applications of microfluidics - jacobs university bremen summer... · 6 - microfluidics &...

Applications of Microfluidics

C. Fütterer, Institut Curie & Fluigent SA, Paris

2 - Microfluidics & Magnetic Beads - C. Fütterer - 23/09/2006

ProgramProgram

•Introducing new technologies,•Electrophoresis,

•Magnetic Particles in Crossed Fields•(Microfluidics and Cells)

•(Microfluidics and Tissues).

Fundamental Applied

Biology/Medicine

Physics/Chemistry

Why Miniaturisation & Integration


Size of Size of Biological ObjectsBiological Objects

nlpl mlμl

?


MicroMicro--FluidicsFluidics: : Defined Flow Defined Flow ProfileProfile

Laminar profile


Many Advantages Many Advantages of Miniaturisation of Miniaturisation

• Size: nanoliters, single cell and molecule manipulations,• Superior flow control (laminar flow),• Speed: diffusion: 1mm -> 15 min, 10μm -> 100ms),• Integration & parallelisation,• Protection against contamination and evaporation,• Kinetics easy to study.


MMicroicroFFluidicluidic CControl ontrol SSystem ystem StartStart--up up ““FluigentFluigent”” (www.(www.fluigentfluigent.com).com)

Industrial prototype

Pressure output

20cm


Very Fast Response in Very Fast Response in MicrochannelMicrochannel

single T4 DNAmolecule

playing with sliding knob

Electrophoresis of Long DNAs N. Minc, K. Dorfman


SeparationSeparation--MechanismsMechanisms of Gelof Gel--ElectrophoresisElectrophoresis::Steric interaction with gel-structure.

Free volume „Reptation“„ Sieve“ No orientation Orientation Ogston, Giddings de Gennes, Zimm, Slater, Noolandi

-

+

Poresizes > 1μm are required for DNA > 1μm !l h l !

μ/μ0∝ exp(−Rg /ξ) ( ) ( )[ ]μμ

ε0

2 2123 2 5= +−N


ArtificialArtificial Matrix: Matrix: SelfSelf--Organisation Organisation of Magneticof MagneticMacroMacro--GelGel

Cooperation with Jérôme Bibette, Pascal Mayer (Bordeaux)

Magnetic microbeadsDiametre = 0.2-0.7 mm, embedded Fe2O3 Particles

H0

r

With homogeneous magnetic field : Dipoles are induced -> Dipole–Dipole interaction:

Magnetic beadcolumns in micro-channel


Need of MicroNeed of Micro--ChannelsChannels

100 μm

Double „T“ channel

42mm

100μm

Soft Plastics Microfluidic-Chips: (PDMS, Whitesides et al.)

Observation

DNA

-

+


Formation of Magnetic Particle MatricesFormation of Magnetic Particle Matrices

Separation: interactionwith obstacles


Observation by Fluorescence Observation by Fluorescence MicroscopyMicroscopy

Channel

Microscope objective

PDMS

Excitation light

PCIntensifiedCCD camera

Emission filter

Dichroicmirror

FluorescentDNAMicrofluidic cell

In magnetic coilOn inverted microscope


0 sec2 sec4 sec6 sec10 sec14 sec

Realisation Realisation ofof DNADNA--plug andplug and migration atmigration at 10V/cm10V/cmElectrophoresisElectrophoresis ExperimentExperiment

Macro-gel

Automatic flow and electrical field control


SeparationSeparationPrecisionPrecision of of Flow Flow Control System:Control System: ReproducibilityReproducibility

Anal. Chem., Electrophoresis

- Superposition of 3 separate runs, separated by 20 mins between runs- Fluorescence intensity was not renormalized between runs

λ-DNA

D1

D2

R

Traditionally: pulsed field gel-electrophoresisneeds at least 12 hours !!


Separation Separation duedue toto Molecular Interaction Molecular Interaction with Obstacleswith Obstacles

CollisionCollision ofof DNADNA--Molecules withMolecules with Single Single MicroMicro--Bead ColumnBead ColumnT4- DNA (167 kbp, 72 μm), E=10 V/cm


Collision: Collision: Length Dependent SlowingLength Dependent Slowing Down of DNADown of DNAT4- DNA (167 kbp, 72 μm), E=10 V/cm


Collision: Collision: Length Dependent SlowingLength Dependent Slowing Down of DNADown of DNAT4- DNA (167 kbp, 72 μm), E=10 V/cm

t =010 μm

t =0.28 s

t =1 s

t =2 s

t =2.76 s

t =4.2 s

E


T4- DNA (167 kbp, 72 μm), E=10 V/cm

120

100

80

60

40

20

Cen

ter o

f Mas

s Po

sitio

n (

μm)

43210-1-2Time (s) Average trapping-time (τ)

T4 = 2.9 sλ = 0.8 sλ-DNA: 48,5 kbp, 21μm

SeparationSeparation--MechanismMechanism::WhyWhy are obstaclesare obstacles requiredrequired forfor separationseparation??


Lattice Monte Carlo Model (K.Lattice Monte Carlo Model (K. DorfmanDorfman,, InstitutInstitutCurie)Curie)

da

a

ad

c ==Π ρ

1~ −Eτ

Collision Probability

Trapping Time*

Mean Velocity ~ EDispersivity ~ ESeparation Resolution ~ E0

Scaling Predictions


First Time:First Time: Quantitative ComparisonQuantitative Comparison with Lattice with Lattice Monte Carlo Model (K. Monte Carlo Model (K. DorfmanDorfman, , InstitutInstitut Curie)Curie)

0

10

20

30

40

50

60

70

80

90

100

0 5 10 15 20 25 30 35 40Electric Field, E

Mea

n V

eloc

ity ( η

m/s

) λ-DNA

2λ-DNA

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

0 5 10 15 20 25 30 35 40

Electric Field Strength, E (V/cm)

Sepa

ratio

n R

esol

utio

n, R

s

L-2L, 17.6

L-2L, 18.6

1.80% Bead Concentration

0.36% Bead Concentration

ReptationPoor hooking interactions

constant plateau

Anal. Chem., Electrophoresis

Mea

n ve

loci

ty

EElectrical Field E (V/cm)


OptimisationOptimisation: : Best Separation ResolutionBest Separation Resolution

< 115 min1.95T2/T437.9/167

Han & Craighead

1.130 min3.3λ/3λ48.5/145.5

Doyle et. al.

2.2150 sec2.2λ/T448.5/167

Present

Resolution in 150 sec

TimeSeparation Resolution

Size DifferenceStudy

Doyle et al., Science 295, 2237 (2002).J. Han & H.G. Craighead, Science 288, 1026-1029 (2000).

Droplets in Microchannels P. Laval, M Chabert


playing with sliding knob

Very Fast Response in Very Fast Response in MicrochannelsMicrochannels::Emusions Emusions ((Rhodia Rhodia SA, SA, PessacPessac))

water

oil

oil


EmulsionsEmulsions, , MicroMicro--ReactorsReactors

Fluorescent DNA moleculesin droplets

Sphere of d=20μm -> 0.4 nl

Applications:-High throughput small volume PCR-Single cell analysis-Micro and Nano(?)particles


Hydrodynamic InstabilitiesHydrodynamic InstabilitiesPhilippe Laval, CF, Philippe Laval, CF, InstitutInstitut CurieCurie

Magnetic Particles inCrossed Magnetic FieldsCF


Crossed Magnetic FieldsCrossed Magnetic Fields

Permanentmagnetic field

Variablemagnetic field

Chip


Bead Bead Stars, Stars, SheetsSheets

4μm


Magnetic Repulsion Magnetic Repulsion in in Turning FieldsTurning Fields

attractive

repulsive

Publication in prep.


NewNew Magnetic Tweezer withMagnetic Tweezer with λλ--DNADNA

4.5μm

1μm

λ-DNA


NewNew Magnetic TweezerMagnetic Tweezer

Without DNA With DNA



~3μ

~7μ

Microfluidics andCells


Cell SortingCell Sorting

•High throughput screening•Concentration of cell type out of mixture•Cancer (e. g. Leucemia)•Diagnosis of raising metastasis formation

Sequencial Methods - Parallel Method


On On ChipChip Cell SeparationCell Separation(T(T--lymphociteslymphocites))

μCP


CD3


Flow Flow Experiment to Experiment to Determine Binding Determine Binding StrengthStrength


Calculation of Involved ForcesCalculation of Involved Forces

F = 6 π r η v ~ 18 * 5 μm * 9.5x10-4 Pa s * 50 μm/40ms~ 10-10 N = 100 pN ~ Biotin-Avidin

Force of immuno-link can be estimated by using precision flow control.

Enzyme Chip N. Minc, Z. Bilkova*, M. Slovakova** Univ. Pardubice, Czec Republic


Beads

Principle:

Proteins(BApNA =uncolored)

Protein-fragments(pNA = yellow)

MassSpectrometer

ProteomicsProteomics:: On Chip On Chip Protein AnalysisProtein Analysis

Trypsin


Magnetic Microparticle PlugMagnetic Microparticle PlugMagnetic „macrogel“ in microfluidic-channel stabilised with permanent magnetic field

Formation of gel

Withoutmagnetic field:

With magnetic field:

S N


Perpendicular Magnetic FieldPerpendicular Magnetic Field

Field-gradients: forces on beads: ∇B2B


Plug CreationPlug Creation


Efficiency, Efficiency, Residential TimeResidential Time

Residence time : (V-Vbeads)/Q

Flowrate Q


Fast Fast OnOn--ChipChip CatalysisCatalysis

10x higher efficiency !


Jean-Louis Viovy,Zuzana Bilkova,Volker Bormuth,Jean-Hugues Codarbox,Kevin Dorfman,Philippe Laval,Nicolas Minc,Eleni Psichari,Annie Rousselet,Marcela Slovakova,

THANKS TO:THANKS TO:

Thanks to Jacques Prost

applications of microfluidics - jacobs university bremen summer... · 6 - microfluidics &...

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