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

[email protected]

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)

[email protected]

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


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


- 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


After deprotection




Reproducibility



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

)




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


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)

modification chimique de surface pour laccrochage de biomolécules bernard bennetau directeur de...

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