Diapositive 1

1
Modification chimique de surface pour
laccrochage de biomolécules
Bernard Bennetau Directeur de Recherche
CNRS LCOO, UMR 5802 CNRS
b.bennetau_at_lcoo.u-bordeaux1.fr
2
PROTECTIVE COATINGS
SURFACE MODIFICATION

• Dinh Haï (PhD)
• F. Choplin (PhD)
• P. Martin (PhD)
• D. Bousbaa (post-doc)

• Permanent members
• Dr B. Bennetau
• Dr J.-P. Pillot
• Dr L. Vellutini
• L. Thomas

• Permanent members
• Dr J.-P. Pillot
• Dr M. Birot

• Dao Thé Minh (PhD)
• Tran Thuy (PhD)
• M. Boutar (post-doc)

• Collaboration
• P. L. Tran, ENS Cachan
• D. Rebière, C. Dejous, IXL, Univ. Bx I
• E. Souteyrand, J. P. Cloarec, E. C. Lyon
• G. Deléris, INSERM, Univ. Bx II
• J. P. Aimé, CPMOH, Univ. Bx I
• B. Desbat, LPCM, Univ. Bx I
• D. Moynet, LI, Univ. Victor Ségalen

• Collaboration
• B. Desbat, LPCM, Univ. Bx I
• Institut de Technologie Tropicale, Hanoï, Viet
  Nam
• N. Pébère (CIRIMAT, Toulouse)

b.bennetau_at_lcoo.u-bordeaux1.fr
3
Modification de surface
Oléophobe / Hydrophobe
Modification chimique de surface propriétés
spécifiques
4
Surface modification for immobilization of
biomolecules
Antibodies
DNA
Biosensor
Blood cells
Bacteria
5
Surface modification for immobilization of
biomolecules
To deposit biomolecules on mineral surfaces
(Si/SiO2 wafers, microscope glass slides), the
first step is the grafting of an organic
film Chemical surface modification chemical
bonding of molecules to a surface in order to
change its chemical or physical properties in a
controlled way.
Why ? - to modify the wettability properties
to improve the biocompatibility between the
substrate and the biomolecules - to introduce on
the surface the required chemical functions (OH,
COOH, NH2...) - the density surface coverage
should be optimized a low density surface
coverage will yield a correspondingly low density
of biomolecules A high surface density may
prevent interactions between immobilized probes
biomolecules and target molecules What are
the properties needed for the organic film ? -
thermal stability - chemical stability (UV,
hydrolysis, acid and bases are often used to
immobilize or to synthesize in situ ,
biomolecules) - activity of the biomolecules
must be preserved
6
Surface modification by silylated coupling agents
Silanization reaction
Surface
Commercially available compounds
 "Idealize surface"
The silanization reaction "capricious " (high
level of irreproducibility)
7
Surface modification for immobilization of
biomolecules

Surface
The true surface is an heterogeneous
surface (roughness, chemical functions)
- Reproducibity problems due to the high number
of relevant parameters (cleaning procedure,
solvents, temperature of reaction,
characterization of the modified surfaces
8
Surface modification by silylated coupling agents
- Synthesis of functionnalized long-chain silanes
? true dense monolayer
- Characterization of the grafted monolayer at
different scale (mm to nm)
Goals - avoid further reactions on the
substrate - obtain a true dense and homogeneous
Self-Assembled Monolayer (SAMs) to protect
siloxanic bonds (Si-O-Si) between the coupling
agents and the surface. - covalent immobilization
may result in better biomolecule activity,
reduced nonspecific adsorption, and greater
stability.
 DNA chips ". GENOME program (CNRS 97/00 OO/O2).
9
Self-Assembled Monolayers (SAMs)
SAMs (silanes on silicon oxides) - driving
force maximization of chain-chain interactions
(VdW) - Stability of the monolayer chain
length dependent. Properties - covalent
bonding headgroups can be SiCl3 -Si(OR)3 -
stable and well-oriented layers - strong and
well protected bonds between the organic film and
the substrate
10
Self-Assembled Monolayers (SAMs)
H2O

n
Octadecyltrichlorosilane (solution)
Covalent bondings
11
OH protected silylated compounds
MgBr
Mg/THF
( )
9
1) 5 CuI
CH3COCl/Et3N
H-SiCl3
O
H
O
C
O
C
H
O
C
O
C
H
C
l
S
i
( )
3
( )
3
3
( )
20
20
20
2) H3O
CH2Cl2
Pt
MeLi/THF
OLi
Br
Br
O
H
( )
( )
11
11
MgBr
Br
Mg/THF
( )
( )
9
9
1) Mg/THF
1) 5 CuI
O
H
Br
( )
( )
20
25
2)
2) H3O
Br
O
H
/ MeLi/THF
MeLi/THF
( )
Br
Br
5
( )
11
3) 5 CuI
CH3COCl/Et3N
H-SiCl3
O
C
O
C
H
C
l
S
i
O
C
O
C
H
3
3
( )
3
( )
25
25
Pt
CH2Cl2
Bennetau B. Bousbaa J. Choplin F. CNRS Patent,
2001, WO/FR01/00139 licensed to ROSATECH.
12
Glass slides "silanized" with Cl3Si-(CH2)n-OCOR
Coll. with B. Desbat (LPCM, UMR 5803 CNRS,
Université Bordeaux I)
Choplin, F. Navarre, S. Bousbaa, J. Babin, P.
Bennetau, B. Bruneel, JL. Desbat, B. Journal
of Raman Spectroscopy 2003, 34, 902.
13
Glass slides "silanized" with Cl3Si-(CH2)n-OCOR
After deprotection
100,2
100,0
99,8
99,6
Transmittance
99,4
99,2
99,0
n 22
98,8
n 27
98,6
3000
2980
2960
2940
2920
2900
2880
2860
2840
2820
2800
Wave number (cm-1)
Coll. with B. Desbat (LPCM, UMR 5803 CNRS,
Université Bordeaux I)
Choplin, F. Navarre, S. Bousbaa, J. Babin, P.
Bennetau, B. Bruneel, JL. Desbat, B. Journal
of Raman Spectroscopy 2003, 34, 902.
14
Glass slides "silanized" with Cl3Si-(CH2)n-OCOR
Reproducibility
Coll. with B. Desbat (LPCM, UMR 5803 CNRS,
Université Bordeaux I)
Choplin, F. Navarre, S. Bousbaa, J. Babin, P.
Bennetau, B. Bruneel, JL. Desbat, B. Journal
of Raman Spectroscopy 2003, 34, 902.
15
Raman cartography
Coll. with B. Desbat (LPCM, UMR 5803 CNRS,
Université Bordeaux I)
Choplin, F. Navarre, S. Bousbaa, J. Babin, P.
Bennetau, B. Bruneel, JL. Desbat, B. Journal
of Raman Spectroscopy 2003, 34, 902.
16
Raman cartography
Coll. with B. Desbat (LPCM, UMR 5803 CNRS,
Université Bordeaux I)
Choplin, F. Navarre, S. Bousbaa, J. Babin, P.
Bennetau, B. Bruneel, JL. Desbat, B. Journal
of Raman Spectroscopy 2003, 34, 902.
17
PEG terminated long-chain alkylsilanes
Bennetau B. Bousbaa J. Choplin F. CNRS patent,
2001, WO/FR01/00139 licensed to ROSATECH.
18
Si/SiO2 wafers "silanized" with
Cl3Si-(CH2)22-(OCH2CH2)3-OH
5 mm
True monolayer
5 mm
Coll. with JP Aimé (CPMOH, UMR 5798 CNRS,
Université Bordeaux I)
Navarre, S. Choplin, F. Bousbaa, J. Bennetau,
B. Nony, L. Aime, J.-P. Langmuir 2001, 17,
4844.
19
Hybridization on SAMs Cl3Si-(CH2)22-EG3-COR
X
Y
Z
Hybridization happens between any two
complementary single stranded molecules
Complement of Y
Complement of X
Complement of Z
Bennetau B. Bousbaa J. Choplin F Souteyrand
E. Martin JR. Cloarec JP. CNRS Patent, 2001,
WO/FR01/00140 licenced to ROSATECH.
20
DNA adsorbed on aminated silicon wafer surfaces
Martin P., Marsaudon S., Thomas L., Desbat B.,
Aimé J-P., Bennetau B. Langmuir 2005, 21,
6934-6943 Nanotechnology 2005, 16, 901907
21
DNA adsorbed on aminated silicon wafer surfaces
5 mm
5 mm
Martin P., Marsaudon S., Thomas L., Desbat B.,
Aimé J-P., Bennetau B. Langmuir 2005, 21,
6934-6943 Nanotechnology 2005, 16, 901907
22
Tumoral cells on microscope glass slides
Surface cells antibodies Cells
(Number)
NH2/EDC MCF7 anti-epithelial
3806
NH2/EDC T47D anti-HER-2
4328
NH2/BS3 MCF7 anti-epithelial
2260
NH2/BS3 MCF7 anti-HER-2
3298
Epoxy surf. T47D anti-HER-2
10060
Epoxy. Surf. MCF7 anti-HER-2
5308
BS3 Bissulfosuccimidylsuberate EDC
Ethyldiethylaminopropylcarbodiimide
23
Tumoral cells on microscope glass slides
3 cm
1.5 cm
- Epoxy groups on the surface - Cells T47D
Bennetau B. Tran L. Brevet FR04/07722, CNRS/ENS
Cachan/Univ Bx I.
24
Biomolecules to detect
Variation fréquence (kHz)
Addition of biological species
VIn
VOut
Génération de londe
Time (min)
Mat piézo-électrique (Quartz)
Love waves
Delayed decay
Antibodies
Sensitive layer
Silylated coupling agents
Guidewave layer (SiO2)
Quartz
Coll. D. Rebière, C. Dejous (IXL) D. Moynet,
E. Pascal (Univ. Bx II) B. Bennetau, JP Pillot,
L Vellutini, L Thomas (LCOO)
25
SURFACE MODIFICATION
PROTECTIVE COATINGS

• Students
• Dinh Haï (PhD)
• F. Choplin (PhD)
• P. Martin (PhD)
• D. Bousbaa (post-doc)

• Permanent members
• Dr B. Bennetau
• Dr J.-P. Pillot
• Dr L. Vellutini
• L. Thomas

• Permanent members
• Dr J.-P. Pillot
• Dr M. Birot

• Students
• Dao Thé Minh (PhD)
• Tran Thuy (PhD)
• M. Boutar (post-doc)

• Collaborations
• P. L. Tran, ENS Cachan
• D. Rebière, C. Dejous, IXL, Univ. Bx I
• E. Souteyrand, J. P. Cloarec, C S, Lyon
• G. Deléris, INSERM, Univ. Bx II
• J. P. Aimé, CPMOH, Univ. Bx I
• B. Desbat, LPCM, Univ. Bx I

• Collaborations
• B. Desbat, LPCM, Univ. Bx I
• Institut de Technologie Tropicale, Hanoï, Viet
  Nam
• N. Pébère (CIRIMAT, Toulouse)

• Financial supports
• Proteomic and Protein Engineering Program, (CNRS
• 2004-2006)
• Nanoscience Program (CNRS, CA 2004)
• PhD grant (Conseil Régional dAquitaine,
  2002/2005)
• BQR (Unviv. Bx 1, 2002/2004)
• Nanobioengineering (ACI CNRS, 2001-2002)
• GENOME Program (CNRS, 1997-2002)

• Financial supports
• ESPOIR Program (MAE, 1999-2003)
• Corrosion-PED Program (CNRS) 2001-2003
• Pôle Aquitaine Matériaux
• Conseil Régional dAquitaine
• SNECMA Moteurs