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Lab on a Chip Integration

© Optolabcard

© Ikerlan

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IntroductionDescription

State of the artObjectivesStrategy

Fabrication and packagingResults overview Exploitation potentialConclusions

Summary

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www.ikerlan.es

Ikerlan is a private research centreThere are 230 researchers working in 12 groups

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• Home: Domestic appliances (Fagor), elevators (Orona), locks (Azbe)• Spare time: Azkoyen coffee machines, vending machines• Travelling: Bilbao, Madrid and Hong-Kong underground (CAF), buses (Irizar)• Working: Fagor CNC and presses, Mecalux and Ulma warehouses• Sports: Orbea cycles, Dikar-Wingroup gyms tools

Introduction

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http://www.cepheid.com/Sites/cepheid/content.cfm?id=165

CEPHEID SOLUTION

•Self Contained Cartridges for Integrated sample preparation - for automated and self-contained testing.

•They use fluorescence method but it is not portable

State of the artThe most remarkable available product

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State of the art

SAMSUNG LTD.:

Jeong-Gun Lee, Kwang Ho Cheong, Nam Huh, Suhyeon Kim, Jeong-Woo Choi and Christopher Ko, Microchip-based one step DNA extraction and real-time PCR in one chamber for rapid pathogen identification, Lab Chip, 2006, 6, 886 - 895.

They report a novel method for efficient DNA extraction and subsequent real-time detection in a single cartridge by combining laser irradiation lysis and magnetic beads.

A remarkable research work

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Dynabeads+Antibody+Bacteria On-chip Lysis and PCR

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Dynabeads+Antibody+Bacteria On-chip Lysis and PCR

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158 bp

CT=19

+ Lysis + qPCRReal sampleconcentration

State of the art

María Agirregabiria et al. Concentration, purification, lysis and real-time PCR on a single SU-8 microchamber for rapid detection of Salmonella spp. In faeces. The 11th International Conference on Miniaturized Systems for Chemistry and Life Sciences, microTAS 2007 Conference, 7-11 October, Paris ( France )

Another remarkable research work

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Objective: to solve a social need

Real food, human, environmental sample

preparation

Integration of Sample preparation and

Detection

Available solutions

Current strategyLab on a Chip

strategy

Microtechnology+

Biology

+

High demandof point of

care devices 1. High throughput fabrication process

2. Few materials involved3. Authentic sample

preparation4. Simple fluidic control5. High sensitivity and

specificity6. Small amount of

measured sample7. Reagents stored within

the Lab on a Chip8. Fluidic microdevices

easy to handle9. Low cost disposable

components10. Regulatory issues

Restrictions

Jesus M.Ruano-Lopez. Fabrication strategies to integrate 3D microfluidic networks with biosensors to manufacture Lab on a Chip devices. Measurement + Control, Vol.40, No. 4, pp. 111-115

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IkerlanIkerlan MicrosMicrosyystem stem Group StrategyGroup Strategy

PProductroduct DevelopmentDevelopment

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Photo of a PCR chamber fabricated

on PyrexM.Agirregabiria et al. Fabrication of SU-8 multilayer

microstructures based on successive CMOS compatible adhesive bonding and releasing steps . Lab on a chip,

Vol. 5, No. 5, pp. 545-552F J Blanco et al. Microfluidic-optical integrated CMOS compatible devices for label-free biochemical sensing,

Journal of Micromechanics and Microengineering, Vol.16, No.5, pp. 1006-1016

Fabrication of Lab on Chips

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Patented: Polymer microfluidic-electrical technology tofabricate flexible devices

© Ikerlan-IK4

Fabrication of flexible Lab on Chips

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Other Polymeric microfluidic chipsHot embossing and Thermal bonding without channel collapsePolymers such as PMMA, PC and COC

M.T.Arroyo, L.J.Fernández, M.Agirregabiria, N.Ibañez, J.Aurrekoetxea and F.J.Blanco. Novel all-polymer microfluidicdevices monolithically integrated within metallic electrodes for SDS-CGE of proteins. Journal of Micromechanics and Microengineering, Vol. 17, No. 7, pp. 1289-1298.

Slide 13

PackagingAfter Kapton releasing, easy electric/fluidic connectionWithout glue/wires, easy device replacement

open/closeScrews

PCB

Top capsule

O-ringsPCR chip

Bottom capsule

External tube

External electriccontact

Spring loadedcontact

Slide 14

Multiple Calibration

3.080.86TCR

23.354.1R (Ω)

StD betweenwafers (%)

StD in thewafer (%)

Average R at 30ºC = 84.36Ω

Average TCR = 1979

∆T = 0.7ºC∆T = 2.53ºC

Slide 15

Sample concentration plus Real Time PCR on a LabonachipCZE and GCE electrophoresis of DNA and proteins within a flex devicePolymeric Microfluidic probesA hybrid integration example of a Lab on a Chip

Ikerlan Microsystem applications

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Sample concentration plus Real Time PCR on a Lab on a Chip

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Experimental methodology

Pyrex

Pyrex

Pyrex

NS

SN

Sample

Pyrex

NS

SN

PCR reagents

Bacteria concentration+ washing

Lysis

PCR mixture loading

qPCR

Magnetic bead

Bacteria

DNA

Slide 18

Dynabeads+Antibody+Bacteria On-chip Lysis and PCR

10.0010.5011.0011.5012.0012.5013.0013.5014.0014.5015.00

0 5 10 15 20 25 30 35

Time [s]

Phot

omul

tiplie

r V

olta

ge [V

]

Dynabeads+Antibody+Bacteria On-chip Lysis and PCR

10.0010.5011.0011.5012.0012.5013.0013.5014.0014.5015.00

0 5 10 15 20 25 30 35

Time [s]

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]

human faecal sample

Swab with real sample

bacteria + beads + antibodies

Salmonella DNA collection Sample concentration Lysis and real-time PCR ON CHIP

Magnet

human faecal sample

Cellsbeads

RNA collection

Lyisisbuffer

Sample concentration

Magnet

RT PCR OFF CHIP

RTPCR ON CHIP IN PROGRESS

PCR Lab on chip results

CT=19

Method and device patented

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CZE and GCE electrophoresis of DNA

and proteins within a flex device

Separation of trypsin inhibitor and gliceraldehyde

2

2,5

3

3,5

4

4,5

0 50 100 150 200Separation time (sec)

Det

ecto

r sig

nal (

mV)

1

2

)

Separación de inhibidor de tripsina y gliceraldehído sin gel, a 343V/cm, a una distancia de 10 mm

6

7

8

9

10

11

12

13

14

15

0 50 100 150 200

Tiempo de separación (segundos)

Seña

l del

det

ecto

r (m

V)

4

6

8

10

12

14

0 20 40 60 80 100 120 140

Tiempo de migración (segundos)

Seña

l del

det

ecto

r (m

V) Visualización del amplificado del genotipo WT de 250 bp, en el dispositivo

miniaturizado

Maria Agirregabiria, Francisco Javier Blanco, Javier Berganzo, AsierFullaondo, Ana María Zubiaga, Kepa Mayora, Jesús Miguel Ruano-López. Sodium dodecyl sulfate-capillary gel electrophoresis of proteins in microchannels made of SU-8 films. Electrophoresis, Vol.27, No. 18, pp. 3627-3634 *In collaboration with The

Basque Country UniversityBiology Dept.

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Amperometric detection of the electrophoresis1. Sample preparation by stacking

2. Integration of detection avoidingcomplex optical detectors

AmperometricSensor*

*In collaboration with Facultad de Química de la Universidad de Oviedo

-3

-1

1

3

5

7

9

11

13

0 15 30 45 60 75 90 105 120 135 150

tiempo (s)

Inte

nsid

ad (n

A)

+500V+750V+1000V+1250V

Vsep (V) ip (nA) tm (s) w1/2 (s)250 4.6 166.5 24500 5.8 76.8 8.7750 5.8 57 5.11000 6.9 46.2 4.51250 6.7 36.3 3.3

Condiciones: Viny = +250 V, tiny = 5 sEd = +0.5 V, Buffer: 50 mM Tris-Bórico pH = 7.0

Analito: Dopamina (DA) – 100 mM

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An Instrument to identify extinct life on Mars

A hybrid integration example of a lab on a

Chip

*In collaboration with INTA-CAB and SENER

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The Life Marker Device should be an instrument capable to identify evidence of extinct life on the exploration of Mars. The approach proposed on this document is based on a well known techniques, protein microrrays, measuring fluorescence.

An Instrument to identify extinct life on Mars

*In collaboration with INTA-CAB and SENER

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20 µm

60 µm 20 µm

100 µm

Integratedelectrodes

Microfluidics

MultipleMicrofluidic channels

Ikerlan Microfluidicprobes

L.J.Fernández, M.Tijero, R.Vilares, J.Berganzo, K.Mayora andF.J.Blanco. SU-8 based microneedle for drug delivery in nanomedicine applications with integrated electrodes. The 11th International Conference on Miniaturized Systems for Chemistry and Life Sciences, microTAS 2007 Conference, 7-11 October, Paris (France).

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Microfluidic probes results

0,0 0,2 0,4 0,6 0,8 1,0 1,20

10

20

30

40

50

60

70

80

90

100Channel dimensions (µm width x µm height)Red 200x25 Blue 150x25 Yellow 125x25Magenta 100x25Green 70x25Grey 50x25

Y=1,75+30,73*X;R=0,99

Y=1,35+41,56*X;R=0,99

Y=1,03+50,23*X;R=0,99

Y=6,6+60,6*X;R=0,98

Y=4,6+72,1*X;R=0,99

Flow

(µl/m

in)

Pressure (bar)

Y=9,0+79,8*X;R=0,98

PC adquisition

system

Flowmeter

DiferentialPressure Sensor

Microfilter (0.2 µm)

Liquidinjectionsystem

Microfluidic chip

Experimental set up

• Microfluidic-electrical flexible/semiflexible polymer

• Integration electrical and chemical functions into tissues.

• Chronic ImplantsHigh Biocompability

Polymer devices can fit to the tissues and deform their shapes as the organs deform

Slide 25

Aguja encapsulada

Dispositivo para experimentación biomédica

Microfluidic probes results

*In collaboration with CNM Barcelona

Patent pending

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Product´s Roadmap of two technologies: Lamination and Embossing

20102006 2007 2008

Lam

inat

ion

Hot

em

boss

ing

PlatformR&D

Embossed Samples

Available Product Pasives

Plataform I+DSU8+Laminates

DeviceqPCR

qPCRCommersialisation

2009

Other devices: CE,µPCR,concentrator

Product´s Roadmap of two technologies: Lamination and Embossing

20102006 2007 2008

Lam

inat

ion

Hot

em

boss

ing

PlatformR&D

Embossed Samples

Available Product Pasives

Plataform I+DSU8+Laminates

Truly Labonchips

LOCCommercialisation

2009

Other devices:

Current situation

Exploitation potential

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Exploitation plan based on Ikerlan Chips

A New Business Model*

• Similar market prices offer a dramatic increase of potential cash flow • Sales price decreasing from 2,5 to 0,7 €/Cm2.• Less dependency on productivity

*In collaboration with the Strategic Innovation Area at Ikerlan

Dry filmsOLED

LOC Labcard

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ConclusionsThe developed fabrication technology proves to be an excellent tool

for rapid prototyping, as well as for mass production of low cost disposable Lab on Chip.

We have developed a set of Lab-on-a-Chips fulfilling the restrictions to be mass produced

A truly Lab on a Chip where bacteria concentration from human faecal sample, lysis and DNA amplification of Salmonella spp. were successfully carried out on a single chip.

The advantages of the miniaturisation in terms of time and cost in comparison with the conventional electrophoresis methods have been proved

It has demonstrated the potential of Ikerlan Probes

A Lab on a Card has been created by integrating a Lab on a chip in a Smartcard

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Acknowledgements

Basque Government and the Provincial Council for its financial support under the ETORTEK program.

Optolabcard european project.

To all the Ikerlan groups involved in our activities and special thanks to all the microsystem team.

OPTOLABCARDwww.optolabcard.com

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Thank you for your attention

It is time for questions

Jesus M. Ruano-López

jmruano@ikerlan.es

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Nerve stimulationPaceMakers

cochlearcatheters

Implants

MicroneedleScalpelProbes

Microsurgery

In Vivo

Point of careCapillary Electrophoresis

DNA deteccion PCRCell studies

Lab-on-a-Chip

ProteomicsGenómicsPharma

Drug screening

Microarrays

In Vitro

BIOMICROTECHNOLOGY

Lab on Chip state of the art

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State of the art

• The 90´s was a decade of incredible microfabrication process developments and transducing mechanism characterisation.

• However, few of these processes were transfered successfully into real applicationsbecause the difficulty of integrating them reliably in a mass-production.

• Due to the maturity of the microtechnology, it is now the moment to prioritise miniaturisation, sample preparation and optical detection

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Planarmicroelectrodearrays (pMEAS)

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http://www.multichannelsystems.com/

http://www.med64.com/products/medprobe.html

http://www.plexoninc.com/

Planar microelectrode arrays (pMEAS)Vital organs and tissues

Bioelectrical phenomena

Electrical activityat cellular level

Long-term monitoring ofthe activity of cell cultures

electronic signal amplification

signal processing software & data logging

to keep cells alive

Commercially available systems

Polymeric and flexible bottom substrates made of SU-8 and PDMS

PDMS

SU8Ti/Au electrodes Flexible MEAS made ofPDMS

+ planar electrodes& SU8 passivation

SU8

SU8Ni electrodes Flexible MEAS made of SU8

+ 3D electrodes& SU8 passivation

Flexible chips with Ni electrodes

Slide 35

Wafers substrate

Dry films

ImmunoPCRRT-PCR

NASBALionprobes

Skin absorption

OLED

Gelification

LOC Labcard Labcard ReaderSyringe

www.smartbiophone.com

LOC

Current Bio-MEMS convergence

Wafer depencencyNo sample prep or limitedBulky equipmentExpensive for dispose

MEMS based onfilms

Molecular biology reactions and biological techniques that simplify the LOC (e.g. gelification)

Ikerlan MST group Bio-MEMS convergence

Band-Aid

Exploitation potential