1

Characterisation and separationCharacterisation and separation

Characterisation:

Single particles:Single particles:Frequency at which DEP force is zero Frequency at which DEP force is zero (cross(cross--over or zeroover or zero--force frequency) force frequency) Most widely usedMost widely usedMost widely usedMost widely used

Particle velocity (dynamic)Particle velocity (dynamic)

Levitation height (balance DEP and gravity)Levitation height (balance DEP and gravity)

Cross over spectrum for a Cross over spectrum for a solidsolid isotropic isotropic spherical particlespherical particle

In this region the particle is always less polarisable than the medium.

C i tSuspending Med. Conductivity

( )( 2 )12 ( )( 2 )

p m p mo

p m p m

fσ σ σ σ

π ε ε ε ε− +

= −− +

Cross-over pointAt low freq. σ dominatesAt high freq. ε dominates

Polymer particle: εp = 2.5; εm=80σp > σm

2

1

orce

EP Membrane

CellCell

Easy method to measure the Easy method to measure the membrane capacitancemembrane capacitance

CELLSCELLS

0 5

0

0.5

Nor

mal

ized

DE

P F

o

-ve

DE

P

+ve

DE

Cytoplasm

Nucleus

1kHz -0.5

Frequency

1MHz 100MHz0.1MHz 10kHz 10MHz

Measuring this point Measuring this point -- zero force, enables single zero force, enables single particles to be uniquely characterised particles to be uniquely characterised -- ffCrossCrossNote this must be done in low conductivity buffer.Note this must be done in low conductivity buffer.

( )2 2 22 4 98Cross m mem mem

mem

f aG a GaC

σπ

= − −

S ifi M b d t G d

σm = suspending medium conductivity

mem o memC dε ε=

Specific Membrane conductance

Specific Membrane capacitance

mem memG dσ=Cell membrane conductivity lies between 10 and 100S/mCell membrane conductivity lies between 10 and 100S/m22

(and can usually be ignored).(and can usually be ignored).

2m

memCross

Cf a

σπ

=If we assume zero membrane conductivity then:

A plot of (fCross x a) vs σm is a straight line, with a slope proportional to CMem.

3

Example Example -- characterising blood cells for characterising blood cells for DEP separation (see later)DEP separation (see later)

T-lymphocytes

Gascoyne and Vykoukal Dielectrophoresis-Based Sample Handling in General-Purpose Programmable Diagnostic Instruments Proc. IEEE, 92 2004

Monocyte

Particle velocity measurementsParticle velocity measurements

[ ] 2Re E∇αυParticle

Field geometry solved numerically.

Instantaneous velocity of

[ ] 2DEP 4

Re Ev ∇=fαυ

4.00E+01

5.00E+01

6.00E+01

Velocity vsdistance

Particle

Instantaneous velocity of particles obtained by analysing video of particle trajectories.

Fit gives the polarisability0.00E+00

1.00E+01

2.00E+01

3.00E+01

0 2 4 6 8 10 12 14 16 18 20

4

Levitation Height Measurements Levitation Height Measurements Under negative DEP a particle is pushed up from an electrodeThis force is balanced by sedimentation (gravity)

( )m

mp gf

ερρ

32

E][Re 2CM

−=∇

This force is balanced by sedimentation (gravity).

Stable levitation occurs when(a)

m

i.e. depends on particle i.e. depends on particle polarisabilitypolarisability

d1 d2Electrolyte

22

3

ydo

DEPVA ed

π⎛ ⎞−⎜ ⎟⎝ ⎠∇ =E

Example of levitation experiment

yy

Glass substrate

0o 180o 0o 180o 0o

0o 90o 180o 270o 0o

Phases of the applied potential signals for DEP

Phases of the applied potential signals for twDEP

Analytical approximationAnalytical approximation

[ ]2

3 Re4

yoDEP d

DEPVA ed

πυα

−=F UPUP

ρυ= ΔF g DOWNDOWN

xx

g ρυ= ΔF g DOWNDOWN

32 /DEPA π=WhereWhere

[ ]2

3

Reln

4DEP oA Vdy

d gα

π ρ⎡ ⎤

= −⎢ ⎥Δ⎢ ⎥⎣ ⎦

Steady-state levitation height

5

Separation Separation technologies technologies ––binary separationbinary separation

B

A

ffCMCM for solid particlesfor solid particles

1

0

0.5

1positive

DEP This is the cross This is the cross over pointover point

Binary Separation

-0.5

Frequency (Hz)1kHz

negative DEP

1MHz 1GHz

SINGLE INTERFACE SINGLE INTERFACE –– One RelaxationOne Relaxation

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INTERDIGITATED ELECTRODESINTERDIGITATED ELECTRODESPlug Plug of particles introduced, field switched on, particle of particles introduced, field switched on, particle equilibrium establishedequilibrium establishedFluid passed across the electrode array, removing particles Fluid passed across the electrode array, removing particles u d passed ac oss t e e ect ode a ay, e o g pa t c esu d passed ac oss t e e ect ode a ay, e o g pa t c esnot held by positive not held by positive DEPDEP

Electrodes

Voltage OFF

Electrodes

Voltage ON

BINARY Separation ON/OFFBINARY Separation ON/OFF

H ldi f f DEP i t

Markx GH, Talary MS, Pethig R. 1994. Separation of viable and non-viable yeast using dielectrophoresis. J. Biotechnol. 32:29–37

Holding force for =pDEP is greater than nDEP in castellated electrodes

BATCH SEPARATION

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Flow through Flow through continuous separationcontinuous separation

−FDEP

+F

FFluid−FDEP

+F

FFluid−FDEP

+F

FFluid

+FDEP FBuoyancy+FDEP FBuoyancy+FDEP FBuoyancy

If we know the forces we can calculate trajectories

Two electrode Two electrode continuous continuous separatorseparator

Focusing electrode Separation electrode

F f F f

• Red cells experience negative DEP

• Green cells positive DEP

Frequency f1 Frequency f2

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Forces experienced by a particle Forces experienced by a particle in a DEP separation system.in a DEP separation system.

d

FBuoyancy

FDragFDEPh

Interdigitated electrode array

d

Flow

y

x

DEP Buoyancyy

F Fu

f+

=2 21 ( )

2o

xo

pu h ylη

= −

Vertical Horizontal

Simulating cell Simulating cell trajectoriestrajectories6x 10-5

T cells

60μm

0 1 2 3 4 5 6 7x 10-3

0

2

4 T cells

B cellsMonocytes

Distance along device (mm)

20μm

40μm

Distance along device (mm)

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“Electro“Electro--smear” smear” –– changing the frequency of changing the frequency of the applied field along device to maximise the the applied field along device to maximise the

trapping efficiency and separationtrapping efficiency and separation

Das et al Dielectrophoretic Segregation of Different Human Cell Types Das et al Dielectrophoretic Segregation of Different Human Cell Types on Microscope Slides Anal. Chem., on Microscope Slides Anal. Chem., 77 77 20052005

Hyperlayer DEP FFFHyperlayer DEP FFFParticles separated by a balance of forces.Particles separated by a balance of forces.Negative DEP force acts upwards ON ALL particlesNegative DEP force acts upwards ON ALL particles

FDEP

FFDEP= Fg

Negative DEP force acts upwards ON ALL particles Negative DEP force acts upwards ON ALL particles --balanced against a downwards acting balanced against a downwards acting gravitationalgravitational force.force.

Fg

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Hyperlayer DEP FFFHyperlayer DEP FFFy

TimeFluid flow

x

Equilibrium position:Equilibrium position:Position at which the (negative) buoyancy force exactly balances the Position at which the (negative) buoyancy force exactly balances the repulsive (negative) DEP force. repulsive (negative) DEP force.

Ignoring hydrodynamic effectsIgnoring hydrodynamic effects

34 ( ) 0aπ ρ ρ + =g F( ) 03 p m DEPaπ ρ ρ− + =g F

where ρp and ρm is the density of the particle and suspending medium respectively.

32 ( )

3

32 y doVe

dπ

π−∇ =EFor interdigitated array we know

2

3

24 Re[ ]ln o m CMV fdyd gε

π π ρ⎡ ⎤

= −⎢ ⎥Δ⎣ ⎦Combining these equations, the equilibrium height is:

d the electrode gap and width

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Example of batch Example of batch separation of blood cellsseparation of blood cells

Wang Wang et alet al. Separation by dielectrophoretic field. Separation by dielectrophoretic field--flowflow--fractionation. Anal. Chem. (2002)fractionation. Anal. Chem. (2002)

QuadrupoleQuadrupole electrodeelectrode

VoldmanVoldman J, J, BraffBraff RA, Toner M, Gray ML, Schmidt MA. 2001. Holding forces RA, Toner M, Gray ML, Schmidt MA. 2001. Holding forces of singleof single--particle dielectrophoretic traps. particle dielectrophoretic traps. BiophysBiophys. J. 80:531. J. 80:531––4141

Cell pushed upwards by nDEP. Gravity pulls cell down into stable position (as for DEP FFF)

12

Opposed Opposed octopoleoctopole electrodeelectrode

Reichle C, Muller T, Schnelle T, Fuhr G. 1999. Electro-rotation in octopole micro cages. J. Phys. D Appl. Phys. 32:2128–35

Cell held by opposing nDEP forces forming a cage.

Ring geometries for multiple traps Ring geometries for multiple traps –– pDEPpDEP point and ringpoint and ring

pDEP attracts particles (in low conductivity buffer) to theconductivity buffer) to the point.

Albrecht et al. 2005. Photo- and electro-patterning of hydrogel-encapsulated living cell arrays. Lab Chip 5

13

nDEPnDEP Ring Traps Ring Traps

PhysiologicalPhysiological mediamediaPhysiological Physiological mediamediaParticles Particles immobilised at immobilised at

trap trap centre centre –– closed trapclosed trapIndividually addressable Individually addressable

TFTTFT, matrix addressing, matrix addressing

Cell trapping and transportingCell trapping and transporting

Addressable electrodes to form rollingAddressable electrodes to form rollingAddressable electrodes to form rolling Addressable electrodes to form rolling cages (cages (pDEPpDEP or or nDEPnDEP))

DEP to deflect particles that flow down a DEP to deflect particles that flow down a micromicro--channelchannelmicromicro channelchannel

14

Transporting cells by switching electrodes Transporting cells by switching electrodes --grid system using grid system using pDEPpDEP

Suehiro J, Pethig R. 1998. The dielectrophoretic movement and positioning of biological cell using a three-dimensional grid electrode system. J. Phys. D Appl. Phys. 31:3298–305

Steering and trapping cells in Steering and trapping cells in electric field cages by nDEP electric field cages by nDEP

SiBiosystems, Bologna

Manaresi N, Romani A, Medoro G, Altomare L, Leonardi A, et al. 2003. A CMOS chip for individual cell manipulation and detection. IEEE J. Solid-State Circuits 38:2297–305

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Changing the potentials on the electrodes sequentially transports cells through a rolling nDEP cage

nDEP is much better than pDEP, because:

• Joule heating is highest close to the electrodes (σE2); cells are trapped away from the electrodes, so the thermal stress is low.

• Cells experience lower field strength (away from electrode edges).(away from electrode edges).

• Cells are trapped by nDEP using high frequencies. The field goes through the cells, the potential dropped across the membrane is low; less electrical stress.

Combining DEP with Combining DEP with hydrodynamic flow for single hydrodynamic flow for single

cell manipulationcell manipulation

First paper describing this concept in detail:First paper describing this concept in detail:

Fiedler S, Shirley SG, Fiedler S, Shirley SG, SchnelleSchnelle T, T, FuhrFuhr G.G.Dielectrophoretic sorting of particles and cells in a Dielectrophoretic sorting of particles and cells in a microsystemmicrosystem. . Anal. Chem. 70:1909Anal. Chem. 70:1909––15 (15 (1998)1998)yy (( ))

16

DEP Focusing in a flow 0V

5V

10V

500μm

Flow

15V

20V

80μm

Electrodes20μm

6μm beads f = 5MHzVelocity 10mms-1

80μm

40μm

Glass Insulator Holmes, Morgan and GrennBiosensors and Bioelectronics 2005

Combining hydrodynamic and DEP forces Combining hydrodynamic and DEP forces sorting at sorting at a junctiona junction

No electric field

17

High speed sorting of 6μm beads

20V 10MHz

∼ Particles in

Gating Electrodes

Focusing Electrodes

Summary:Summary:DEP DEP movement and separation depends movement and separation depends on: on:

Size (volume)Size (volume)Dielectric properties (frequencyDielectric properties (frequency--dependent):dependent):Dielectric properties (frequencyDielectric properties (frequency dependent):dependent):

Cell membrane capacitance, surface charge Cell membrane capacitance, surface charge densitydensityParticle mass densityParticle mass density

andandSuspending medium (conductivity, viscosity, Suspending medium (conductivity, viscosity, pe mitti it )pe mitti it )permittivity)permittivity)However, DEP force on the particle cannot be However, DEP force on the particle cannot be decoupled from the intrinsic particle propertiesdecoupled from the intrinsic particle properties

Therefore, active sorting methods are being developed

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Reference material Reference material DielectrophoresisDielectrophoresis, H.A. Pohl, Cambridge University Press, Cambridge, UK, , H.A. Pohl, Cambridge University Press, Cambridge, UK, 19781978

ElectromechanicsElectromechanics of Particles, T.B. Jones, Cambridge University Press, New of Particles, T.B. Jones, Cambridge University Press, New York, 1995York, 1995

AC AC ElectrokineticsElectrokinetics: Colloids and : Colloids and NanoparticlesNanoparticles, H. Morgan and N. G. Green , H. Morgan and N. G. Green Research Studies Press Ltd, Research Studies Press Ltd, BaldockBaldock, Hertfordshire, UK, 2003, Hertfordshire, UK, 2003

AC AC ElectrokineticsElectrokinetics: a survey of sub: a survey of sub--micrometre particle dynamics, N.G. micrometre particle dynamics, N.G. Green, A. Ramos and H. Morgan, J. Phys D: Green, A. Ramos and H. Morgan, J. Phys D: ApplAppl Phys, Vol.33, p632Phys, Vol.33, p632--641, 641, 20002000VoldmanVoldman J. Electrical Forces For J. Electrical Forces For MicroscaleMicroscale Cell Manipulation Cell Manipulation AnnuAnnu. Rev. . Rev. Biomed. Eng. 2006. 8:425Biomed. Eng. 2006. 8:425––54 54