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WO2006128644A1 - Revetement polymere et fonctionnalisation de surfaces solides - Google Patents

Revetement polymere et fonctionnalisation de surfaces solides Download PDF

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Publication number
WO2006128644A1
WO2006128644A1 PCT/EP2006/005047 EP2006005047W WO2006128644A1 WO 2006128644 A1 WO2006128644 A1 WO 2006128644A1 EP 2006005047 W EP2006005047 W EP 2006005047W WO 2006128644 A1 WO2006128644 A1 WO 2006128644A1
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WO
WIPO (PCT)
Prior art keywords
polymer
alkyl
glass
coating
silicon
Prior art date
Application number
PCT/EP2006/005047
Other languages
English (en)
Inventor
Giovanna Pirri
Marcella Chiari
Original Assignee
Giovanna Pirri
Marcella Chiari
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Giovanna Pirri, Marcella Chiari filed Critical Giovanna Pirri
Publication of WO2006128644A1 publication Critical patent/WO2006128644A1/fr

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54353—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals with ligand attached to the carrier via a chemical coupling agent
    • C—CHEMISTRY; METALLURGY
    • C03—GLASS; MINERAL OR SLAG WOOL
    • C03C—CHEMICAL COMPOSITION OF GLASSES, GLAZES OR VITREOUS ENAMELS; SURFACE TREATMENT OF GLASS; SURFACE TREATMENT OF FIBRES OR FILAMENTS MADE FROM GLASS, MINERALS OR SLAGS; JOINING GLASS TO GLASS OR OTHER MATERIALS
    • C03C17/00—Surface treatment of glass, not in the form of fibres or filaments, by coating
    • C03C17/28—Surface treatment of glass, not in the form of fibres or filaments, by coating with organic material
    • C03C17/30—Surface treatment of glass, not in the form of fibres or filaments, by coating with organic material with silicon-containing compounds
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
    • G01N33/545—Synthetic resin

Definitions

  • 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.
  • block polymers brushes are produced by RAFT polymerization to generate substrates for microarray s. Background of the invention
  • molecules carrying -C(S)SR groups are chain transfer agents for radical polymerization of the type described in WO 98/01478.
  • 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 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.
  • the invention provides a method for coating a plastic, glass or silicon oxide surface with "brush"block polymers, which comprises:
  • 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 ( ⁇ , ⁇ '-azo-bis-isobutyronitryle)
  • step IV optionally, elongating the polymer with further blocks obtained by polymerization of the mixtures according to step II.
  • the thiol-bearing organosilane is 3- mercaptopropyltrimethoxysilane
  • the dithiobenzoic derivative is 2-cyanoprop- 2-yl dithiobenzoate
  • the monomers are selected from the group comprising N,N-dimethylacrylamide, glicidylmethacrylate, N-acryloylsuccinimide, 2-vinyl-4,4'-dimethylazalactone.
  • the resultant polymeric coating has the formula 1 :
  • the resultant polymeric coating has the formula 2
  • the resultant polymeric coating has the formula 3
  • 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.
  • 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.
  • 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 improvement obtained by this invention concerns the way surface living chains are generated.
  • 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.
  • 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.
  • 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.
  • the SH group initiates polymer chains that grow mediated by ZC(S)SR groups
  • 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.
  • Benzoic acid 600 mg, 5.13 mmol was dissolved in dry and degassed toluene.
  • P 4 Si 0 550 mg, 1.2 mmol
  • AIB ⁇ 2.5 gr , 15 mmol
  • 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 60 0 C 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 60 0 C 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.
  • 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 60 0 C 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 6O 0 C 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 60 0 C and the polymerization was carried out for 16 hours. Cooled slides were washed and dried as reported above. Poly(DMA-b-NAS) graft polymerization.
  • GMA glycidyl methacrylate
  • AIBN 6.5 mM
  • 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 60 0 C and the polymerization was carried out for 16 hours. Cooled slides were washed and dried as reported above.
  • PhC(S)SC(CH 3 ) 2 CN living chains
  • Hybridization density After spotting the slides with a 10 ⁇ M 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 50 0 C 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 50 0 C and briefly rinsed with water.
  • oligonucleotide complementary to the one spotted on the surface, at a 1.0 ⁇ M concentration (2.5 ⁇ L per cm 2 of cover slip) was dissolved in the hybridization buffer (5X SSC, 0.1% SDS and 2% BSA), and immediately applied to microarrays.
  • 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/cm 2 by using a standard curve of fluorescence made from a serial dilution of known amounts of fluorescent oligonucleotide (in the 0.025 to 15 ⁇ M concentration range). The data reported are replicates of 400 spots.
  • Silanated surface by ⁇ -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)

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Food Science & Technology (AREA)
  • General Physics & Mathematics (AREA)
  • Cell Biology (AREA)
  • Biotechnology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Pathology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Geochemistry & Mineralogy (AREA)
  • Materials Engineering (AREA)
  • Organic Chemistry (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)

Abstract

L'invention concerne un procédé destiné à revêtir du verre, de la silice, de l'oxyde de silicium ou d'autres matériaux avec des polymères en brosse obtenus par polymérisation RAFT. Les surfaces revêtues sont utilisées pour immobiliser des biomolécules, notamment dans des applications associées à la technologie des microréseaux.
PCT/EP2006/005047 2005-05-30 2006-05-26 Revetement polymere et fonctionnalisation de surfaces solides WO2006128644A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP05011600 2005-05-30
EP05011600.3 2005-05-30

Publications (1)

Publication Number Publication Date
WO2006128644A1 true WO2006128644A1 (fr) 2006-12-07

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102030482A (zh) * 2010-10-13 2011-04-27 中国科学院化学研究所 一种纳米级图案化的二元聚合物刷的制备方法
CN102432745A (zh) * 2011-11-28 2012-05-02 苏州大学 一种含有环氧和叔胺双官能团的活性共聚物的制备方法
CN114907606A (zh) * 2022-06-21 2022-08-16 华东理工大学 一种用于活性固载蛋白的聚合物刷表面改性方法

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998001478A1 (fr) * 1996-07-10 1998-01-15 E.I. Du Pont De Nemours And Company Polymerisation presentant des caracteristiques vivantes
WO2000043539A2 (fr) * 1999-01-25 2000-07-27 Biochip Technologies Gmbh Immobilisation de molecules sur des surfaces par des brosses polymeres
US20030108879A1 (en) * 2001-01-10 2003-06-12 Symyx Technologies, Inc. Polymer brushes for immobilizing molecules to a surface or substrate having improved stability
WO2003083040A2 (fr) * 2001-07-30 2003-10-09 Sts Biopolymers, Inc. Matrices polymeres de greffe

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998001478A1 (fr) * 1996-07-10 1998-01-15 E.I. Du Pont De Nemours And Company Polymerisation presentant des caracteristiques vivantes
WO2000043539A2 (fr) * 1999-01-25 2000-07-27 Biochip Technologies Gmbh Immobilisation de molecules sur des surfaces par des brosses polymeres
EP1144677B1 (fr) * 1999-01-25 2006-07-26 Micronas Holding GmbH Immobilisation de molecules sur des surfaces par des brosses polymeres
US20030108879A1 (en) * 2001-01-10 2003-06-12 Symyx Technologies, Inc. Polymer brushes for immobilizing molecules to a surface or substrate having improved stability
WO2003083040A2 (fr) * 2001-07-30 2003-10-09 Sts Biopolymers, Inc. Matrices polymeres de greffe

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
PRUCKER O ET AL: "Polymer Layers through Self-Assembled Monolayers of Initiators", LANGMUIR, AMERICAN CHEMICAL SOCIETY, NEW YORK, NY, US, vol. 14, no. 24, 1998, pages 6893 - 6898, XP002157970, ISSN: 0743-7463 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102030482A (zh) * 2010-10-13 2011-04-27 中国科学院化学研究所 一种纳米级图案化的二元聚合物刷的制备方法
CN102432745A (zh) * 2011-11-28 2012-05-02 苏州大学 一种含有环氧和叔胺双官能团的活性共聚物的制备方法
CN102432745B (zh) * 2011-11-28 2013-10-09 苏州大学 一种含有环氧和叔胺双官能团的活性共聚物的制备方法
CN114907606A (zh) * 2022-06-21 2022-08-16 华东理工大学 一种用于活性固载蛋白的聚合物刷表面改性方法
CN114907606B (zh) * 2022-06-21 2023-11-17 华东理工大学 一种用于活性固载蛋白的聚合物刷表面改性方法

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