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.

Point-of-care Analysis for Non-invasive Diagnosis of Oral cancer (PANDORA): A technology-development proof of concept diagnostic accuracy study of dielectrophoresis in patients with oral squamous cell carcinoma and dysplasia.

BACKGROUND: Delays in the identification and referral of oral cancer remain frequent. An accurate and non-invasive diagnostic test to be performed in primary care may help identifying oral cancer at an early stage and reduce mortality. Point-of-care Analysis for Non-invasive Diagnosis of Oral cancer (PANDORA) was a proof-of-concept prospective diagnostic accuracy study aimed at advancing the development of a dielectrophoresis-based diagnostic platform for oral squamous cell carcinoma (OSCC) and epithelial dysplasia (OED) using a novel automated DEPtech 3DEP analyser. METHODS: The aim of PANDORA was to identify the set-up of the DEPtech 3DEP analyser associated with the highest diagnostic accuracy in identifying OSCC and OED from non-invasive brush biopsy samples, as compared to the gold standard test (histopathology). Measures of accuracy included sensitivity, specificity, positive and negative predictive value. Brush biopsies were collected from individuals with histologically proven OSCC and OED, histologically proven benign mucosal disease, and healthy mucosa (standard test), and analysed via dielectrophoresis (index test). RESULTS: 40 individuals with OSCC/OED and 79 with benign oral mucosal disease/healthy mucosa were recruited. Sensitivity and specificity of the index test was 86.8% (95% confidence interval [CI], 71.9%-95.6%) and 83.6% (95% CI, 73.0%-91.2%). Analysing OSCC samples separately led to higher diagnostic accuracy, with 92.0% (95% CI, 74.0%-99.0%) sensitivity and 94.5% (95% CI, 86.6%-98.5%) specificity. CONCLUSION: The DEPtech 3DEP analyser has the potential to identify OSCC and OED with notable diagnostic accuracy and warrants further investigation as a potential triage test in the primary care setting for patients who may need to progress along the diagnostic pathway and be offered a surgical biopsy.

High-throughput, low-loss, low-cost, and label-free cell separation using electrophysiology-activated cell enrichment.

Currently, cell separation occurs almost exclusively by density gradient methods and by fluorescence- and magnetic-activated cell sorting (FACS/MACS). These variously suffer from lack of specificity, high cell loss, use of labels, and high capital/operating cost. We present a dielectrophoresis (DEP)-based cell-separation method, using 3D electrodes on a low-cost disposable chip; one cell type is allowed to pass through the chip whereas the other is retained and subsequently recovered. The method advances usability and throughput of DEP separation by orders of magnitude in throughput, efficiency, purity, recovery (cells arriving in the correct output fraction), cell losses (those which are unaccounted for at the end of the separation), and cost. The system was evaluated using three example separations: live and dead yeast; human cancer cells/red blood cells; and rodent fibroblasts/red blood cells. A single-pass protocol can enrich cells with cell recovery of up to 91.3% at over 300,000 cells per second with >3% cell loss. A two-pass protocol can process 300,000,000 cells in under 30 min, with cell recovery of up to 96.4% and cell losses below 5%, an effective processing rate >160,000 cells per second. A three-step protocol is shown to be effective for removal of 99.1% of RBCs spiked with 1% cancer cells while maintaining a processing rate of ∼170,000 cells per second. Furthermore, the self-contained and low-cost nature of the separator device means that it has potential application in low-contamination applications such as cell therapies, where good manufacturing practice compatibility is of paramount importance.

Accurate quantification of apoptosis progression and toxicity using a dielectrophoretic approach.

A loss of ability of cells to undergo apoptosis (programmed cell death, whereby the cell ceases to function and destroys itself) is commonly associated with cancer, and many anti-cancer interventions aim to restart the process. Consequently, the accurate quantification of apoptosis is essential in understanding the function and performance of new anti-cancer drugs. Dielectrophoresis has previously been demonstrated to detect apoptosis more rapidly than other methods, and is low-cost, label-free and rapid, but has previously been unable to accurately quantify cells through the apoptotic process because cells in late apoptosis disintegrate, making cell tracking impossible. In this paper we use a novel method based on light absorbance and multi-population tracking to quantify the progress of apoptosis, benchmarking against conventional assays including MTT, trypan blue and Annexin-V. Analyses are performed on suspension and adherent cells, and using two apoptosis-inducing agents. IC50 measurements compared favourably to MTT and were superior to trypan blue, whilst also detecting apoptotic progression faster than Annexin-V.

Factors affecting particle collection by electro-osmosis in microfluidic systems.

Alternating-current electro-osmosis, a phenomenon of fluid transport due to the interaction between an electrical double layer and a tangential electric field, has been used both for inducing fluid movement and for the concentration of particles suspended in the fluid. This offers many advantages over other phenomena used to trap particles, such as placing particles at an electrode centre rather than an edge; benefits of scale, where electrodes hundreds of micrometers across can trap particles from the molecules to cells at the same rate; and a trapping volume limited by the vortex height, a phenomenon thus far unstudied. In this paper, the collection of particles due to alternating-current electro-osmosis driven collection is examined for a range of particle concentrations, inter-electrode gap widths, chamber heights and media viscosity and density. A model of collection behaviour is described where particle collection over time is governed by two processes, one driven by the vortices and the other by sedimentation, allowing the determination of the maximum height of vortex-driven collection, but also indicates how trapping is limited by high particle concentrations and fluid velocities. The results also indicate that viscosity, rather than density, is a significant governing factor in determining the trapping behaviour of particles.

Continuous flow nanoparticle concentration using alternating current-electroosmotic flow.

Achieving real-time detection of environmental pathogens such as viruses and bacterial spores requires detectors with both rapid action and a suitable detection threshold. However, most biosensors have detection limits of an order of magnitude or more above the potential infection threshold, limiting their usefulness. This can be improved through the use of automated sample preparation techniques such as preconcentration. In this paper, we describe the use of AC electroosmosis to concentrate nanoparticles from a continuous flow. Electrodes at an optimized angle across a flow cell, and energized by a 1 kHz signal, were used to push nanoparticles to one side of a flow cell, and to extract the resulting stream with a high particle concentration from that side of the flow cell. A simple model of the behavior of particles in the flow cell has been developed, which shows good agreement with experimental results. The method indicates potential for higher concentration factors through cascading devices.

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.

Rapid, automated measurement of dielectrophoretic forces using DEP-activated microwells.

Dielectrophoresis (DEP) is a physical effect that generates a force on polarisable particles experiencing a non-homogeneous electric field; studying the effect as a function of frequency allows the determination of the electrical properties of that particle, i.e., the electrical permittivity and conductivity. In the past, DEP-based techniques applied to the measurement of one or several cells at a time have been subject to many sources of noise, which result in an ambiguous or inaccurate result. However, improvements are possible by generating more information from the experiments. In this paper, we present a rapid automated system that measures the DEP spectrum from a large population of cells with a low level of noise using the microwell electrodes, based on a method of analysis that provides additional information about the electrical properties of the cells and a new theoretical approach was developed to obtain accurate, bias-free results in <5 min.

Ten-Second Electrophysiology: Evaluation of the 3DEP Platform for high-speed, high-accuracy cell analysis.

Electrical correlates of the physiological state of a cell, such as membrane conductance and capacitance, as well as cytoplasm conductivity, contain vital information about cellular function, ion transport across the membrane, and propagation of electrical signals. They are, however, difficult to measure; gold-standard techniques are typically unable to measure more than a few cells per day, making widespread adoption difficult and limiting statistical reproducibility. We have developed a dielectrophoretic platform using a disposable 3D electrode geometry that accurately (r2 > 0.99) measures mean electrical properties of populations of ~20,000 cells, by taking parallel ensemble measurements of cells at 20 frequencies up to 45 MHz, in (typically) ten seconds. This allows acquisition of ultra-high-resolution (100-point) DEP spectra in under two minutes. Data acquired from a wide range of cells - from platelets to large cardiac cells - benchmark well with patch-clamp-data. These advantages are collectively demonstrated in a longitudinal (same-animal) study of rapidly-changing phenomena such as ultradian (2-3 hour) rhythmicity in whole blood samples of the common vole (Microtus arvalis), taken from 10 µl tail-nick blood samples and avoiding sacrifice of the animal that is typically required in these studies.

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.

Rapid determination of antibiotic resistance in E. coli using dielectrophoresis.

In recent years, infections due to antibiotic-resistant strains of bacteria such as methicillin-resistant Staphylococcus aureus and ciprofloxacin-resistant Escherichia coli are on the rise, and with them the demand for rapid antibiotic testing is also rising. Conventional tests, such as disc diffusion testing, require a primary sample to be tested in the presence of a number of antibiotics to verify which antibiotics suppress growth, which take approximately 24 h to complete and potentially place the patient at severe risk. In this paper we describe the use of dielectrophoresis as a rapid marker of cell death, by detecting changes in the electrophysiology of the cell caused by the administration of an antibiotic. In contrast to other markers, the electrophysiology of the cell changes rapidly during cell death allowing live cells to be distinguished from dead (or dying) cells without the need for culturing. Using polymyxin B as an example antibiotic, our studies indicate that significant changes in cell characteristics can be observed as soon as 1 h passes after isolating a culture from nutrient broth.

Rapid determination of nanowire electrical properties using a dielectrophoresis-well based system.

The use of high quality semiconducting nanomaterials for advanced device applications has been hampered by the unavoidable variability in the growth of one-dimensional (1D) nanomaterials such as nanowires (NWs) and nanotubes, resulting in highly variable electrical properties across the population. Therefore, assessment of the quality of nanomaterials is vital for the fabrication of high-performance and reliable electronic devices. The controllable selection of high quality NWs has been recently demonstrated using a dielectrophoretic (DEP) NW assembly method; however, no convenient, rapid method has been adopted for the characterization of nanomaterial semiconducting properties. In this study, we solve this challenge with a low-cost, industrially scalable method for the rapid analysis of the electrical properties of inorganic single crystalline NWs, by identifying key features in the DEP frequency response spectrum (1 kHz - 20 MHz). NWs dispersed in anisole were characterized using a three-dimensional DEP chip (3DEP) in 60 seconds, and the resultant spectrum demonstrated a sharp change in NW response to DEP signal in 1 MHz - 20 MHz frequency rage. The 3DEP analysis, confirmed by field-effect transistor (FET) data, indicates that NWs with higher quality are collected at high DEP signal frequency range such as above 10 MHz. These results show that platforms such as the 3DEP, can be used for the characterization of rod-shaped nanoscale particles where the dipole moment is sufficiently large. It also shows that the 3DEP can be used to assess heterogeneous nanoparticle mixtures and identify nanomaterials with superior conductivity properties. The proposed methodology can be applied to any type of 1D nanomaterials. The 3DEP analysis coupled with dielectrophoretic assembly for the deposition of NWs at selected signal frequencies, leads to a reproducible fabrication of high quality NW FET devices.

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.

Simultaneous Tunable Selection and Self-Assembly of Si Nanowires from Heterogeneous Feedstock.

Semiconducting nanowires (NWs) are becoming essential nanobuilding blocks for advanced devices from sensors to energy harvesters, however their full technology penetration requires large scale materials synthesis together with efficient NW assembly methods. We demonstrate a scalable one-step solution process for the direct selection, collection, and ordered assembly of silicon NWs with desired electrical properties from a poly disperse collection of NWs obtained from a supercritical fluid-liquid-solid growth process. Dielectrophoresis (DEP) combined with impedance spectroscopy provides a selection mechanism at high signal frequencies (>500 kHz) to isolate NWs with the highest conductivity and lowest defect density. The technique allows simultaneous control of five key parameters in NW assembly: selection of electrical properties, control of NW length, placement in predefined electrode areas, highly preferential orientation along the device channel, and control of NWs deposition density from few to hundreds per device. Direct correlation between DEP signal frequency and deposited NWs conductivity is confirmed by field-effect transistor and conducting AFM data. Fabricated NW transistor devices demonstrate excellent performance with up to 1.6 mA current, 10(6)-10(7) on/off ratio and hole mobility of 50 cm(2) V(-1) s(-1).

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.

Biophysical characteristics reveal neural stem cell differentiation potential.

BACKGROUND: Distinguishing human neural stem/progenitor cell (huNSPC) populations that will predominantly generate neurons from those that produce glia is currently hampered by a lack of sufficient cell type-specific surface markers predictive of fate potential. This limits investigation of lineage-biased progenitors and their potential use as therapeutic agents. A live-cell biophysical and label-free measure of fate potential would solve this problem by obviating the need for specific cell surface markers. METHODOLOGY/PRINCIPAL FINDINGS: We used dielectrophoresis (DEP) to analyze the biophysical, specifically electrophysiological, properties of cortical human and mouse NSPCs that vary in differentiation potential. Our data demonstrate that the electrophysiological property membrane capacitance inversely correlates with the neurogenic potential of NSPCs. Furthermore, as huNSPCs are continually passaged they decrease neuron generation and increase membrane capacitance, confirming that this parameter dynamically predicts and negatively correlates with neurogenic potential. In contrast, differences in membrane conductance between NSPCs do not consistently correlate with the ability of the cells to generate neurons. DEP crossover frequency, which is a quantitative measure of cell behavior in DEP, directly correlates with neuron generation of NSPCs, indicating a potential mechanism to separate stem cells biased to particular differentiated cell fates. CONCLUSIONS/SIGNIFICANCE: We show here that whole cell membrane capacitance, but not membrane conductance, reflects and predicts the neurogenic potential of human and mouse NSPCs. Stem cell biophysical characteristics therefore provide a completely novel and quantitative measure of stem cell fate potential and a label-free means to identify neuron- or glial-biased progenitors.

Dielectrophoresis as a cell characterisation tool.

Dielectrophoresis (DEP) is a technique which offers label-free measurement of cell electrophysiology by monitoring its movement in non-uniform electric fields. In this chapter, the theory underlying DEP is explored, as are the implications of the development of equipment for taking such measurements. Practical considerations such as the selection of a suspending medium are also discussed.

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.

Dielectrophoresis-activated multiwell plate for label-free high-throughput drug assessment.

Dielectrophoresis (DEP) offers many advantages over conventional cell assays such as flow cytometry and patch clamp techniques for assessing cell electrophysiology as a marker for cancer studies and drug interaction assessment. However, despite the advantages offered by DEP analysis, uptake has been low, remaining largely in the academic arena, due to the process of analysis being time-consuming, laborious, and ultimately allowing only serial analysis on small numbers of cells. In this paper we describe a new method of performing DEP analysis based on laminate manufacturing methods. These use a three-dimensional "well" structure, similar in size and pitch to conventional microtiter well plates, but offer electrodes along the inner surface to allow easy measurement of cell properties through the whole population. The result can then be determined rapidly using a conventional well-plate reader. The nature of the device means that many electrodes, each containing a separate sample, can be tested in parallel, while the mode of observation means that analysis can be combined with simultaneous measurement of conventional fluorimetric well-based assays. Here we benchmark the device against standard DEP assays, then show how such a device can be used to (a) rapidly determine the effects both of ion channel blockers on cancer cells and antibiotics on bacteria and (b) determine the properties of multiple subpopulations of cells within a well simultaneously.