Lab-On-Chip based proteing profiling for CANcer

DIAgnosis Priority 2.5.2. Micro/nano based sub-system

Funded by EC contract FP6-034202

Nano2life annual meeting,

Champéry (Switzerland)

9-11 January 2008

Pierre GRANGEAT (1) , Blanca JORDAN RODRIGUEZ (2),

and all the LOCCANDIA partners(1) CEA-LETI, MINATEC, pierre.grangeat@cea.fr

(2) ATOS ORIGIN, blanca.jordan@atosorigin.com

DTBS/STD/08E-02

Healthcare Objective: cancer diagnosis

• Detecting proteins in human blood plasma

• Early detection of a protein panel applied to pancreatic cancer

• High sensitivity window (~1-1000 picomolar concentration)

Funded by EC contract FP6-034202

LOCCANDIA2

Technical objectives

• To be able to determine low concentration cancer markers in the window of classic cancer marker– Targeted concentration range: 1 to 1000 pmole/L or 1 to

1000 ng/mL• To quantify a multiparametric set of markers to improve the

measurement specificity• To use chromatography nano-columns to improve the

sensitivity and the throughput• To use electrospray ionisation for a soft on-line ionisation• To use a mass spectrometry characterisation:

– to get a specific, sensitive and semi-quantitative recognition

– to distinguish isoforms when the sensitivity is appropriate

Funded by EC contract FP6-034202

LOCCANDIA3

Micro-nano technology objective: lab-on-chip

• Point of care :

Full system from blood plasma sample to diagnostic information

• Lab-on-Chip

Miniaturized integrated components to increase sensitivity (nano-LC, nano-ESI) and throughput

Funded by EC contract FP6-034202

LOCCANDIA4

Silicium technology for optimized microfluidics

Funded by EC contract FP6-034202

≈ 1mm

800µm

1,5 µm

MS Nano-LC separation

Techno 3 wafersOutlet 5 X50 µm2

5 LOCCANDIA

LOCCANDIA analysis chain

Blood plasma sample

Blood sample

preparation

Lab-on-Chip & mass

spectrometryInformation technology

Selected protein mixture

Peptides spectrogram

Diagnostic information

BIO

Material flow Information flow

NANO INFO

Patient Doctor

Funded by EC contract FP6-034202

LOCCANDIA6

Project Goals (I): Blood sample preparation

– Preparation of proteins to make synthetic proteins mixture using standards protocol.

– Production of the specific antibodies.

– Design of the affinity columns and the quality control of the proteins using MALDI-MS.

Funded by EC contract FP6-034202

LOCCANDIA7

Blood plasma sample

Blood sample

preparation Lab-on-Chip & mass

spectrometry

Selected protein mixture

BIO

Material flow

Patient

Project Goals (II): Lab-on-chip and mass spectrometry

•The objectives are:– to provide new batches of our current microsystems– to improve the chromatography-electrospray microsystem technology

Funded by EC contract FP6-034202

LOCCANDIA8

Lab-on-Chip & mass

spectrometrySelected protein mixture

Peptides spectrogram

Material flow

NANO

•Two interconnected modules will be designed:– a protein digestion module– a liquid chromatography-electrospray ionisation module

Project Goals (III): Information technology

To build an Integrated Clinico-Proteomics Environment (ICPE):

• a Proteomic Information Management System (PIS) for sample information management

• a Clinical Information System (CIS) for patient information management to allow clinical evaluation

• an Information and data mediation infrastructure including preprocessing, reconstruction, visualization, protein/peptide identification and data analysis modules

Funded by EC contract FP6-034202

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Information technology

Peptides spectrogram

Diagnostic information

Information flow

INFO

Doctor

LOCCANDIA Information Management System Moke-Up

Funded by EC contract FP6-034202

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Example of profile reconstruction results on a non-optimized nano-LC microsystem

• 10 mixtures of Cytochrom C and RnaseB at 4 concentrations :

• 2 mixtures at 0.0 µmol/l• 3 mixtures at 0.2 µmol/l• 2 mixtures at 0.8 µmol/l • 3 mixtures at 0.4 µmol/l

• Estimated concentration for prediction mixtures (0.4 µmol/l):• PLS (Partial Least Square): 0.344 µmol/l (13,9 % of

error)• N-PLS (Multiway PLS): 0.357 µmol/l (10,7 % of error)

Funded by EC contract FP6-034202

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For calibration

For prediction

Results of profile reconstruction using chemometrics approaches

Funded by EC contract FP6-034202

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450 500 550 600 650 7000

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

200 400 600 800 1000 1200 1400 16000

0.05

0.1

0.15

0.2

XIC (Base Peak Intensity) Projection on m/z

Retention time (s) Mass / Charge (Da)

Major peptide of

CytoC (478Da, 1560

s)

200 400 600 800 1000 1200 1400 1600

1

1.5

2

2.5

3

3.5

4

x 105 TIC (Total Ion Current) Projection on m/z

Retention time (s) Retention time (s)450 500 550 600 650 700

0.5

1

1.5

2

2.5

3

3.5

x 106

Major peptide of

CytoC (478Da, 1560

s)

Mass / Charge (Da)

Exp

eri

men

tal m

ixtu

re1st

N-P

LS

extr

acte

d

facto

r

Project Goals (IV): validation

Funded by EC contract FP6-034202

LOCCANDIA13

• validation on a cohort of patients combining patients with different state of pancreatic cancer, patient with confounding disease and healthy volunteers

• comparison with state of the art techniques of in vitro diagnosis and MALDI-mass spectrometry measurement

• targeted performances:

– to detect low abundant proteins simultaneously within complex mixtures

– to operate the full analysis chain in less than 12 hours

– to improve the sensitivity and specificity using a protein panel

Main research outcomes

Funded by EC contract FP6-034202

LOCCANDIA14

• an optimized chromatographic-electrospray lab-on-chip dedicated to protein profiling for cancer diagnosis

• an Integrated Clinico-Proteomics Environment supporting the integrated device and the diagnosis

• a proof-of-concept of this innovative lab-on-chip technology and the associated analysis chain for cancer diagnosis

List of participants• Atos Origin sae, Spain – ATOS

• Commissariat à l’Energie Atomique, France – CEA-LETI

• DIGILAB BIOVISION GmbH, Germany – DBVN

• Foundation for Research and Technology, Greece – FORTH

• University of Münster, Germany – WWU

• Swiss Institute of Bioinformatics, Switzerland – SIB

• Geneva Bioinformatics, Switzerland - GeneBIO

Funded by EC contract FP6-034202

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ATOS

FORTH

Uni Muenster, Biovision

CEA-LETI

SIB, Gene Bio

WWU and DBVN

CEA-Leti

SIB, GeneBIO

ATOS ORIGIN

FORTH

Research Teams• ATOS: Blanca Jordán, José F. Esteban, Manuel Perez

• CEA-LETI: Pierre Grangeat, Laurent Gerfault, Caroline Paulus, Grégory Strubel, Florence Ricoul, Emeline Mery, Frédérique Mittler, Caroline Fontelaye, Françoise Vinet, Nicolas Sarrut, Olivier Constantin

• DBVN: Harald Tammen, Karl Schorn

• FORTH: Dimitris Kafetzopoulus, Manolis Tsiknakis, Sophie Kaforou, Hara Roumpaki, George Potamias, Haris Kondylakis, Manolis Kalaitz, Vangelis Kritsotakis

• WWU: Jürgen Schnekeburger, Verena Schick, Jasna Peter-Katalinic, Laura Bindila, Daniela Hahn, Rainer Ossig

• SIB: Frédérique Lisacek

• GeneBIO:Pierre-Alain Binz

Funded by EC contract FP6-034202

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Contact details

http://www.loccandia.eu

Blanca Jordán RodríguezProject ManagerAtos Research and InnovationAtos Origin Spain

blanca.jordan@atosorigin.comPhone: +34 91 214 93 22Fax: +34 91 754 3252 (Att. Blanca Jordán)

Pierre GrangeatScientific DirectorCEA/Grenoble, FranceLETI-DTBS

pierre.grangeat@cea.frPhone: +33 4 38 78 43 73Fax: +33 4 38 78 54 56 (Att. Pierre Grangeat)

Funded by EC contract FP6-034202

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Funded by EC contract FP6-034202

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