Transcript Lab-On-Chip based proteing profiling for CANcer DIAgnosis

Lab-On-Chip based proteing
profiling for CANcer
DIAgnosis
Priority 2.5.2. Micro/nano based sub-system
Pierre GRANGEAT
(1)
, Blanca JORDAN RODRIGUEZ
(2),
and all the LOCCANDIA partners
(1) CEA-LETI, MINATEC, [email protected]
(2) ATOS ORIGIN, [email protected]
Nano2life annual meeting,
Champéry (Switzerland)
9-11 January 2008
DTBS/STD/08E-02
Funded by EC contract FP6-034202
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)
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Funded by EC contract FP6-034202
LOCCANDIA
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
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Funded by EC contract FP6-034202
LOCCANDIA
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
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Funded by EC contract FP6-034202
LOCCANDIA
Silicium technology for optimized microfluidics
MS
Nano-LC separation
1,5 µm
≈ 1mm
Techno 3 wafers
800µm
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Funded by EC contract FP6-034202
Outlet 5 X50 µm2
LOCCANDIA
LOCCANDIA analysis chain
Blood plasma sample
Selected
protein
mixture
Blood
sample
preparation
Peptides
spectrogram
Lab-on-Chip
& mass
spectrometry
Diagnostic
information
Information
technology
Patient
Doctor
BIO
NANO
Material flow
Information flow
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Funded by EC contract FP6-034202
INFO
LOCCANDIA
Project Goals (I): Blood sample preparation
Blood plasma sample
Selected
protein
mixture
Blood
sample
preparation
Patient
BIO
Lab-on-Chip
& mass
spectrometry
–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.
Material flow
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Funded by EC contract FP6-034202
LOCCANDIA
Project Goals (II):
Lab-on-chip and mass spectrometry
Selected
protein
mixture
•The objectives are:
– to provide new batches of
our current microsystems
– to improve the
chromatography-electrospray
microsystem technology
Lab-on-Chip
& mass
spectrometry
NANO
Material flow
Peptides
spectrogram
•Two interconnected modules will be designed:
– a protein digestion module
– a liquid chromatography-electrospray
ionisation module
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Funded by EC contract FP6-034202
LOCCANDIA
Project Goals (III): Information
technology
To build an Integrated
Clinico-Proteomics
Environment (ICPE):
Diagnostic
information
a Proteomic Information
Management System (PIS) for
sample information management
•
Information
technology
Doctor
Peptides
spectrogram
• an Information and data
mediation infrastructure including
preprocessing, reconstruction,
visualization, protein/peptide
identification and data analysis
modules
INFO
Information flow
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Funded by EC contract FP6-034202
• a Clinical Information System
(CIS) for patient information
management to allow clinical
evaluation
LOCCANDIA
LOCCANDIA Information Management
System Moke-Up
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Funded by EC contract FP6-034202
LOCCANDIA
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
For calibration
• 3 mixtures at 0.2 µmol/l
• 2 mixtures at 0.8 µmol/l
For prediction
• 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)
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Funded by EC contract FP6-034202
LOCCANDIA
Experiment
al mixture
Results of profile reconstruction using
chemometrics approaches
TIC (Total Ion Current)
5
x 10
6
x 10
Projection on m/z
3.5
4
3
3.5
Major
peptide of
CytoC
(478Da, 1560
s)
2.5
3
2
2.5
1.5
2
1
1.5
0.5
1
1st N-PLS
extracted factor
200
400
600
800
1000
1200
1400
1600
450
Retention time (s)
XIC (Base Peak Intensity)
500
550
700
0.7
0.6
0.15
Major
peptide of
CytoC
(478Da, 1560
s)
0.5
0.4
0.1
0.3
0.2
0.05
0.1
400
600
800
1000
1200
Retention time (s)
1400
1600
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Funded by EC contract FP6-034202
650
Projection on m/z
0.8
0.2
0
200
600
Mass / Chargetime
(Da) (s)
Retention
0
450
500
550
600
650
Mass / Charge (Da)
700
LOCCANDIA
Project Goals (IV): validation
• 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
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Funded by EC contract FP6-034202
LOCCANDIA
Main research outcomes
• an optimized chromatographicelectrospray lab-on-chip
dedicated to protein profiling for
cancer diagnosis
• an Integrated ClinicoProteomics 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
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Funded by EC contract FP6-034202
LOCCANDIA
List of participants
and
DBVN
UniWWU
Muenster,
Biovision
• Atos Origin sae, Spain – ATOS
CEA-LETI
CEA-Leti
SIB, Gene Bio
SIB, GeneBIO
• Commissariat à l’Energie Atomique,
France – CEA-LETI
ATOS ORIGIN
ATOS
FORTH
FORTH
• 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
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Funded by EC contract FP6-034202
LOCCANDIA
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 PeterKatalinic, Laura Bindila, Daniela Hahn, Rainer Ossig
• SIB: Frédérique Lisacek
• GeneBIO:Pierre-Alain Binz
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Funded by EC contract FP6-034202
LOCCANDIA
Contact details
http://www.loccandia.eu
Blanca Jordán Rodríguez
Project Manager
Atos Research and Innovation
Atos Origin Spain
[email protected]
Phone: +34 91 214 93 22
Fax: +34 91 754 3252 (Att. Blanca Jordán)
Pierre Grangeat
Scientific Director
CEA/Grenoble, France
LETI-DTBS
[email protected]
Phone: +33 4 38 78 43 73
Fax: +33 4 38 78 54 56 (Att. Pierre Grangeat)
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Funded by EC contract FP6-034202
LOCCANDIA
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Funded by EC contract FP6-034202
LOCCANDIA