EP1732963A2 - Hochmolekulargewichtpolymer auf basis von n,n-dimethylacrylamid - Google Patents
Hochmolekulargewichtpolymer auf basis von n,n-dimethylacrylamidInfo
- Publication number
- EP1732963A2 EP1732963A2 EP05739585A EP05739585A EP1732963A2 EP 1732963 A2 EP1732963 A2 EP 1732963A2 EP 05739585 A EP05739585 A EP 05739585A EP 05739585 A EP05739585 A EP 05739585A EP 1732963 A2 EP1732963 A2 EP 1732963A2
- Authority
- EP
- European Patent Office
- Prior art keywords
- polymer
- solid support
- molecules
- chosen
- polymers
- Prior art date
- Legal status (The legal status 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 status listed.)
- Withdrawn
Links
- 229940088644 n,n-dimethylacrylamide Drugs 0.000 title description 9
- YLGYACDQVQQZSW-UHFFFAOYSA-N n,n-dimethylprop-2-enamide Chemical compound CN(C)C(=O)C=C YLGYACDQVQQZSW-UHFFFAOYSA-N 0.000 title description 9
- 229920006158 high molecular weight polymer Polymers 0.000 title 1
- 229920000642 polymer Polymers 0.000 claims abstract description 95
- 239000007787 solid Substances 0.000 claims abstract description 56
- 239000000178 monomer Substances 0.000 claims abstract description 35
- 238000000034 method Methods 0.000 claims abstract description 33
- 238000006243 chemical reaction Methods 0.000 claims abstract description 28
- 239000000126 substance Substances 0.000 claims abstract description 16
- 239000000523 sample Substances 0.000 claims description 37
- 239000000243 solution Substances 0.000 claims description 33
- 229920001577 copolymer Polymers 0.000 claims description 26
- 102000004169 proteins and genes Human genes 0.000 claims description 22
- 108090000623 proteins and genes Proteins 0.000 claims description 21
- 239000002904 solvent Substances 0.000 claims description 20
- 238000002360 preparation method Methods 0.000 claims description 18
- 125000001424 substituent group Chemical group 0.000 claims description 14
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 13
- 229910052757 nitrogen Inorganic materials 0.000 claims description 13
- 108020004707 nucleic acids Proteins 0.000 claims description 13
- 102000039446 nucleic acids Human genes 0.000 claims description 13
- 150000007523 nucleic acids Chemical class 0.000 claims description 13
- HEMHJVSKTPXQMS-UHFFFAOYSA-M sodium hydroxide Substances [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 13
- HRPVXLWXLXDGHG-UHFFFAOYSA-N Acrylamide Chemical compound NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 claims description 10
- 102000004190 Enzymes Human genes 0.000 claims description 10
- 108090000790 Enzymes Proteins 0.000 claims description 10
- 108091034117 Oligonucleotide Proteins 0.000 claims description 10
- 238000001179 sorption measurement Methods 0.000 claims description 10
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 claims description 9
- 230000000295 complement effect Effects 0.000 claims description 9
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 8
- 125000000391 vinyl group Chemical group [H]C([*])=C([H])[H] 0.000 claims description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 8
- 238000007334 copolymerization reaction Methods 0.000 claims description 7
- -1 antibodies Proteins 0.000 claims description 6
- 239000011521 glass Substances 0.000 claims description 6
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- 230000008929 regeneration Effects 0.000 claims description 6
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- 102000036639 antigens Human genes 0.000 claims description 3
- 108091007433 antigens Proteins 0.000 claims description 3
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 claims description 3
- 150000004676 glycans Chemical class 0.000 claims description 3
- 239000007788 liquid Substances 0.000 claims description 3
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 claims description 3
- 229920001542 oligosaccharide Polymers 0.000 claims description 3
- 150000002482 oligosaccharides Chemical class 0.000 claims description 3
- 229920001282 polysaccharide Polymers 0.000 claims description 3
- 239000005017 polysaccharide Substances 0.000 claims description 3
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 claims description 2
- 150000003926 acrylamides Chemical class 0.000 claims description 2
- 125000000217 alkyl group Chemical group 0.000 claims description 2
- 238000003795 desorption Methods 0.000 claims description 2
- 229910052700 potassium Inorganic materials 0.000 claims description 2
- 229920002554 vinyl polymer Polymers 0.000 claims description 2
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 claims 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 claims 1
- 239000000908 ammonium hydroxide Substances 0.000 claims 1
- 125000004123 n-propyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])* 0.000 claims 1
- KWYUFKZDYYNOTN-UHFFFAOYSA-M potassium hydroxide Substances [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 claims 1
- 229910052708 sodium Inorganic materials 0.000 claims 1
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 20
- 102000004142 Trypsin Human genes 0.000 description 18
- 108090000631 Trypsin Proteins 0.000 description 18
- 239000012588 trypsin Substances 0.000 description 18
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 12
- 229960002685 biotin Drugs 0.000 description 10
- 235000020958 biotin Nutrition 0.000 description 10
- 239000011616 biotin Substances 0.000 description 10
- NIXOWILDQLNWCW-UHFFFAOYSA-N acrylic acid group Chemical group C(C=C)(=O)O NIXOWILDQLNWCW-UHFFFAOYSA-N 0.000 description 9
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 7
- 230000008569 process Effects 0.000 description 7
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 6
- 239000000872 buffer Substances 0.000 description 6
- CVSVTCORWBXHQV-UHFFFAOYSA-N creatine Chemical compound NC(=[NH2+])N(C)CC([O-])=O CVSVTCORWBXHQV-UHFFFAOYSA-N 0.000 description 6
- 230000029087 digestion Effects 0.000 description 6
- SMZOUWXMTYCWNB-UHFFFAOYSA-N 2-(2-methoxy-5-methylphenyl)ethanamine Chemical compound COC1=CC=C(C)C=C1CCN SMZOUWXMTYCWNB-UHFFFAOYSA-N 0.000 description 5
- 108020004414 DNA Proteins 0.000 description 5
- 230000015572 biosynthetic process Effects 0.000 description 5
- 238000001514 detection method Methods 0.000 description 5
- 239000011541 reaction mixture Substances 0.000 description 5
- 238000003786 synthesis reaction Methods 0.000 description 5
- 238000000018 DNA microarray Methods 0.000 description 4
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 4
- 238000013459 approach Methods 0.000 description 4
- 238000007306 functionalization reaction Methods 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 239000011780 sodium chloride Substances 0.000 description 4
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 3
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- WHNWPMSKXPGLAX-UHFFFAOYSA-N N-Vinyl-2-pyrrolidone Chemical compound C=CN1CCCC1=O WHNWPMSKXPGLAX-UHFFFAOYSA-N 0.000 description 3
- 108010090804 Streptavidin Proteins 0.000 description 3
- ROOXNKNUYICQNP-UHFFFAOYSA-N ammonium persulfate Chemical compound [NH4+].[NH4+].[O-]S(=O)(=O)OOS([O-])(=O)=O ROOXNKNUYICQNP-UHFFFAOYSA-N 0.000 description 3
- 239000007822 coupling agent Substances 0.000 description 3
- 229960003624 creatine Drugs 0.000 description 3
- 239000006046 creatine Substances 0.000 description 3
- VYFYYTLLBUKUHU-UHFFFAOYSA-N dopamine Chemical compound NCCC1=CC=C(O)C(O)=C1 VYFYYTLLBUKUHU-UHFFFAOYSA-N 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
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- 125000003396 thiol group Chemical group [H]S* 0.000 description 3
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 2
- SXGZJKUKBWWHRA-UHFFFAOYSA-N 2-(N-morpholiniumyl)ethanesulfonate Chemical compound [O-]S(=O)(=O)CC[NH+]1CCOCC1 SXGZJKUKBWWHRA-UHFFFAOYSA-N 0.000 description 2
- AIBUFXJASFNIGU-UHFFFAOYSA-N 2-(dichloromethylidene)oxolane Chemical compound ClC(Cl)=C1CCCO1 AIBUFXJASFNIGU-UHFFFAOYSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- QOSSAOTZNIDXMA-UHFFFAOYSA-N Dicylcohexylcarbodiimide Chemical compound C1CCCCC1N=C=NC1CCCCC1 QOSSAOTZNIDXMA-UHFFFAOYSA-N 0.000 description 2
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
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- HRZFUMHJMZEROT-UHFFFAOYSA-L sodium disulfite Chemical compound [Na+].[Na+].[O-]S(=O)S([O-])(=O)=O HRZFUMHJMZEROT-UHFFFAOYSA-L 0.000 description 2
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- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 2
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- 125000004178 (C1-C4) alkyl group Chemical group 0.000 description 1
- KZMAWJRXKGLWGS-UHFFFAOYSA-N 2-chloro-n-[4-(4-methoxyphenyl)-1,3-thiazol-2-yl]-n-(3-methoxypropyl)acetamide Chemical compound S1C(N(C(=O)CCl)CCCOC)=NC(C=2C=CC(OC)=CC=2)=C1 KZMAWJRXKGLWGS-UHFFFAOYSA-N 0.000 description 1
- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 description 1
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- 239000004033 plastic Substances 0.000 description 1
- 229920000729 poly(L-lysine) polymer Polymers 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- NHARPDSAXCBDDR-UHFFFAOYSA-N propyl 2-methylprop-2-enoate Chemical compound CCCOC(=O)C(C)=C NHARPDSAXCBDDR-UHFFFAOYSA-N 0.000 description 1
- 125000001436 propyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 108060006633 protein kinase Proteins 0.000 description 1
- 238000000425 proton nuclear magnetic resonance spectrum Methods 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 238000010526 radical polymerization reaction Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 238000010517 secondary reaction Methods 0.000 description 1
- 230000028043 self proteolysis Effects 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 229910052710 silicon Inorganic materials 0.000 description 1
- 239000010703 silicon Substances 0.000 description 1
- 229940001584 sodium metabisulfite Drugs 0.000 description 1
- 239000004296 sodium metabisulphite Substances 0.000 description 1
- 229910000679 solder Inorganic materials 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 229920001059 synthetic polymer Polymers 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- JOYRKODLDBILNP-UHFFFAOYSA-N urethane group Chemical group NC(=O)OCC JOYRKODLDBILNP-UHFFFAOYSA-N 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F265/00—Macromolecular compounds obtained by polymerising monomers on to polymers of unsaturated monocarboxylic acids or derivatives thereof as defined in group C08F20/00
- C08F265/10—Macromolecular compounds obtained by polymerising monomers on to polymers of unsaturated monocarboxylic acids or derivatives thereof as defined in group C08F20/00 on to polymers of amides or imides
Definitions
- the present invention relates to high molecular weight statistical polymers based on N, N-dialkylacrylamide and monomers functionalized laterally by molecules of interest.
- the present invention relates to N, N-DIMETHYLACRYLAMIDE AND MONOMERS FUNCTIONALIZED BY PROBE MOLECULES AND USES THEREOF. , when used for the preparation of solid supports comprising at least one surface on which said polymers are adsorbed.
- the present invention also relates to the solid supports thus modified and to their process for obtaining, as well as to their various uses, in particular for the immobilization of molecules of interest or the carrying out of chemical, biochemical or biological reactions.
- the biological molecules are immobilized on the support via a bond covalent so that the molecules do not detach from the support during the use of the chips, especially during the washing steps.
- this immobilization is generally carried out in two distinct stages: - a first stage of functionalization of the supports which consists in a chemical modification of their surface by grafting of synthetic molecules
- Another approach consists in immobilizing ON via the copolymerization of ON functionalized by acrylamide with a crosslinked polyacrylamide (Rehman FN, et al., Nucleic Acid Research, 1999, 27 (2), 649-655).
- the gels can be prepared by the techniques of conventional polymerization and well known to those skilled in the art such as radical polymerization.
- the surfaces thus prepared have a high density of probes stably attached.
- the surfaces of these supports on the other hand, have the drawback of being permanently modified and therefore cannot be easily regenerated. It has also already been proposed, in particular by Southern E. et al, Nat.
- 5,695,936 describes a method of detecting a nucleotide sequence of interest using a nucleotide probe labeled with a tracer.
- a reagent consisting of a copolymer based on N-vinylpyrrolidone or maleic anhydride, having a molecular mass of between 5000 and 400,000 g / mol and comprising lateral substituents of oligonucleotide nature.
- the immobilization of this copolymer on the surface of the support is carried out in a fairly complicated manner via a complex between the solid support, a probe labeled with a tracer, a reagent and a target nucleic acid.
- US Patent 5,453,461 also describes biologically active polymers of formula P- (A) q in which P represents a linear, branched or crosslinked polymer formed for example from acrylic monomers, A represents a biologically active section and can for example be biotin or an ON comprising from 1 to 80 nucleotide units and q is an integer equal to 1 or 2. Depending on the structure of these polymers, A is always positioned at the end of the polymer chain and not sideways.
- 5,723,344 describes the preparation of copolymers formed from an N-vinylpyrrolidone monomer and a second monomer comprising a reactive function allowing the attachment by covalent bond of a biological ligand capable of forming a complex with a target molecule such as for example an antigen / antibody complex, polynucleotide / polynucleotide, polynucleotide / nucleic acid, antibody / hapten, hormone / receptor, as well as their capacity to adsorb on the surface of solid supports.
- Such copolymers have a molecular weight generally between 1000 and 500,000, preferably between 10,000 and 250,000 and contain 25 to 70% of units derived from N-vinylpyrrolidone.
- the supports thus prepared can be used for the immobilization of target molecules in solution, in particular in methods for detecting and assaying nucleotide sequences.
- the layers of polymers adsorbed on such supports nevertheless have the disadvantage of being very unstable, due to the relatively small molecular size of these copolymers and the high percentage of lateral substituents (30 to 75%).
- American patent US Pat. No. 6,692,914 also describes sensors made up of a substrate, the surface of which comprises a plurality of chains of segmented polymers (block polymers) in the form of a brush comprising a water-soluble or water-dispersible segment and at least one chosen probe. among biological molecules, linked to said segment.
- trypsin can be attached to the surface of reactors and undergoes, in this case, very little autolysis compared to trypsin in solution.
- the fixation of the trypsin is carried out on quartz or silica microbeads which are then inserted into microcapillaries which serve as reactors for trypsin.
- the fixation of trypsin is carried out by means of a Schiff base reduction reaction between a residual primary amino group of trypsin and an aldehyde group carried by the surface (see in particular Muilin C, in “Methods in Enzymology", Colowick, SP, Caplan NO Eds., Académie Press, New York, 1987, Vol 136).
- Such methods are however long and laborious and are not entirely satisfactory.
- after immobilization of the trypsin on the surface of the supports there remain parts of the surface which have not been modified and which it is necessary to saturate in order to minimize the non-specific reactions of adsorption of molecules. in solution (peptides, proteins for example).
- the present invention firstly relates to a water-soluble statistical polymer (not segmented) comprising probe molecules as lateral substituents, characterized by the fact: - that it results from the copolymerization of a monomer A of
- the water-soluble statistical polymers in accordance with the present invention and as defined above are capable of physically adsorbing stably to the surface of solid supports, and thus make it possible to lead to solid supports comprising at least one surface functionalized by probe molecules such as for example nucleic acid or protein chips on which it is then possible to immobilize target molecules of interest while minimizing the adsorption of other molecules in solution.
- probe molecules such as for example nucleic acid or protein chips
- the polymers in accordance with the invention do not lead to the formation of a brush of polymers adsorbed by one of their ends on the surface of the support.
- the term “small organic molecule” is understood to mean organic molecules preferably having a molecular weight less than or equal to 1000 g / mole.
- said polymer has a molecular mass greater than or equal to 1.10 g / mole. The inventors have indeed found that the stability of surfaces functionalized by such polymers was even greater when the polymers used had such a molecular weight.
- N, N- [dialkyl (C] -C 4 ) acrylamide] constituting the copolymers in accordance with the invention, mention may be made of methyl, ethyl, w-propyl and zz-butyl groups, the methyl group being very particularly preferred.
- monomer A the
- N, N-dimethylacrylamide is therefore very particularly preferred.
- monomers B mention may in particular be made of vinyl monomers and acrylamides such as acrylic and methacrylic acids, N-aminoalkyl acrylamides, N-aminoalkyl methacrylamides, aminoalkyl acrylates and aminoalkyl methacrylates, in which the alkyl group may represent for example the ethyl, propyl, butyl, pentyl, or hexyl group.
- the probe molecules present in the polymer as lateral substituents are preferably chosen from active biological molecules having a limited lifespan, such as enzymes.
- the degree of incorporation of the probe molecules is between 1 and 20% by number relative to the total number of monomeric units of monomer A of N, N- [dialkyl (Cj -
- the polymers in accordance with the invention and as described above can be prepared according to conventional copolymerization techniques well known to those skilled in the art, such as, for example, radical copolymerization, by reacting the monomers A as defined above. above with monomers Bl or B2 as defined above in a suitable solvent such as water, in the presence of a polymerization activator such as for example a red / ox couple such as the ammonium persulfate / sodium metabisulfite couple.
- a polymerization activator such as for example a red / ox couple such as the ammonium persulfate / sodium metabisulfite couple.
- the preparation of the monomers B2 can also be carried out according to conventional methods and well known to those skilled in the art, for example by reacting, under anhydrous conditions in a suitable organic solvent such as dichloromethylene, tetrahydrofuran (THF), dimethylformamide ( DMF) or dimethylsulfoxide (DMSO), a probe molecule containing a carboxylic group (biotin for example) with the vinyl group of a copolymerized monomer such as for example N-aminopropyl methacrylamide, in the presence of a coupling agent such as for example the dicyclohexyl carbodiimide; the reaction of a carboxyl group and of an ine group being in fact well known for a long time in the literature, in particular in the article by Khorana HG et al, Chem.
- a suitable organic solvent such as dichloromethylene, tetrahydrofuran (THF), dimethylformamide ( DMF) or dimethylsulfoxide (DM
- trypsin modified with a vinyl group can be prepared according to the method described by Plate NA et al, Polymer Science USSR, 1989, 31, 216-219.
- the polymers according to the invention can be characterized by classical analytical techniques such as refractive index diffusion exclusion chromatography and detection by light scattering or by viscosimetry in order to collect information on the size of the polymers or even by magnetic resonance techniques nuclear (NMR) to gather information on their structure.
- NMR magnetic resonance techniques nuclear
- the present invention also relates to the use of at least one water-soluble statistical polymer as described above for the preparation of a solid support comprising at least one surface functionalized by probe molecules, and in particular for the preparation of chips proteins or nucleic acids and in particular DNA.
- a process for the preparation of a solid support comprising at least one surface functionalized with a water-soluble statistical polymer in accordance with the invention characterized in that it comprises the following steps: '' a solid support comprising at least one surface to be functionalized, with a solution in a compatible solvent of at least one water-soluble random polymer in accordance with the invention and as defined above, - incubation of said surface with said polymer solution for a time sufficient for the adsorption of the polymer on the surface of the solid support, - rinsing of the support solid using a polymer-free solvent to obtain a solid support comprising at least one surface functionalized by an adsorbed layer of polymers.
- the solid supports which can be functionalized according to this method are preferably chosen from the supports comprising at least one surface of the silica type and its derivatives such as glass and quartz, or any other material covered with silica, glass or quartz.
- the term “compatible solvent” is understood to mean any solvent allowing the dissolution of the polymer, not causing any alteration of the probe molecules which it comprises as lateral substituent and for which the surface of the solid support to be functionalized will have an affinity lower than that which it will have for the polymers confused with the invention.
- the polymers in solution may spontaneously adsorb on the surface of the support.
- the first factor is the level of interaction between the surface of the solid support and the monomeric units constituting the polymer, that is to say the amount of energy necessary for the adsorption of the polymer (forces of attraction).
- the second factor is the reduction in the conformational states of the polymer on the surface of the solid support, that is to say the reduction in chain entropy. This is due to the impenetrability of the surface for the monomeric units of the polymer and corresponds to the energy which tends to repel the polymer from the surface of the solid support (repulsive forces). The thickness of the polymer layer adsorbed on the surface of the solid support will be greater the greater the repulsive forces.
- the solidity of the adsorption of the polymer layer on the surface of the solid support will therefore depend on the ratio between the forces of attraction and the repulsive forces.
- the duration of the incubation of the solid support with the polymers in solution is preferably between 1 and 60 minutes approximately, and even more preferably between 5 and 40 minutes approximately.
- the quantity of polymers in solution is between approximately 0.001% and 5% by weight relative to the volume of the polymer solution and even more preferably between approximately 0.1% and 2% ( weight / volume).
- the solvents used to rinse the support after adsorption of the polymers and before their use are preferably chosen from the solvents described above and used to make the polymer solution.
- the solvent used to rinse the support is identical to that used to make the polymer solution.
- the present invention also relates to the solid supports obtained by implementing the preparation process in accordance with the invention, said supports being characterized by the fact that they comprise at least one surface functionalized by an adsorbed layer of water-soluble carrier polymers of probe molecules as lateral substituents as defined above.
- Such supports can in particular be in the form of studs, channels, capillaries, reactors or reaction chambers such as, for example, the devices usually used to carry out enzymatic reactions (enzymatic digestion).
- the adsorption of the polymers on the surface of the solid support is carried out via the monomers A of N, N- [dialkyl (C] -C 4 ) a_crylamide].
- These solid supports are in particular protein chips and in particular enzyme, peptide or polypeptide chips, acid chips nucleic acid and in particular DNA or RNA, oligosaccharide or polysaccharide chips.
- the thickness of the polymer layer is generally between 1 and 100 nm.
- the thickness of the polymer layers can be measured for example by elipsometry or using more sophisticated techniques using evanescent waves (Allain C. and ⁇ /., Phys. Rev. Leti, 1982, 49, 1694) or the neutron scattering (Barnett K. et al, "The effects of Polymers on Dispersion Stability", Tadros,
- biochemical refers to reactions, processes and protocols which involve at least one substrate and its enzyme.
- biochemical reaction can be used within the framework of the present invention to designate the methods of amplification of nucleic acids such as polymerase chain reactions (PCR), the determination of a genotype such as microsequencing, or alternatively nucleic acid sequencing.
- PCR polymerase chain reactions
- biochemical also includes other types of enzyme-catalyzed reactions such as digestion of proteins by proteases, cleavage of DNA by nucleases, phosphorylation of molecules by kinases, isomerization of molecules by isomerases, the conversion of dopamine to norepinephrine by dopamine hydrolase, etc.
- chemical is used to denote reactions, methods and processes in which there is at least one step involving a reaction which is not catalyzed by an enzyme.
- the term “chemical” can be used to designate syntheses of organic or inorganic molecules, degradation reactions in which one of the steps is not catalyzed by an enzyme as well as the chemical reactions catalyzed by the ultra violet radiation.
- the term “biological” is used to designate reactions of which at least one of the stages involves a living organism such as a cell, a culture of cells, a cluster of cells. adherent, a mono- or multicellular organism, parts of tissues or organs.
- the term “biological” used in the context of the present invention therefore includes mono- or pluricellular eukaryotic organisms, as well as prokaryotic organisms such as bacteria and viruses.
- the subject of the invention is also a method for immobilizing target molecules and for screening complementary target molecules in solution or for carrying out biochemical, chemical or biological reactions on a solid support, characterized in that it comprises at least the following steps: a) bringing a solid support into contact with at least one surface functionalized by an adsorbed layer of water-soluble random polymers carrying probe molecules as lateral substituents with a liquid sample capable of containing target molecules, during sufficient time to carry out said immobilization or said reaction, b) desorption of the polymer layer from the surface of the support by rinsing the support with an alkaline solution or a solvent, c) optionally, repeating steps a) and b) above.
- the alkaline solutions which can be used to carry out the stage of regeneration of the support can be chosen from solutions of sodium hydroxide, potassium or ammonium.
- the solvents which can be used to carry out the stage of regeneration of the support are preferably chosen from solvents for which the adsorbed polymers have more affinity than for the surface on which they have previously been adsorbed.
- step b) of regeneration of the support is carried out using a sodium hydroxide solution.
- the invention also comprises other arrangements which will emerge from the description which follows, which refers to an example of the preparation of a copolymer of N, N-dimethylacrylamide and acrylic acid comprising trypsin as a probe molecule, to an example of the preparation of a glass capillary comprising a surface functionalized by such a polymer and to the use of such a capillary to carry out a creatine digestion reaction, to an example of preparation of a copolymer of N, N-dimethylacrylamide and biotin functionalized by a vinyl group, to an example of preparation of a solid support comprising a surface modified by such a copolymer and its use for the immobilization of target molecules biotinylated by through streptavidin. It should be understood, however, that these examples are given solely by way of illustration of the subject of the invention, of which they do not in any way constitute a limitation.
- the mixture is then stirred for 20 minutes under strong bubbling with nitrogen in order to remove the dissolved oxygen.
- the reaction mixture is then brought to a temperature of 32 ° C. using a water bath. 1% by mole of ammonium peroxydi sulphate and 0.1% by mole of sodium metabisulphite relative to the amount of monomers are added in order to initiate the copolymerization reaction.
- the reaction is carried out for 1 hour and 30 minutes, the mixture becoming highly viscous after approximately 45 minutes.
- the reaction medium is then diluted to 500 ml with MilliQ water, acidified to a pH of 3 with 3 N hydrochloric acid, then ultrafiltered on membranes of 1,000,000 g / mol and finally lyophilized.
- the product of the reaction was characterized by proton NMR and acid-base assay to determine the rate of incorporation of acrylic acid.
- a copolymer of P (DMA-s-AA) is obtained in which for an initial fraction of
- the grafting protocol used is based on the EDC / NHS couple (ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride /
- the reaction medium is stirred at room temperature for 15 minutes then filtered on a Microcon® membrane having a threshold of 100,000 g / mole cut off to remove unreacted NHS and EDC.
- the reaction mixture is then centrifuged at 10,000 rpm for 1 hour and 15 minutes.
- the reaction mixture is then heated at a temperature of 4 ° C followed by addition of 2.4 mg (1., 04.10 "7 moles) of a trypsin solution at 2 mg / ml in 0.1 M MES, 0 , 5 M NaCl, pH 6.
- the mixture is then maintained at a temperature of
- reaction mixture is diluted to 300 ml by addition of MilliQ water then ultrafiltered on membranes having a cutoff threshold of 100,000 Da in order to remove the unreacted trypsin, the secondary reaction product (NHS) and to desalt the medium.
- the reaction mixture is then concentrated to a volume of 50 to 100 ml and then lyophilized.
- EXAMPLE 2 Preparation of a glass capillary Functionalized AR COPOLYMER CARRIER TRYPSIN AS MOLECULE PROBE
- the trypsin copolymer as prepared above in Example 1 is suspended in NH 4 HCO 3 buffer 25 mM ( pH 8) filtered through a 0.22 ⁇ m filter, at a rate of 3% (w / y) of trypsinized copolymer / ml of buffer.
- a capillary 20 cm long and 75 ⁇ m in diameter (Polymicro®) is treated by passing the following solutions: NaOH 3 N; 25 mM NH 4 HCO 3 buffer; 0.2 M HCl; Buffer and finally the solution of P (D As-AA) trypsin.
- EXAMPLE 3 PREPARATION OF A COPOLYMER OF NN-DIMETHYLACRYLA1MIDE AND BIOTIN FUNCTIONALIZED BY A VINYL GROUP
- This example illustrates the synthesis of a copolymer of N, N-dimethylacrylamide and biotin functionalized with a vinyl group.
- the functionalization of biotin is carried out by reaction of a carboxylic group of biotin with N-aminopropyl methacrylamide containing a vinyl group.
- the reaction is carried out under anhydrous condition in an organic solvent such as dichloromethylene, tetrahydrofuran (THF), dimethylformamide (DMF), dimethylsulfoxide (D SO), using a coupling agent such as dicycloh exyl carbodiimide.
- an organic solvent such as dichloromethylene, tetrahydrofuran (THF), dimethylformamide (DMF), dimethylsulfoxide (D SO)
- a coupling agent such as dicycloh exyl carbodiimide.
- the copolymerization of biotin with the monomer of N, N-dimethylacrylamide is carried out under the same conditions as those described above in Example 1 by reacting 2.7 g (0.0272 moles) and 1.2 g ( 0.0042 moles) of biotin modified by a vinyl group.
- a copolymer is obtained which is purified by ultrafiltration using a threshold cut-off filter of 100,000 Da and then lyophilized.
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- Chemical & Material Sciences (AREA)
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- Chemical Kinetics & Catalysis (AREA)
- Medicinal Chemistry (AREA)
- Polymers & Plastics (AREA)
- Organic Chemistry (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Addition Polymer Or Copolymer, Post-Treatments, Or Chemical Modifications (AREA)
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FR0402630A FR2867479B1 (fr) | 2004-03-15 | 2004-03-15 | Polymere de haut poids moleculaire a base de n,n- dimethylacrylamide et de monomeres fonctionnalises par des molecules sondes et ses utilisations |
PCT/FR2005/000573 WO2005100424A2 (fr) | 2004-03-15 | 2005-03-10 | Polymere de haut poids moleculaire a base de n,n-dimethylacrylamide |
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US (1) | US20070178465A1 (de) |
EP (1) | EP1732963A2 (de) |
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WO2011140590A1 (en) * | 2010-05-10 | 2011-11-17 | Bio-Layer Pty Ltd | Binding systems |
US11529610B2 (en) | 2012-09-17 | 2022-12-20 | W.R. Grace & Co.-Conn. | Functionalized particulate support material and methods of making and using the same |
RU2015114330A (ru) | 2012-09-17 | 2016-11-10 | У.Р. Грейс Энд Ко.-Конн. | Хроматографические среды и устройства |
SG11201605712SA (en) | 2014-01-16 | 2016-08-30 | Grace W R & Co | Affinity chromatography media and chromatography devices |
ES2929099T3 (es) | 2014-05-02 | 2022-11-24 | Grace W R & Co | Material de soporte funcionalizado y métodos de fabricación y uso de material de soporte funcionalizado |
BR112017026193B1 (pt) | 2015-06-05 | 2021-09-14 | W.R. Grace & Co-Conn | Adsorventes, método de produção dos adsorventes e uso dos adsorventes |
CN112816688B (zh) * | 2021-01-04 | 2022-05-13 | 深圳市柏明胜医疗器械有限公司 | 一种酶标板及其制备方法和应用 |
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FR2811083B1 (fr) * | 2000-06-30 | 2002-11-22 | Inst Curie | Milieu liquide non-thermosensible pour l'analyse d'especes au sein d'un canal |
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FR2867479B1 (fr) | 2007-11-02 |
FR2867479A1 (fr) | 2005-09-16 |
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