The present invention relates to the immobilisation of biopolymers to hydrophilised surfaces, and to such pro¬ ducts. The hydrophilic layer which is coupled to a solid surface via a polyethelene imine, has low spontaneous adsorption. The immobilisation of proteins and other biopolymers to solid surfaces is an established technique within a number of applications, such as solid phase diagnostics, analysis with biosensors, affinity chromatography, extra¬ corporeal therapy, and bio-organic synthesis. In all of these cases, the biopolymer is bonded to a solid surface, whereupon its biological activity is utilised for a spe¬ cific purpose. Examples of such applications are solid phase diagnostics, extracorporeal therapy, biological synthesis, and treatment of implants. In solid phase diagonstics, an antibody is frequently immobilised to a plastic surface, usually consisting of polystyrene. When in contact with a body fluid, the immo¬ bilised antibody bonds any antigen that may be present. The antibody-antigen complex is then detected by means of a labelled antibody. The labelling may be in the form of a radioactive isotope, a fluorescent group, or an enzyme conjugate.

In extracorporeal therapy, a biologically active sub¬ stance is bonded to a chamber through which the patient's blood is conducted. A current example of extracorporeal therapy is hemoperfusion across an immobilised immunosti- mulating substance. Interferons and interleukins are exam¬ ples of such substances. Examples of diseases that can be treated by this technique are cancer and AIDS. In bio-organic synthesis, use is made of enzymes for producing organic compounds. An appropriate use for bio- organic synthesis are lipid transformations, i.e. trans-

forming a lipid, usually a triglyceride, into another. Most enzymes are expensive, and frequent reuse is neces¬ sary to ensure good process economy. In view hereof, the use of immobilised enzymes is of interest to most large- scale enzymatic processes.

In the treatment of implants, a biopolymer is bonded to the surface which comes into contact with biological tissue. The biopolymer, for example collagen, promotes tissue growth and stimulates cell colonisation on the implant, resulting in an increased biocompatibility. This technique can be utilised also for in vitro treatment of cell culture dishes to improve cell adhesion.

As mentioned above, the immobilisation of proteins to both organic and inorganic surfaces is today a well-estab- lished technique (see chapter 4 in "Principles of Immobi¬ lization of Enzymes", Handbook of Enzyme Biotechnology, Second Edition, Ellis Horwood Limited, 1985), and it is possible to bond a large amount of protein to the surface while retaining adequate biological activity. However, it has been found that most solid surfaces are so designed that they adsorb spontaneously proteins and other biopolymers. Adsorption in aqueous solution is promoted primarily by two types of physical forces, elec¬ trostatic attraction and hydrophobic interaction. Most surfaces are, at normal pH, negatively charged, but usual¬ ly contain also hydrophobic domains. A protein usually has both positive, negative and hydrophobic seats, which means that a protein is attracted to most surfaces, on the one hand by electrostatic attraction between positive seats and negatively charged groups in the surface and, on the other hand, by hydrophobic interaction between hydrophobic domains of the protein and the surface. This is described in, for example, "Surface and Interfacial Aspects of Bio- medical Polymers", Ed. J.D. Andrade, Plenum Press 1985, Vol. 2, p 81.

This nonspecific adsorption is an undesired pheno¬ menon for the above-mentioned applications. In solid phase diagnostics, it results in an impaired sensitivity and a shorter life of the diagnostic kit. In both extracorporeal therapy and in bio-organic synthesis, spontaneous adsorp¬ tion causes impaired activity and a shorter product life. One way of drastically reducing the adsorption pro¬ teins and other biopolymers on solid surfaces is to pro¬ vide the surfaces with a layer of an uncharged hydrophilic polymer. One example of a polymer that has been used for this purpose is polyethylene glycol (see C.-G. Gδlander, "Preparation and Properties of Functionalised Polymer Surfaces", Dissertation, Royal Institute of Technology, Stockholm 1986), but other substances, such as polysaccha- rides, for example dextran, cellulose ethers and starch; polyvinyl alcohol; and neutral silica sol have also been used for this purpose.

By coating the surface with a layer of the uncharged hydrophilic polymer, both electrostatic attraction and hydrophobic interaction can be avoided.

One way of attaching polyethylene glycol tails to a solid polymer surface is first to subject the surface to so-called acidic etching, then to adsorb a cationic poly¬ mer, polyethylene imine, to the surface, and finally to react a reactive polyethylene glycol derivative to avail¬ able amino groups in the polyethylene imine layer. This technique has been described in Prog. Colloid Poly . Sci. 74, 113-119 (1987). During the acidic etching which is carried out with potassium permanganate in concentrated sulphuric acid, carboxylic acid and sulphonic acid groups as well as sulphuric acid esters are formed on the sur¬ face. To this higly negatively charged polymer surface, the cationic polyethylene imine is bonded very strongly by electrostatic forces. Furthermore, it is likely that salt bonds between amino groups in the polyethylene imine and carboxylate and sulphonate groups on the surface upon drying gradually are transformed into amide bonds, which

gives an even stronger bond of the polyethylene imide to the surface.

Even though hydrophilised surfaces made by the tech¬ nique described in the above paper give an improved repel- lency of biopolymers, the adsorption thereof is still much too high for a number of applications.

Hydrophilised surfaces of this type are of great interest to, inter alia, the above-mentioned applications of immobilised proteins. To covalently bond protein to such a surface, it is necessary to introduce into the hydrophilic layer reactive functional groups serving as anchoring points for the protein. However, it has proved extremely difficult to covalently bond protein to thoroughly hydrophilised surfaces, even if the surfaces contain a high concentration of reactive groups. The hydrophilic surface does not attract the protein. On the contrary, it acts as a repellent because it is energet¬ ically unfavourable for a protein in aqueous solution to approach such a surface. As a result, the amount of immo- bilised protein usually will be low, regardless of whether it is an antibody for solid phase diagnostics, an immuno- stimulating substance for extracorporeal therapy, or an enzyme for bio-organic synthesis.

Thus, there is a need for improved methods of immobi- using biopolymers to hydrophilic layers, as well as a need for making the hydrophilic layer even more highly developed to give low spontaneous adsorption. A thoroughly developed hydrophilic layer, on the other hand, renders the introduction of desirable biopolymers more difficult. According to the invention, it has now proved pos¬ sible to improve the immobilisation of desirable polymers, while simultaneously obtaining a thoroughly developed hydrophilic layer of low spontaneous adsorption. According to the invention, this is achieved by a) causing a water-soluble conjugate consisting of nonionic hydrophilic polymer chains covalently bonded to a polyethylene imine and at least partly to biopolymers, to

react with a solid surface with anionic groups capable of reacting with the amino group; or b) causing a solid surface with anionic groups cap¬ able of reacting with amino groups, to react with a poly- ethylene imine substituted by nonionic hydrophilic polymer chains, whereupon the biopolymer is caused to react with reactive groups of the nonionic hydrophilic polymer chains in the presence of a reaction medium having a dielectri¬ city constant which is less than 10% of the dielectricity constant of pure water; or c) causing a solid surface with anionic groups cap¬ able of reacting with amino groups, to react with poly¬ ethylene imine substituted by an anionic hydrophilic polymer chain deriving from a nonionic hydrophilic polymer having a cloud point which is at least 5°C above the tem¬ perature at which the final product is to be used, and which furthermore contains biopolymer-reactive groups, whereupon the biopolymer is covalently bonded in per se known manner to the reactive groups of the nonionic poly- mer in a water-base reaction medium at a temperature which is more than 5°C below the cloud point of the nonionic hydrophilic polymer in the reaction medium.

By this method, there is obtained a solid surface coated with a hydrophilic outer layer having covalently bonded biopolymers and consisting of nonionic hydrophilic chains which also are bonded to a polyethylene imine which, via its amino groups, is bonded to anionic groups in the solid surface. The method makes it possible to readily introduce hydrophilic nonionic polymer chains in such an amount that they constitute at least 50%, based on the weight of the polyethylene imine, whereby low spon¬ taneous adsorption is ensured.

By first forming the water-soluble conjugate or the polyethylene imine derivative with hydrophilic nonionic polymer chains, and allowing these to adsorb to the nega¬ tively charged surface, a dense and thick hydrophilic layer is obtained.

According to the invention, the water-soluble conju¬ gate can be synthesised by first causing the polyethylene imine to react with a nonionic hydrophilic polymer having groups capable of reacting with the amino groups. Examples of such groups are oxirane rings, aldehyde groups, sul¬ phonic acid esters, tresylate, mesylate, tosylate, cya- huric chloride, carbonyl imidazole, and active carboxylic acid esters. The ratio of reactive amino groups in the polyethylene imine to reactive groups in the nonionic polymer is adjusted such that the latter is bonded with a low number of bonds.

An alternative way of attaching hydrophilic nonionic polymer chains to the polyethylene imine is to add to the latter ethylene oxide, or ethylene oxide and propylene oxide, butylene oxide and/or tetrahydrofuran, to the desired chain length. In the event that a copolymerisation is carried out, the reactants can be distributed randomly or in blocks, or in a combination thereof. When alkoxyla- tion is over, terminal hydroxyl groups are transformed into any of the above-mentioned reactive groups.

The biopolymer is then bonded in per se known manner to polyethylene imine derivatives by reaction between reactive groups on the hydrophilic nonionic polymer chains and functional groups on the biopolymer. The number of biopolymers bonded to each hydrophilic nonionic polymer chain may vary within wide limits depending on the type of biopolymer and hydrophilic nonionic polymer and on the desired degree of immobilisation. Usually, at least 5% of the hydrophilic polymer chains have covalently bonded biopolymers. In the event that the nonionic hydrophilic polymer chains can have two or more groups reactive with the biopolymer, for example when they are derived from cellulose ethers, the number of covalently bonded bio¬ polymers in. each chain may be more than one. Many such coupling reactions for biopolymers, such as proteins, are described in literature, for example in Macromol. Chem. Phys. 25 (1985) pp. 325-373. Bonding of the biopolymers is

usually carried out in water as reaction medium and is made easier by the fact that the polyethylene imine deri¬ vative is dissolved in the water and not applied to a solid surface. According to the invention, the immobili- sation can be further promoted if it is carried out -n the reaction environment and under the conditions set forth in the production alternatives b) and c) and which will be described in more detail hereinafter. When bonding is over, the remaining reactive groups are reacted on the hydrophilic nonionic polymer chains in some suitable man¬ ner, for example by reaction with 2-mercaptoethanol and 2-aminoethanol, resulting in a water-soluble conjugate suitable for application to a solid surface with anionic groups. Bonding of the biopolymers may also be carried out by first coupling the hydrophilic nonionic polymer to the biopolymer and then reacting the resulting product in the above-mentioned manner with the polyethylene imine.

For bonding proteins and peptides, use is preferably made of amino, thiol or phenolic hydroxyl groups reacting by nucleophilic attack with the electrophilic reactive groups at the ends of the polyethylene glycol chains. Examples of such groups are epoxides, aldehydes, sulphonic acid esters, such as tresylate, mesylate and tosylate, cyanuric chlorides, carbonyl imidazoles and carboxylic acid esters. Glycoproteins and carbohydrates can be bonded inversely by attaching suitable groups, such as amino groups, to the ends of the polyethylene glycol chains, and causing them to react with aldehyde groups or carboxylic acid groups originally present or generated in the poly- saccharide, for example by periodate oxidation. This tech¬ nique is described in US Patent 4,217,338.

The resulting soluble conjugate is then made to adsorb to a negatively charged solid surface. Examples of suitable surfaces are those which have a natural negative net charge, for example silica and glass, or those in which negative charges have been generated by chemical or

physical means. Negative charges can be induced on organic polymer surfaces by, for example, acidic etching, i.e. treatment with potassium permanganate in concentrated sul¬ phuric acid, or by plasma- or radiation-induced grafting of an anionic component, such as acrylic acid or meth- acrylic acid. Examples of organic polymers suitable for this purpose are polystyrene, polyvinyl chloride, poly¬ ethylene, polymethyl methacrylate, polycarbonate, poly- sul one and cellulose acetate. The method of immobilising biopolymer to a surface by first bonding it in solution to a water-soluble polymer and then adsorbing the soluble conjugate to the surface, has been described before. Bonding to bovine serum albumin is a technique occasionally used for proteins and pep- tides, and PCT/SE88/00243 describes the use of a hydro- phobated water-soluble polymer, especially hydrophobated uncharged polysaccharide, for this purpose. The present invention, however, discloses a novel and improved prin¬ ciple. By bonding the biopolymer to the surface via a dense layer of hydrophilic nonionic polymer chains, there is obtained an uncharged hydrophilic background surface to which very little nonspecific adsorption occurs, simul¬ taneously as the biopolymer which is anchored to spacer arms reaching far into the water phase, has high access- ibility to, for example, antibody-antigen reactions.

The method of the present invention imparts to the layer of hydrophilic nonionic polymer chains a very high density, far higher than is obtained by direct bonding of hydrophilic nonionic polymers to a solid surface. The dif- ficulty of obtaining a closely packed hydrophilic layer is that the individual hydrophilic nonionic polymers repel each other. The same repellency occurs of course also when polyethylene imine is reacted with hydrophilic nonionic polymer derivatives in solution. However, by first gene- rating the graft polymer between hydrophilic nonionic polymers and polyethylene imine in solution, then bonding the biopolymer, and finally adsorbing this conjugate with

the polyethylene imine down to the solid surface, all hydrophilic nonionic polymer chains - and this applies both to those which have bonded the biopolymer and those which have a free end group - are forced over to the water side. In the two-dimensional perspective, the number of hydrophilic nonionic polymer chains towards the water side will then be twice as large as when the conjugate was dissolved in water.

According to the invention, it is also possible first to coat the solid surface containing anionic groups with the above-mentioned polyethylene imine derivative which consists of a polyethylene imine substituted by nonionic hydrophilic polymer chains, and then to bond the biopoly¬ mers via reactive groups in the nonionic hydrophilic polymer chains. By this technique, bonding takes place in the presence of a reaction medium having a dielectricity constant of less than 10%, preferably less than 5%, of the dielectricity constant of pure water. In the event that the nonionic hydrophilic polymer chains derive from a nonionic hydrophilic polymer having a cloud point which is at least 5°C above the temperature at which the final pro¬ duct is to be used, the biopolymers can also be bonded in a water-based reaction medium at a temperature which is more than 5°C below the cloud point of the nonionic hydro- philic polymer in the reaction medium. These methods of introducing biopolymers are described in Swedish Patent Applications 9002909-1 and 904397-8, respectively.

An especially preferred form of reaction medium hav¬ ing a low dielectricity constant is a microemulsion. The amount of water in the microemulsion usually is 0.5-25% by weight, preferably 1-15% by weight.

The nonpolar reaction medium and the hydrophobic com¬ ponent in the microemulsion usually are an aliphatic hydro carbon, such as hexane or nonane, or a broader distillatio fraction, such as petroleum ether 60-80. The hydrophobic component of the microemulsion usually constitutes 63-98.5. by weight.

The surface-active component usually is a combination of a surface-active compound and a so-called auxiliary tenside. The surface-active substance may be anionic, cationic, amphoteric or nonionic, while the auxiliary ten- side usually is an alcohol or a low-molecular alkylene oxide adduct. Examples of conventional substances of this type are butanol, pentanol, hexanol, ethylene glycol mono- butyl ether and diethylene glycol monobutyl ether. The amount of surface-active component usually constitutes 0.5-20% by weight of the weight of the microemulsion.

It has been found especially advantageous to use a surface-active compound capable of forming microemulsions in the absence of an auxiliary tenside. Surface-active compounds having this ability are certain nonionic com- pounds which as hydrophilic group have a polyalkylene glycol chain produced by polymerisation of ethylene oxide or by combinations of ethylene oxide and propylene and/or butylene oxide, as well as certain ionic compounds having the ionic hydrophilic group in a non-terminal position on the hydrocarbon chain.

As regards nonionic tensides, the preferred hydro¬ philic part is a polyethylene glycol chain which, in the case most preferred, has a length of between 3 and 8 ethy¬ lene oxide units on an average. The hydrophobic part may derive from hydroxyl compounds or carboxy1 compounds con¬ taining an alkyl chain which consists of 8-20 carbon atoms, or of an alkyl aryl group which consists of 9-24 carbon atoms in all. Examples of such compounds are ethylene oxide adducts of nonyl phenol, octyl phenol and fatty alcohols.

The preferred ionic tensides are anionic groups, such as sulphonate, sulphate, carboxylate, phosphate and phos- phonate, sulphonate being especially preferred. If desired, these tensides may also contain alkylene oxide groups, such as ethylene oxide, as coupling agents between the anionic group and the hydrophobic group. The hydro¬ phobic part may consist of an alkyl chain consisting of

10-22 carbon atoms, or of an alkyl aryl group consisting of 9-24 carbon atoms in all. A few ether, ester or amide bonds may occur in the hydrophobic part. Examples of suit¬ able ionic compounds are di(2-ethylhexyl)sulphosuccinate and carboxymethylated nonyl phenol ethoxilates containing 1-4 ethylene oxide groups.

As mentioned above, it is also possible to utilise the unusual dependence on temperature exhibited by some nonionic water-soluble polymers and to immobilise the bio- polymers in aqueous environment. Thus, polyalkylene gly- cols and nonionic cellulose ethers exhibit a decreasing water solubility at elevated temperature. The mechanism behind this dependence on temperature has still not been fully explained, but it is assumed that the conformation of the ethylene oxide groups is changed in connection with an increase in temperature, making the ethylene oxide groups increasingly hydrophobic in character and thus less soluble in water. At a given temperature, the water solu¬ bility of the polymer is so low that the solution is phase-separated. This temperature is usually termed the cloud point of the solution. Polyalkylene glycols and cel¬ lulose ethers can both be produced with defined cloud points, and especially useful are the polymers whose cloud points lie within the range 10-100°C, preferably 30-50°C. The nonionic hydrophilic polymer shall be hydrophilic at the temperature at which the protein-coated surface is used. It is selected such that the cloud point is at least 5°C, preferably at least 10°C, above the temperature at which the coated surface is used. A preferred protein immobilisation temperature is from 3°C below the floccula- tion temperature of the nonionic hydrophilic polymer in the reaction medium, to 50°C.

Examples of suitable polyalkylene glycols are those in which ethylene oxides and alkylene oxides having 3-4 carbon atoms, or tetrahydrofuran, are randomly distri¬ buted or distributed in blocks. Especially suitable are polyalkylene glycols having a molecular weight of

2,000-10,000 and containing one or more blocks of polyoxy propylene and polyoxy ethylene having a molecular weight of 300-3,000. Other types of suitable polyalkylene glycols are adducts of ethylene oxide in combination with higher alkylene oxides, or tetrahydrofuran with a dihydroxy _t or polyhydroxy compound, such as glycerol or pentaerythritol. The cellulose ethers preferably have such a degree of polymerisation that a 1% aqueous solution thereof has a viscosity of 10-10,000 cP, preferably 30-5,000 cP, mea- sured according to Brookfield, LV, 12 rpm at 20°C. They may comprise hydrophobic hydrocarbon groups, such as methyl, ethyl, propyl, butyl, benzyl and higher hydro¬ carbon groups having 8-24 carbon atoms, or polar hydroxyl groups, such as hydroxyethyl, hydroxypropyl and hydroxy- butyl, or mixtures of hydrocarbon groups and polar groups. Examples of suitable cellulose ethers are methyl cellu¬ lose, ethyl cellulose, hydroxyethyl cellulose, hydroxy¬ propyl cellulose, methyl hydroxyethyl cellulose, methyl hydroxypropyl cellulose, ethyl hydroxyethyl cellulose and benzyl ethyl hydroxyethyl cellulose. Alkyl hydroxyalkyl cellulose is the preferred cellulose ether.

The polyethylene imine with the hydrophilic polymer chains suitably consists of a polyethylene imine skeleton having a molecular weight of 10,000-1,000,000, preferably from 50,000 to 500,000, which to at least 90% by weight is built up of the units -C ₂ H ₄ NH-, -C ₂ H ₄ N and -C ₂ H ₄ NH ₂ in which preferably less than 20% of the reactive hydrogens of the imino and amino groups are substituted by hydro¬ philic polymer chains and, optionally, other substituents, such as alkyl groups, or hydroxyl group-containing groups utilised upon grafting of the hydrophilic polymer chains. The hydrophilic polymer chains suitably derive from non¬ ionic alkylene oxide adducts, such as polyethylene glycol or randomly distributed or block-distributed polyalkylene glycols between ethylene oxide and alkylene oxides having 3-4 carbon atoms, or tetrahydrofuran. Other types of alky¬ lene oxide adducts are adducts of ethylene oxide,

optionally in combination with higher alkylene oxides or tetrahydrofuran, with a dihydroxy or polyhydroxy compound, such as glycerol and pentaerythritol. Polysaccharides, such as dextran and starch; cellulose ethers, such as methyl cellulose, methyl hydroxypropyl cellulose, or ethyl hydroxyethyl cellulose; and polyvinyl alcohol are other suitable hydrophilic polymers. The hydrophilic polymer chains are water-soluble, and their molecular weight usually is from 400 to 200,000, preferably from 1,000 to 100,000.

The present invention will be further illustrated by the following Examples.

Example 1 A polyethylene plate having the dimensions 2 x 2 cm was washed in 70% ethanol for 3 min. in an ultrasonic bath. The plate was air-dried and then oxidised for 30 sec. in 2 g/1 KMnO./H ₂ S0 ₄ . The plate was rinsed with distilled water. A solution containing 5% of human albumin and 10% of an epoxidised polyethylene glycol (PEG), obtained by add¬ ing 320 ethylene oxide units to di(trimethylol propane), was reacted for 15 h at 30°C at pH 7.0, whereupon pH was adjusted to 9.5 with sodium hydroxide. Polyethylene imine (PEI) was added to a final concentration of 0.07% and reacted at 45°C under agitation. After 2.5 h, the poly¬ ethylene plate was placed in the solution, and the albumin-PEG-PEI complex was allowed to adsorb for 2 h at 40°C. Parallelly, only the PEG-PEI adduct was synthesised as above, and a polyethylene plate was hydrophilised. Unreacted epoxides were reacted with mercaptoethanol. Also added to the reacted PEG-PEI adduct was albumin, and the copolymer with free albumin was allowed to adsorb to a polyethylene plate. The thickness of the PEG layer was determined by ellipsometry to 27 nm by first measuring the thickness of a layer of PEI alone and the thickness of the

PEG-PEI layer, whereupon the former value was subtracted from the latter.

The amount of bonded albumin was detected by ELISA technique with peroxidase-conjugated antibodies against albumin. The amount of adsorbed protein is proportional to the adsorbency at 490 nm. The results were as follows.

PEG/PEI,

Sample OD 495 nm % by weight

Immobilised albumin accord¬ ing to the invention 1.266 78

Control, without albumin 0.054 78

Control, with albumin 0.178 78

The results show that the plate according to the invention has a high content of albumin, and that the hydrophilic layer has a low spontaneous adsorption of biopolymer.

Example 2

A 96-well microtiter plate of polystyrene was grafted with crotonic acid and gamma-radiation.

A solution containing 0.07% polyethylene imine and 10% of a tresylated polyethylene glycol of molecular weight 7,000 was reacted for 2 h at 37°C and pH 9.0. Immu- noglobulin G (IgG) was added up to a concentration of 1% and allowed to react for a further 3 h at 37°C. The solu¬ tion was dispensed to the mictrotiter plate and allowed to adsorb for 2 h at 40°C. Parallelly, only the PEG-PEI adduct was synthesised as above, and a microtiter plate was hydrophilised. Unreacted tresylate groups were reacted with 1 M sodium hydroxide.

The amount of bonded IgG was detected by ELISA tech¬ nique with peroxidase-conjugated antibodies against IgG. The following results were obtained.

PEG/PEI, Sample OP 495 nm % by weight

Immobilised IgG according to the invention 0.983 80

Control, without IgG 0.026 80

The results show a high immobilisation of IgG and a low spontaneous adsorption to the hydrophilic layer.

Example 3

A PVC plate having the dimensions 2 x 6 cm was acti¬ vated in the same way as the polyethylene plate of Exam¬ ple 1.

A solution containing 2% fibrinogen and 5% of a block polymer of ethylene oxide and propylene oxide having a cloud point of 35°C and equipped with epoxide groups at both ends was allowed to react for 8 h at 37°C and at pH 7.0. Then, pH was adjusted to 9.5 with sodium hydroxide, and polyethylene imine (PEI) was added to a final concentration of 0.03%. The reaction was allowed to proceed for 2.5 h at 45°C under agitation, whereupon the activated PVC plate was immersed in the solution, and the fibrinogen-block polymer-PEI complex was allowed to adsorb at 40°C for 1 h.

As reference, use was made of a PVC plate hydrophi¬ lised with the block polymer-PEI adduct without protein in which any remaining epoxide groups were reacted by treat¬ ment with 1M HC10 4. for 1 h. The thickness of the block polymer layer was determined by ellipsometry to 30 nm by first measuring the thickness of the layer of PEI and the thickness of the block polymer-PEI layer, whereupon the former value was subtracted from the latter.

The amount of bonded fibrinogen was detected by ELISA technique with peroxidase-conjugated antibodies against fibrinogen.

Block Polymer/PEI Sample OP 495 nm % by weight

Immobilised fibrinogen according to the invention 1.605 75

Control, without fibrinogen 0.070 75

The results show a high immobilisation of fibrinogen and low spontaneous adsorption.