AU752230B2

AU752230B2 - Hydrophilic polymer coatings on hydrophobic or hydrophobized surfaces for biotechnological applications

Info

Publication number: AU752230B2
Application number: AU20544/99A
Authority: AU
Inventors: Gunnar Brink; Manuela Gassner; Silke Gotz; Erik Wischerhoff
Original assignee: FRANZ SCHLEICHER DR
Current assignee: FRANZ SCHLEICHER DR
Priority date: 1997-12-17
Filing date: 1998-12-17
Publication date: 2002-09-12
Anticipated expiration: 2018-12-17
Also published as: EP1098940A1; DE59805458D1; AU2054499A; DE19756193A1; EP1098940B1; WO1999031189A1

Description

HYDROPHILIC POLYMER COATINGS ON HYDROPHOBIC OR HYDROPHOBICIZED SURFACES FOR BIOTECHNOLOGICAL

USES

t The invention under consideration refers to coatings made of hydrophilic, neutral polymers with hydrophobic groups on hydrophobic or hydrophobicized surfaces and methods for their production. More precisely, the invention refers to coatings of the aforementioned type for biotechnological uses, which prevent a nonspecific adsorption of organic, in particular, biological material on the surfaces of apparatuses, for example, sensors. If plastic parts are used in apparatuses or sensors which come into contact with organic material, a modification of the surfaces is necessary to prevent nonspecific adsorption, which up to now has been time and cost intensive. Among other things, the methods described in the following are known for this purpose. Polymer surfaces can be hydrophilicized by plasma treatment Chem. Phys. 97:12835 (1993)). The surfaces are oxidized thereby, wherein hydroxy and carboxyl groups are formed. These surfaces do not have very defined functionalization and are not suitable for preventing nonspecific adsorption without further treatment. In Biomaterials 18:845 (1997), polystyrene Ssurfaces are functionalized with carboxyl groups, in that first an adsorption of a salt of undecenoic acid takes place, which is followed by an argon plasma treatment. A charged surface is formed hereby which is hydrophilic, but which tends once again to nonspecific adsorption via electrostatic interactions. Another treatment possibility consists in the adsorption of polyelectrolytes (Macromolecules 30:1752- 1757 (1997)) on oxidatively pretreated surfaces. Here also, charged surfaces with *the disadvantages described above are formed. The adsorption of polyelectrolytes can take place without a previous surface treatment Colloid Interface Sci.

183:18-25 (1996), Chem. Mater. 8:1575-1578 (1996), J. Chem. Phys. 99:13065- 13069 (1995)). In this way, however, a charged surface with its inherent disadvantages is also produced. Furthermore, the resistance of such systems, which are adsorbed exclusively via hydrophobic interactions, is very limited, in comparison to various solvents, and does not ensure their mechanical stability (Macromol. Chem. Phys. 198:3239 (1997)). With the previous methods used, such as the dextranization of surfaces (Biosensors and Bioelectronics 11:579 (1996) -,ournal of Biomedical Materials Research 18:953 (1984)) it was not possible to coat untreated plastic surfaces. Nor was it possible to completely dispense with organic solvents in the methods, which, however, is a matter of concern with respect to environmental incompatibility of the methods and moreover generates costs. In functionalization by means of epoxy groups (Journal of Biomedical Materials Research 18:953 (1984)), the polymer solutions used must also have a very high concentration in order to arrive at satisfactory results. However, this leads, on the one hand, to hard-to-handle solutions because of their high viscosity and, on the other hand, to an increase in costs. In surface modification with aminodextran and EDC (Biosensors and Bioelectronics 11:579 (1996)), expensive additional reagents are required and the coating must contain amino groups, whose capacity for electrostatic interaction represents an inherent disturbance source. The problem of the invention under consideration is to produce hydrophobic or hydrophobicized objects which are modified by a coating such as those made of plastics, glass, quartz, silicon, silver, or gold, which are essentially not subjected to a nonspecific adsorption of hydrophobic or charged substances.

Pure glass, quartz, silicon, silver, or gold surfaces are, in fact, not hydrophobic when they are completely clean, but have the tendency, for example, to adsorb any organic impurities in the room air and thus to become hydrophobic. The object is to prevent this nonspecific adsorption. In addition, a nonspecific charge interaction is also to be prevented by an appropriate coating with glass, quartz, and silicon surfaces. Another problem of the invention is to make available coatings of the aforementioned type, which can be further functionalized and thus are suitable for use in biotechnological apparatuses. Receptors of biosensors on an affinity basis (affinity sensors) must, for example, be modified in such a manner that only one specific substance is adsorbed selectively on them from a complex multicomponent system. The adsorbed substance quantity can be measured via surface plasmon resonance. In affinity chromatography, the active Separation medium must be conditioned in such away that only the substance to be analyzed is selectively bound, whereas nonspecific adsorption is suppressed as much as possible using the coating in accordance with the invention. During the measurement of very small material quantities to be analyzed, for example, in the microliter range and below, it is of the greatest importance that the material to be analyzed does not adhere to vessel walls and the inside of hoses and that 4 J dnificant fractions are lost in this way. It is therefore desired that the adsorption of the material to be analyzed is minimized. Thus, nonspecific adsorption can be suppressed in vessels and hoses which are lined with the coating in accordance with the invention. Furthermore, the coating in accordance with the invention can also be used as a matrix for solid phase syntheses, after further functionalization.

Another problem is to make available a method which makes possible the application of these coatings on hydrophobic or hydrophobicized surfaces in a simple and low-cost, but environmentally friendly manner, and which leads to mechanically stable coatings which are also stable with respect to various solvents. The aforementioned problems are solved by the coating of hydrophobic or hydrophobicized surfaces with hydrophilic, neutral polymers, which have hydrophobic groups. In particular, the invention under consideration makes available an object with a hydrophobic or hydrophobicized surface and a coating which is applied on it and is made of a polysaccharide which has hydrophobic groups. Furthermore, a method of the production of the object is made available which is characterized in that a hydrophobic surface of the object which is to be coated is immersed in a solution of a polysaccharide with hydrophobic groups or a glass, quartz, silicon, or gold surface which is to be coated, is first hydrophobicied and then immersed into the solution of the polysaccharide. The objects of the invention with the surface modified by the applied coating can be used as a biotechnological matrix or instrument or as a matrix for biochemical solid phase synthesis, for example, solid phase protein synthesis. A polysaccharide which has hydrophobic groups is used for the coating. A polysaccharide made of glucose units is preferred. Dextran, pullulan or inulin are more preferable; dextran is particularly preferred. The hydrophobic groups of the polysaccharide essentially make possible, by means of their hydrophobic characteristic, the adsorption on the surfaces to be functionalized, in that a predominant number of the hydrophobic groups in the polymer point to the hydrophobic or hydrophobicized surface and thus hydrophobic interactions arise between the hydrophobic groups and the surface. The polymer is therefore predominantly bound to the surface via hydrophobic interactions. The hydrophobic groups can be crosslinked.

Hydrophobic groups which can be crosslinked by irradiation, for example, with UV light, are more preferable. A particularly preferred hydrophobic group is the 4vinylbenzyl group which, for example, can be introduced into it via the partial fiiherification of 4-vinylbenzyl chloride with hydroxy groups of the polymer. It can be crosslinked by irradiation with UV light of the wavelength 254 nm. If the coating consists of dextran, 1 to 20% of the available hydroxyl groups can be preferably substituted with 4-vinylbenzyl groups. Figure 1 shows a preferred embodiment of a polysaccharide. The ratio of m to n varies from 5:1 to 20:1. R is selected from hydrogen and a hydrophobic group, for example, the 4-vinylbenzyl group, provided that at least one R is a hydrophobic group. The polysaccharide in Figure 1 is a statistical copolymer--that is, the sequence of the monomeric units is random. The coating in accordance with the invention can be applied on various surfaces, wherein a distinction is made between hydrophobic and hydrophobicized surfaces.

The hydrophobic surfaces may be surfaces of plastic, for example, polyethylene, "***polypropylene, polycarbonate (for example, Makrolon®) and thermoplastic olefin polymers of anamorphous structure (for example, Topas®). The hydrophobicized surfaces may be silver, gold, glass, quartz, or silicon surfaces. If we are dealing with glass, quartz, or silicon surfaces, then they are hydrophobicized that is, the surfaces are subjected to a hydrophobicizing treatment. This can, for example, be carried out with a polycation which has hydrophobic groups. Only after hydrophobicizing does the adsorption of the polysaccharide then take place.

Alternately, other methods can be used for the hydrophobicization, for example, a treatment with alkoxyalkylsilanes. They are, however, mostly more complicated and more tedious than the adsorption of a polycation with hydrophobic groups.

With silver or gold surfaces, a hydrophobicization can be attained by treatment with an alkyl thiol or with a negatively charged thiol, such as mercaptopropyl sulfonate, onto which, in turn, a polycation with hydrophobic groups can then be applied. Preferably, a polycation is used for the hydrophobicization, which can be crosslinked via the hydrophobic groups. The crosslinking is preferably brought about by radiation. The hydrophobic groups of the polycation are preferably 4vinylbenzyl groups. A particularly preferred embodiment of the polycation with hydrophobic groups is poly(methacrylic acid-(3-dimethylaminopropylamide)), which was quaternized 100% with 4-vinylbenzyl chloride. A crosslinking of the coating via the hydrophobic groups of the polysaccharide and a crosslinking of any crosslinkable polycations present via the hydrophobic groups increases both the mechanical stability and also the solvent resistance of the coating. If the crosslinking is brought about by radiation, then it is also possible to crosslink only -oartial regions of the coating and the polycation. For this purpose, radiationimpermeable masks are used which cover the region which is not to be crosslinked. In addition, a functionalization of the coating is possible both via the hydroxyl groups and also via the hydrophobic groups which are not crosslinked.

o: Some of the hydroxyl groups can, for example, be brought to reaction with S..bromoacetic acid, wherein carboxyl groups are introduced into the coating which can be subjected to esterification or amidation in turn. This procedure can be useful, in particular with solid phase protein synthesis. Moreover, thio groups can be introduced on the noncrosslinked 4-vinylbenzyl groups via radical reactions. In particular, the functionalized coatings can be used-for the production of biosensors or for other uses in diagnosis or in screening methods. For the production of the coated object whose coated surface is essentially not subjected to a nonspecific adsorption of hydrophobic or charged substances, the hydrophobic or hydrophobicized surface of the object is merely immersed into a solution of a polysaccharide with hydrophobic groups. Preferably, it is an aqueous solution. The immersion time can be between 8 and 48 h and is, typically ca. 24 h. The method can be carried out in a temperature range of 1 to 50°C, preferably at room temperature. The concentration of the polysaccharide in the solution varies from 0.001 to 0.1 mol-L- 1 preferably from 0.01 to 0.05 mol-L 1 and, 0.02 mol-L- 1 is particularly preferred. If the polysaccharide contains crosslinkable hydrophobic groups, then a coating preferably follows wherein the groups are crosslinked and thus the mechanical stability of the coatings is increased. The type of surfaces to be used and the polysaccharide were already described in detail above. As additional investigations showed, this simple method is suitable for the application of the coating in accordance with the invention not only for polysaccharides, but very generally for neutral hydrophilic polymers with hydrophobic groups. The invention under consideration is thus to make available a method for the production of a coating which is essentially not subjected to nonspecific adsorption of hydrophobic substances, on a surface of an object which is characterized in that the surface, optionally after a previous hydrophobicization, is immersed in a solution of a neutral hydrophilic polymer with hydrophobic groups. The neutral hydrophilic polymer preferably refers to polymers with hydroxy or polyoxyalkylene groups. The hydrophobic groups and their preferred embodiments are those previously described. More preferred polymers with hydroxy groups are polysaccharides of the type described above. The type of polymers with polyoxyalkylene groups is not particularly limited. For example, polyalkylene derivatives_ such as polymethacrylic acid derivatives, for example, polymethacrylic amide or polymethacrylic acid ester, can be used. The polyoxyalkylene groups are optionally bound to the basic polymer structure via the carboxy or amide groups of the polymers as side chains. Polyoxyethylene groups are particularly well suited as polyoxyalkylene groups. Particularly preferred polymers are the compounds with the following formulas I, II, III, and IV.

0 0 0 oo S.

S.

S

.5

S

5.55 j* S S *IUSS S S S* 555.5 S S S S

S.

SSSS

S

5555

S

S. S S S

S.

5 0 0$

CH

NH

H-IC-N

I I)

S

S S

S.

*555

S

*SSS

S S

S

5S5~ 5555 N H

H

3

C-N

fl 0

(III)

(IV)

8 in which x:y varies from 20:1 to 5:1, and n varies from ca. 50 to ca.1000.

The invention is explained further by the following examples.

EXAMPLES

Reference example Synthesis of 4-vinylbenzyl-substituted dextran (VBD): 2.00 g Dextran (dextran T500 from Pharmacia, 37 mmol hydroxyl groups) are dissolved in a mixture of mL water and 10 mL dimethyl sulfoxide and mixed with 0.384 g potassium hydroxide (6.74 mmoi). When these are dissolved, 1.029 g 4-vinylbenzyl chloride and 2 drops nitrobenzene are added. The mixture is stirred at room temperature for 4 days. Subsequently, the polymer is precipitated in 400 mL acetone, wherein the work must be carried out with the exclusion of light.

Yield: 2.04 g (98%) *HNMR specrum 1 H-NMR spectrum (D 2 0): 0 0

U

0* 0* 0d r,u v xy= 181 CY -1 b c I H.o e

HO

I OH I t- x 6 [ppm]: 7.55 7.25 Harom), 6.85 6.65 Ha), 5.8 Hc), 5.3 Hb), 4.92 (He), 4.55 (Hd, partially masked by HDO), 4.2-3.3 (several m, H atoms of the polysaccharide backbone) Th sequence of the monomeric units is random; therefore it is a statistical :c lymer. The functionalization with the hydrophobic group is not carried out ."i exclusively on the 4-positions of the saccharide units, but rather, more or less statistically on all available hydroxyl groups.

*6 Example 1 .i Coating of plastics. In the first step, the plastic surface to be coated is cleaned by immersion in concentrated sulfuric acid for ca. 30 sec. Afterwards, rinsing is carried out with demineralized water three times, and subsequently the plastic is immersed in an aqueous solution VBD (2 x 10 2 mol-L-1) for 24 h. Afterwards, rinsing with demineralized water is carried out three times. Then, the photo crosslinking of the applied layer is undertaken by UV radiation at 254 nm for sec. Figure 2 shows UV spectra of a layer of the photoreactive polymer on •Topas®. The success of the photoreaction is documented by the decline of the absorption at 254 nm (broken-line measurement curve). Comparative measurements of nonspecific adsorption processes were carried out with a confocal microscope. The surface is hereby brought into contact with a solution of a fluorescence-label protein. With the aid of a microscope, the protein concentration is recorded as a function of the distance from the surface, in that the fluorescent intensity is determined in the individual focal plane. An almost uniform brightness profile is obtained as a function of the distance from the surface. This i: indicates that nonspecific adsorption does not take place on the functionalized surface. The surface depicted on the left in Figure 3 represents the concentration profile of a 500 nm fluorescence-label beef serum albumin (BSA-Texas Red) solution, on a coated Topas@ surface, recorded with a confocal microscope, wherein light areas correspond to BSA concentrations. To the right in Figure 3, the uncoated reference surface is depicted. The vertical area depicted corresponds to approximately 2 pm.

Example 2 Coating of silver, gold, glass, quartz, or silver surfaces. The surfaces are cleaned according to generally common methods (Ber. Bunsenges. Phys. Chem. 100:1033 (1996), Journal of Biomedical Materials Research 18:953 (1984)). The cleaned old or silver surfaces are then immersed for ca. 12 h in an aqueous 10 mercaptopropylsulfonate solution (2 x 10- 2 mol.L') and subsequently placed in a solution (2 x 10- 2 mol.L of poly(methacrylic acid-(3-dimethylaminopropylamide)), which was quaterized 100% with 4-vinylbenzyl chloride for min. Without previously treating with the mercaptopropylsulfonate solution, the cleaned glass, quartz, or silicon surfaces are placed in a solution (2 x 2 mol.L-1 of poly(methacrylic acid-3dimethylaminopropylamide)), which was quaternized 100% with 4-vinylbenzyl chloride for 20 min. After rinsing three times with an alytically pure water, the material is immersed in an aqueous solution VBD (2 x 10- 2 mol.L 1 for 24 h. Subsequently, rinsing with demineralized water is carried out three times. Then, the photocrosslinking of the applied layers is undertaken by means of UV irradiation at 254 nm for 60 sec. Figure 4 shows the structural formula of poly(methacrylic acid-(3dimethylaminopropylamide)), quaternized with 4-vinylbenzyl chloride.

In this specification, except where the context requires otherwise, the words "comprise", "comprises", and "comprising" mean "include", "includes", and "including", respectively, ie when the invention is described or defined as comprising specified features, various embodiments of the same invention may also include additional features.

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Claims

1. Object with a hydrophobic or hydrophobicized surface and a coating made of a polysaccharide, which has hydrophobic groups applied on the surface, wherein the coating is crosslinked by radiation via the hydrophobic groups.

2. Object according to Claim 1, characterized in that the polysaccharide is bound to the surface via hydrophobic interactions.

3. Object according to one of Claims 1 or 2, characterized in that the polysaccharide is a dextran with 15 hydrophobic groups.

4. Object according to one of Claims 1 to 3, characterized in that the hydrophobic groups are 4- vinylbenzyl groups. "o

5. Object according to one of Claims 1 to 4, S" characterized in that the coating is made of a dextran in which 1 to 20% of the available hydroxyl groups are substituted with 4-vinylbenzyl groups.

6. Object according to one of Claims 1 to characterized in that the surface is made of a hydrophobic plastic.

7. Object according to one of Claims 1 to characterized in that the surface comprises glass, quartz, silicon, silver, or gold and is hydrophobicized before the Aapplication of the polysaccharide. H:\Shona1\Keep\Speci\20544-99 Speci 4/07/02 12

8. Object according to Claim 7, characterized in that the hydrophobicization of glass, quartz, or silicon surfaces is carried out by treatment with a polycation which has hydrophobic groups.

9. Object according to Claim 7, characterized in that the hydrophobicization of silver or gold surfaces is carried out by treatment with an alkyl thiol or with a negatively charged thiol on which a polycation with hydrophobic groups is then applied.

Object according to one of Claims 8 or 9, characterized in that the polycation is crosslinked via 15 the hydrophobic groups.

11. Object according to Claim 10, characterized in that the crosslinking is brought about by radiation. 20

12. Object according to one of Claims 8 to 11, characterized in that the hydrophobic groups are 4- vinylbenzyl groups.

13. Object according to one of Claims 8 to 12, characterized in that the polycation with hydrophobic groups is poly(methacrylic acid-3- dimethylaminopropylamide), which was quaternized, 100% with 4-vinylbenzyl chloride.

14. Object according to one of Claims 1 to 13, characterized in that the coating and optionally any crosslinkable polycation present is crosslinked in partial areas or on the entire surface.

H:\Shona1\Keep\Speci\20544-99 Speci 4/07/02 13 Object according to one of Claims 1 to 14, characterized in that some of the hydroxyl groups of the coating are functionalized.

16. Method for the production of a coating which is essentially not subjected to a non specific adsorption of hydrophobic or charged substances on a surface of an object, characterized in that the surface, optionally after a previous hydrophobicization, is immersed in a solution of polysaccharide with hydrophobic groups and subsequently the coating is crosslinked by radiation via hydrophobic groups. 15

17. Method according to Claim 16 for the production of a coated object according to one of Claims 1 to 14, charaeterized in that a hydrophobic surface of the object to be coated is immersed in a solution of a polysaccharide with hydrophobic groups, or a glass, quartz, silicon, 20 silver, or gold surface to be coated is first hydrophobicized and is subsequently immersed in the solution of the polysaccharide.

18. Method according to one of Claims 16 or 17, characterized in that the solution is an aqueous solution.

19. Method according to one of Claims 16 to 18, characterized in that any crosslinkable groups in the coating obtained are crosslinked in partial areas or on the entire surface. Use of an object according to one of Claims 1 to T as a biotechnological matrix or instrument.

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21.- Use of an object according to one of Claims 1 to as a matrix for the biochemical solid phase synthesis.

22. Use of an object according to one of Claims 1 to as a affinity sensor.

23. Use of an object according to one of Claims 1 to as an active separation medium in affinity chromatography in which x y varies from 20:1 to 5:1, and n varies from ca. 50 to ca. 1000.

24. Objects with a hydrophobic or hydrophobicized surface and a coating made of a polysaccharide, uses of 15 said objects or methods for the production of said coating, substantially as hereinbefore described with reference to the examples and/or drawings. 20 Dated this 4 t h day of July 2002 BIOTUL AG By their Patent Attorneys o GRIFFITH HACK Fellows Institute of Patent and Trade Mark Attorneys of Australia H,\Shona1\Keep\Speci\20544-99 Speci 4/07/02