Modification chimique de surface pour laccrochage de biomolécules Bernard Bennetau Directeur 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 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/SiO 2 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 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) 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, reduced nonspecific adsorption, and greater stability. - 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

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 –SiCl 3 ; -Si(OR) 3 - stable and well-oriented layers - strong and well protected bonds between the organic film and the substrate Self-Assembled Monolayers (SAMs)

n H2OH2O + Covalent bondings Octadecyltrichlorosilane (solution)

OH protected silylated compounds Br MeLi/THF ( ) 11 1) 5% CuI MgBr Mg/THF ( ) 9 Br ( ) 9 BrOH ( ) 11 Br ( ) 20 Br MeLi/THF ( ) 11 1) 5% CuI 2) H 3 O + Mg/THF ( ) 9 Br ( ) 9 Br ( ) 11 OCOCH 3 ( ) 20 CH 3 COCl/Et 3 N CH 2 Cl 2 OCOCH 3 ( ) 25 H-SiCl 3 Pt OCOCH 3 Cl 3 Si ( ) 20 OCOCH 3 Cl 3 Si ( ) 25 / MeLi/THF 3) 5% CuI 1) Mg/THF BrOH ( ) 5 2) OH ( ) 20 OH ( ) 25 2) H 3 O + H-SiCl 3 CH 3 COCl/Et 3 N CH 2 Cl 2 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 = ,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 Transmittance Wave number (cm ) a CH 2 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 Cl 3 Si-(CH 2 ) n -OCOR

,6 98,8 99,0 99,2 99,4 99,6 99,8 100,0 100,2 Transmittance 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 Cl 3 Si-(CH 2 ) 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 Cl 3 Si-(CH 2 ) n -OCOR Coll. with B. Desbat (LPCM, UMR 5803 CNRS, Université Bordeaux I) Wave numbers (cm -1 ) ,0 99,2 99,4 99,6 99,8 100,0 100,2 Transmittance

Raman cartography 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. 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. PEG terminated long-chain alkylsilanes

Coll. with JP Aimé (CPMOH, UMR 5798 CNRS, Université Bordeaux I) 5 m True monolayer Navarre, S.; Choplin, F.; Bousbaa, J.; Bennetau, B.; Nony, L.; Aime, J.-P.; Langmuir 2001, 17, Si/SiO 2 wafers "silanized" with Cl 3 Si-(CH 2 ) 22 -(OCH 2 CH 2 ) 3 -OH

Hybridization on SAMs [ Cl 3 Si-(CH 2 ) 22 -EG 3 -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 Z Complement of Y

Martin P., Marsaudon S., Thomas L., Desbat B., Aimé J-P., Bennetau B. Langmuir 2005, 21, ; 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, ; Nanotechnology 2005, 16, 901–907 5 m DNA adsorbed on aminated silicon wafer surfaces

Institut d'Alembert – IFR 121 Applications des Lasers et Molécules aux Biotechnologies et Réseaux de Télécommunication Surfacecells antibodies Cells (Number) Epoxy surf.T47D anti-HER Epoxy. Surf.MCF7 anti-HER NH 2 /BS3MCF7 anti-epithelial 2260 NH 2 /BS3MCF7 anti-HER NH 2 /EDCMCF7 anti-epithelial 3806 NH 2 /EDCT47D anti-HER Tumoral cells on microscope glass slides BS3 : Bis[sulfosuccimidyl]suberate EDC : 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) V In Génération de londe Réception de londe Biomolecules to detect Love waves Mat piézo-électrique (Quartz) Delayed decay V Out Variation fréquence (kHz) Time (min) Addition of biological species Quartz Sensitive layer Guidewave layer (SiO 2 ) 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 ) Nanoscience Program (CNRS, CA 2004) PhD grant (Conseil Régional dAquitaine, 2002/2005) BQR (Unviv. Bx 1, 2002/2004) Nanobioengineering (ACI CNRS, ) GENOME Program (CNRS, ) 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, ) Corrosion-PED Program (CNRS) Pôle Aquitaine Matériaux Conseil Régional dAquitaine SNECMA Moteurs Permanent members Dr J.-P. Pillot Dr M. Birot Students Dao Thé Minh (PhD) Tran Thuy (PhD) M. Boutar (post-doc)