Microfluidique digitale

Dimension: px

Commencer à balayer dès la page:

Download "Microfluidique digitale"

Bernard Sénéchal
il y a 8 ans
Total affichages :

1 2008 Microfluidique digitale Yves Fouillet CEA / LETI-Minatec 1

2 Plan Introduction Fonction fluidiques Electromouillage Principe physique Les filières technologiques Architectures et exemples d applications Quelques études récentes Conclusions/Perspectives/discussions 2

3 Microfluidique digitale Avatar = volumes fluides isolés Une plateforme Une règle du jeux Des opérations élémentaires Architecture Des entrées/sorties Des zones de stockages Des poubelles Une interface Un score 3

4 Les microsystèmes pour la biologie Sampling Sampling Sample Sample preparation preparation Detection Detection L.O.C Miniaturisation Intégration Automatisation Portabilité & autonomie Réduction des temps Réduction des coûts Meilleur sensibilité Haut débit 4

C Miniaturisation Intégration Automatisation Portabilité &

5 Pompes et valves externes Vanne de sélection Pompe V>10µl EDI Tampon1 Tampon2 Lavage 6 V1 1 2 P1 Passive Chip Réservoirs Sample 5 4 V>µl 3 V2 Chip Loop Poubelle Pompe Vanne d injection 5

6 Carte microfluidique 1 Pathogenic Bacteria Detection Cells hybridation Cells Concentration/ Lyse (1ml+10µl+20µl) purification PCR (20µl) Motorola, Liu et col Mix Hybridation buffer DNA Microarray Detection Valves en paraffine (5) Closed-open et Open-closed-Open 6

7 Carte microfluidique 2 BioMerieux - Leti joint team, SPS, SNPE Biomed Microdevices (2008) 10:35 45 Marquage (biotine) /clivage Hybridation Lavage Stain (streptavidine Lavage + HRP) R. Chimie luminescence Detection with Active Pixel Sensor (APS) photodetector & chemiluminescence valves Pyrotechnique (16) Open closed Open Vannes 3 voies 7

8 Technologie souple (PDMS) Quake Groupe, Science 16, 2005: Vol Multistep synthesis of radiolabeled imaging probe F18 concentration + transfert de phase H 2 0 Acétone nitrile Radioflurination (reaction Glucose) Transfert de phase (Acétone nitrile H 2 0) Déprotection valves hydraulique (~40) 8

transfert de phase H 2 0 Acétone nitrile Radioflurination (reaction

9 9

10 Analyse fonctionnelle simplifiée Fonctions fluidiques Outils Déplacement de fluide Aiguillage Dosage Mélange Stockage (réactifs pré-stockés, incubation) Pompes, géné. de pression valves boucle+valves (+filtres) Boucle+pompe, piezo chambres Concentration/purif./changement de buffer Lyse, élution, PCR Détection Compatibles avec billes, colonnes Thermique Puce ADN, lecteur fluo. Autres 10

de pression valves boucle+valves (+filtres) Boucle+pompe, piezo chambres Concentration/purif.

11 Microfluidique en gouttes Segmented flow Raindance technology Manipulation individuel de goutte S.A.W. Lévitation magnétique Actuation électrostatique 11

12 V Force Electrostatique l ε 1 d b=unité W = ε 1l 2d V 2 Degré de liberté : ε V ε 2 ε1 ε r ( ε F DEP 2d 2 ε 1 V ε 2 > ε 1 ) V 2 liquide dielectrique L-DEP 12

13 Electrostatic actuation of droplet First experiment (1998) : Collaboration with J.P. Le Pesant from Osmooze US 4,636,785 (1983) Localized actuation of droplets Individual handling No movable part, No external pumps 2D motion, Splitting, coalescence 0 +V Dielectric Liquid 13

Le Pesant from Osmooze US 4,636,785 (1983) Localized actuation of

14 V Force Electrostatique l ε 1 d b=unité W = ε 1l 2d V 2 ε Degré de liberté : Degré de liberté : l ε 2 ε1 ε r ( ε F DEP 2d 2 ε 1 L-DEP V ε 2 > ε 1 ) V liquide dielectrique 2 l 2 1V CV F e ε = 2d 2 V Electromouillage liquide Conducteur 2 14

15 γ ElectroWetting On Dielectric (EWOD) θ (0) V = 0 γ sg γ γ sl γ γ θ + sg = cos (0) γ sl f e θ (V ) V 0 γ sg γ sl f e + γ γ cosθ + γ sg = (0) Eq. Lippmann Young B. Berge 1993 cosθ = cosθ + ( V ) ( 0) L apparente mouillabilité est contrôlée par un jeu de potentiel électrique C V 2γ EWOD est un phénomène d origine électromécanique 15 2

Berge 1993 cosθ = cosθ + ( V ) ( 0) L apparente mouillabilité est contrôlée par un

16 Courbe d électromouillage Mesure d angles Courbe cosθ = cosθ + ( V ) ( 0) C V 2γ 2 16

17 Droplet displacement 0 EWOD displacement Counter electrode Gold wire(30µm) 800µm*800µm droplet ( 0.8 µl) Si3N4 : 0.4µm + teflon like : 1µm 0 0 V 0 0 Bavière R. et al. Microfluidics and nonofluidics 17

18 Technologie Un plaque (ouvert) Deux plaques (fermé) Filière : Glass, silicium, PCB, Polymère Leti Advanced Liquid Logic Inc Univ. Toronto Microfabrication des electrodes par les techniques les plus standards Process spécifique : Couche diélectrique et hydrophobique + packaging 18

Toronto Microfabrication des electrodes par les techniques les plus

19 SiOC instead of Teflon-AF SiOC is a standard material used as a dielectric layer in CMOS technology Deposited by PECVD (Plasma Enhanced Chemical Vapor Deposition) Conformal growth techniques SiOC compatible with fluorocarbon solvent (ex FC70) SiOC Material properties is equivalent to Teflon Material Teflon SiOC SiN Dielectric coefficient ε teflon ε SiOC ε SiN = 2 = 2.75 = 6.3 Dielectric strength D teflon = V / m 8 D SiOC = 1.6 to V / m D SiN = 10 9 V / m Thikness control on 200mm wafer Voltage reduction (Sin : 600nm) SiOC Thickness : 1µm working voltage 50V 200nm working voltage 20V Contact angle hysteresis 5 to 10 6 to 10 Standard MEMS Foundery : Full 200 mm wafers equipment & clean-room requirements 19

2 = 2.75 = 6.3 Dielectric strength D teflon = 1.510 8 V / m 8 D SiOC = 1.6 to 4.

20 EWOD Technology on silicon Standard microtechnology (200mm) IC fab are already available Benefit of the know-how and techniques of microelectronics for dielectric and hydrophobic layer SIN (PECVD) as dielectric layer and SiOC for hydrophobic layer Clean-room environment (minimum defects) Limitation of breakdown and electrolysis and voltage reduction Good thermal conductivity of Silicon bulk CMOS technology few dollars /cm2 chip (~cm), droplet from pl, nl to µl Hydrophobe (SiOC) Isolant (SiN) Métal (Au) 20

Clean-room environment (minimum defects) Limitation of breakdown and electrolysis and voltage reduction Good thermal

21 Droplet dispensing Principle : Optimization - Chip filling - Reproducibility - Bubble free spreading pinching Results EWOD (64nl) intra-experiment : C.V. 0.5 % inter-experiment : C.V. 3.3 % Research Eppendorf 0.25µl : C.V 6% 21

22 Droplets mixing Coalescence & Displacement Y. Fouillet, et al. Microfluidics and Nanofluidics, vol. 4, pp , Results Mixing is complete in a few seconds Only 15 moves 22

23 Chip architecture 1 Area of electrodes 2 Area of elementary blocks Advanced Liquid Logic Inc Lab Chip, 2008, 8,

24 Input/Output Chip architecture Bus Storage Processor : Generic architecture Standardization of elementary design : Dispensing Mixing Storage (reagents, sample, reactions) Integration of several validated elementary functions in a single chip Simple chip manufacturing : Size optimization Switch number optimization Nb of reservoirs : Y. Fouillet, et al. Microfluidics and Nanofluidics, vol. 4, pp ,

25 Example of Fluidic validation Dispensing (CV<3%) Storage Mixing (few sec) 25

26 Ex : Real time PCR Biological assays : PCR PCR mix : Primers DNTP Enzymes DNA template Thermo-cycling Cycle 1, cycle2 Cycle3.. Detection : FRET* homogeneous phase detection Enzyme Enzyme F Q Polymerase DNA Extension F Q hυ PCR Probe degradation Enzyme Droplet fluorescence increase if genes are amplified * Fluorescence resonance energy transfer Led 1 : 492 (Fam) Led 2 : 538 (Vic) Filter 517 to 554 Optical detection 1,2 1 E. Coli Puce : 500 copies/goutte Tube (ml) Chip (64nl) fluo normalisée 0,8 0,6 0,4 0,2 Tube : copies/µl Puce : 5 copies/goutte Tube : 100 copies/µl ,2 nbre de cycles 26

27 EWOD Microfluidic platform 27

28 Magnetic beads Supematant Adding particles with specific coating Mixing Capture Beads concentration Pellet Splitting Beads(pellet) and droplet Concentration Washing Elution 28

29 Magnetic beads & EWOD Ex: Removing supernatant from magnetic beads pellets Permanent magnet 29

30 Example : fully integrated protocol Injection : Blood (250nl) + Lysis buffer + Beads Lyses DNA capture Wash X3 Elution qpcr 130nl Field of application : Diagnostic or fundamental research Small apparatus for laboratory, Single use Aim : To purify and to analyze DNA from a droplet of blood (<µl) for genotyping study (Detection of a mutation) Protocol with a commercial kit Chemical lysis of cells DNA capture on magnetic beads DNA amplification by PCR Input : Droplet of blood tube Chip Time of sample prep 40 min 40 min Time of essay 65 min 40 min Price of sample prep reagents 1 euro 0,025 euro Price of PCR reagents 1 euro 0,025 euro fluorescence (u.a.) 1,2 1,15 1,1 1,05 1 0,95 SNip2CHIP (Europeen project) EuroNanoForum2009 on June 15, Prague CFTR Genotyping extraction et PCR VIC puce extraction et PCR FAM puce extraction & PCR VIC tube extraction & PCR FAM tube Cycles number -The whole protocol from sample prep to essay was performed on EWOD chip -Same result as standard tube, with time reduction and coast reduction 30 1,3 1,1 0,9

31 Single cells gene expression Droplet handling with Cells qrt-pcr on chip mrna equivalent to 2 single cells Towards single cells gene expression preparation and analysis on ewod lab on chip. Rival A., Delattre C., Fouillet Y, Gidrol Vinet F., Jary D Nanobio Europe, Grenoble,

32 Approche Locale AC-EWOD Thèse : Malk Rachid q s θ Iso potentiels 0 π/4 π/2 Toute est concentré à la ligne triple! θ 32

33 AC-EWOD Malk,R. Davoust L. Fouillet.Y. Eurosensor 2009 Vue de profile Vue de dessus Visualisation avec des particules fluorescentes Visualisation stroboscopique 100Hz 3KhZ Oscillations Brassage 33

34 . Bien plus qu un packman. S.K. Cho University of Pittsburgh Beads,cells Water Bubble Piezo 34

Documents pareils

841 000 Capital Head Offices : 30 chemin des Mouilles 69290 GREZIEU LA VARENNE - FRANCE SIRET : 497 802 165 000 17 TVA intracee : FR 3649780216500017

1 CATALOGUE MATERIALS ET CONSUMABLES May 2015 V6 841 000 Capital Head Offices : 30 chemin des Mouilles 69290 GREZIEU LA VARENNE - FRANCE SIRET : 497 802 165 000 17 TVA intracee : FR 3649780216500017 Tel