DE19853815C2 - Use of a surface-modified plastic product - Google Patents

Description

Known surface-modified plastic products are too different Chen purposes used.

In US patent 4352884 modified plastic surfaces are described which are coated with a copolymer consisting of two different Mo nomertypes of the acrylate type is constructed and that in the immobilization of Biomolecules can be used. Among the two monomers Types fall into monomer units with functional groups, e.g. B. carboxyl or Amino groups to which the biomolecules can bind. The other mono In contrast, mertyp has no specific or unspecific binding egg properties for biomolecules, with biomolecules essentially all Substances should be understood that are involved in biological / biochemical reaction are involved. It can in particular be proteins, e.g. B. enzymes, and act nucleic acids or nucleotides etc.

The coating with the copolymer is used in the US patent 4352884 known products ensure that the non-specific Bindun  against the inclined plastic surface is completely covered. The disadvantage is al lerdings the purely adsorptive nature of the coating, so that a permanent Stability of the coating cannot be guaranteed. Beyond that the use of acrylates because of their low hydrolysis stability for many Applications problematic.

There are other art used to immobilize biomolecules fabric products in which polymers are firmly coupled to plastic surfaces are. For example, For example, U.S. Patent 5,043,278 discloses a method of immobilization tion of biomolecules via polyethylene oxide spacers on the plastic surface chen. In U.S. Patent 5258041, polyethylene oxide is over a photoche mixed initiated coupling process. Both mentioned known ver However, driving does not allow targeted control of the surface coverage density (with biomolecules).

US Pat. No. 5,476,509 describes further surface-modified plastics. Here is the surface by graft copolymerization of monomer mixtures modified from ionic and nonionic monomers. This kind of modifi Adornment allows the adjustment of the surface coverage density, disadvantageous However, here is the ionic character of the immobilization, which leads to confor Mation change of the bound biomolecules can lead and beyond is unsuitable for media with high ionic strengths.

Finally, EP 0 872 512 discloses modified polymer substrates which for the production of medical technology products or hygiene articles nen. The modification can be carried out, inter alia, by graft copolymerization of two different olefinically unsaturated monomers on pretreated poly mer substrate take place, it being z. B. acrylic or Methacrylic compounds can act.

The object of the invention is the field of application of the last-mentioned polymer expand substrate.

The object is achieved with a use according to claim 1.

Then a surface-modified plastic product for immobilization tion of biomolecules used, which on the surface by means of solid in the we considerable covalent bond copolymers with hydrophilic properties Shafts, the copolymer of two different Monomerty pen is built, of which the one monomer type 1 one or more bin has sites where biomolecules can bind while the other Monomer type 2 no specific binding properties for the biomolecule and both types of monomers are selected so that they do not enter into non-specific interactions with the biomolecule.

The copolymer used according to the invention is usually made up of two built up different monomer units, one of which under Mono type 1 and the other falls under type 2 monomer. In special application two or more different monomer units can also occur that are placed under one of the monomer types, usually monomer type 1 fall. This will be discussed further below.

The plastic products used according to the invention have a permanent modified surface, usually copolymers the whole art shield the surface of the fabric and in this way unspecific bindings of the Prevent biomolecules with the surface. The types of monomers used are designed so that they are essentially hydrophilic in nature non-specific interactions with biomolecules,  where monomer type 2 also serves as a spacer and thus the access Availability and availability of the biomolecules bound to monomer type 1 increases.

With regard to the spacer function in particular, it looks advantageous staltung of the invention that the ratio of monomer type 2 to 1 minde is at least 1: 1. This ensures that statistically between mono meren with z. B. always functional binding groups of monomer type 1 Spacer monomer of type 2 is provided, which, as mentioned above, the Zu mobility of the bound biomolecules is increased.

Further configurations relate to those used in the invention Monomer types provided for plastic products. Basically, as Monomer types of any kind of polymerizable molecules are used. Important is only that the monomer types or the copolymers formed from them hydro possess phile surface properties to non-specific interactions to avoid with the biomolecules.

Monomer types which can be used according to the invention preferably belong to the group of the hydrophilic vinyl units. Acrylamide or Methacrylamide derivatives used because they are not as easy to hydrolyze as e.g. B. Acrylates (which, however, at least in principle also for the Invention appropriate procedures are suitable). However, the use of Vinyl saccharides, e.g. B. vinyl dextran or derivatized with vinyl groups Chitosan.

As mentioned several times above, monomer type 1 is equipped with binding sites for biomolecules. The binding sites can e.g. B. functional groups such as COOH, SH, NH ₂ , epoxy or NR ⁺ ₄ groups or the same. A suitable in this context monomer type 1 with COOH groups is e.g. B. acrylic acid.

However, there may also be binding sites with much more specific properties ten are provided. In this context it is envisaged that the Mo nomertype 1 contains special "binding molecules" at its binding sites such as e.g. B. nitrilotriacetic acid, peptides or oligo dT. In this context ge suitable monomer types 1 would be z. B. vinyl nitrilotriacetic acid or vinyl dT Units.

In a further embodiment it is provided that in particular in the monomer type 1 may contain a polyethylene oxide spacer. These spacers that are so are arranged that they protrude from the main chain in the finished copolymer in monomer type 1, the binding sites at their free ends. In this way increases the mobility and thus the distributability of the binding sites over the plastic surface.

Furthermore, as mentioned above, the invention is not limited to that only two different monomers are used, each under one of the Monomer types fall. It is quite possible that an Erfin usable monomer type 1 two or more different mono covers units that immobilize different biomolecules, where again the concentration ratio between monomer types 1 and 2 is advantageously set so that monomer type 2 as a spacer between the different monomer units of type 1 can serve.

Plastic products covered by the invention with correspondingly modified In particular, PCR reaction vessels, microtiter plates or also filter surfaces, e.g. B. membranes.  

Modified plastic reaction vessels can e.g. B. serve for enrichment or isolation of biomolecules. So it is z. B. conceivable to use appropriately modified special reaction vessels, the streptavidin (monomer type 1 = vinyl-R-COOH), mRNA (monomer type 1 = vinyl-dT _n ), peptides (monomer type 1 = vinyl-R-COOH-) or DNA bind (monomer type 1 = vinyl unit with NR ₄ ⁺ groups).

The modified plastic products can just as well be used as a party fabric carrier for z. B. Use enzyme reactions. It would be conceivable, for. B. in Zu connection with PCR reactions, reaction vessels with immobilized Taq To provide polymerase.

Of course, the plastic products mentioned are not on vessels and the like limited. As mentioned below, Saatpo Lymerisation also modified beads can be produced.

Appropriate modified implants could be used in the field of medical technology anticoagulant and / or fibrinolytic properties the. Corresponding implants have modified surfaces on which the monomer type 1 used for the modification two different mono may contain mer units (with different functional groups) that each specifically anticoagulant biomolecules, e.g. B. heparin or hirudin, or fibrinolytic bio-molecules, e.g. B. bind urokinase.

Modified plastic surfaces usable within the scope of the invention create yourself in different ways.  

One possibility is to place the copolymer on the plastic surface Arrange ways of graft copolymerization. With this configuration, too be activated accordingly in front of the plastic surface, e.g. B. with high toxic radiation such as plasma, corona, X-rays or the like. After activation, the copolymer can then polymerize to the surface become.

Another particularly preferred in connection with the graft copolymerization A photoinitiator can be activated on the plastic surface to absorb. After activation with a Ge mix of monomer types 1 and 2 and then coated with UV Radiation initiated a radical polymerization at the interface. A such heterogeneous, photo-initiated graft copolymerization has the advantage that one about the setting of different parameters, such as. B. Concentration Ratios of the mixture of monomer types, duration of polymerization or tax tion of the adsorption of the photoinitiators on the plastic surface in simple How to adjust the density and number of copolymers bound.

In particular, by means of the graft copolymerization mentioned, the invention can be achieved surface modification according to the invention with the Eigen described above achieve in a simple manner and permanently. In contrast to known ones Modification processes that work with graft copolymerization enables the inventive method at the same time an unproblematic setting the surface coverage density and a permanent connection of biomole cool.

However, it is also possible to bind the copolymer to the plastic surface in a plasma polymerization step. With this out  design the plastic surface with a gaseous mixture Monomer types 1 and 2 contacted under plasma polymerization conditions.

Furthermore, it is also possible that so-called polymer Beads are used. In the raw state, such beads usually have an egg ne outer layer that is not fully polymerized. Man can further polymerize z. B. the questionable Polymerize copolymers and then receive beads with a functionalized upper area.

The invention will be explained in more detail below with the aid of a few examples. The references M1, 2 etc. or V1, 2 etc. in the examples refer to the am Table attached at the end of the description.  

1. Monomer synthesis 1.1. Synthesis of methacrylic acid N-hydroxysuccinimide ester (M1)

0.05 mol of methacrylic acid (Fluka) was mixed with 0.05 mol of dicyclohexylcarbodiimide (DCC, Fluka) and 0.05 mol N-hydroxysuccinimide (NHS, Aldrich) in 150 ml dried THF stirred at 0 ° C for 2 h. After removal of the urea Precipitate and addition of the inhibitor hydroquinone monomethyl ether (0.005%) was concentrated in vacuo, mixed with n-hexane and close to the boil point filtered. Finally the filtrate was dried in vacuo.

The product was used as a starting material for derivatizations or as a function lized comonomer used for the graft copolymerization.

1.2. Synthesis of α-amino-ω-methacryloylamino polyethylene oxide (M2)

It was 0.05 mol Bisaminopolyethylenoxid:; placed in 500 ml DMF (Jeffamine ^® -PEGs Mr = 600, 900, 2000 Fluka). Subsequently, 0.01 mol of methacrylic acid chloride (Fluka), dissolved in a little DMF, was added dropwise at 0 ° C. and the mixture was stirred for 2 h. Finally, the product was separated from the excess bisaminopo ethylene oxide by chromatography and purified.

1.3. Synthesis of α-methoxy-ω-methacryloylamino polyethylene oxide (M3)

0.05 mol of α-aminopolyethylene oxide (Huntsman, USA) was dissolved in 500 ml of DMF, mixed with 0.06 mol of triethylamine (Fluka) presented. Subsequently at 0 ° C 0.05 mol of methacrylic acid chloride, dissolved in a little DMF, added dropwise and for 2 h touched. Finally, the product was purified by chromatography.  

1.4. Synthesis of α-carboxyl-ω-methacryloylamino polyethylene oxide (M4)

0.05 mol of α-carboxyl-ω-aminopolyethylene oxide (from Shearwaters, Enschede, NL) was placed in 500 ml Tris (pH = 8.6). Subsequently, 0.05 mol at 0 ° C Methacrylic acid-N-hydroxysucchinimide ester, dissolved in a little DMF, was added dropwise and stirred for 2 h. Finally, the product was chromatographed nigt.

1.5. Synthesis of vinyl oligo (dT) ₂₀ (M5)

0.01 mol ω-amino-oligo- (dT) ₂₀ (from Tib Molbiol, Berlin), modified with a PEG spacer, was placed in 100 ml Tris (pH = 8.6). Subsequently, 0.05 mol of methacrylic acid N-hydroxysuccinimide ester, dissolved in a little DMF, was added dropwise at 0 ° C. and the mixture was stirred for 2 h. Finally, the product was chromatographically cleaned.

2. Graft copolymerization on PCR tubes

The photoinitiator was adsorbed on the inner surface of the PCR tubes. For this purpose, a methanolic benzophenone solution (100 mM) incubated. After removing the solution, the PCR tubes were ge at 60 ° C dries. The vessels thus prepared were washed with an aqueous one with benzo phenon-saturated monomer mixture (10% by weight) and covered with UV radiation (1 <300 nm) graft-copolymerized for 1-30 min. According to the polyme The PCR tubes were used for the removal of homopolymers with Was rinsed. The monomer mixtures listed in Table 1 were mono mere used.  

3. Immobilization on surface-functionalized PCR tubes 3.1. Derivatization of surface-functionalized PCR tubes

The coating became the basis for the derivatizations on the solid phase performed according to V2. PCR modified with a coating according to V2 Vessels were washed with 300 µl of an aqueous 0.005 mM EDC (PBS buffer: pH = 7.4) Bisaminopolyethylenoxid solution (10%) added. After 1 h at RT the solution was removed and the PCR tube was washed with water.

3.2 Immobilization of Oligo-dT on Surface-Functionalized PCR Vessels

A PCR tube coated with the monomer mixture according to V1 was placed under Add 10 mM EDC (N- (3-dimethylaminopropyl) -N'-ethyl-carbodiimide hydrochloride, Aldrich) in 300 µl PBS buffer (pH = 7.4) with 300 ng ω-phosphatyl Oligo-dT (Tib Molbiol, Berlin) added for 30 min. After the reaction was over rinsed with water.

3.3. Immobilization of streptavidin to surface functionalized PCR tubes

A PCR tube coated with the monomer mixture V1 was added under Zuga be from 10 mM EDC (N- (3-dimethylaminopropyl) -N'-ethylcarbodiimide hydrochloride, Aldrich) in 300 ul PBS buffer (pH = 4.8). After the reaction one is rinsed with water and then with 5 µg streptavidin in PBS (pH = 7.4) incubated. Finally, it was rinsed again with water.  

table

Claims (8)

1. Use of a surface-modified plastic product for im mobilization of biomolecules, which on its surface by means of solid copolymers arranged essentially covalently with hydro has phile properties, the copolymer of two under different types of monomers is built, one of which is a monomer type 1 has one or more binding sites at which biomolecules can bind, while the other monomer type 2 has no specific Has binding properties for the biomolecule and both monomers types are still selected so that they do not cause any unspecific changes interact with the biomolecule. 2. Use according to claim 1 according to claim 13, characterized net that the surface-modified plastic product is a microtiter plate, filter surface or a reaction vessel. 3. Use according to claim 1 or 2, characterized in that the The ratio of monomer type 2 to monomer type 1 is at least 1: 1.   4. Use according to any one of the preceding claims, characterized ge indicates that monomer type 1 and / or monomer type 2 hydrophilic Vinyl units, especially acrylamide or methacrylamide derivatives are. 5. Use according to any one of the preceding claims, characterized in that the binding sites in monomer type 1 have functional groups, in particular COOH, SH, NH ₂ , epoxy or NR ⁺ ₄ groups. 6. Use according to any one of the preceding claims, characterized ge indicates that the binding sites in monomer type 1 are special bin have molecules. 7. Use according to any one of the preceding claims, characterized ge indicates that monomer type 1 and / or 2 polyethylene oxide spacers hold. 8. Use according to any one of the preceding claims, characterized ge indicates that monomer type 1 has at least two different mo has monomer units with different binding properties for biomolecules.