Lab-On-Chip development for diagnostic applications

Transcription

1 Lab-On-Chip development for diagnostic applications Smart Systems Integration Conference 2009 March 10th, 2009 Claude Vauchier 1

2 CEA LETI: biochips activities facilities Microfabrication Biotechnologies Clean room 150 m² Laboratories 1000 m² spotting electrochemistry in-situ syntheses fluorescence detection chemical engineering biology chemistry electrochemistry microfluidics bio packaging Silicon Plastic/Silicon Glass/Silicon 2

3 Lab on chip development: the challenge of integration Microtechnologies & microfluidics are necessary to miniaturize and integrate all the steps of a biological or chemical analysis Sample Sample collection collection (blood, (blood, water, water, air, air, biopsy ) biopsy ) Extraction Extraction concentration concentration purification purification Molecular Molecular recognition recognition Detection Detection Data Data treatment treatment Miniaturisation Batch fabrication, low sample or reagent volume, reproducibility, sensitivity Integration Sequential and/or parallel reactions (biological protocol) Automatisation Automated device point of care 3

4 microfluidics toolbox for lab on chip applications 3 ways for fluid handling architecture: Monophasic microfluidics Droplets in channel Digital microfluidics Technology Microfluidics circuits with etched channel. Specific packaging steps (low T C) Microfluidics circuits (glass or PDMS) Microdroplets are actuated on microelectrodes pathway Microfluidics Fluid actuation Mono-phasic Hydrodynamics force electro-osmotic force Bi-phasic Hydrodynamics biphasic (air/water; oil/water; air/ionic liquid) Electrostatic, SAW Valve or pump architecture Thermopneumatic actuation; passive valves; mechanical; External pump No need dispensing Capillaries, wells Capillaries Integrated reservoirs 4

5 Droplet handling with electrode areas V = 0 V Hydrophobic surface V = 80 V Hydrophilic surface EWOD Principle (ElectroWetting On Dielectric) 0 droplet 0.8µl counter electrode (gold wire ø 20µm) f e electrode areas (800µm x 800µm) 0 0 V 0 0 J. Lee and C. J. Kim, Journal of Microelectromechanical Systems, M. G. Pollack, R. B. Fair, and A. D. Shenderov, Applied Physics Letters, Stroboscopic visualization loop frequency : 10 hz Light pulse frequency 1/0.101~9.9 hz 1 image/ 1ms droplet velocity : 80mm/s (100 loops are needed to get the complete video) Electrowetting induced transportation is extremely reproducible 5

6 Fabrication microtechnology 200mm Silicon fab. Standard microtechnology (IC) 2.3 cm Synchronized actuation of Fluidic handling (all by EWOD ) Definition of an Assembly language Optical detection (2 colors+ccd) Data acquisition Thermal control (peltier) or on chip heating & sensors Bus & reservoirs (In/Out or storage) Addressing of electrodes is multiplexed I.R Camera Wells Counter electrode Interconnections Cover Polycarbonate or glass Counter electrode (ITO) Hydrophobic layer (SiOC) Silicon Oil Driving electrodes Contacts pads Chip Silicon 1 st metallization Al: Dielectric Si 3 N 4 2 nd metallization Al : Dielectric Si 3 N 4 : Spacer : Ordyl Hydrophobic layer SiOC Integration of fluidic protocol in a compact Instrumentation 6

7 Example of Fluidic validation Validation, optimization of basic operation : Chip Filling Dispensing (CV<3%) Mixing (few sec) Storage 7

8 Applications based on digital microfluidics platform Environment monitoring on-site pathogen detection for water or air sample (LETI) Electronic paper (Philips) Digital Microfluidics EWOD In-vitro diagnostic (LETI) Liquid microlens (Varioptics, Philips, ST) Chemistry organic syntheses with Ionic Liquid solvent (LETI) Life-science Integrated RT-PCR equipment (LETI) 8

9 Applications to biological analysis Sampling Sample preparation analysis Air 30l/min. to 10 ml Concentration DNA amplification Liquid 1 l to 10ml Blood ml - µl Purification Detection 10 ml to 2 µl EWOD Platform 9

10 real time PCR integration on-chip Adapted to parallel analysis of low volume sample Amplification: specific fluorescent detection in each droplet Time: 35 mn Amplification X sample + mix PCR - PCR reaction validated in droplet - fluidic automatic control Software control - dried primers and probes 10

11 Basic validation : real time PCR + + Cycle 2 Cycle fluorescence (u.a.) fluorescence (u.a.) 1,8 1,7 1,6 1,5 1,4 1,3 1,2 1,1 1 0,9 1, ,8 cycles (n ) 1,7 1,6 1,5 1,4 1,3 1,2 1,1 1 0, ,2 cycles (n ) Tube (ml) Chip (64nl) E. Coli -0, Pathogens are detected in 64 nl droplet volume with same Ct as 10µl tube A few number of copies per droplet are detected In progress : packaging, embedded reagent, thermal cycling fluorescence (a.u.) 1 0,8 0,6 0,4 0,2 0 Chip: 500 copies/droplet Tube: copies/µl Chip: 5 copies/droplet Tube: 100 copies/µl cycles n 11

12 Example 1: Validation of qrt-pcr for gene expression On chip: 64nL per droplet - mrna from 65 human cells Droplets during PCR Cycle 1 Cycle 40 In tube: 10µL mrna from 10,000 human cells Normalized Fluorescence Intensity (Chip) 1,27 1,22 1,17 1,12 1,07 1,02 Droplet 1 Droplet 2 Droplet 3 Droplet 4 Tube 1,8 1,7 1,6 1,5 1,4 1,3 1,2 1,1 1 Normalized Fluorescence Intensity (Tube) 0, Cycle 0,9 12

13 Example 2 : fully integrated protocol Field of application : Medical diagnostic or research Small apparatus for laboratory, Single use Drop of blood (1µl) Objective : concentration, purification and DNA analysis from a droplet of blood (<µl) for genotyping study Beads (Detection of a mutation) concentration Biological protocol analysis with a commercial kit Chemical lyses of cells DNA capture on magnetic beads DNA amplification by PCR Input : Droplet of blood Output : Data (ex CT) Permanent magnet Sample prep Assay Supernatant Supernatant extraction with electrowetting Detection DNA (few nl) PCR amplification (60nl) Data Blood handling Pellet extraction Re-suspension 13

14 Fully integrated protocol (2) Preparative area Sample preparation steps (washing, elution) Sample + reagents Pure DNA droplet Analytic area (PCR) Génotypage CFTR sur Puce EWOD Validation on blood droplet for genotyping application fluorescence(u.a.) 1,2 1,15 extraction et PCR VIC puce 1,1 1,05 extraction et PCR FAM puce 1 0, nombre cycles (n ) 14

15 Genotyping in 130nl droplets (3) Diagnosis of cystic fibroses (Mucoviscidosis). A.Rival PhD (X.Gidrol & D. Jary) A. C. Reymann fluorescence (u.a.) 1,2 1,15 1,1 1,05 1 0,95 CFTR Genotyping extraction et & PCR VIC puce chip extraction et & PCR FAM puce tube extraction & PCR VIC VIC tube extraction & PCR FAM tube Cycles number 1,3 1,1 0,9 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 The whole protocol from sample prep to assay was performed on EWOD chip Same result as standard tube, with time reduction and coast reduction Next steps : packaging optimization (Cover, embedded reagents.) automation (magnet handling) 15

16 Others applications Enzymatic reactions studies Cells sorting F 5 Q 3 + Fpg Fapy N-glycosylase Solution (of E. coli) with Enzyme Q F [Fpg]=1µM Reaction product (nm) Solution with 200 substrate µM Tube 0.25µM Chip 0.5µM Tube 0.5µMChip 0.75µM Tube 0.75µM Chip with enzyme time (sec) without enzyme 16

17 Summary EWOD: a new microfluidics approach for Lab-on-chip development Application of digital microfluidics platform in the field of biological analysis represents a clear opportunity to develop new tools for: in-vitro diagnostics (blood analysis) one shot detection life science (RT PCR module) environment monitoring (pathogen detection for air and water detection) Bioprotocol in the microliter range (few nl to few µl) Middle throughput (less than one droplet/seconds) few analyses/day with complex protocol autonomous concept adapted to on-site detection Sampling and sample preparation from raw sample can be integrated Nice opportunity for industrial partner to develop specific application with a strong IP 17

18 THANK YOU FOR YOUR ATTENTION! Plan to join us on June 22 nd -23 rd, 2009 at Minatec for the 11 th Leti Annual Review For more information : 18