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
Dielectrophoresis (DEP) was used to examine a panel of MCF-7 cell lines comprising parental MCF-7 cells and MDR derivatives: MCF-7TaxR (paclitaxel-resistant, P-glycoprotein (P-gp) positive), MCF-7DoxR (doxorubicin-resistant MRP2 positive) plus MCF-7MDR1 (MDR1 transfected, P-gp positive). MCF-7DoxR and MCF-7MDR1 were broadly cross-resistant to natural product anticancer agents, whereas MCF-7TaxR cells were not, contrary to P-gp expression. Whilst DEP revealed modest membrane changes in MDR sub-lines, we saw significant changes in their cytoplasmic conductivity: MCF-7TaxR
Labeed F, Hughes MP, Hoettges K, Fatoyinbo HO (2004) AC Electrokinetics of Particles, In: Dorf RC (eds.), Handbook of Engineering CRC Press
Humphries A, Lister TS, Wright PA, Hughes MP (2013) Determination of the thermal and physical properties of black tattoo ink using compound analysis, Lasers in Medical Science 28 (4) pp. 1107-1112
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.
Hoettges KF, Hughes MP, Cotton A, Hopkins NAE, McDonnell MB (2003) Optimizing particle collection for enhanced surface-based biosensors, IEEE ENGINEERING IN MEDICINE AND BIOLOGY MAGAZINE 22 (6) pp. 68-74 IEEE-INST ELECTRICAL ELECTRONICS ENGINEERS INC
Mulhall HJ, Cardnell A, Hoettges KF, Labeed FH, Hughes MP (2015) Apoptosis progression studied using parallel dielectrophoresis electrophysiological analysis and flow cytometry., Integrative biology : quantitative biosciences from nano to macro Royal Society of Chemistry
Apoptosis is characterised by many cellular events, but the standard Annexin-V assay identifies two; the transfer of the phospholipid phosphatidylserine (PS) from inner to outer leaflets of the plasma membrane, acting as an "eat me" signal to macrophages, and the permeabilisation of the plasma membrane. In this paper we compare the results from the Annexin-V assay with electrophysiology data obtained in parallel using dielectrophoresis, which highlights two changes in cell electrophysiology; a change in cytoplasmic conductivity which correlates with PS expression, and a membrane conductance spike that correlates with permeabilisation. Combining results from both methods shows a strong inverse relationship between conductivity and PS externalisation. One mechanism which may explain this correlation is related to intracellular Ca(2+), which is known to increase early in apoptosis. PS expression occurs when enzymes called scramblases swap external and internal phospholipids, and which are usually activated by Ca(2+), whilst the change in cytoplasmic conductivity may be due to K(+) efflux from intermediate conductance (IK) ion channels that are also activated by Ca(2+).
Hughes MP, Ogin SL, Hoettges KF, Wattingham R (2005) Device for Dielectrophoretic Manipulation of Particles,
Mohamad AS, Jeynes JC, Hughes MP (2014) Dielectrophoretic response of DNA shows different conduction mechanisms for poly(dG)-poly(dC) and poly(dA)-Poly(dT) in solution., IEEE Trans Nanobioscience 13 (1) pp. 51-54
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.
Labeed FH, Hughes MP, Charlton P, Coley HM (2002) Assessment of modulator action on drug-resistant and sensitive cancer cells using dielectrophoretic methods, BRITISH JOURNAL OF CANCER 86 pp. S22-S23 NATURE PUBLISHING GROUP
Morgan H, Hughes MP, Green NG (1999) Separation of submicron bioparticles by dielectrophoresis., Biophys J 77 (1) pp. 516-525
Submicron particles such as latex spheres and viruses can be manipulated and characterized using dielectrophoresis. By the use of appropriate microelectrode arrays, particles can be trapped or moved between regions of high or low electric fields. The magnitude and direction of the dielectrophoretic force on the particle depends on its dielectric properties, so that a heterogeneous mixture of particles can be separated to produce a more homogeneous population. In this paper the controlled separation of submicron bioparticles is demonstrated. With electrode arrays fabricated using direct write electron beam lithography, it is shown that different types of submicron latex spheres can be spatially separated. The separation occurs as a result of differences in magnitude and/or direction of the dielectrophoretic force on different populations of particles. These differences arise mainly because the surface properties of submicron particles dominate their dielectrophoretic behavior. It is also demonstrated that tobacco mosaic virus and herpes simplex virus can be manipulated and spatially separated in a microelectrode array.
Hughes MP, Hoettges KF (2005) Dielectrophoresis for drug discovery and cell analysis: novel electrodes for high-throughput screening, BIOPHYSICAL JOURNAL 88 (1) pp. 172A-172A
Hughes MP (2002) Dielectrophoretic behavior of latex nanospheres: low-frequency dispersion., J Colloid Interface Sci 250 (2) pp. 291-294
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.
Fatoyinbo HO, McDonnell MC, Hughes MP (2014) Dielectrophoretic sample preparation for environmental monitoring of microorganisms: Soil particle removal., Biomicrofluidics 8 (4)
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.
Hughes MP (2004) Numerical simulation of dielectrophoretic ratchet structures, JOURNAL OF PHYSICS D-APPLIED PHYSICS 37 (8) PII S0022-3727(04)69865-2 pp. 1275-1280 IOP PUBLISHING LTD
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. © 2007 IOP Publishing Ltd.
Selvakumaran J, Keddie JL, Ewins DJ, Hughes MP (2008) Protein adsorption on materials for recording sites on implantable microelectrodes, JOURNAL OF MATERIALS SCIENCE-MATERIALS IN MEDICINE 19 (1) pp. 143-151 SPRINGER
Hughes MP (2000) AC electrokinetics: applications for nanotechnology, NANOTECHNOLOGY 11 (2) pp. 124-132 IOP PUBLISHING LTD
Mulhall HJ, Hughes MP, Kazmi B, Lewis MP, Labeed FH (2013) Epithelial cancer cells exhibit different electrical properties when cultured in 2D and 3D environments., Biochim Biophys Acta 1830 (11) pp. 5136-5141
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.
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.
Mulhall HJ, Labeed FH, Kazmi B, Costea DE, Hughes MP, Lewis MP (2011) Cancer, pre-cancer and normal oral cells distinguished by dielectrophoresis., Analytical and bioanalytical chemistry 401 (8) pp. 2455-2463
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.
Apoptosis, or programmed cell death, is a vital cellular process responsible for causing cells to self-terminate at the end of their useful life. Abrogation of this process is commonly linked to cancer, and rapid detection of apoptosis in vitro is vital to the discovery of new anti-cancer drugs. In this paper, we describe the application of the electrical phenomenon dielectrophoresis for detecting apoptosis at very early stages after drug induction, on the basis of changes in electrophysiological properties. Our studies have revealed that K562 (human myelogenous leukemia) cells show a persistent elevation in the cytoplasmic conductivity occurring as early as 30 minutes following exposure to staurosporine. This method therefore allows a far more rapid detection method than existing biochemical marker methods.
Mohamad AS, Hughes MP (2016) Characterization of Impedance Spectroscopy for Single-Walled Carbon Nanotubes with Ionic Liquid of Dielectrophoretic Assembly Method, INTERNATIONAL CONFERENCE FOR INNOVATION IN BIOMEDICAL ENGINEERING AND LIFE SCIENCES, ICIBEL2015 56 pp. 279-282 SPRINGER
Hoettges KF, McDonnell MB, Hughes MP (2003) Use of combined dielectrophoretic/electrohydrodynamic forces for biosensor enhancement, Journal of Physics D: Applied Physics 36 (20)
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 (H1 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.
Hughes MP, Archer S, Morgan H (1999) Mapping the electrorotational torque in planar microelectrodes, Journal of Physics D: Applied Physics 32 (13) pp. 1548-1552
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 ¼m × 400 ¼m 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.
Fatoyino HO, Huebner Y, Hoettges KF, Hughes MP, Labeed FH (2006) Applications of Dielectrophoretic / electrohydrodynamic electrodes for concentration of biological nanoparticles, 2006 NSTI Nanotechnology Conference and Trade Show - NSTI Nanotech 2006 Technical Proceedings 2 pp. 275-277
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.
Hughes MP, Hoettges KF (2008) Bacterial manipulation by dielectrophoresis, In: Zourob M, Elwary S, Turner APF (eds.), Principles of Bacterial Detection Springer
Hughes MP, Morgan H, Rixon FJ (2001) Dielectrophoretic manipulation and characterization of herpes simplex virus-1 capsids., Eur Biophys J 30 (4) pp. 268-272
The dielectrophoretic behaviour of the capsids of herpes simplex virus type-1 has been measured over a range of conductivities of KCl solutions, with and without the addition of mannitol. The dielectrophoretic response of the capsids was recorded by measuring the frequency corresponding to zero dielectrophoretic force. The data were analysed using a multi-shelled model, and the permittivity and conductivity of the particles estimated. The capsid was modelled as a porous protein shell through which suspending medium passes, an inner chamber containing suspending medium in equilibrium with the outside, and a central core of protein (the scaffold). Capsids suspended in KCl without mannitol exhibited a different behaviour to those suspended in KCl with mannitol.
Graham KA, Mulhall HJ, Labeed FH, Lewis MP, Hoettges KF, Kalavrezos N, McCaul J, Liew C, Porter S, Fedele S, Hughes MP (2015) A dielectrophoretic method of discrimination between normal oral epithelium, and oral and oropharyngeal cancer in a clinical setting., Analyst 140 (15) pp. 5198-5204
Despite the accessibility of the oral cavity to clinical examination, delays in diagnosis of oral and oropharyngeal carcinoma (OOPC) are observed in a large majority of patients, with negative impact on prognosis. Diagnostic aids might help detection and improve early diagnosis, but there remains little robust evidence supporting the use of any particular diagnostic technology at the moment. The aim of the present feasibility first-in-human study was to evaluate the preliminary diagnostic validity of a novel technology platform based on dielectrophoresis (DEP). DEP does not require labeling with antibodies or stains and it is an ideal tool for rapid analysis of cell properties. Cells from OOPC/dysplasia tissue and healthy oral mucosa were collected from 57 study participants via minimally-invasive brush biopsies and tested with a prototype DEP platform using median membrane midpoint frequency as main analysis parameter. Results indicate that the current DEP platform can discriminate between brush biopsy samples from cancerous and healthy oral tissue with a diagnostic sensitivity of 81.6% and a specificity of 81.0%. The present ex vivo results support the potential application of DEP testing for identification of OOPC. This result indicates that DEP has the potential to be developed into a low-cost, rapid platform as an assistive tool for the early identification of oral cancer in primary care; given the rapid, minimally-invasive and non-expensive nature of the test, dielectric characterization represents a promising platform for cost-effective early cancer detection.
Hughes MP, Dean Astumian R (2006) Dielectrophoretic ratchets for linear and rotary nanomotors, 2006 NSTI Nanotechnology Conference and Trade Show - NSTI Nanotech 2006 Technical Proceedings 3 pp. 377-380
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.
Hughes MP, Hoettges KF (2010) Microengineering in biotechnology, Humana Press
Hughes MP (2000) AC Electrokinetics: Applications for Nanotechnology, Nanotechnology 11 pp. 124-132
Abdallat RG, Ahmad Tajuddin AS, Gould DH, Hughes MP, Fatoyinbo HO, Labeed FH (2013) Process development for cell aggregate arrays encapsulated in a synthetic hydrogel using negative dielectrophoresis, Electrophoresis 34 (7) pp. 1059-1067
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%. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
WANG XB, HUGHES MP, HUANG Y, BECKER FF, GASCOYNE PRC (1995) NONUNIFORM SPATIAL DISTRIBUTIONS OF BOTH THE MAGNITUDE AND PHASE OF AC ELECTRIC-FIELDS DETERMINE DIELECTROPHORETIC FORCES, BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS 1243 (2) pp. 185-194 ELSEVIER SCIENCE BV
HUGHES MP, WANG XB, BURT JPH, PETHIG R, WATKINS LR (1994) SIMULATION OF TRAVELING ELECTRIC-FIELD MANIPULATION OF BIOPARTICLES, SECOND INTERNATIONAL CONFERENCE ON COMPUTATION IN ELECTROMAGNETICS (384) pp. 48-51 INST ELECTRICAL ENGINEERS
Humphries A, Lister TS, Wright PA, Hughes MP (2012) Finite element analysis of thermal and acoustic processes during laser tattoo removal, Lasers in Surgery and Medicine 45 (2) pp. 108-115
Background and Objective Q-switched laser therapy is commonly used for the removal of tattoos. However, despite ever increasing demand for this intervention, a better understanding of the mechanisms that result in pigment reduction is required in order to optimise outcomes and reduce the number of treatment episodes. Study Design A finite element analysis computer simulation was developed to model the fragmentation response of ink granules during irradiation of a professional black tattoo using a Q-switched Nd:YAG laser. Thermal and acoustic mechanisms were considered, allowing the optimal laser settings to be predicted throughout the course of treatment. Changes in the thermal properties of the ink during heating were taken into account to improve the reliability of the results obtained. Results The simulated results are in close agreement with clinical observations. Thermal fragmentation was shown to be the dominant mechanism in pigment reduction when using a 6 nanoseconds pulse at 1,064 nm. In order to provide maximum clearance whilst maintaining acceptable levels of tissue thermal damage, later treatments were shown to benefit from higher fluence levels than initial treatments. Larger spot diameters were also preferable throughout the course of treatment. Conclusions The results from the simulation build upon previous work carried out in the field, applying ink thermal coefficients which vary with temperature for the first time. These results compliment clinical knowledge, suggesting that a proactive increase in fluence during a course of treatments is likely to improve the response to laser therapy. © 2012 Wiley Periodicals, Inc.
Kadri NA, Hoettges KF, Hughes MP (2008) Microelectrode Fabrication Using Indium Tin Oxide (ITO) For Microfluidic Devices Employing Dielectrophoresis, IFMBE Proceedings 21 (1-2) pp. 719-722 SPRINGER
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.
Hughes MP, Morgan H, Flynn MF (1999) The dielectrophoretic behavior of submicron latex spheres: Influence of surface conductance, JOURNAL OF COLLOID AND INTERFACE SCIENCE 220 (2) pp. 454-457 ACADEMIC PRESS INC
Fatoyinbo HO, Hughes MP, Martin SP, Pashby P, Labeed FH (2007) Dielectrophoretic separation of Bacillus subtilis spores from environmental diesel particles, JOURNAL OF ENVIRONMENTAL MONITORING 9 (1) pp. 87-90 ROYAL SOC CHEMISTRY
Mohamad AS, Jeynes JC, Hughes MP (2012) Dielectrophoresis of poly at and poly GC DNA Nanomanipulation, Proceedings of the IEEE Conference on Nanotechnology
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.
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.
Hughes MP, Flynn MF, Morgan H (1999) Dielectrophoretic measurements of sub-micrometre latex particles following surface modification, ELECTROSTATICS 1999 163 pp. 81-84 IOP PUBLISHING LTD
Selvakumaran J, Hughes MP, Keddie JL, Ewins DJ (2002) Assessing biocompatibility of materials for implantable microelectrodes using cytotoxicity and protein adsorption studies, 2ND ANNUAL INTERNATIONAL IEEE-EMBS SPECIAL TOPIC CONFERENCE ON MICROTECHNOLOGIES IN MEDICINE & BIOLOGY, PROCEEDINGS pp. 261-264
HUGHES MP, WANG XB, BECKER FF, GASCOYNE PRC, PETHIG R (1994) COMPUTER-AIDED ANALYSES OF ELECTRIC-FIELDS USED IN ELECTROROTATION STUDIES, JOURNAL OF PHYSICS D-APPLIED PHYSICS 27 (7) pp. 1564-1570 IOP PUBLISHING LTD
Abdul Razak MA, Hoettges KF, Fatoyinbo HO, Labeed FH, Hughes MP (2013) Efficient dielectrophoretic cell enrichment using a dielectrophoresis-well based system., Biomicrofluidics 7 (6)
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.
Mulhall HJ, Abdallat R, Liang X, Fedele S, Lewis MP, Porter S, Tsinkalovsky O, Johannessen AC, Hughes MP, Costea DE, Labeed FH (2010) Rapid detection of oral cancer: Electrophysiological characterization by dielectrophoresis technology, Technical Proceedings of the 2010 NSTI Nanotechnology Conference and Expo 3 pp. 35-38
Green NG, Hughes MP, Monaghan W, Morgan H (1997) Large Area Multilayered Arrays for Dielectrophoretic Fractionation, Microelectronic Engineering 35 pp. 421-424
Labeed FH, Chin S, Broche LM, Coley HM, Thomas H, Hughes M (2005) Rapid assessment of biophysical changes in K562 cells during apoptosis, determined using dielectrophoresis, BIOPHYSICAL JOURNAL 88 (1) pp. 565A-565A BIOPHYSICAL SOCIETY
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.
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.
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.
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.
Faraghat S, Hoettges K, Steinbach M, Van Der Veen DR, Brackenbury W, Henslee E, Labeed F, Hughes MP (2017) High-Throughput, Low-Loss, Low-Cost and Label-Free Cell Separation using Electrophysiology Activated Cell Enrichment (EPACE), Proceedings of the National Academy of Sciences 114 (8) pp. 4591-4596
National Academy of Sciences
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.
Cell separation is an important component of modern medicine, with both clinical and research applications. Clinically, it is often desirable to isolate cell subpopulations providing focused treatment; on the research side, cell isolation is necessary for studies underpinning many discoveries in cell biology, further enabling research in areas such as regenerative medicine and cancer therapy. Cell separation requirements include high throughput, purity and recovery.
Three cell separators dominate: fluorescence and magnetic-activated cell sorting and density-gradient centrifugation. Despite gold-standard establishing performances, they can be improved in affordability, throughput, and label-free cell separation implementation. A technology with potential to offer the next rotation of gold-standard cell separators is Dielectrophoresis, DEP.
Two DEP cell separators are presented. The first, the Syringe Separator (SS), uses 3D-electrodes on a low-cost, disposable chip and a DEP field perpendicular to fluid flow; one cell type is passed through whilst the other is retained and subsequently recovered. Two-pass protocols achieved a 96.4% recovery at over 200,000 cells/second with <7% loss. Additionally, a three-step protocol removed 99.1% of RBCs spiked with cancer cells (100:1). Other SS implementations include hitherto unachieved separation of high and low quality nanowires and T-cell isolation.
The second employs a novel electrode geometry termed the Canyon. Using a novel electrode fabrication method (Plotter-Canyon printing), Canyons were built of alternating layers of metal and non-metal. Cellular solutions flow through the Canyon directed to one of two outlets, one for each of the negative or positive DEP cell subpopulations. The Canyon cell separator achieved an 84% recovery and 10% loss at ~2,000 cells/second.
We have demonstrated that DEP cell separators can be built to perform cell separations with high purity, rivalling established separators, at significantly higher throughput and recovery. The SS and Canyons are cheap, easy-to-operate and offer a stepwise improvement in conventional cell separation capabilities.
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.
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.
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.
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.
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.
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 (pÂ0.001). Post-operative responders to fasciotomy have no significant differences in IMCP from asymptomatic controls (p=0.182).
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.
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.
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.
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.
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. Stratified medicine offers an option with many of the benefits without the associated costs; rather than treatments being unique to an individual, patients are grouped (stratified) according to whether they respond to a given treatment, allowing the treatment to be selected from an appropriate panel, and 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, it is necessary to develop tests that can rapidly assess whether a treatment will be effective. Here we examine the use of dielectrophoresis to determine whether cancer drugs like Iressa, Cisplatin and MTX (metateroxide) are efficatious by treating cancer cell lines (one expressing EGFR, one not) with the drug Iressa, which indicates discernable difference. We also examine the use in assessing the efficacy of combination therapies, including both chemotherapeutic and radiotherapeutic interventions. Results suggest that a DEP-based assay could potentially be used in a standard pathology laboratory setting, where biopsy cells can be screened at low cost and high speed, enabling the selection of an appropriate course of treatment from the outset, increasing patient survival rates whilst reducing time and cost for the health provider. K562 is suspension cell line, butHN5 and T47D cell lines are both adherent cells and need to be detached before any experiment. Prior to the investigation of cancer treatments, the effect of various detachment methods on these cell lines were studied to find out whether these methods could impact the electrophysiological properties of the cells. The results in investigation of detachment methods exhibit that unlike chemical substances, a harvesting method significantly alters the values of dielectric parameters.
Chronic Exertional Compartment Syndrome (CECS) presents as exertional pain in the lower limb presumed to be a result of elevated intramuscular compartment pressure (IMCP) although this has never been proven. Doubt exists regarding the validity of the diagnostic criteria for CECS, the role of IMCP and the outcomes from surgical management. An alternative biomechanical condition, Anterior Biomechanical Overload Syndrome (ABOS), was proposed to account for the symptoms of CECS and a programme of gait re-education (GRE) was introduced although no primary research has been carried out to investigate the predisposing biomechanical and anthropometric factors for CECS.
Case-control studies investigated the anthropometric, biomechanical and IMCP differences between CECS cases and asymptomatic controls. A post-surgical study evaluated the role of IMCP and a longitudinal study investigated the effectiveness of GRE and the nature of resultant biomechanical changes.
Cases were significantly shorter than controls with increased plantarflexion at toe off and an increased rate of plantarflexion after heel strike. IMCP levels were significantly higher in cases than controls allowing for the extraction of diagnostic criteria for CECS. Surgical responders had similar IMCP to controls but significantly lower IMCP than non-responders. The biomechanical symptoms of ABOS were not replicated. GRE made changes to gait but these did not correspond to those identified in the CECS case-control study.
The intrinsic role of IMCP in CECS has been confirmed allowing for improved diagnostic criteria. Use of these criteria should allow for improved patient selection for surgery and improved outcomes for CECS. Novel insights to the biomechanical and anthropometric differences are provided allowing for the proposal of a new pathophysiological model whereby extrinsic training conditions impact upon intrinsic risk factors leading to CECS. These studies do not support the existence of ABOS or the use of GRE in the management of CECS.
The brain is a very delicate, but sophisticated and complex organ of the body. During life, the brain grows and develops, matures, and then ages like all other organs of the body. This maturation and ageing comes with various physiological and anatomical changes which have an impact on the background activity of the brain. Brain activity can be recorded using various techniques including magnetoencephalography and magnetic resonance imaging, and these recordings combined with signal processing can be useful to characterise the changes in the brain that can be a result of the ageing process. By analysing various aspects of the brain, such as functional and effective connectivity, entropy and complexity, the state of the resting brain at different ages can be understood with greater detail. Furthermore, these analyses can be used to investigate the resting state brain networks that are present in the brain, as well as their topology. This information can be combined with network analysis techniques such as graph theory and used to understand the manner in which the brain both matures and ages.
This research made use of two magnetoencephalogram (MEG) databases recorded from both males and females. The first, containing resting state MEGs (rMEGs) from 220 healthy volunteers (aged 7-84), was used as the main database in this thesis to investigate the effects of age on rMEG signals throughout life. The aim of this research was to make use of rMEG signals and signal processing techniques to determine the effects of healthy ageing on the brain throughout life. It was hypothesised that the effects of age are identifiable using advanced signal processing techniques. Thus, the effects of age on linear interactions, causality, synchronisation, information flow, entropy and complexity, were investigated in the 148 MEG channels lying over 5 brain regions (anterior, central, left lateral, posterior, and, right lateral) using multiple linear and non-linear analysis techniques (namely: Pearson?s correlation, coherence, Granger causality, phase slope index, rho index, transfer entropy, synchronisation likelihood, Lempel-Ziv complexity, permutation Lempel-Ziv complexity, permutation entropy, and, modified permutation entropy). Additionally, graph theory principles were used to evaluate different network components such as integration (global efficiency), segregation (clustering coefficient and modularity), centrality (betweenness), and resilience (strength and assortativity) so as to obtain an understanding of the construct of the resting brain network. Moreover, complex network analysis was also used to determine the overall network topology of the brain network and how this changed at different stages in life. Gender effects were also studied so as to identify if there were any significant differences between males and females at different stages of life. Results from these analyses showed that the healthy resting brain has low effective and functional connectivity, relatively low complexity and entropy, as well as no significant detectable direction of information flow. Therefore this showed that there is very little synchronous or simultaneously occurring information in the rMEG time series. Thus, during rest, the brain resembles a system in limbo/phase transition, with low effective and functional connectivity, relatively low complexity and entropy, and no significant detectable direction of information flow.
The second database used in this research project was obtained during a collaboration visit to the Cognitive and Computational Neuroscience laboratory at the Centre for Biomedical Technology- Universidad Politécnica de Madrid (CTB-UPM). This database was made up of rMEGs recorded from 199 healthy volunteers (aged 60-80), and the focus of this additional set of analyses was to identify differences between the rMEG signals recorded from healthy individuals, those with subjective cognitive decline as well as those with