Modification chimique de surface pour laccrochage de biomolécules Bernard Bennetau Directeur de...
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Transcript of Modification chimique de surface pour laccrochage de biomolécules Bernard Bennetau Directeur de...
Modification chimique de surface pour l’accrochage de biomolécules
Bernard BennetauDirecteur de Recherche CNRS
LCOO, UMR 5802 CNRS
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
• Dinh Haï (PhD) • F. Choplin (PhD)• P. Martin (PhD)• D. Bousbaa (post-doc)
SURFACE MODIFICATION
Permanent members• Dr B. Bennetau• Dr J.-P. Pillot• Dr L. Vellutini• L. Thomas
PROTECTIVE COATINGS
Collaboration• B. Desbat, LPCM, Univ. Bx I• Institut de Technologie Tropicale, Hanoï, Viet Nam• N. Pébère (CIRIMAT, Toulouse)
Permanent members• Dr J.-P. Pillot• Dr M. Birot
• Dao Thé Minh (PhD)• Tran Thuy (PhD)• M. Boutar (post-doc)
Modification chimique de surface = propriétés spécifiques
Oléophobe / Hydrophobe
F
FF
F
*
F F
F F
*n
npolymérisation
PTFE
Modification de surface
Bacteria
DNA
Blood cells
Surface modification for immobilization of biomolecules
Antibodies
Biosensor
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
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.
Surface modification by silylated coupling agents
Si OOMeO
MeOMeO
OH OH
O
Si
O
Si
O
Si
O
Si
OH
O
OH
OH
OH
O
O
O
O
O
Si
O
Si
O
Si
O
Si
O
O
O
O
O
O
O
O
O
SiO
Si OOH
OOH
Si
Si
O
O
O
O
OOH
OOH
OOH
OOH
OOH
OOH
2 )
1)
Surface
Commercially available compounds "Idealize surface"
Silanization reaction
The silanization reaction = "capricious " (high level of irreproducibility)
The true surface is an heterogeneous surface(roughness, chemical functions)
Si OOMeO
MeOMeO
OH OH
O
Si
O
Si
O
Si
O
Si
OH
O
OH
OH
OH
O
O
O
O
O
Si
O
Si
O
Si
O
Si
O
O
O
O
O
O
O
O
O
Si OO
O
Si OOH
OOH
Si O
Si OOH
OH
O
O
O
O
O
OH
2 )
1)
Surface
Surface modification for immobilization of biomolecules
- Reproducibity problems due to the high number of relevant parameters : (cleaning procedure, solvents, temperature of reaction, characterization of the modified surfaces…
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, reducednonspecific adsorption, and greater stability.
X3Sin
O
Si
O
OH
COOROCOR
18<n<27
- Synthesis of functionnalized long-chain silanes true dense monolayer
- Characterization of the grafted monolayer at different scale (m to nm)
« DNA chips ". GENOME program (CNRS 97/00; OO/O2).
Surface modification by silylated coupling agents
O
Si
Fct
O
O
Si
Fct
O
O
Si
Fct
O Si
Fct
O
O OSi
Fct
O
OSi
Fct
O
OH
O
Si
Fct
OH
SiO2
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
Self-Assembled Monolayers (SAMs)
O
Si O
O
Si O
O
Si O Si
O
O OSi
O
OSi
O
OH
O
Si OH
SiO2
Self-Assembled Monolayers (SAMs)
Si
Fct
O
O OHSi
Fct
O
OSi
Fct
O
OH
O
Si
Fct
OH OHO
Si
Fct
OH OH
SiO2
Si
Fct
OH
O OHSi
Fct
OH
OSi
Fct
OH
OH
O
Si
Fct
O
O
Si
Fct
O
O
Si
Fct
O Si
Fct
O
O OSi
Fct
O
OSi
Fct
O
OH
O
Si
Fct
OH
SiO2
Si
Fct
ClClCl
nH2O
Si
Fct
OH
OHOH
+
OHOH OHOHOH
SiO2
Covalent bondings
Octadecyltrichlorosilane (solution)
OH protected silylated compounds
BrMeLi/THF( ) 11
1) 5% CuI
MgBrMg/THF ( )9Br
( )9
Br OH( )11
Br( )20
Br BrMeLi/THF( )11
1) 5% CuI
2) H3O+
Mg/THF ( )9Br
( )9
Br Br( )11
OCOCH3( )20
CH3COCl/Et3N
CH2Cl2
OCOCH3( )25
H-SiCl3
Pt
Pt
OCOCH3Cl3Si( )20
OCOCH3Cl3Si( )25
/ MeLi/THF
3) 5% CuI
1) Mg/THF
Br OH( )5
2)
OH( )20
OH( )25
2) H3O+
H-SiCl3CH3COCl/Et3N
CH2Cl2
OLi
MgBr
Bennetau B.; Bousbaa J.; Choplin F.CNRS Patent, 2001, WO/FR01/00139; licensed to ROSATECH.
Coll. with B. Desbat (LPCM, UMR 5803 CNRS, Université Bordeaux I)
n = 16
n = 27
3000 2980 2960 2940 2920 2900 2880 2860 2840 2820 280098,9
99,0
99,1
99,2
99,3
99,4
99,5
99,6
99,7
99,8
99,9
100,0
100,1
Tra
nsm
itta
nce
Wave number (cm-1
)
a CH2
s
n = 22
Choplin, F.; Navarre, S.; Bousbaa, J.; Babin, P.; Bennetau, B.; Bruneel, JL.; Desbat, B. Journal of Raman Spectroscopy 2003, 34, 902.
Glass slides "silanized" with Cl3Si-(CH2)n-OCOR
3000 2980 2960 2940 2920 2900 2880 2860 2840 2820 280098,6
98,8
99,0
99,2
99,4
99,6
99,8
100,0
100,2
Tra
nsm
itta
nce
Wave number (cm-1)
n = 16 n = 22 n = 27
Choplin, F.; Navarre, S.; Bousbaa, J.; Babin, P.; Bennetau, B.; Bruneel, JL.; Desbat, B. Journal of Raman Spectroscopy 2003, 34, 902.
After deprotection
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.
Reproducibility
Glass slides "silanized" with Cl3Si-(CH2)n-OCOR
Coll. with B. Desbat (LPCM, UMR 5803 CNRS, Université Bordeaux I)
Wave numbers (cm-1)
3000 2980 2960 2940 2920 2900 2880 2860 2840 2820 2800
99,0
99,2
99,4
99,6
99,8
100,0
100,2
Tra
nsm
itta
nce
Raman cartography
26002700280029003000310032003300
dark arealight area
0
100
200
300
400
500
Wavenumbers (cm -1 )
Ram
an s
ign
al (
arb
itra
ry u
nit
)
Choplin, F.; Navarre, S.; Bousbaa, J.; Babin, P.; Bennetau, B.; Bruneel, JL.; Desbat, B. Journal of Raman Spectroscopy 2003, 34, 902.
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.
26002700280029003000310032003300
light area
dark area
-50
0
50
100
150
200
250
300
350
Wavenumbers (cm -1)
Ram
an s
igna
l (ar
bitr
ary
unit
)
Raman cartography
Coll. with B. Desbat (LPCM, UMR 5803 CNRS, Université Bordeaux I)
Bennetau B.; Bousbaa J.; Choplin F.CNRS patent, 2001, WO/FR01/00139; licensed to ROSATECH.
I
1. SOCl22. NaI
( )20
O OH
HOC2H4OH THF, TBABNaOH
( )20
O OCOCH3
ClCOCH3, Et3N CH2Cl2
( )20
O O OH
H(OC2H4)3OH
( )20
( )2
O O OCO( )20
( )2
ClCOCH3, Et3N CH2Cl2
H-SiCl3 Pt
O OCOCH3Cl3Si( )20
O O OCOMeCl3Si ( )20
( ) 2
H-SiCl3 Pt
OH( )20
Br OLiMeLi/THF( )11
1°) 5% CuI
2°) H3O+
MgBrMg/THF( )9
Br( )9
Br OH( )11
OCOCH3( )20
CH3COCl/Et3N
CH2Cl2
H-SiCl3
Pt
OCOCH3Cl3Si( )20
CH3
PEG terminated long-chain alkylsilanes
Coll. with JP Aimé (CPMOH, UMR 5798 CNRS, Université Bordeaux I)
5 m
5 mTrue monolayer
Navarre, S.; Choplin, F.; Bousbaa, J.; Bennetau, B.; Nony, L.; Aime, J.-P.; Langmuir 2001, 17, 4844.
Si/SiO2 wafers "silanized" with Cl3Si-(CH2)22-(OCH2CH2)3-OH
Hybridization on SAMs [Cl3Si-(CH2)22-EG3-COR]
Bennetau B.; Bousbaa J.; Choplin F; Souteyrand E.; Martin JR.; Cloarec JP.CNRS Patent, 2001, WO/FR01/00140; licenced to ROSATECH.
CGTCTCTGGGCT
CGTCTCCGGGCT
CGTCTCAGGGCT
X
Z
Y
Hybridization happens between any two complementary single stranded molecules
Complement of X Complement of ZComplement of Y
99.2
99.6
100
2820286029002940
Tra
nsm
ittan
ce (
%)
Wavenumber (cm-1) -2
-1
0
1
2
3
4
0 200 400 600 800 1000
Height (nm)
Size (nm)
Cl3Si Me
Cl3Si N
O
O
Cl3Si NH2
17 21 21
OTS N-protected AHTS AHTS
Martin P., Marsaudon S., Thomas L., Desbat B., Aimé J-P., Bennetau B.Langmuir 2005, 21, 6934-6943; Nanotechnology 2005, 16, 901–907
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, 901–907
5 m
5 m
DNA adsorbed on aminated silicon wafer surfaces
Institut d'Alembert – IFR 121Applications des Lasers et
Molécules aux Biotechnologies et Réseaux
de Télécommunication
Surface cells antibodies Cells (Number)
Epoxy surf. T47D anti-HER-2 10060
Epoxy. Surf. MCF7 anti-HER-2 5308
NH2/BS3 MCF7 anti-epithelial 2260
NH2/BS3 MCF7 anti-HER-2 3298
NH2/EDC MCF7 anti-epithelial 3806
NH2/EDC T47D anti-HER-2 4328
Tumoral cells on microscope glass slides
BS3 : Bis[sulfosuccimidyl]suberateEDC : Ethyldiethylaminopropylcarbodiimide
Tumoral cells on microscope glass slides
Bennetau B.; Tran L.Brevet FR04/07722, CNRS/ENS Cachan/Univ Bx I.
- Epoxy groups on the surface- Cells T47D
1.5 cm
3 cm
Coll. : D. Rebière, C. Dejous (IXL); D. Moynet, E. Pascal (Univ. Bx II);B. Bennetau, JP Pillot, L Vellutini, L Thomas (LCOO)
VIn
Génération de l’onde
Réception de l’onde
Biomolecules to detect
Love waves
Mat piézo-électrique (Quartz)
Delayed decay
VOut
Va
ria
tio
n f
réq
ue
nc
e (
kH
z)
Time (min)
0 20 40 60 80 100
-10
-8
-6
-4
-2
0Addition of biological species
Quartz
Sensitive layer
Guidewave layer (SiO2)
Antibodies
Silylated coupling agents
BORDEAUX
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
Financial supports• Proteomic and Protein Engineering Program, (CNRS 2004-2006)• Nanoscience Program (CNRS, CA 2004)• PhD grant (Conseil Régional d’Aquitaine, 2002/2005)• BQR (Unviv. Bx 1, 2002/2004)• Nanobioengineering (ACI CNRS, 2001-2002) • GENOME Program (CNRS, 1997-2002)
Students• Dinh Haï (PhD) • F. Choplin (PhD)• P. Martin (PhD)• D. Bousbaa (post-doc)
SURFACE MODIFICATION Permanent members• Dr B. Bennetau• Dr J.-P. Pillot• Dr L. Vellutini• L. Thomas
PROTECTIVE COATINGS
Collaborations• B. Desbat, LPCM, Univ. Bx I• Institut de Technologie Tropicale, Hanoï, Viet Nam• N. Pébère (CIRIMAT, Toulouse)
Financial supports• ESPOIR Program (MAE, 1999-2003)• Corrosion-PED Program (CNRS) 2001-2003• Pôle Aquitaine Matériaux• Conseil Régional d’Aquitaine • SNECMA Moteurs
Permanent members• Dr J.-P. Pillot• Dr M. Birot
Students• Dao Thé Minh (PhD)• Tran Thuy (PhD)• M. Boutar (post-doc)