WO2006128644A1

WO2006128644A1 - Polymer coating and functionalization of solid surfaces

Info

Publication number: WO2006128644A1
Application number: PCT/EP2006/005047
Authority: WO
Inventors: Giovanna Pirri; Marcella Chiari
Original assignee: Giovanna Pirri; Marcella Chiari
Priority date: 2005-05-30
Filing date: 2006-05-26
Publication date: 2006-12-07

Abstract

Disclosed is a method for coating glass, silica, silicon-oxide or other materials with brush polymers obtained by RAFT polymerization. The coated surfaces are used to immobilize biomolecules especially for microarray technology applications.

Description

POLYMER COATING AND FUNCTIONALIZATION OF SOLID SURFACES

The present invention provides a method for coating glass, silica, silicon-oxide or other materials with brush polymers consisting of one or more blocks one of which is covalently bound to the substrate. The coated surfaces provide sensors able to detect the presence and quantity of biomolecules in biological fluids in different formats including microarray technology. The method used for producing the polymeric coating according to the invention utilizes radical polymerization techniques that allow to control coating architecture and employ monomers with functional groups that do not require activation prior to binding the biological molecule. According to the present invention, block polymers brushes are produced by RAFT polymerization to generate substrates for microarray s. Background of the invention

In a number of situations it is important to coat the surface of a substrate or of an analytical device with a polymeric coating able to protect the surface or to confer to it the desired properties. The same surface modification can be achieved through physical adsorption or through a chemical binding of a polymer. In the case of substrates that are in contact with biological fluids (for instance contact lenses) the adhesion between the substrate and the coating must be strong and irreversible. In order to improve the strength of the interaction between polymeric species and support surface, this latter can be treated e.g. using the procedures described in US patents no. 4663232, 4311537, 4595632 and 4589964. Also analytical applications require substrates functionalized by suitable coating. An example is given by the microarray technology where the immobilization of biological molecules on different substrates is crucial (Nature Genetics Supplement VoI 21, 1999). In recent years there has been a wide spreading of this technique in different fields of medical and biological research. The methods and materials employed for microarray surface coating determine the success of the technique. US patents no. 5981734, 5861247, 5932711, 6180770, 6121027, 5858653, 5741551, 5512329, 6833276 deal with the problem of irreversibly immobilizing biological probes on glass or other surfaces maintaining characteristics of homogeneity, stability, reproducibility of the coating, high probe grafting density, and absence of non-specific interactions between biological molecules and coated surfaces. The production of polymeric coatings made of low polydispersity chains (Id close to 1) that are still susceptible of radical polymerization can be achieved by different mechanisms of living radical polymerization starting from surfaces on which radical initiators have been immobilized. In particular, the radical addition fragmentation transfer (RAFT) mechanism takes place as shown in the scheme reported below:

I. I 2R nM R - ~ P ^•

III. R- + Monomer — P_n

P₁; + Monomer ► P_1n

V. I, R, P₁₁, P Dead polymers

wherein molecules carrying -C(S)SR groups are chain transfer agents for radical polymerization of the type described in WO 98/01478. There are few examples of immobilization of molecules on surfaces coated with polymer block generated by a RAFT mechanism. In these cases the polymeric coating is achieved by binding radical initiators to the surface. The derivatized surface is immersed in a solution containing: 1) dithiobenzoic derivatives of the type ZC(S)SR wherein Z is, for example, a phenyl group substituted with an tertiary alkyl or with a phenyl group, 2) a radical initiator such as AIBN and 3) allyl monomers. The polymerization of allyl monomers in these conditions leads to formation of a polymeric coating made of living polymer chains terminated by -SC(S)Z groups able to reinitiate the polymerization in the presence of a radical initiator and of a second monomer (WO 98/01478). Description of the invention

The present invention relates to the coating and functionalization of various type of solid surfaces (glass, silica, silicon oxide) with polymer chains having controlled architecture, particularly useful for the immobilization of biological molecules such as nucleic acids, peptides and proteins. More precisely the invention provides polymer "brushes", i.e. polymer chains originating from the surface containing one or more blocks which are constituted by homopolymers or copolymers one of which is in direct contact with the surface and the others are exposed to the sample solution. The external polymer block carries functional groups directly involved in the immobilization of the biological molecules, e.g. DNA, proteins, polysaccharide molecules and synthetic molecules such as peptides, oligonucleotides or carbohydrates. The immobilization can occur on a portion of the surface or on the entire surface. The coated surface will be preferably used in protein and DNA microarray techniques.

In a first embodiment, the invention provides a method for coating a plastic, glass or silicon oxide surface with "brush"block polymers, which comprises:

I) derivatizing said surface with a thiol-bearing organosilane by reaction with compounds of general formula R'R²R³Si-(CR⁴R⁵)_nSH wherein R¹, R² and R³ are independently -OCH₃, -OCH₂CH₃ or Cl, R⁴ is hydrogen and R⁵ is H or CH₃ n is an integer from 2 to 10;

II) attaching to said glass surface a first polymer block obtained through polymerization of a mixture containing: a) monomers of general formula (I) or (II): [CH₂=C(R^I)C(O)NR^IIR^πi] (I) [CH₂=C(R^I)C(O)OR^1V] (II) wherein R¹ is H or CH₃; R^π,R^iπ,R^IV can be independently H or a linear or branched Cl -C 12 alkyl chain, optionally interrupted by O, N or S, said alkyl chain optionally bearing functional groups which are selected from epoxy, aldehyde, episulphide, aziridine, ester, thioester, N-hydroxy succinimide ester, pentafluorophenyl ester, dithioester, azalacton, lacton, lactide, anhydride, alogen, cyanate, isothicyanate, double bonds, amino, thiol, hydrazine, methoxy, ethoxy, hydroxyl, trimethoxysilyl, -OR^V, -C(O)-OR^VI wherein R^v is a C1-C6 alkyl or heterocyclic ring and R^VI is a C1-C6 alkyl or phenyl optionally substituted with -OH, -SH₅-NH₂₅-CH=O, oxirane. b) dithiobenzoic derivatives of general formula:

_/S — R

wherein R is selected from (Cl -C 12) alkyl or substituted alkyl, aliphatic ring or 5 to 10 membered (hetero)aromatic ring optionally substituted; alkylthio, alkoxy, dialkylamino optionally substituted; organometallic compound; Z is selected from hydrogen, chlorine, (C l-C12)alkyl, 5 to 10 membered (hetero)aryl optionally substituted; alkylthio, alkoxycarbonyl, arylossicarbonyl, carboxy; acyloxy or carbamoyl, optionally substituted; cyano; dialkyl or diaryl phosphonate or phoshinate; c) a radical initiator, prefereably AIBN (α,α'-azo-bis-isobutyronitryle)

III) whashing the coated surface with an organic solvent selected from dimethylformamide (DMF), tetrahydrofuran (THF), chloroform (CHCl₃ ) or mixtures thereof;

IV) optionally, elongating the polymer with further blocks obtained by polymerization of the mixtures according to step II.

Preferably the thiol-bearing organosilane is 3- mercaptopropyltrimethoxysilane, the dithiobenzoic derivative is 2-cyanoprop- 2-yl dithiobenzoate and the monomers are selected from the group comprising N,N-dimethylacrylamide, glicidylmethacrylate, N-acryloylsuccinimide, 2-vinyl-4,4'-dimethylazalactone.

In one preferred embodiment of the invention, the resultant polymeric coating has the formula 1 :

1-

for /V-acryloyloxy succinimide

for glycidyl methacrylate

wherein the first and the second block are homopolymers, the molecular weight of the polymer ranging from 10³ to 10⁶. In another preferred embodiment of the invention, the resultant polymeric coating has the formula 2

2.

R= H, R¹= for W-acryloyloxy succinimide

R= CH₃, R

for glycidyl methacrylate

wherein the first block is a poly(DMA) homopolymer and y=l-99, 2=99-1 mole%, the molecular weight of the polymer ranging from 10³ to 10⁶.

In another preferred embodiment of the invention, the resultant polymeric coating has the formula 3

3.

R= H, R'= for Λ/-acryloyloxy succinimide

for glycidyl methacrylate

wherein the graft polymer is a living random copolymer, X= 1-99 mole%,

Z=99-l%, the molecular weight of the polymer ranging from 10³ to 10⁶. In a further embodiment, the invention provides a method for the immobilization of biological molecules on the surface of a plastic, glass or silicon support, which comprises coating said surface as above described and subsequently contacting the coated surface with a solution containing the biological molecules, in suitable conditions for the covalent binding of the biological molecules to the polymers.

In a further embodiment, the invention provides a plastic, glass or silicon-oxide support for the immobilization of biological molecules, having at least one surface coated in accordance with the invention. Preferably the support is in form of a multiwell plate, bead, test tube, flask, microscope slide, silicon wafer, silicon membrane or bead.

The need of excluding false positive in experiments of molecular recognition on solid phases requires an effective screening of the surface underneath in order to prevent non specific interactions of the biomolecules with the support. The growth of a first polymeric layer with high affinity for the substrate, according to the present invention, allows to achieve an efficient screening of the surface thus reducing the number of false positive tests.

The initiation of living or dead polymer chains on the surface of glass or silicon surfaces has been achieved through grafting of a radical initiator on the surface (Prucker, O. and Ruhe, J.; Langmuir 1998, 14(24), 6893-6898; Macromolecules , 1998 31(3), 592-601; Macromolecules, 1998, 31(3), 602-613;). In the literature, the chains of block polymers obtained by a RAFT mechanism are grafted to the surfaces by anchoring a radical initiator on the surface and by carrying out the polymerization in the presence of solution initiators, monomers and dithiobezoic esters. In this way living segments of different living polymers are generated, for instance polystyrene and polydimethylacrylamide, which are able to propagate the chain by addition of a second polymeric segment. One of the functional groups that is widely used to generate radicals is the diazo group. The functionalization of a surface with a diazo group is a complex process that requires asymmetric reagents bearing two distinct functionalities. One end of the molecule must react with substrate silanols and the other must bear the group which upon activation produce radicals. The initiators introduced by Ruhe (O. Prucker and J. Ruhe, "Polymer Layers Through Self- Assembled Monolayers of Initiators", vol. 14, No. 24, p. 6893-6898, American Chemical Society, Oct. 30, 1998) are asymmetric diazo compounds that bear a trichloro silane function at one end, which is reactive towards glass. This type of compounds forms self assembled monolayers on the surface and provide glass surface with a high density of immobilized radical initiators resulting in a high initiation efficiency. However, the difficulty of producing asymmetric diazo compounds with a high yield discourages their use in industrial applications.

The improvement obtained by this invention concerns the way surface living chains are generated. Unlike the methods reported in the literature, where the radical initiators are covalently bound to the surface silanols, the method of the invention provides the immobilization of mercato groups and chain transfer agents on the surface by organosilanization. The mercapto groups are (i) precursors of -SC(S)Z, formed in situ through transesterification reaction and (ii) in the presence of monomers, ditiobenzoic derivatives, of the type reported in 2b, and soluble radical initiators such as AIBN, originate living chains that are terminated by a -SC(S)Z group.

The method provided by the invention is advantageous in that the synthesis of mercapto bearing organosilane is much easier than that of organosilane bearing radical initiators such as diazo or -SC(S)Z groups. The following examples demonstrate that the density of mercapto groups on a surface derivatized according to the invention, is sufficient to provide a number of living chains that is adequate for the proposed application. In particular, the following experimental conditions have been optimized: a) mercapto group surface density, b) type of solvent, c) type and concentration of monomers, d) type and concentration of CTA in solution. The experimental conditions reported in the examples have been chosen to balance the ratio between living and dead chains bound to the surface. By contacting a high thiol density glass surface obtained by organosilanization with 3-mercapto propyl trimethoxysilane with a solution containing, besides monomers, dithiobenzoic groups in solution, acting as CTA, and radical initiators (AIBN) the following reactions take place simultaneously:

1. the SH group transesterificates with the group ZC(S)SR;

2. the SH group initiates polymer chains that grow mediated by ZC(S)SR groups;

3. the SH group terminates the growth of polymer chains.

In the following scheme the hypothetical mechanism of initiation of living chains from the surface is reported:

AIBN, calorβ

The mechanism of radical polymerization reported above can, for instance, be used to bind to the substrate a first layer of homopolymer chains constituted by an allylic monomer such as N,N-dimethylacrylamide. This first polymeric segment, being living, can reinitiate the polymerization to form a second segment made of a homo- or co-polymeric segment comprising an electrophilic monomers, such as the glycidyl methacrylate or the N-hydroxy succinimide ester.

Example 1

Silanization process Or gano silanization with (3-mercaptopropyl)trimethoxy silane. In order to introduce mercapto groups on the glass surface, the slides were covered with a 10% v/v (3-mercapto-2,2-dialkyl-propyl)trimethoxy silane solution in dry tetrahydrofuran (THF) and left in this solution for 12 h at room temperature under nitrogen. After the reaction was completed, the slides were washed with THF, with acetone and then dried under nitrogen in oven at 60⁰C for 30 minutes.

Example 2

2-cyanoprop-2-yl dithiobenzoate synthesis (1)

Benzoic acid (600 mg, 5.13 mmol) was dissolved in dry and degassed toluene. P₄Si₀ (550 mg, 1.2 mmol) and AIBΝ (2.5 gr , 15 mmol) were added to this solution, under stirring and nitrogen. The reaction flask was warmed at

110⁰C for 1 h. After cooling, the solution was poured in cold cyclohexane and then filtered. A crude sample, containing the cyanoisopropyl dithiobenzoate

(R_f 0.8 in CHCl₃), was recovered and purified by flash chromatography first on silica (CH₂Cl₂) and then on neutral alumina (petroleum benzine/CH₂Cl₂

9/1) with an overall yield of 40%. ¹H-NMR (δ ppm, CDCl₃): 7.93 (d, 2H, o-ArH), 7.56 (dd, lH, /?-ArH), 7.4 (dd, 2H, w-ArH), 1.95 (s,6H, 2 CH₃).

(1). Dureault, A.; Gnanou, Y.; Taton, D.; Destarac, M.; Leising, F. Angew. Chem. Int. Ed. 2003, 42, 2869 - 2872. Example 3

Procedure of derivation of glass slides with graft/block polymers

PoIy(DMA) graft homopolymerization. A polymerization solution was prepared by dissolving N₁N- dimethylacrylamide (DMA, 1.0 M), cyanoisopropyl dithiobenzoate (5.0 mM) and AIBN (1 mM) in degassed and dry DMF. The ratio [CTA]/[AIBN]= 5/1. The slides derivatized with mercapto groups (from example 1) were dipped in this solution, under nitrogen atmosphere. The reactor containing the slides was sealed and the polymerization carried out at 60⁰C in an oxygen free environment. After 48 hours the slides were cooled and washed with DMF at room temperature, with THF and with chloroform, then dried in a oven at 60⁰C for 10 minutes.

The free polymer in solution was characterized by GPC (in THF as eluent, in polystyrene units) and molecular weight values and polydispersity indexes were reported in Table 1. The surface was characterized by tensiometry measurements, listed in Table 2.

PoIy(DMA) copolymer graft polymerization. A polymerization solution was prepared by dissolving in DMF (degassed and dry) N,N-dimethylacrylamide (DMA, 0.9 M) and the N-acryloyloxy succinimide (0.1M), AIBΝ (1 mM) and cyanoisopropyl dithiobenzoato (5 mM), in a ratio [CTA]/[AIBΝ]= 5/1 were added to the monomer solution. . The slides derivatized with mercapto groups (from example 1) were dipped in this solution, under nitrogen atmosphere. The reactor containing the slides was sealed and the polymerization carried out at 60⁰C in an oxygen free environment. After 48 hours the slides were cooled and washed with DMF at room temperature, with THF and with chloroform, then dried in a oven at 6O⁰C for 10 minutes.

Poly(DMA-b-GMA) graft polymerization. In order to introduce the epoxide groups, a solution of glycidyl methacrylate (GMA, 0.7 M) and AIBN (6.5 mM) was prepared in dry and degassed DMF. The grafted poly(DMA)- SC(S)Ph slides were placed in a reactor filled with this monomer solution. The reactor was heated at 60⁰C and the polymerization was carried out for 16 hours. Cooled slides were washed and dried as reported above. Poly(DMA-b-NAS) graft polymerization. In order to introduce N- hydroxysuccinimide ester groups, a solution of N-acryloyloxy succinimide (NAS, 0.7 M) and AIBN (6.5 mM) was prepared in dry and degassed DMF. The grafted poly(DMA)-SC(S)Ph slides were placed in a reactor filled with this monomer solution. The reactor was heated at 60⁰C and the polymerization was carried out for 16 hours. Cooled slides were washed and dried as reported above.

Table 1. Molecular weights of polymers in solution

Example 4

Characterization of coatings obtained by tensiometry measurements

Tensiometry measurements were carried out to assess the presence of dithiobenzoic group at the end of polymer chains. For this reason, contact angles were measured either for dead chains of polyDMA grafted obtained by free radical polymerization or for growth in presence of dithiobenzoate

PhC(S)SC(CH₃)₂CN (living chains).

These measurements highlight a trend to produce an increase in contact angle values for polyDMA terminated by a dithiobenzoic group (phenyl group) with respect to the non-living polyDMA. The surface derivatized by a block polymer poly(DMA-b-GMA) was compared with a surface silanated by (3-glycidyloxypropyl) trimethoxysilane

(γ-GPS). Table 2. Contact angles measured by tensiometry

Static CA (°)/ σ (N)"

Entry Solvent/T [CTA]

Group ratio H₂O CH₂I₂ (°C) [AIBN]

1 Thiol (γ-MPS) THF/RT — 66.4/1.4 36.9/ 1.6 (17) (21)

2 g-pDMA-SC(S)Ph DMF/60 5/1 72.0/2.8 — (10)

3 g-pDMA DMF/60 — 56.0/1.8 — (17)

4 g-pGMA DMF/60 ~ 58.4/4.8 — (17)

5 g-p(DMA-i-GMA) DMF/60 5/1 53.0/1.0 — (12)

6 Glass — — 13.0/3.4 58.8/1.6 (17) (16) b. Number of measurements. Entry descriptions:

1) glass silanated by 3-mercaptopropyl trimethoxysilane (γ-MPS);

2) glass coated by poly(dimethylacrylamide) initiated by AIBN in presence of dithiobenzoic ester (CTA), grafted on the glass as reported in Example 1;

3) glass coated by poly(dimethylacrylamide) initiated by AIBN grafted on the glass, as reported in Example 1 ;

4) glass coated by poly(glycidyl methacrylate) initiated by AIBN in presence of (dithiobenzoic ester) CTA, grafted on the glass as reported in Example 1 ;

5) glass coated by poly(dimethylacrylamide) ended by the dithiobenzoic group -SC(S)Z and restarted in presence of AIBN and 3-glycidyl methacrylate; 6) glass functionalized by (3-glycidyloxypropyl) trimethoxysilane;

7) Glass with no treatment Example 5 Hybridization experiment Oligonucleotides deposition and immobilization. 23-mer oligonucletides bearing an amino group in 3' position, lyophilized an desalted, were dissolved in sodium phosphate buffer 150 mM at a concentration of 10 μM (pH 8.5). A 10 μM solution of this oligonucleotide was printed on coated slides to form 10 X 10 spots subarrays using a QArray² spotter from Genetix (Hampshire, UK). Printed slides, in an uncovered storage box, were placed in a sealed chamber, saturated with NaCl, and incubated overnight at room temperature.

Hybridization density. After spotting the slides with a 10 μM solution of 23 mer 5' amino-modified oligonucleotide as reported above, the residual reactive groups of the coating were blocked by dipping the printed slides in 5OmM ethanolamine/0.1% SDS/0.1 M Tris pH 9.0 at 50⁰C for 15 min. After discarding the blocking solution, the slides were rinsed two times with water and shaken for 15 min in 4X SSC/0.1 % SDS buffer, pre-warmed at 50⁰C and briefly rinsed with water.

An oligonucleotide, complementary to the one spotted on the surface, at a 1.0 μM concentration (2.5 μL per cm² of cover slip) was dissolved in the hybridization buffer (5X SSC, 0.1% SDS and 2% BSA), and immediately applied to microarrays. The slides, placed in a hybridization chamber, were transferred to a humidified incubator at a temperature of 65°C for 4 hours.

After hybridization, the slides were first washed with 4X SSC at room temperature to remove the cover slip, then with 2X SSC/0.1% SDS at hybridization temperature for 5 minutes. This operation was repeated two times and was followed by two washing steps with 0.2X SSC and 0.1X SSC for 1 minute at room temperature. The slides were scanned with a Scan Array Express fluorescence scanner from Packard Bioscience (USA) at 22% laser power and 64% PMT.

The row data of spot fluorescence intensity were converted to molecules/cm²by using a standard curve of fluorescence made from a serial dilution of known amounts of fluorescent oligonucleotide (in the 0.025 to 15 μM concentration range). The data reported are replicates of 400 spots.

Description of the Figure

Silanated surface by γ-MPS (mercapto groups): polymerization carried out starting from monomer and CTA in solution (AIBN as initiator).

Silanated and transesterificated surface: polymerization carried out starting from dithiobenzoic groups on surface and monomer and CTA in solution (AIBN as initiator)

Claims

1. Method for coating a plastic, glass or silicon oxide surface with block polymers, which comprises: I) derivatizing said surface with a thiol-bearing organosilane by reaction with compounds of general formula R¹R²R³Si-(CR⁴R⁵X₁SH wherein R',R² and R³ are independently -OCH₃, -OCH₂CH₃ or Cl, R⁴ is hydrogen and R⁵ is H or CH_3, n is an integer from 2 to 10;

II) attaching to said glass surface a first polymer block obtained through polymerization of a mixture containing: a) monomers of general formula (I) or (II): [CH₂=C(R¹OC(O)NR¹¹R¹¹¹] (I) [CH₂=C(R^π)C(O)OR^IV] (II) wherein R' is H or CH₃; R^π,R^iπ,R^IV can be independently H or a linear or branched Cl -C 12 alkyl chain, optionally interrupted by O, N or S₅ said alkyl chain(s) optionally bearing functional groups which are selected from epoxy, aldehyde, episulphide, aziridine, thioester, ester, N-hydroxy succinimide ester, pentafluorophenyl ester, dithioester, azalacton, lacton, lactide, anhydride, alogen, cyanate, isothiocyanate, double bonds, amino, thiol, hydrazine, methoxy, ethoxy, hydroxyl, trimethoxysilyl, -OR^V,

-C(O)-OR^VI wherein R^VI is a C1-C6 alkyl or heterocyclic ring optionally containing one or more nitrogen atoms and R^VI is a C1-C6 alkyl or phenyl containing one or more reactive substituents selected from -OH,

-SH₅-NH₂₅-CH=O, oxirane. b) dithiobenzoic derivatives of general formula:

/ S- R

C

\ wherein R is selected from (Cl -C 12) alkyl or substituted alkyl, aliphatic ring or 5 to 10 membered (hetero)aromatic ring optionally substituted; alkylthio, alkoxy, dialkylamino optionally substituted; organometallic compound; Z is selected from hydrogen, chlorine, (Cl-C12)alkyl, 5 to 10 membered

(hetero)aryl optionally substituted; alkylthio, alkoxycarbonyl, arylossicarbonyl, carboxy; acyloxy or carbamoyl, optionally substituted; cyano; dialkyl or diaryl phosphonate or phoshinate; c) a radical initiator, prefereably AIBN (α,α'-azo-bis-isobutyronitryle) III) whashing the coated surface with an organic solvent selected from dimethylformamide (DMF), tetrahydrofuran (THF), chloroform (CHCl₃ ) or mixtures thereof;

IV) optionally, elongating the polymer with further blocks obtained by polymerization of the mixtures according to step II. 2. Method according to claim 1, wherein the thiol-bearing organosilane is 3-mercaptopropyltrimethoxysilane.

3. Method according to claim 1, wherein the monomers are selected from the group comprising N.N-dimethylacrylamide, glicidylmethacrylate, Ν-acryloylsuccinimide,

2-vinyl-4,4'-dimethylazalactone. 4. Method according to claim 1, wherein the dithiobenzoic derivative is 2-cyanoprop-2-yl dithiobenzoate. 5. Method acording to claim 1, wherein the coating polymer has formula 1 :

for W-acryloyloxy succinimide

for glycidyl methacrylate wherein the first and the second block are homopolymers, the molecular weight of the polymer ranging from 10³ to 10⁶.

6. Method acording to claim 1, wherein the coating polymer has formula 2:

R= H, R¹= for Λ/-acryloyloxy succinimide

R= CH₃, R ^'= > *-^/-~~ for glycidyl methacrylate

wherein the first block is a poly(DMA) homopolymer and y=l-99, z=99-l mole%, the molecular weight of the polymer ranging from 10³ to 10⁶.

7. Method acording to claim 1, wherein the coating polymer has formula 3:

3.

R= H, R¹= for W-acryloyloxy succinimide

for glycidyl methacrylate

wherein the graft polymer is a living random copolymer, X= 1-99 mole%, Z=99-l%, the molecular weight of the polymer ranging from 10³ to 10⁶.

8. A method for the immobilization of biological molecules on the surface of a plastic, glass or silicon support, which comprises coating said surface according to claims 1-8, and subsequently contacting the coated surface with said biological molecules in conditions allowing their covalent binding to the polymers.

9. A plastic, glass or silicon-oxide support for the immobilization of biological molecules, having at least one surface coated according to claims 1-8.

10. A support according to claim 9, which is in form of a multiwell plate, bead, test tube, flask, microscope slide, silicon wafer, silicon membrane or bead.

1 1 The use of a support according to claim 10, for the immobilization of biological molecules.

12. The use of a support according to claim 10, for the manufacture of microarrays for solid phase hybridization techniques.