1. Modification chimique de surface pour laccrochage de biomolécules Bernard Bennetau Directeur de Recherche CNRS LCOO, UMR 5802 CNRS b.bennetau@lcoo.u-bordeaux1.fr

2. 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) b.bennetau@lcoo.u-bordeaux1.fr

3. Modification chimique de surface = propriétés spécifiques Oléophobe / Hydrophobe Modification de surface

4. Bacteria DNA Blood cells Surface modification for immobilization of biomolecules Antibodies Biosensor

5. 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.

6. Surface modification by silylated coupling agents Surface Commercially available compounds "Idealize surface" Silanization reaction The silanization reaction = "capricious " (high level of irreproducibility)

7. 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…

8. 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

9. 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)

10. n H2OH2O + Covalent bondings Octadecyltrichlorosilane (solution)

11. 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.

12. Coll. with B. Desbat (LPCM, UMR 5803 CNRS, Université Bordeaux I) n = 16 n = 27 30002980296029402920290028802860284028202800 98,9 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

13. 30002980296029402920290028802860284028202800 98,6 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)

14. 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 ) 30002980296029402920290028802860284028202800 99,0 99,2 99,4 99,6 99,8 100,0 100,2 Transmittance

15. 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)

16. 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)

17. Bennetau B.; Bousbaa J.; Choplin F. CNRS patent, 2001, WO/FR01/00139; licensed to ROSATECH. PEG terminated long-chain alkylsilanes

18. 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, 4844. Si/SiO 2 wafers "silanized" with Cl 3 Si-(CH 2 ) 22 -(OCH 2 CH 2 ) 3 -OH

19. 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

20. 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

21. 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 DNA adsorbed on aminated silicon wafer surfaces

22. 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-2 10060 Epoxy. Surf.MCF7 anti-HER-2 5308 NH 2 /BS3MCF7 anti-epithelial 2260 NH 2 /BS3MCF7 anti-HER-2 3298 NH 2 /EDCMCF7 anti-epithelial 3806 NH 2 /EDCT47D anti-HER-2 4328 Tumoral cells on microscope glass slides BS3 : Bis[sulfosuccimidyl]suberate EDC : Ethyldiethylaminopropylcarbodiimide

23. 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

24. 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) 020406080100 -10 -8 -6 -4 -2 0 Addition of biological species Quartz Sensitive layer Guidewave layer (SiO 2 ) Antibodies Silylated coupling agents BORDEAUX

25. 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 dAquitaine, 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 dAquitaine SNECMA Moteurs Permanent members Dr J.-P. Pillot Dr M. Birot Students Dao Thé Minh (PhD) Tran Thuy (PhD) M. Boutar (post-doc)