Professor Michael Hughes
Originally from tiny Holy Island of the northwest coast of Wales, Mike Hughes was inspired to a life in science by the popular TV series Cosmos, in which the presenter (Carl Sagan) looked at the interconnected nature of the different aspects of science. Faced with a choice between engineering and life sciences, he chose to do a degree the former but was able to combine both in his PhD of a newly emerging field of bioelectronics. Following a brief period of postdoctoral study in Glasgow, Prof Hughes was appointed as a Lecturer in Biomedical Engineering at the University of Surrey in 1999, rising to the rank of Professor by 2008. During that time, he has become preeminent in the field of bioelectronics; he has published over 80 journal papers on the topic (cited over 5000 times), as well as two books and five patents. He was also Editor in Chief of one of the leading journals in the field, IEEE Tranactions on Nanobioscience, for 6 years. His research has also led to the formation of a pair of companies (DEPtech and DEParator) making technology developed in his group at Surrey.
University roles and responsibilities
- Director, Centre for Biomedical Engineering
- Examinations Officer, Department of Mechanical Engineering Sciences
My research is in the area of bioelectronics, most specifically in a phenomenon of dielectrophoresis (DEP). DEP is the name given to micro- or nano-particle movement due to the interaction with certain kind of electrical field; the speed and direction of movement depends on the resistance and capacitance of the particle and the frequency of the electric field; analysing the response as a function of frequency allows those properties to be measured, whilst mixtures of particles with different properties can be separated at frequencies where their responses are different. Whilst my group has looked at a range of particles including proteins, DNA, nanowires, carbon nanotubes and clay, the primary focus has been on cells - including cancer, stem cells, bacteria and yeast.
My principal line of research is the development of new technology to exploit the DEP effect. We have developed a new chip technology - called the DEP-Well - which has dramatically increased the speed, accuracy and throughput of technology. For example, we have built a tool for measuring the mean electrical properties of 20,000 cells in 10 seconds (current gold-standard technology measures one cell in 5 hours); and have recently build the world's fastest cell separator, capable of sorting 200,000 cells per second. Both of these technologies require no special chemicals, needing only a low-cost disposable chip; both have also been commercialisd, with the former now being sold as the DEPtech 3DEP and the latter as the DEParator.
We have used the technology to meet a number of scientific needs, including an electrophysiology-based test for oral cancer and a new method for sorting blood cells. Investigations using the technology have given new insights into cell death and responses to anti-cancr treatment.
Chondrogenic progenitor cells (CPCs) may be used as an alternative source of cells with potentially superior chondrogenic potential compared to mesenchymal stem cells (MSCs), and could be exploited for future regenerative therapies targeting articular cartilage in degenerative diseases such as osteoarthritis (OA). In this study, we hypothesised that CPCs derived from OA cartilage may be characterised by a distinct channelome. First, a global transcriptomic analysis using Affymetrix microarrays was performed. We studied the profiles of those ion channels and transporter families that may be relevant to chondroprogenitor cell physiology. Following validation of the microarray data with quantitative reverse transcription-polymerase chain reaction, we examined the role of calcium-dependent potassium channels in CPCs and observed functional large-conductance calcium-activated potassium (BK) channels involved in the maintenance of the chondroprogenitor phenotype. In line with our very recent results, we found that the KCNMA1 gene was upregulated in CPCs and observed currents that could be attributed to the BK channel. The BK channel inhibitor paxilline significantly inhibited proliferation, increased the expression of the osteogenic transcription factor RUNX2, enhanced the migration parameters, and completely abolished spontaneous Ca events in CPCs. Through characterisation of their channelome we demonstrate that CPCs are a distinct cell population but are highly similar to MSCs in many respects. This study adds key mechanistic data to the in-depth characterisation of CPCs and their phenotype in the context of cartilage regeneration.
In 1966, Herbert Pohl and Ira Hawk published the first demonstration of dielectrophoresis of living and dead yeast cells; their paper described how the different ways in which the cells responded to an applied nonuniform electric field could form the basis of a cell separation method. Fifty years later, the field of dielectrophoretic (DEP) cell separation has expanded, with myriad demonstrations of its ability to sort cells on the basis of differences in electrical properties without the need for chemical labelling. As DEP separation enters its second half-century, new approaches are being found to move the technique from laboratory prototypes to functional commercial devices; to gain widespread acceptance beyond the DEP community, it will be necessary to develop ways of separating cells with throughputs, purities and cell recovery comparable to gold-standard techniques in life sciences, such as fluorescence- and magnetically-activated cell sorting (FACS and MACS, respectively). In this paper the history of DEP separation is charted, from a description of the work leading up to the first paper, to the current dual approaches of electrode-based and electrodeless DEP separation, and the path to future acceptance outside the DEP mainstream is considered.
The characterisation of healthy ageing of the brain could help create a fingerprint of normal ageing that might assist in the early diagnosis of neurodegenerative conditions. This study examined changes in resting state MEG permutation entropy due to age and gender in a sample of 220 healthy participants (98 males and 122 females, ages ranging between 7 and 84). Entropy was quantified using normalised permutation entropy and modified permutation entropy, with an embedding dimension of 5 and a lag of 1 as the input parameters for both algorithms. Effects of age were observed over the 5 regions of the brain i.e. anterior, central, posterior, and left and right lateral, with the anterior and central regions containing the highest permutation entropy. Statistically significant differences due to age were observed in the different brain regions for both genders, with the evolutions described using the fitting of polynomial regressions. Nevertheless, no significant differences between the genders were observed across all ages. These results suggest that the evolution of entropy in the background brain activity, quantified with permutation entropy algorithms, might be considered an alternative illustration of a ‘nominal’ physiological rhythm.
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.
Dielectrophoresis (DEP) has increasingly been used for the assessment of the electrical properties of molecular scale objects including proteins, DNA, nanotubes and nanowires. However, whilst techniques have been developed for the electrical characterisation of frequency-dependent DEP response, biomolecular study is usually limited to observation using fluorescent markers, limiting its applicability as a characterisation tool. In this paper we present a label-free, impedance-based method of characterisation applied to the determination of the electrical properties of colloidal protein molecules, specifically Bovine Serum Albumin (BSA). By monitoring the impedance between electrodes as proteins collect, it is shown to be possible to observe multi-dispersion behaviour. A DEP dispersion exhibited at 400 kHz is attributable to the orientational dispersion of the molecule, whilst a second, higher-frequency dispersion is attributed to a Maxwell-Wagner type dispersion; changes in behaviour with medium conductivity suggest that this is strongly influenced by the electrical double layer surrounding the molecule.
The use of high quality semiconducting nanomaterials for advanced device applications has been hampered by the unavoidable growth variability of electrical properties of one-dimensional nanomaterials, such as nanowires and nanotubes, thus highlighting the need for the characterization of efficient semiconducting nanomaterials. In this study, we demonstrate a low-cost, industrially scalable dielectrophoretic (DEP) nanowire assembly method for the rapid analysis of the electrical properties of inorganic single crystalline nanowires, by identifying key features in the DEP frequency response spectrum from 1 kHz to 20 MHz in just 60 s. Nanowires dispersed in anisole were characterized using a three-dimensional DEP chip (3DEP), and the resultant spectrum demonstrated a sharp change in nanowire response to DEP signal in 1–20 MHz frequency range. The 3DEP analysis, directly confirmed by field-effect transistor data, indicates that nanowires of higher quality are collected at high DEP signal frequency range above 10 MHz, whereas lower quality nanowires, with two orders of magnitude lower current per nanowire, are collected at lower DEP signal frequencies. These results show that the 3DEP platform can be used as a very efficient characterization tool of the electrical properties of rod-shaped nanoparticles to enable dielectrophoretic selective deposition of nanomaterials with superior conductivity properties.
One of the bottlenecks for cell therapy development is the need to isolate specific cells, be it stem cells with specific differentiation fates, or specific white cells from a blood cell sort. However, the nature of the application means that the separation method should ideally be label-free and GMPcompliant, as well as achieving appropriate levels of throughput and cell recovery. One emergent field in cell separation is dielectrophoresis, an electrostatic method that has the potential to meet this growing need. Recent commercial developments mean that for the first time, this technique will be more widely available to the cell therapy sector.
Bladder cancer is the 9th most common cancer worldwide. Diagnosing bladder cancer typically involves highly invasive cystoscopy, with followup monitored using uteroscopy. Molecular methods have been developed as an adjunct to this, but tend to be expensive or require expert operator input. Here we present a study of the use of dielectrophoresis (DEP) of voided cells from eight cancer-presenting patients and eight healthy controls as an alternative low-cost and operator-independent method of bladder cancer detection. This study suggests that there are statistically significant differences (p=0.034) between characteristics of the DEP spectrum of clinical samples, and that using this marker we were able to obtain sensitivity of 75% and specificity of 87.5%, in line with many molecular methods; exclusion of samples where a DEP spectrum is not present (due to low cell counts) shows this can be improved by increasing the cell collection rate. As samples were analyzed a day after collection, we suggest that the method may be amenable to a centralized mail-in analysis service.
This study reports the effect of an increasing ion dose on both the electrical activation yield and the characteristic properties of implanted bismuth donors in silicon. A strong dependence of implant fluence is observed on both the yield of bismuth donors and the measured impurity diffusion. This is such that higher ion concentrations result in both a decrease in activation and an enhancement in donor migration through interactions with mobile silicon lattice vacancies and interstitials. Furthermore, the effect of implant fluence on the properties of the Si:Bi donor bound exciton, D0X, is also explored using photoluminescence (PL) measurements. In the highest density sample, centers corresponding to the PL of bismuth D0Xs within both the high density region and the lower concentration diffused tail of the implanted donor profile are identifiable.
© 2012 John Wiley & Sons, Ltd. Dielectrophoresis (DEP) is a non-invasive cell analysis method that uses differences in electrical properties between particles and surrounding medium to determine a unique set of cellular properties that can be used as a basis for cell separation. Cell-based therapies using skeletal stem cells are currently one of the most promising areas for treating a variety of skeletal and muscular disorders. However, identifying and sorting these cells remains a challenge in the absence of unique skeletal stem cell markers. DEP provides an ideal method for identifying subsets of cells without the need for markers by using their dielectric properties. This study used a 3D dielectrophoretic well chip device to determine the dielectric characteristics of two osteosarcoma cell lines (MG-63 and SAOS-2) and an immunoselected enriched skeletal stem cell fraction (STRO-1 positive cell) of human bone marrow. Skeletal cells were exposed to a series of different frequencies to induce dielectrophoretic cell movement, and a model was developed to generate the membrane and cytoplasmic properties of the cell populations. Differences were observed in the dielectric properties of MG-63, SAOS-2 and STRO-1 enriched skeletal populations, which could potentially be used to sort cells in mixed populations. This study provide evidence of the ability to characterize different human skeletal stem and mature cell populations, and acts as a proof-of-concept that dielectrophoresis can be exploited to detect, isolate and separate skeletal cell populations from heterogeneous bone marrow cell populations.
This study focuses on the resting state network analysis of the brain, as well as how these networks change both in topology and location throughout life. The magnetoencephalogram (MEG) background activity from 220 healthy volunteers (age 7-84 years), was analysed combining complex network analysis principles of graph theory with both linear and non-linear methods to evaluate the changes in the brain. Granger Causality (GC) (linear method) and Phase Slope Index (PSI) (non-linear method) were used to observe the connectivity in the brain during rest, and as a function of age by analysing the degree, clustering coefficient, efficiency, betweenness, modularity and maximised modularity of the observed complex brain networks. Our results showed that GC showed little linear causal activity in the brain at rest, with small world topology, while PSI showed little information flow in the brain, with random network topology. However, both analyses produced complementary results pertaining to the resting state of the brain.
Dielectrophoresis is an electrical phenomenon that occurs when a polarisable particle is placed in non-uniform electrical fields. The magnitude of the generated force is dependent upon the electrophysiological make-up of the particle, therefore the specific DEP profile may be attained for any polarisable particles based on the intrinsic electrical properties alone. Any changes to these parameters may be detected by observing the corresponding DEP spectra. Despite having the advantages of being non-invasive, DEP applications are still not widely used due to the time-consuming processes involved. This study presents the preliminary outcomes in the development of a semi-automated DEP-based cell characterisation tool that allowed concurrent DEP experiments to be conducted serially, thus significantly reducing the time taken to complete the required sets of experiments. The results showed that the system is capable of producing a DEP spectrum for K562 leukaemic cells between the 10 kHz to 1 MHz range in less than 10 minutes, when recorded at eight points per decade.
Dielectrophoresis (DEP) is the induced motion of polarizable particles in non-uniform electric fields. Used for many years for the manipulation of particles from cell-scale to macromolecules, we present here the application of the technique for manipulation of DNA containing only adenine-thymine (poly AT) bases, and that of DNA containing only cytosine-guanine (poly GC), using microfabricated electrode structures. Poly AT was stained with DAPI and JOJO-1 for poly GC DNA. It was found that there were differences between the frequency-dependent DEP behavior of the two molecules; when looking at the difference between the two types on crossover frequency (the point where DEP changes from attractive to repulsive), it was found that they varied by up to a factor of 2. This points to possible insigns in the charge conduction mechanism in different DNA forms, as well as potential new mechanisms for gene separations and sequencing. © 2012 IEEE.
Dielectrophoresis (DEP) is a phemomenon of induced particle motion in non-uniform electric fields. In this paper, we will describe how the phenomenon can be used to design electrokinetic ratchet devices to provide both linear and rotary motion. Such devices exploit Brownian motion through the imposition of an asymmetrical potential energy field, to provide motion. Since Brownian motion is a prerequisite, such devices are most applicable to the transport of nanoparticles. In this paper, we present the results of modelling of such devices capable of linear translation, and of particular novelty, rotary motion and show the torque of the rotary motor is of the order of that generated by a bacterial motor.
Dielectrophoretic and electrohydrodynamic forces have been demonstrated in the literature to cause movement of particles across the surface of planar electrodes when exposed to low-frequency (≈1 kHz) electric fields. In this paper we describe the development of this phenomenon for collection of particles, covering a range of sizes, out of a liquid and focusing them at the centre of a novel electrode consisting of large interlocking circles. The volume of analyte across which this effect is observed is significantly larger than has been reported for conventional dielectrophoretic arrays. By altering the experimental conditions, particles can either be collected or cycled across the surface and then removed. This technique offers great scope for enhancement of surface-based detection methods.
In this article, which is related to the biomedical signal processing applications area of the workshop, we present a system for positioning a single-neuron inside each micro-well of a 4-by-4 planar microelectrode array (MEA). Neurons are moved toward the electrode sites of the MEA (located at the bottom of the wells) using dielectrophoresis. The system utilizes the image acquisition and processing capabilities of MATLAB to detect the presence of a neuron inside each micro-well and stop the dielectrophoretic force, thus preventing more cells from being loaded. This method provides a fast, simple and relatively inexpensive way for loading cells on MEAs embedded with micro-wells for the purpose of acquiring and processing action potentials from geometrically defined biological neural networks at the single-cell level. Recordings from neurons that were positioned using this system have been obtained and are presented. © 2013 IEEE.
In cancer, multidrug resistance (MDR) is the simultaneous resistance of tumor cells to different natural product anticancer drugs that have no common structure. This is an impediment to the successful treatment of many human cancers. A common correlate of MDR is the overexpression of a membrane protein, P-glycoprotein. Many studies have shown that MDR can be reversed after the use of substrate analogs, called MDR modulators. However, our understanding of MDR modulation is incomplete. In this article, we examine the electrical properties of the human leukemic cells (K562) and its MDR counterpart (K562AR) using dielectrophoresis and flow cytometry (with a membrane potential sensitive dye, DIOC5), both before and after treatment with XR9576 (a P-glycoprotein-specific MDR-reversal agent). The results show significant differences in the cytoplasmic conductivity between the cell lines themselves, but indicate no significant changes after modulation therapy. We conclude that the process of MDR modulation is not associated with changes in the electrical properties of cancer cells. Moreover, the results demonstrate that using the flow cytometry method alone, with MDR cells, may produce artifactual results—whereas in combination with dielectrophoresis, the results show the role of MDR modulators in preventing drug efflux in MDR cells.
A major problem for surface-based detection techniques such as surface plasmon resonance and quartz crystal microbalances is that at low concentrations, diffusion is an insuffi cient driving force to bring colloidal submicron-scale particles to the detection surface. In order to overcome this, it has previously been demonstrated that a combination of dielectrophoresis and AC-electro-hydrodynamic fl ow can be used to focus cell-sized particles from suspension onto a large metal surface, in order to improve the detection capabilities of such systems. In this paper we describe how the combination of these two phenomena, using the so-called “zipper” electrode array, can be used to concentrate a wide range of nanoparticles of biological interest, such as infl uenza virus, dissolved albumin, and DNA molecules as well as latex beads of various sizes. We also demonstrate that the speed at which particles are transported towards the centre of the electrode pads by dielectrophoresis and electro-hydrodynamic fl ow is not related to the particle size for colloidal particles.
Background Patients with Chronic Exertional Compartment Syndrome (CECS) have pain during exercise that usually subsides at rest. Diagnosis is usually confirmed by measurement of intramuscular compartment pressure (IMCP) following exclusion of other possible causes. Management usually requires fasciotomy but reported outcomes vary widely. There is little evidence of the effectiveness of fasciotomy on IMCP. Testing is rarely repeated post-operatively and reported follow-up is poor. Improved diagnostic criteria based on pre-selection and IMCP levels during dynamic exercise testing have recently been reported. Objectives 1. To compare IMCP in 3 groups, one with classical symptoms and no treatment and the other with symptoms of CECS who have been treated with fasciotomy and an asymptomatic control group. 2. Establish if differences in IMCP in these groups as a result of fasciotomy relate to functional and symptomatic improvement. Methods Twenty subjects with symptoms of CECS of the anterior compartment, 20 asymptomatic controls and 20 patients who had undergone fasciotomy for CECS were compared. All other possible diagnoses were excluded using rigorous inclusion criteria and MRI. Dynamic IMCP was measured using an electronic catheter wire before, during and after participants exercised on a treadmill during a standardised 15-minute exercise challenge. Statistical analysis included t-tests and ANOVA. Results Fasciotomy results in reduced IMCP at all time points during a standardised exercise protocol compared to pre-operative cases. In subjects responding to fasciotomy there is a significant reduction in IMCP below that of pre-operative groups (p˂0.001). Post-operative responders to fasciotomy have no significant differences in IMCP from asymptomatic controls (p=0.182). Conclusion Fasciotomy reduces IMCP in all patients. Larger studies are required to confirm that the reduction in IMCP accounts for differences in functional outcomes and pain reductions seen in post-operative patients with CECS. Key Findings 1. Post-fasciotomy subjects demonstrate lower IMCP values compared to pre-operative symptomatic subjects at all time points. 2. Subjects who improve function and reduce pain after faSCiotomy demonstrate no Significant differences in dynamic IMCP measurement to normal controls.
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.
The electrostatic manipulation of nanoparticles using nonuniform electric fields (dielectrophoresis) has proved a useful method of investigating the movement of charge around colloidal particles. While previous work has explained many of the ways in which particle behavior deviates from that predicted by classical Maxwell-Wagner interfacial polarization theory, there exists an additional, anomalous polarization mechanism observed in media of high conductivity, causing an unexpected observation of positive dielectrophoresis. Here this is suggested that this may be explained in terms of the polarization of the Stern layer.
In recent years, infections due to antibiotic-resistant strains of bacteria such as methillicin-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.
Despite the widespread use of laser therapy in the removal of tattoos, comparatively little is known about its mechanism of action. There is a need for an improved understanding of the composition and thermal properties of the tattoo ink in order that simulations of laser therapy may be better informed and treatment parameters optimised. Scanning electron microscopy and time-of-flight secondary ion mass spectrometry identified that the relative proportions of the constituent compounds of the ink likely to exist in vivo are the following: carbon black pigment (89 %), carvacrol (5 %), eugenol (2 %), hexenol (3 %) and propylene glycol (1 %). Chemical compound property tables identify that changes in phase of these compounds lead to a considerable reduction in the density and thermal conductivity of the ink and an increase in its specific heat as temperature increases. These temperature-dependent values of density, thermal conductivity and specific heat are substantially different to the constant values, derived from water or graphite at a fixed temperature, which have been applied in the simulations of laser therapy as previously described in the literature. Accordingly, the thermal properties of black tattoo ink described in this study provide valuable information that may be used to improve simulations of tattoo laser therapy. © 2012 Springer-Verlag London Ltd.
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 whilst 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/second with >3% cell loss. A two-pass protocol can process 300,000,000 cells in under 30 minutes, with cell recovery of up to 96.4% and cell losses below 5%, an effective processing rate >160,000 cells/second. A three-step protocol is shown to be effective for removal of 99.1% of RBCs spiked with 1% cancer cells, whilst maintaining a processing rate of ~170,000 cells/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 GMP compatibility is of paramount importance. Significance statement. Cell separation is a fundamental process in biomedicine, but is presently complicated, cumbersome and expensive. We present a technique that can sort cells at a rate equivalent to or faster than gold-standard techniques such as FACS and MACs, but can do label-free and with very low cell loss. The system uses dielectrophoresis (DEP) to sort cells electrostatically, using a novel electrode chip that eschews microfabrication in favour of a laminate drilled with 397 electrode-bearing wells. This high level of parallelisation makes the system immune to the bubbles that limit labs-on-chip, whilst also increasing capacity and throughput to unprecedented levels, whilst the chip is cheap enough to be disposable, preventing inter-separation contamination.
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.
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.
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
In this article, electrode structures that combine dielectrophoretic effects with electrohydrodynamic fluid flow to concentrate particles on active sensor surfaces were presented. To optimize the collection effect on a surface, a novel electrode configuration called zipper electrodes has been developed. The local enrichment effect of these electrodes is such that particles at local concentration of 5×103 spores/mL can be collected using a single electrode pad. The fluid flow induced in the bulk flow is an efficient mechanism especially for very small particles, since the dielectrophoretic forces working on these particles are normally very small and do not penetrate the liquid as far as the vortex induced by combined dielectrophoresis/fluid flow does. The bulk flow makes it also a very versatile method that can extract a wide range of particles out of the liquid.
Although the subject of some scrutiny over the years, the mechanism of conduction in DNA has not yet been resolved, with competing theories suggesting either electronic and ionic conduction mechanisms. In this paper we use dielectrophoresis to determine the electrical properties of poly(dG)-poly(dC) (GC) and poly(dA)-poly(dT) (AT) DNA in solution. The molecules show different conduction mechanisms; GC DNA exhibits conduction primarily through the molecule, whereas in AT DNA conduction through the counterion cloud surrounding the molecule in solution is more significant.
The use of a single implantable microprobe to detect multiple, bi-directional action potential velocities is demonstrated. By using multiple electrode sites along the direction of propagation to record the same action potentials, the velocities of action potentials from both sensory and motor neurons can be determined.
An investigation has been performed into the biophysical properties of the enveloped mammalian virus, herpes simplex virus type 1 (HSV-1). The dielectrophoretic behaviour of the virus particles was measured as a function of applied frequency (over the range 100 kHz-20 MHz) and conductivity of the suspending medium (over the range 1-100 mS m(-1)). The dielectric properties of the virus were determined from the dielectrophoretic data using the smeared-out shell model. The data suggest that the intact particle has a surface conductance of 0.3 nS, an internal and membrane permittivity of 75varepsilon(o) and 7.5varepsilon(o), respectively, an internal conductivity of approximately 0.1 S m(-1) and a zeta potential of 70 mV. The dielectric properties were measured for intact, fresh virus particles and also for particles following exposure to various modifying agents, such as treatment with enzymes, ionophores and ageing. It is shown that the observed changes in the dielectrophoretic spectrum, and the variations in the dielectric properties of the virus concur with the expected physiological effects of these agents.
The dielectrophoretic collection spectra of antibiotic-sensitive and antibiotic-resistant strains of Staphylococcus epidermidis have been determined. These indicate that in the absence of antibiotic treatment there is a strong similarity between the dielectric properties of sensitive and resistant strains, and that there is a significant difference between the sensitive strains before and after treatment with the antibiotic streptomycin after 24 h exposure. This method offers possibilities for the assessment of bacterial resistance to antibiotics.
Dielectrophoresis (DEP) is a phenomenon of induced particle motion in non-uniform electric fields. The effect is frequency dependent; by monitoring the motion of particles in AC fields and analyzing the change in motion with frequency, it is possible to determine the electrical properties of single cells in lab-on-a-chip systems. By combining two common electrokinetic phenomena dielectrophoresis and electrohydrodynamic fluid flow - we demonstrate that it is possible to manipulate, concentrate and trap particles from cell to molecular scale, and show how the trapping phenomenon is not related to particle size. We also discuss application of the phenomenon, from particle preconcentration in sensor systems to the deposition of particles on sensor surfaces.
Dielectrophoresis (DEP) has been widely employed as one of the techniques in developing various micro-electromechanical systems (MEMS). The manipulation of particles using DEP forces is dependent upon the particles’ dipole moment that is produced in the presence of an electric field gradient. The paper documents the fabrication processes employed at the University of Surrey in developing the microelectrodes to be used in generating the said field gradient.
This work is a comprehensive approach to bacterial detection requiring a thorough knowledge of the subject and an effective integration of other disciplines in ...
Dielectrophoresis (DEP) is a phemomenon of induced particle motion in non-uniform electric fields. The effect is frequency dependent; by monitoring the motion of particles in AC fields and ananlysing the change in motion with frequency, it is possible to determine the electrical properties of nanoparticles in lab-on-a-chip systems. In this paper, we demonstrate how DEP can be used to determine the ratio of semiconducting and metallic carbon nanotubes in solution, by monitoring the frequency-dependent impedance change between two electrodes as a function of energising frequency.
Previous studies have indicated that the variations in torque induced in particles in electrorotation electrode arrays are sufficiently large to cause errors in electrorotation measurements. In order to avoid this, experimenters usually study particles bounded by an arbitrary region near the centre of the electrodes. By simulating the time-dependent electric field for polynomial electrodes, we have assessed the variation in torque across the centre of the array. By considering both the variation in applied torque and the dielectrophoretic force in the electrode chamber, the optimal conditions for electrorotation experiments have been determined. Further to this, by comparing the torque variation across the electrode chamber for a number of common electrode designs, a comparison of the suitability of each electrode design for multiparticle electrorotation analysis has been made.
In this paper we show how simplifying assumptions can be used to extract useful data from the dielectrophoretic collection spectrum, in particular for the cytoplasm, and hence determine the properties of multiple populations of cells within a sample. Specifically, the observation of the frequencies of onset of dielectric dispersion allows the identification and enumeration of populations of cells according to cytoplasmic conductivity, with particular relevance to the determination of the action of drugs for high-throughput screening applications.
Implantable microelectrodes have the potential to become part of neural prostheses to restore lost nerve function after nerve damage. The initial adsorption of proteins to materials for implantable microelectrodes is an important factor in determining the longevity and stability of the implant. Once an implant is in the body, protein adsorption takes place almost instantly before the cells reach the surface of an implant. The aim of this study was to identify an optimum material for electrode recording sites on implantable microelectrodes. Common materials for electrode sites are gold, platinum, iridium, and indium tin oxide. These, along with a reference material (titanium), were investigated. The thickness and the structure of adsorbed proteins on these materials were measured using a combination of atomic force microscopy and ellipsometry. The adsorbed protein layers on gold (after 7 and 28 days of exposure to serum) were the smoothest and the thinnest compared to all the other substrate materials, indicating that gold is the material of choice for electrode recording sites on implantable microelectrodes. However, the results also show that indium tin oxide might also be a good choice for these applications.
The influence of the Stern layer conductance on the dielectrophoretic behavior of sub-micrometer-sized latex spheres is examined. The dielectrophoretic response of the particles is measured and analyzed in terms of a model of surface conductance divided into discrete components related to the structure of the double layer. The effect of both co- and counterions in the bulk solution on the Stern layer conductance is demonstrated.
The phenomena of dielectrophoresis and electrorotation, collectively referred to as AC electrokinetics, have been used for many years to study, manipulate and separate particles on the nanotechnology, that is the precise manipulation of particles on the nanometre scale. In this paper we present the principles of AC electrokinetics for particle manipulation, review the current state of AC Electrokinetic techniques for the manipulation of particles on the nanometre scale, and consider how these principles may be applied to nanotechnology
A major problem for surface-based detection techniques such as surface plasmon resonance and quartz crystal microbalances is that at low concentrations, diffusion is an insufficient driving force to bring colloidal submicron-scale particles to the detection surface. In order to overcome this, it has previously been demonstrated that a combination of dielectrophoresis and AC-electro-hydrodynamic flow can be used to focus cell-sized particles from suspension onto a large metal surface, in order to improve the detection capabilities of such systems. In this paper we describe how the combination of these two phenomena, using the so-called "zipper" electrode array, can be used to concentrate a wide range of nanoparticles of biological interest, such as influenza virus, dissolved albumin, and DNA molecules as well as latex beads of various sizes. We also demonstrate that the speed at which particles are transported towards the centre of the electrode pads by dielectrophoresis and electro-hydrodynamic flow is not related to the particle size for colloidal particles.
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. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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.
Maturation and ageing, which can be characterised by the dynamic changes in brain morphology, can have an impact on the physiology of the brain. As such, it is possible that these changes can have an impact on the magnetic activity of the brain recorded using magnetoencephalography. In this study changes in the resting state brain (magnetic) activity due to healthy ageing were investigated by estimating the complexity of magnetoencephalogram (MEG) signals. The main aim of this study was to identify if the complexity of background MEG signals changed significantly across the human lifespan for both males and females. A sample of 177 healthy participants (79 males and 98 females aged between 21 and 80 and grouped into 3 categories i.e., early-, mid- and late-adulthood) was used in this investigation. This investigation also extended to evaluating if complexity values remained relatively stable during the 5 min recording. Complexity was estimated using permutation Lempel-Ziv complexity, a recently introduced complexity metric, with a motif length of 5 and a lag of 1. Effects of age and gender were investigated in the MEG channels over 5 brain regions, i.e., anterior, central, left lateral, posterior, and, right lateral, with highest complexity values observed in the signals recorded by the channels over the anterior and central regions of the brain. Results showed that while changes due to age had a significant effect on the complexity of the MEG signals recorded over 5 brain regions, gender did not have a significant effect on complexity values in all age groups investigated. Moreover, although some changes in complexity were observed between the different minutes of recording, due to the small magnitude of the changes it was concluded that practical significance might outweigh statistical significance in this instance. The results from this study can contribute to form a fingerprint of the characteristics of healthy ageing in MEGs that could be useful when investigating changes to the resting state activity due to pathology.
A major problem for surface-based detection techniques such as surface plasmon resonance and quartz crystal microbalances is that at low concentrations, diffusion is an insufficient driving force to bring colloidal submicron-scale particles to the detection surface. In order to overcome this, it has previously been demonstrated that a combination of dielectrophoresis and AC-electro-hydrodynamic flow can be used to focus cell-sized particles from suspension onto a large metal surface, in order to improve the detection capabilities of such systems. In this paper we describe how the combination of these two phenomena, using the so-called “zipper” electrode array, can be used to concentrate a wide range of nanoparticles of biological interest, such as influenza virus, dissolved albumin, and DNA molecules as well as latex beads of various sizes. We also demonstrate that the speed at which particles are transported towards the centre of the electrode pads by dielectrophoresis and electro-hydrodynamic flow is not related to the particle size for colloidal particles.
Most oral cancers are oral squamous cell carcinomas (OSCC) that arise from the epithelial lining of the oral mucosa. Given that the oral cavity is easily accessible, the disease lends itself to early detection; however, most oral cancers are diagnosed at a late stage, and approximately half of oral cancer sufferers do not survive beyond five years, post-diagnosis. The low survival rate has been attributed to late detection, but there is no accepted, reliable and convenient method for the detection of oral cancer and oral pre-cancer. Dielectrophoresis (DEP) is a label-free technique which can be used to obtain multi-parametric measurements of cell electrical properties. Parameters such as cytoplasmic conductivity and effective membrane capacitance (C(Eff)) can be non-invasively determined by the technique. In this study, a novel lab-on-a-chip device was used to determine the cytoplasmic conductivity and C(Eff) of primary normal oral keratinocytes, and pre-cancerous and cancerous oral keratinocyte cell lines. Our results show that the electrical properties of normal, pre-cancerous and cancerous oral keratinocytes are distinct. Furthermore, increasing C (Eff) and decreasing cytoplasmic conductivity correlate with disease progression which could prove significant for diagnostic and prognostic applications. DEP has the potential to be used as a non-invasive technique to detect oral cancer and oral pre-cancer. Clinical investigation is needed to establish the reliability and temporal relationship of the correlation between oncologic disease progression and the electrical parameters identified in this study. To use this technique as an OSCC detection tool in a clinical setting, further characterisation and refinement is warranted.
A method is described whereby dielectrophoresis of algal cells is used to perform rapid water quality analysis, specifically detecting the presence of CuSO4. The dielectric collection spectrum of the fresh water alga Selenastrum capricornutum was determined for a range of concentrations of CuSO4 from 25 mg L(-1) to 0.25 mg L(-1) for exposure times of 15 min and 18 h. In all cases increasing the concentration of CuSO4 reduced cell collection, but a step reduction was observed in collection between 2 mg L(-1) and 5 mg L(-1). This method has potential for forming a rapid, low-cost test for water quality with broad specificity and significantly reduced analysis time compared to current methods.
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.
The field of nanotechnology is inherently multidisciplinary-in its principles, in its techniques, and in its applications-and meeting its current and future challenges will require the kind of approach reflected in this book.
At least one-third of the authors are expected to be from Europe and Japan. Since this encyclopedia is the first book in this emerging and potentially large field, the market is expected to be large and steady.
BACKGROUND: Many drug development and toxicology studies are performed using cells grown in monolayers in well-plates and flasks, despite the fact that these are widely held to be different to cells found in the native environment. 3D, tissue engineered, organotypical tissue culture systems have been developed to be more representative of the native tissue environment than standard monolayer cultures. Whilst the biochemical differences between cells grown in 2D and 3D culture have been explored, the changes on the electrophysiological properties of the cells have not. METHODS: We compared the electrophysiological properties of primary normal oral keratinocytes (nOK) and cancerous abnormal oral keratinocytes (aOK), cultured in standard monolayer and reconstituted 3D organotypical tissue cultures. The electrophysiological properties of populations of the cells were analysed using dielectrophoresis. The intracellular conductivity of aOK was significantly increased when grown in organotypical cultures compared to counterpart cells grown in monolayer cultures. RESULTS: 3D cultured aOK showed almost identical intracellular conductivity to nOK also grown in organotypical cultures, but significantly different to aOK grown in monolayers. The effective membrane capacitance of aOK grown in 3D was found to be significantly higher than nOK, but there was no significant difference between the electrophysiological properties of nOK grown in 2D and 3D cultures. GENERAL SIGNIFICANCE: This work suggests that factors such as cell shape and cytoplasmic trafficking between cells play an important role in their electrophysiology, and highlights the need to use in vitro models more representative of native tissue when studying cell electrophysiological properties.
Whilst personalized medicine (where interventions are precisely tailored to a patient’s genotype and phenotype, as well as the nature and state of the disease) is regarded as an optimal form of treatment, the time and cost associated with it means it remains inaccessible to the greater public. A simpler alternative, stratified medicine, identifies groups of patients who are likely to respond to a given treatment. This allows appropriate treatments to be selected at the start of therapy, avoiding the common “trial and error” approach of replacing a therapy only once it is demonstrated to be ineffective in the patient. For stratification to be effective, tests are required that rapidly predict treatment effectiveness. Most tests use genetic analysis to identify drug targets, but these can be expensive and may not detect changes in the phenotype that affect drug sensitivity. An alternative method is to assess the whole-cell phenotype by evaluating drug response using cells from a biopsy. We assessed dielectrophoresis to assess drug efficacy on short timescales and at low cost. To explore the principle of assessing drug efficacy we examined two cell lines (one expressing EGFR, one not) with the drug Iressa. We then further explored the sensitive cells using combinations of chemotherapeutic and radiotherapeutic therapies. Our results compare with known effects of these cell/treatment combination, and offer the additional benefit over methods such as TUNEL of detecting drug effects such as cell cycle arrest, which do not cause cell death.
The frequency-dependent dielectrophoretic behaviour of an enveloped mammalian virus, herpes simplex virus type 1 is described. It is demonstrated that over the range 10 kHz-20 MHz, these viral particles, when suspended in an aqueous medium of conductivity 5 mS m(-1), can be manipulated by both positive and negative dielectrophoresis using microfabricated electrode arrays. The observed transition from positive to negative dielectrophoresis at frequencies around 4.5 MHz is in qualitative agreement with a simple model of the virus as a conducting particle surrounded by an insulating membrane.
A design for an electric nanomotor driven by ac electric fields is presented and evaluated. The motor induces torque by means of a rotating electric field, which induces a dipole in the rotor; the interactions between field and dipole are responsible for both the generation of torque and the repulsion of the rotor, which stabilizes its position without contact and removes the need for bearings. The applied electric field is generated by square-wave pulses, allowing the direct computer control of the device without any digital-to-analogue requirement; the amount of torque generated can be controlled precisely by varying the number of clock cycles between digital state changes. The technology is scaleable, and a motor consisting of a rotor 1μm long and 100 nm diameter is shown to be capable of generating approximately 10-15 N m-1, equivalent to a bacterial flagellar motor.
The dielectric properties of a polarizable particle can be characterized by measuring the frequency dependence of the torque in a rotating electric field. Measurements performed using planar electrodes indicate a spatial variation in the torque across the dimensions of the array. In this paper the variation in rotation rate of elliptical latex beads was measured in 203 discrete positions within a 400 μm × 400 μm polynomial electrorotation electrode array. It is shown that torque variations across the electrode array can exceed 50% of the mean value at the centre. Data averaging and smoothing were performed to reveal trends that match theoretical predictions made using numerical models. The results indicate that the torque depends on variations in both the magnitude and phase of the electric field.
Asymmetrical periodic ('ratchet') potential energy structures have a number of applications, including the dielectrophoretic rectification of Brownian motion, the implementation of quantum tunnelling devices, and as a model of the action of molecular motors such as muscles. The effectiveness of such devices is dependent on the asymmetry of the potential energy, not that of the potential energy generating structures. Using empirical analysis of simulations of electric field ratchets, this paper derives empirical expressions describing, and optimizing, electric field ratchets in terms of the electrode dimensions.
Dielectrophoretic collection rates have been used to measure the dielectric properties of particles. For accurate collection-rate measurements it is important to be able to discriminate between particles collecting on an electrode and particles that are in the bulk solution. In this paper we demonstrate how evanescent-imaging methods can be used to observe only those particles that are in the plane of the electrode array, so that significant improvements in the signal-to-noise ratio compared with conventional imaging methods are achieved.
AC electrokinetics have played an important role in the advancement of BioMEMS research. Although the various AC electrokinetics methods offers a myriad of advantages in conducting bioparticle electrophysiology, the uptake of the technology has been limited only to research centres. One reason is due to the time-consuming procedures involved. This report documents the development of a programmable, multiple frequency signal generator that may be used in any AC electrokinetics experimental setup in conducting parallel electrophysiology assays. The development employs commercially available tools and components, which hopefully encourages the uptake of the various AC electrokinetics methods in conducting BioMEMS research.
The force produced by the flagella of the bacterium Salmonella typhimurium has been measured using negative dielectrophoretic methods. The bacteria are held in a force funnel, produced using a nonuniform electric field. When the motor force is balanced against an opposing negative dielectrophoretic force the bacteria become motionless. Numerical simulations have been used to estimate the electric field gradient in the electrodes. Together with experimental observations of bacterial motion the data gives a value of the force produced by the bacterial motor to be 0.37 pN.