Rapid Cell Analysis and Separation Using Dielectrophoresis

W.B. Betts & A.P. BrownCell Analysis Ltd

Institute for Applied BiologyUniversity of York

DielectrophoresisScientific Background

Dielectrophoresis (DEP) Dielectrophoresis is observed when particles are

placed in a non-uniform electric field applied to electrodes

Particles move toward the electrodes independently of the direction of applied field

Movement is determined by dielectric properties (conductivity & permittivity) of particles, not simply by their charge (as in electrophoresis)

Dielectric Force (F) = pv (E.)E

where:p = polarisability of suspended particlev = volume of the particleE = local root mean square electric field = del vector operator (i.e. E . represents the

divergence, or non-uniformity of the electric field)

Dielectrophoretic Collection If the polarisability of particle › than the suspending

medium positive dielectrophoresis will be observed (in a non-uniform electric field)

Convergence of field lines causes uneven charge alignment in the particle inducing it to move towards regions of greatest field intensity

Dielectrophoresis allows cells to concentrate at the electrodes

Effects of Uniform & Non-uniform Electric Fields on Charged and Neutral Particles

Dielectrophoretic Collection of Cells

Electric field switched off Electric field switched on

DEP Collection Spectra The polarisability of particles (thus direction &

magnitude of dielectrophoretic force) varies as a function of the magnitude and frequency of the applied electric field

When cell collection is observed over a range of frequencies the DEP collection spectrum is distinctive for that cell type

Dielectric properties of all materials (including cells) have characteristic frequency dependent components

Typical Dielectrophoretic Collection Spectra

Advanced Dielectrophoretic Systems and Electrodes

DEP Systems - Electrodes

Microelectrodes are often manufactured using photolithographic methods

Many DEP system configurations utilise microelectrode arrangements

This permits the design of electrodes with µm dimensions

Electrodes are located within a chamber constructed on glass slide/silicon wafer

Microscope slide

Glass channel cover

Suspension in

Suspensionout

Electrode tabs(connected to functiongenerator)

Electrode bars

Photoresist/stickytape channel

Dielectrophoretic Electrode & Chamber Design

Dielectrophoresis Systems

System can analyse low cell concentrations, including individual cells

Earlier spectrophotometric system enabled rapid DEP measurements with good repeatability

But high cell concentrations were required (e.g. >108 cells/ml of resuspended sample)

More recent system incorporates an image analysis detection facility allowing detection with same rapidity as spectrophotometric method

DielectrophoresisSystem Design

Automated Recirculating Dielectrophoretic Analytical System

Computer controlled, flow through, recirculating system developed at York provides detailed spectra < 10 min

Dielectrophoretic collection of cells on electrodes can be dramatically rapid

Complete analysis over wide frequency range is no longer laborious & time-consuming

Measurements lend easily to automation

DEP System Development

Quantification of particle and cell collection using and an integrated impedance measurement

Further reduction of electrode dimensions (greatly submicron, for analysis of viruses, viroids, prions, subcellular organelles, DNA, proteins, etc.)

Further system miniaturisation (laboratory on a chip) Integration of dielectrophoresis with other techniques

(e.g. Capillary Electrophoresis, PCR, DNA Hybridisation, Microfluidics, Optical tweezers)

Rapid, automated and integrated pre-dielectrophoresis sample preparation

Measurements of Dielectrophoretic Collection

Direct light microscopical measurement

Counting cells on photomicrographs Determining voltage required to hold a single

cell against a gravitational force Measuring dielectrophoretic velocity using

quasi-elastic light scattering Spectrophotometric measurement systems

 Image Analysed microscopyImpedance measurements

Advanced DEP Electrode & Chamber Design

Suspension in Medium in

Glass substrate

Glass cover slip

DEP-impedanceelectrodes

Conductivityprobeelectrodes

Photoresistchannel wall

Impedance Measurements

Technique uses a twin channel electrode system with suspension in one channel and medium alone in other channel. Electrical differences between the two channels is attributed to particle DEP collection.

Visualisation of virus sized particles difficult due to inefficient resolution of light microscope.

The use of electrical methods (impedance) to detect DEP collection are more appropriate and sensitive.

Impedance Measurement of DEP CollectionParticles collected by DEP displace suspending medium between the electrodes, changing local average permittivity (with corresponding change in capacitance):

Cav ≈ nv (p -m)

V(where C is the capacitance change, av is average permittivity change, n is number of particles collecting, v is the volume of a particle, V is the local volume surrounding the electrodes and is complex permittivity of particle or medium respectively)

Image Analysis Versus Impedance Measurement of DEP Collection

Grid Electrodes

Flow-through grids for large volume processing

Column Electrode Arrangement

Column electrode designed for “dielectrophoretic chromatography”

DielectrophoresisApplications

DEP Adjunc

t

Microbiological Applications of Dielectrophoresis

Microbial identificatio

n

Microbialenumeration

Biocide sensitivity

testing

Viable butnon-culturable

analysis

Microbial viability

assessmentCE, PCR, DNA hybridisation

Microbialenrichment

AnalyticalMicrobiolog

y

Antibiotic sensitivity testing

Escherichia coli

DEP Spectra of Chlorinated E. coli 39323

DEP Spectra of Tetracycline Treated E. coli

Cryptosporidium parvum

Sporozoite excysting

Oocyst

DEP Spectra of Chlorinated and Ozonated Cryptosporidium Oocysts

Stem, tumour and “T ” cells from bone marrow & peripheral

blood

Haematological Applications of Dielectrophoresis

•Analysis of abnormal white cells•Analysis of platelet microparticles•Analysis of immature platelets•Analysis of foetal red cells•Analysis of nucleated red blood cells•Analysis of blood product contamination

Separation

Analysis Clinical in vitro

DEP Spectral Analysis of Tumour Cells

Sample Viability (%)Neat 97.09 %SG medium 97.20 %0 (Background) 97.13 %1 (Uncollected cells) 97.27 %2 (Eluted cells) 97.16 %3 (Repelled at 10 kHz) 97.13 %4 (Field off) 97.16 %

Viability of stem cells after various treatments

Dr Andy Brown (Haematology Co-ordinator) Dr Keith Gregory (Microbiology Co-ordinator)Dr Keith Milner (Bioelectronics Co-ordinator) Dr Robert Anthony (Clinical Co-ordinator)

Dr Adrian Harrison (Laboratory Manager) Dr Ka Lok Chan (Bioelectronics Scientist)Mr Richard Beal BSc (Project Scientist) Mr Mark Ripley BSc (Project Scientist)

Mr Neil Bennett BSc (Haematology Technician); Mrs Irene Watson BSc (Microbiology Technician)Mrs Kerri Eagle-Moore (Laboratory Technician)

Dr Andrew Jack (Consultant Haematologist, Leeds GeneraI Infirmary)Ms Anne English (Medical Laboratory Scientific Officer, Leeds GeneraI Infirmary)

Dr Gary O’Neill (Public Health Microbiologist, Yorkshire Water plc) Dr Sandy Anderson (Consultant Microbiologist, York District Hospital)

Dr Lee Bond (Consultant Haematologist, York District Hospital)Dr Duncan Allsopp (Dept of Electronics, Bath University)

Dr Marcos Rodrigues (Dept of Computer Science, Hull University)

Contributing Research Team

Financial Support & Collaborating Companies

BBSRC (Ropa) DTI Smart (1997 Feasibility; 1998 Foresight; 1999 Development)

3i

National Power Dalgety Yorkshire Water

Berlex Bioscience

FLLU (Leeds General Infirmary) British Technology Group