasmic conductivity that depends on the cycling of cytoplasmic K+ levels. Using pharmacological intervention and ion replacement, we show that inhibition of K+ transport abolishes RBC electrophysiological rhythms. Our results suggest that in the absence of conventional transcription cycles, RBCs maintain a circadian rhythm in membrane electrophysiology through dynamic regulation of K+ transport.

Dielectrophoretic sample preparation for environmental monitoring of microorganisms: Soil particle removal.

Detection of pathogens from environmental samples is often hampered by sensors interacting with environmental particles such as soot, pollen, or environmental dust such as soil or clay. These particles may be of similar size to the target bacterium, preventing removal by filtration, but may non-specifically bind to sensor surfaces, fouling them and causing artefactual results. In this paper, we report the selective manipulation of soil particles using an AC electrokinetic microfluidic system. Four heterogeneous soil samples (smectic clay, kaolinitic clay, peaty loam, and sandy loam) were characterised using dielectrophoresis to identify the electrical difference to a target organism. A flow-cell device was then constructed to evaluate dielectrophoretic separation of bacteria and clay in a continous flow through mode. The average separation efficiency of the system across all soil types was found to be 68.7% with a maximal separation efficiency for kaolinitic clay at 87.6%. This represents the first attempt to separate soil particles from bacteria using dielectrophoresis and indicate that the technique shows significant promise; with appropriate system optimisation, we believe that this preliminary study represents an opportunity to develop a simple yet highly effective sample processing system.

Process development for cell aggregate arrays encapsulated in a synthetic hydrogel using negative dielectrophoresis.

Spatial patterning of cells is of great importance in tissue engineering and biotechnology, enabling, for example the creation of bottom-up histoarchitectures of heterogeneous cells, or cell aggregates for in vitro high-throughput toxicological and therapeutic studies within 3D microenvironments. In this paper, a single-step process for creating peelable and resilient hydrogels, encapsulating arrays of biological cell aggregates formed by negative DEP has been devised. The dielectrophoretic trapping within low-energy regions of the DEP-dot array reduces cell exposure to high field stresses while creating distinguishable, evenly spaced arrays of aggregates. In addition to using an optimal combination of PEG diacrylate pre-polymer solution concentration and a novel UV exposure mechanism, total processing time was reduced. With a continuous phase medium of PEG diacrylate at 15% v/v concentration, effective dielectrophoretic cell patterned arrays and photo-polymerisation of the mixture was achieved within a 4 min period. This unique single-step process was achieved using a 30 s UV exposure time frame within a dedicated, wide exposure area DEP light box system. To demonstrate the developed process, aggregates of yeast, human leukemic (K562) and HeLa cells were immobilised in an array format within the hydrogel. Relative cell viability for both cells within the hydrogels, after maintaining them in appropriate iso-osmotic media, over a week period was greater than 90%.

Efficient dielectrophoretic cell enrichment using a dielectrophoresis-well based system.

Whilst laboratory-on-chip cell separation systems using dielectrophoresis are increasingly reported in the literature, many systems are afflicted by factors which impede "real world" performance, chief among these being cell loss (in dead spaces, attached to glass and tubing surfaces, or sedimentation from flow), and designs with large channel height-to-width ratios (large channel widths, small channel heights) that make the systems difficult to interface with other microfluidic systems. In this paper, we present a scalable structure based on 3D wells with approximately unity height-to-width ratios (based on tubes with electrodes on the sides), which is capable of enriching yeast cell populations whilst ensuring that up to 94.3% of cells processed through the device can be collected in tubes beyond the output.

Real-time cell electrophysiology using a multi-channel dielectrophoretic-dot microelectrode array.

Dielectrophoresis (DEP) has been used for many years for the analysis of the electrophysiological properties of cells. However, such analyses have in the past been time-consuming, such that it can take 30 min or more to collect sufficient data to make valid interpretations from a single DEP spectrum. This has limited the application of the technology to a rapid tool for non-invasive, label-free research in areas from drug discovery to diagnostics. In this paper we present the development of a programmable, multi-channel DEP system for rapid biophysical assessment of populations of biological cells. A new assay format has been developed for continuous near-real-time monitoring, using simultaneous application of up to eight alternating current electrical signals to independently addressable dot microelectrodes in an array format, allowing a DEP spectrum to be measured in 20 s, with a total cycle time between measurements of 90 s. To demonstrate the system, human leukaemic K562 cells were monitored after exposure to staurosporine and valinomycin. The DEP response curves showed the timing and manner in which the membrane properties changed for the actions of these two drugs at the early phase of induction. This technology shows the great potential for increasing our understanding of the role of electrophysiology in drug action, by observing the changes in electrical characteristics as they occur.

A bio-analytical system for rapid cellular electrophysiological assays.

In this paper, the use of non-uniform ac electric fields on biological cells for bioanalysis, through multiple, independently configurable channels is presented. The programmable system has been used to obtain the dielectrophoretic spectra of cells in near real time, within 90 seconds. This is a significant improvement on existing dielectrophoretic techniques as simultaneous parallel measurement of the dielectrophoretic forces at different frequencies has potential of revealing subtle changes to the electrophysiology of cells, as they occur. The results show that with continuous on-chip monitoring, cells exposed to a chemical agent that induces apoptosis begin to exhibit a spectrum that differs from untreated cells, as indicated from shifts in the observed crossover frequency values.

Rapid-on-chip determination of dielectric properties of biological cells using imaging techniques in a dielectrophoresis dot microsystem.

Dielectrophoresis is a technique whereby polarisable particles are manipulated by non-uniform alternating electric fields. A specific application of this technique is deducing the dielectric properties of cells from analysis of the dielectrophoretic spectrum of that particular cell population. We have developed a new microelectrode geometry consisting of two parallel electrode planes, one of which is patterned with arrays of circular apertures or 'dots'. The radial symmetry of the dots means that the polarisability of the particles within the dot can be directly related to change shifts in light transmission through the dot, and quantified from analysis of digital images. We have validated our system using well-characterised cell types and found a high degree of agreement to published data. Furthermore, we have observed that at high particle concentrations, electrostatic inter-particle repulsion causes spontaneous, rapid particle re-dispersion over the dot volume upon removal of an applied electric field. This allows the automated acquisition of a spectrum of 26 data points in approximately 15 min.

An integrated dielectrophoretic quartz crystal microbalance (DEP-QCM) device for rapid biosensing applications.

A factor limiting the detection time of biological particles using a quartz crystal microbalance (QCM) system is the kinetics of the particles arriving within the sensing region of the crystal surface. A device has been developed which, for the first time, combines ac electro-kinetic particle manipulation with simultaneous acoustic sensing on an electrode surface. We have termed this device a dielectrophoretic quartz crystal microbalance (DEP-QCM). Particles within the system are rapidly driven by electro-hydrodynamic and dielectrophoretic forces on to the crystal surface. Frequency shift analysis of mass-loaded DEP-QCM, induced by fluid motion, has shown significant improvements in rates of detection based on particle concentration, with steady-state responses established by a factor of five times faster than other quartz crystal microbalance surface loading techniques described in the literature. Comparisons of the static fluid case for QCM devices revealed that particles with a concentration of less than 10(8) nano-spheres/ml could not be detected within a 1h time period when allowed to sediment.

Dielectrophoretic separation of Bacillus subtilis spores from environmental diesel particles.

Isolation of pathogenic bacteria from non-biological material of similar size is a vital sample preparation step in the identification of such organisms, particularly in the context of detecting bio-terrorist attacks. However, many detection methods are impeded by particulate contamination from the environment such as those from engine exhausts. In this paper we use dielectrophoresis--the induced motion of particles in non-uniform fields--to successfully remove over 99% of diesel particulates acquired from environmental samples, whilst letting bacterial spores of B. subtilis pass through the chamber largely unimpeded. We believe that such a device has tremendous potential as a precursor to a range of detection methods, improving the signal-to-noise ratio and ultimately improving detection rates.

A high-throughput 3-D composite dielectrophoretic separator.

Dielectrophoresis has great potential to offer a range of diverse fields from bioprocessing to clinical medicine, but is hampered by low throughput rates to the micrometer scale of the electrodes required to generate highly nonuniform fields. Here we describe a novel approach to electrode construction, using a drilled laminated structure to form channels bearing electrodes 30 microm across and 150 microm apart. Since these electrodes appear along all sides of the drilled bore, the trapping efficiency is improved over conventional devices. We have developed and demonstrated a separator capable of sorting a 50:50 mixture of viable and nonviable yeast cells into an 86:14 mixture at 25 mLhr(-1).