lecture 2 handout - us
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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.
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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
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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
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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)
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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
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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
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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 (( ))
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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
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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
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