WO2017215683A1

WO2017215683A1 - Method of preparation of a substrate containing carboxybetaine groups and bound bioactive substances which is resistant against undesirable deposition from biological media

Info

Publication number: WO2017215683A1
Application number: PCT/CZ2017/050025
Authority: WO
Inventors: Hana LISALOVA; Eduard Brynda; Ivana VISOVA; Milan Houska; Frantisek SURMAN; Katerina MRKVOVA; Xue CHADTOVA SONG; Jiri Homola
Original assignee: Ustav Fotoniky A Elektroniky Av Cr VVI; USTAV MAKROMOLEKULARNI CHEMIE AV CR VVI
Current assignee: Ustav Fotoniky A Elektroniky Av Cr VVI; USTAV MAKROMOLEKULARNI CHEMIE AV CR VVI
Priority date: 2016-06-17
Filing date: 2017-06-16
Publication date: 2017-12-21
Anticipated expiration: 2018-12-17
Also published as: CZ307026B6; CZ2016361A3

Abstract

The present invention relates to a method of preparation of a substrate surface containing carboxybetaine groups with bound bioactive substances, having resistance to undesirable deposition of biological media components on the substrate surface, comprising the steps of chemical activation of carboxybetaine groups on the surface of the substrate by converting the carboxybetaine carboxyl group into an active ester, covalent binding of a bioactive substance to some of the active esters, and reaction of the product from the previous step with an acid of general formula (I) NH₂-(CH₂-CH₂-0)_n-CH₂-COOH, where n = 1 to 4, wherein the acid of general formula (I) reacts with those active esters, which have not undergone covalent bonding of the bioactive substance.

Description

Method of preparation of a substrate containing carboxybetaine groups and bound bioactive substances which is resistant against undesirable deposition from biological media

Field of Art

The present invention relates to a method of preparation of a substrate surface containing carboxybetaine functional groups with bound bioactive substances, which significantly increases surface resistance against undesirable biological deposition upon contact with biological media. Background Art

At contact of virtually all common materials with biological media, biological deposition (so-called "fouling") on their surface takes place, which begins with the adsorption of biological molecules, especially proteins. Depending on composition of the medium, cells and microorganisms can adhere on them, followed by other biological processes such as blood coagulation, inflammatory and immune reactions, or formation of bacterial biofilms. Resulting biological deposits may impair the function of materials and equipment that work in biological media such as body fluids, cell- containing media, food and media from biological production, and from biological environment in general. The problem is particularly critical for materials used in contact with blood serum, plasma, or blood. Therefore, surfaces that prevent non-specific formation of biological deposits in biological media and simultaneously facilitate binding of bioactive elements enabling the specific interaction of the surface with target components of the biological environment are very important for many biotechnological and medical applications. Such applications include biosensors, membranes and particles for separation and accumulation of biological agents and cells, drug carriers and diagnostic particles applied to blood stream, blood-contacting materials and cell carriers or scaffolds for tissue engineering.

Currently, the most resistant "antifouling" surfaces are considered those prepared by grafting hydrophilic electroneutral polymers such as non-ionogenic (poly(oligo(hydroxy ethylene glycol) methacrylate) (polyHOEGMA), poly(2-hydroxyethyl methacrylate) polyHEMA, poly(3- hydroxypropyl methacrylate) (polyHPMA), poly(N-(2-hydroxypropyl methacrylamide) (polyHPMAA), and zwitterionic poly(carboxybetaine methacrylate) (polyCBMA), and poly(carboxybetaine acrylamide) (polyCBAA) from the substrate surface (the "grafting from" method) using the surface -initiated atom transfer radical polymerization (SI-ATRP). The resulting so-called polymer brush is a layer of densely arranged polymer chains bonded with one end to the surface. Brushes made of polyCBAA, polyCBMA, and polyHPMAA are the only ones effectively suppressing even deposition from undiluted blood plasma and serum.

An alternative method is to graft polymer chains prepared by polymerization in solution to a surface, or the so-called "grafting to" method. Smaller density of polymer chains achieved in the brushes prepared by this method versus brushes prepared by the "grafting from" method gives the surface a weaker resistance to biological deposition.

Even weaker resistance, particularly against plasma and serum deposition, provide frequently used coatings of carboxymethyl dextran or self-assembled monolayers (SAMs) from a mixture of (CH₂)n(EO)₄0H and (CH₂)n(EO)₆COOH molecules where EO is an ethylene oxide unit. Their surface is formed by densely arranged oligoethylene oxides containing in certain positions carboxyl groups used for binding of bioactive substances. Functionalization of non-ionogenic polymer chains by binding of bioactive substances uses activation of hydroxyl groups in their side chains. The residual products of this activation, which remain on the polymers after functionalization, greatly aggravate the resistance of brushes against biological deposition. Surfaces with desired biological activity and better resistance to deposition were prepared by binding bioactive substances to polyCBMA or polyCBAA brushes that have zwitterionic carboxybetaine as side groups of polymer chains. Brushes of polyCBMA and polyCBAA with bioactive substances bound to side carboxybetaine groups of polymers are included, among others, in US20140370567 and US20130244249. Patent application PV 2015-313 describes poly(HPMAA-co-CBMAA) brushes grafted via copolymerization of HPMAA monomers and carboxybetaine acrylamide (CBMAA) from surfaces of various substrates including polymer nanoparticles and binding of bioactive substances to their activated carboxybetaine groups. PCT/CZ2016/050011, which relates to PV 2015-313, also includes the preparation of brushes from poly(HPMAA-co-CBMAA) prepared by polymerization in a solution and covalently grafted onto the surface of substrate. The state of the art also include hydrogels of poly(HEMA-co-CBMAA) copolymers resistant to biological deposition (Kostina et al., Biomacromolecules 2012,13,4164- 4170) or sorbents whose surface is modified by grafting of carboxybetaine zwitterions (WO2014165421 Al).

Bioactive substances containing one or more amino groups are bound to the carboxybetaine groups virtually exclusively by reaction with the carboxybetaine zwitterion carboxylate which is first activated into an intermediate product easily reacting with the nucleophilic amino group of the bound bioactive substance. This activation is mainly accomplished by reacting with l-ethyl-3-(3- dimethylaminopropyl)carbodiimide (EDC) in the presence of N-hydroxysuccinimide (NHS) or its derivatives, resulting in the formation of an active NHS ester (EDC/NHS activation). The bioactive substance is subsequently bonded by reaction of its amino group with an active NHS ester formed by activating the carboxyl in the carboxybetaine group.

After binding of bioactive substances, it is necessary to deactivate the residual active NHS esters in order to avoid, when the surface is in contact with complex solutions, the non-specific deposition, or binding of biological substances, especially proteins, by covalent bonds of their amino groups. NHS esters formed from free carboxyl groups, e.g. on the surface of the above-mentioned SAMs, are deactivated back to carboxyl groups using spontaneous hydrolysis in aqueous media.. Another traditional method is the deactivation of residual active NHS esters by ethanolamine binding, for example deactivation of functionalized carboxymethyl dextran by incubation with a solution of ethanolamine (US 5561069 A). Several documents describe the use of glycine as a deactivating agent for deactivating of active NHS esters in activated carboxybetaine groups. US 7 943 370 B2 describes the preparation of a substrate surface containing carboxybetaine groups by converting carboxybetaine into active ester, covalent binding of a bioactive substance and deactivating unreacted active esters with glycine or β-alanine. WO 2009/130 233 Al describes the preparation of halogenated surfaces with a potential content of carboxybetaine groups by converting the carboxyl group to an active ester, covalent binding of a bioactive substance and deactivating the unreacted active esters with glycine. Nonpatent documents by Chou Y.-N. et al., Acta Biomaterialia 2016, 40, 31-37 and Vaisocherova H. et al., Biosensors and Bioelectronics 2014, 51, 150-157 describe the preparation of a substrate surface containing carboxybetaine groups by converting carboxybetaine into active ester, covalent binding of bioactive substance and deactivating unreacted active esters using glycine.

The current state of the art of technology provides a method of deactivating the aforementioned active esters using amino acids, especially glycine. However, the present invention describes a significantly more efficient method of deactivating active esters on the substrate surface. Compared to the current state of the art, the use of this more efficient method of deactivation leads, in most cases, to complete or almost complete restoration of the original (prior to activation and binding of bioactive substances) ability of the surface to withstand non-specific deposition from biological media. Disclosure of the Invention

The present invention relates to a method for preparing surfaces which by reacting with an acid of general formula (I) regenerate carboxyl groups from active NHS esters remaining after binding of bioactive substances.

Parts of a product or device, the surface of which in a biological medium has an active function to perform that could be affected by biological deposition, and whose surface contains carboxybetaine groups, may be from organic and inorganic materials. They may have any morphology such as particles, membranes, pipes, hoses, wafers, porous material, and fibre webs and may have various uses in contact with biological media, such as biosensors, affinity particles and membranes for separation and accumulation of biological agents, targeted drug delivery carriers, biomaterials for tissue engineering, and antithrombogenic materials for contact with blood. The present invention relates on the fact that immediately after the activation of carboxybetaine groups and binding of bioactive substances, the substrate surface is incubated with a solution of an acid of general formula (I). During this incubation, the terminal amino group of this molecule reacts with active esters that were not consumed in the prior binding of bioactive substances. The terminal carboxyl group in the part of the molecule that replaced the active ester forms a new zwitterion with the quaternary nitrogen cation left over after activation of the original carboxyl group. The above procedure on one hand neutralizes the reactivity of the residual active esters while at the same time compensates the positive charge of quaternary nitrogen cations, thereby increasing the resistance of the surface against biological deposition. The technical effect of this method for preparing substrate surface containing carboxybetaine functional groups takes shape particularly, but not only, in high density polymer brushes containing carboxybetaine side groups. Small molecules of activating reagents can penetrate between the polymer chains and react with carboxybetaine s throughout the whole volume of the brush, while for binding of large bioactive substances, such as proteins, only activated carboxybetaine s near the surface of the layer are available. It has been found that the deactivation of active esters by the acid of the present invention is significantly more effective than the deactivation conventionally carried out by glycine or by other methods. Even with 10 times lower concentrations of the acid according to the present invention, the deactivation efficiency was comparable or slightly higher than deactivation by glycine. Using the same concentrations of glycine and acid according to the present invention, the deactivation efficiency of the present invention was significantly higher. This more efficient method of deactivation leads, in most cases, to a complete or almost complete restoration of the original ability of the surface to resist nonspecific deposition from biological media. The subject of the present invention is a method of preparation of a substrate surface containing carboxybetaine groups, with increased resistance to undesirable deposition of biological media components on the substrate surface, comprising the following steps:

a) chemical activation of carboxybetaine groups on the surface of the substrate by converting the carboxybetaine carboxyl group into active ester;

b) covalent binding of a bioactive substance by reacting of its amino group with the active ester prepared in step a);

wherein following step b), step c) is carried out in which the product of step b) reacts with an acid of general formula (I)

NH₂-(CH₂-CH₂-0)„-CH₂-COOH (I),

where n = 1 to 4, wherein the acid of general formula (I) reacts with those active esters, which have not undergone covalent bonding of the bioactive substance in step b).

Carboxybetaine groups can either be directly a part of the substrate material or may be attached to the substrate surface, e.g., as a part of molecules grafted onto the substrate surface, or the substrate can be coated with a layer containing these groups, e.g. a polymer brush prepared by grafting from the substrate surface using surface initiated polymerization ("grafting from") or prepared by grafting of the polymer chains prepared by polymerization in solution to surface ("grafting to").

Carboxybetaine groups are well known to a person skilled in the art, and are generally defined as neutral chemical groups containing a quaternary ammonium cation that carries no hydrogen atom and a negatively charged carboxyl group not directly adjacent to quaternary ammonium cation (IUPAC, Compendium of Chemical Terminology).

In one preferred embodiment, the acid of general formula (I) is (2-aminoethoxy)acetic acid (AEAA).

In one preferred embodiment, concentration of the acid of general formula (I) in step c) of the method according to the present invention is in the range of from 0.5 M to 2 M, more preferably in the concentration range of from 0.7 M to 1.5 M, most preferably at the concentration of 1 M. The pH of step c) is preferably neutral or basic in the range of from pH 7 to 9, more preferably from pH 7 to 8.5, most preferably at pH 8. For the purposes of the present invention, the substrate is an object which is resistant to the deposition of biological media components or which is to be coated with a polymeric layer giving it resistance to the deposition of biological media components. A polymer brush can be grafted to it or it can be coated with a polymer. A substrate thus can be:

(1) An object whose surface is covered by a polymer brush layer ("grafted from") by live radical polymerization, see PV 2015-313 and PCT/CZ2016/050011,

(2) An object whose surface is covered by a layer of polymers prepared in solution and subsequently attached to the surface by covalent bonding or by physical adsorption ("grafted to"), see PCT/CZ2016/050011,

(3) An object whose surface is covered by polymerization initiated by radicals formed by chemical or physical activation of the surface of the object,

(4) An object whose surface is coated with an adhesive polymeric layer deposited from a solution (polymeric coating),

(5) An object whose surface can be modified by binding to a molecule containing carboxybetaine zwitterions,

(6) An object containing carboxybetaine zwitterions in its structure, such as a polymer gel, where some monomer units contain carboxybetaine zwitterions.

The polymeric layers formed on objects (1), (2), (3), and (4) always contain at least one homopolymer or copolymer containing carboxybetaine zwitterions in side chains. The layers on objects (2) and (4) may contain polymers containing carboxybetaine zwitterions in a mixture with hydrophilic polymers, preferably selected from polyHPMAA, polyHOEGMA, polyHEMA, and polyHPMA.

Preferably, the layers on objects (1), (2), (3), and (4) are formed from a homopolymer selected from the group comprising polyCBMAA, polyCBMA, and polyCBAA or from a copolymer poly(A-co- B), where A is a monomelic unit selected from the group comprising HPMAA, HOEGMA, HEMA, HPMA, and B is a monomelic unit at a concentration from 1 to 99 mol%, selected from the group comprising CBMAA, CBMA, CBAA.

The shape, dimensions, morphology, and chemical nature of the substrate are not critical. They may be planar or differently shaped objects, tubes, fibres, particles, membranes, microparticles, nanoparticles, porous materials, metals, silicon, silicate- or aluminosilicate-based materials (such as glass), polymers, inorganic materials, and the like.

The active carboxybetaine ester is a product of a reaction of carboxyl group of carboxybetaine with N-substituted carbodiimides, i.e. O-acylurea, and/or a product of a reaction of carboxyl group of carboxybetaine with N-substituted carbodiimides and N-hydroxysuccinimide (NHS) or derivatives thereof. Preferably, active esters are NHS ester or sulfo-NHS ester. In a preferred embodiment, step c) is carried out by first rinsing the product of step b) with a solution or buffer, which was used as the solvent in step b), preferably water, an aqueous NaCl or PBS, followed by rinsing with a buffer, which will be used in the next step for incubation, preferably PBS buffer. Then the product is incubated with a solution of aminoethoxyacetic acid of general formula (I) in a buffer (preferably PBS buffer). Then it is rinsed with the buffer previously used for incubation, and then rinsed with a solution into which it is then deposited for storage or with water and dried. In a preferred embodiment, the bioactive substance of the present invention is a substance containing at least one NH₂ group selectively interacting with the target component of the biological medium. The bioactive substance may have affinity for the target component. Typically, the bioactive substance is a natural antibody, antigen, lectin and the cellular receptor and their synthetic analogues and parts prepared by recombinant techniques as well as synthetic oligopeptide sequences, nucleic acids and portions thereof, and synthetic oligonucleotide sequences and aptamers. The bioactive substance can catalyse chemical conversion of the target substance, such as enzyme, coenzyme, and their synthetic analogues. Alternatively, the bioactive substance can induce a biological response, such as an anticoagulant including heparin, proteins and oligopeptide sequences responding to cellular integrins, growth factors, hormones, and derivatives of drugs and vitamins. Bioactive substances may also include nanoparticles functionalized by the NH₂ group, in particular metallic, polymeric, and silicon nanoparticles, as well as nanoparticles based on metal oxides, or polymeric nanoparticles with magnetic core. Preferably, the bioactive substance is a substance with affinity for the target component, selected from the group consisting of antibody, antigen, lectin, cellular receptor and analogues thereof, as well as parts prepared by recombinant techniques, synthetic oligopeptide sequences, nucleic acids and parts thereof, synthetic oligonucleotide sequences, and aptamers; a substance catalysing chemical transformation of the target substance, selected from the group comprising enzymes, coenzymes, and synthetic analogues thereof; a substance that induces biological response, selected from the group comprising anticoagulants, proteins and oligopeptide sequences reacting with cellular integrins, growth factors, hormones, and drugs.

The biological medium for the purposes of the present invention is a fluid containing biological agents, that is biomolecules and their associates (proteins, saccharides, polysaccharides, lipids, nucleic acids, lipoproteins, glycoproteins, organelles etc.), viruses, cells, microorganisms, and fragments thereof. The biological medium is, for example, blood and other body fluids, blood plasma and serum, tissue extracts, cell lysates and suspensions, as well as food extracts. In one embodiment, the carboxybetaine groups are contained in a polymeric layer on the substrate surface as a polymer brush, prepared by surface polymerization or grafting of polymers to the substrate surface, or a polymer coating, preferably having a thickness ranging between 5 nm and 5 um.

In one preferred embodiment, the polymeric layer containing carboxybetaine groups is a polymer brush of poly(carboxybetaine methacrylamide) (polyCBMAA), poly(carboxybetaine methacrylate) (polyCBMA), poly(carboxybetaine acrylamide) (polyCBAA) or copolymer poly (A-co-B), where A is a monomeric unit from the group of N-(2-hydroxypropyl) methacrylamide (HPMAA), 2- hydroxyethyl methacrylate, 3-hydroxypropyl methacrylate and oligo(hydroxy ethylene glycol) methacrylate, and B is a monomeric unit from the group of carboxybetaine methacrylamide, carboxybetaine methacrylate and carboxybetaine acrylamide at a concentration of from 1 to 99 mol%, especially poly(HPMAA-co-CBMAA/xmol%), where x (molar concentration of CBMAA) ranges between 1 and 99 mol%. Preferably, this layer has a thickness in the range of from 5 nm to 5 um.

In one embodiment, the substrate is selected from the group comprising particles, porous membranes, cell carriers ("scaffolds"), and biosensors.

Particles are preferably from material selected from the group containing gold, silver, magnetic materials, silicon, S1O2, and polymers falling within the definitions of substrate (1) to (6) and preferably have their diameter of from 5 nm to 1 mm. Particles with bioactive substances bound on activated carboxybetaine groups on the surface of particles including the inner surfaces of porous particles are applicable as carriers for targeted therapy and in vivo diagnostics, separation and accumulation of biological agents, and for enzymatic catalysis in bioreactors.

Porous membranes are membranes designed for affinity separation of biological substances from biological media.

Cells carriers or scaffolds, well known to a person skilled in the art, are used for example for tissue engineering such as:

http ://www. wikiskripta.eu/index.php/Tk%C3 %A 1 %C5 %88ov%C3 %A9_in%C5 %BEen%C3 %BDr stv%C3%AD).

In one embodiment, the substrate is a biosensor detection surface for direct detection or multistage detection of analytes in complex biological media, such as by optical or mass biosensors. Preferably, the detection surface is coated with a polyCBMAA, polyCBMA, polyCBAA or poly (HPMAA-co-CBMAA) polymer brush with bound bioreceptors. Bioreceptors in this application are bioactive agents that have selective affinity for the target components of the analysed medium.

In one embodiment, the substrate is intended for contact with blood in vitro, ex vivo, and in vivo.

The method of preparation of a substrate surface containing carboxybetaine groups according to the present invention, which has an increased resistance to undesirable deposition of biological media components on the surface of the substrate, provides a very rapid, cheap, and effective mitigation of problems with deposition of undesirable components of biological media. In contrast to the current solutions of the background art (use of glycine), the claimed use of (2-aminoethoxy)acetic acid is more effective, as illustrated in the following comparative examples.

Brief description of figures Fig. 1 : Diagram of activation of carboxybetaine groups (a), binding of bioactive substance (b), and incubation with an aqueous solution of (2-aminoethoxy)acetic acid (AEAA) (c).

Fig. 2: Example of detection of carcinoembryonic antigen (CEA, 10 ug/mL) in undiluted blood plasma using an SPC biosensor coated with a polyCBAA brush functionalized by anti-CEA binding and subsequently deactivated by AEAA. The figure shows the compensated reference data that was obtained by subtracting the SPR response from the reference channel with anti-CEA incubated in undiluted plasma from the measuring channel with anti-CEA incubated in undiluted blood plasma with added CEA (10 μ^ιηΐ,).

Fig. 3: Example of detection of Salmonella typhimorium (1 ^χ 10⁷ CFU/mL) in a homogenized cucumber solution using a SPR biosensor coated with a polyCBAA brush functionalized by anti- Salm binding and subsequently deactivated by AEAA. The figure shows reference compensatory data obtained by subtracting the SPR response from the reference channel with anti-Salm incubated in a homogenized cucumber solution from the anti-Salm measuring channel incubated in Salmonella-added homogenized cucumber solution (1 ^χ 10⁷ CFU/mL). Examples

Example 1 : Method of treating a surface of the substrate containing activated carboxybetaine functional groups by reaction with (2-aminoethoxy)acetic acid

Fig. 1 shows the method of treating a surface of the substrate containing carboxybetaine functional groups. In step (a), the carboxybetaine groups are chemically activated by converting the carboxyl groups of carboxybetaine into an active NHS ester or sulfo-NHS ester by reaction with l-ethyl-3- (3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) or sulfo NHS. In step (b), incubation with a solution containing the bioactive substances (BAS) takes place during which these substances are covalently bound to some of the active esters formed in step (a). In step (c), a reaction of those active NHS esters or sulfo-NHS esters with (2-aminoethoxy)acetic acid (AEAA) takes place, which did not react with bioactive substance in step b). During the sample incubation with AEAA aqueous solution, the AEAA molecules are covalently bound to the surface by a permanent amide bond.

Comparative Example 1: Regeneration of resistance of EDC/NHS activated polyCBAA brushes against biological deposition by a reaction with deactivating agents

The golden surface of a chip for measuring of the surface plasmon resonance (SPR), a 50 nm gold- plated glass plate, was incubated with a 1 mM solution of co-mercaptoundecyl bromoisobutyrate initiator in ethanol and left overnight in the dark. From the initiator layer immobilized on the golden surface, a polyCBAA brush from the CBAA monomer solution of the catalyst mixture containing CuCl, CuCl₂ and Me₄Cyclam was grafted using the SI ATRP method.

The chip covered with the polyCBAA brush was rinsed with water and mounted into the SPR sensor chamber with four flow microfluidic channels. Carboxybetaine groups of brush in the 2^nd, 3^rd, and 4^th channels were activated by reaction with an aqueous solution of N-hydroxysuccinimide (NHS, 0.1 M) and l-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC; 0.5 M) for 20 minutes with 10 mM NaCl at 20 °C (step (a) in Example 1). After rinsing with water for 5 minutes, the activated brush was exposed to deactivation buffer (10 mM sodium borate + 10 mM imidazole + 10 mM NaCl, pH 8) for 30 minutes or 40 minutes in the 2^nd channel, to the solution of (2- aminoethoxy)acetic acid (1 M, water pH 7, 30 minutes) in the 3^rd channel, and to the glycine solution (1 M, water pH 7, 30 minutes) in the 4^th channel at 20 °C (step (c) in Example 1).

Specifically, a SPR sensor was prepared in this way, in which the 1^st channel contained an inactivated brush; the 3^rd channel contained the brush after deactivation by (2-aminoethoxy)acetic acid; and the 4^th channel contained the brush after deactivation by glycine. After brush modification in each channel, the brush was always rinsed for 5 minutes with water and 10 minutes with PBS. The SPR resonance wavelength (λι) was measured, the surface was incubated with undiluted blood plasma for 10 minutes, rinsed with PBS, measured λ₂, incubated with high ionic strength solution (PBS* ¹, PBS containing 0.75 M NaCl, pH 7.4) for 5 minutes, rinsed with PBS, and measured λ₃. The size of biological deposits in ng/cm² was calculated from the differences λ₂ - λ_! and λ₃ - In the same way, biological deposits from undiluted cerebrospinal fluid were measured on other chips. The results are shown in Table 1. Table 1 : Deposition from undiluted human blood plasma and cerebrospinal fluid on polyCBAA brushes activated by EDC/NHS and deactivated by deactivating agents.

PBS, 0.75 M NaCl, pH 7.4

*² (2-aminoethoxy)acetic acid (AEAA), 1M, pH 7, 30 minutes

3 glycine, 1M, pH 7, 30 minutes

< LOD below the detection limit of the SPR method

The effect of the reaction of EDC/NHS activated polyCBAA brush with AEAA solution was compared with that of a known reaction of activated carboxyl groups with glycine solution of the same concentration. Deposition from undiluted blood plasma on activated polyCBAA brushes after deactivation by AEAA was 7 times lower, and deposition from undiluted cerebrospinal fluid was 39 times lower than after deactivation by glycine. The effect of AEAA is therefore significantly higher than that of glycine. Moreover, the resulting values of deposits from both tested complex biological media after deactivation by AEAA (1M, pH 7, 30 minutes) are comparable to the original size of deposits from the same media prior to activation. These results demonstrate a unique complete regeneration of the surface resistance to non-specific deposition from biological media after activation and deactivation using AEAA. Comparative Example 2: Reaction of functionalized polyCBAA brushes with (2- aminoethoxy)acetic acid increases their resistance to biological deposition

Activation of the polyCBAA brush, binding of bioactive substances, and incubation with deactivating agents (step (b) in Example 1) was carried out in four microfluidic channels. The brush was activated by EDC/NHS as described in Comparative example 1. After rinsing with water, the brush was incubated with an antibody against carcinoembryonic antigen solution (anti- CEA, 50 μg/ml, 10 mM borate buffer, pH 8) for 15 minutes, the achieved level of bound antibody was 200 ng/cm² according to SPR, or with solution of NH₂-DNA oligonucleotide probes (NH₂- DNA-ON, 22-mer, 2 μΜ, 10 mM borate buffer, pH 8), the level of bound probes was 75 ng/cm² according to SPR, or with anti-Salmonella typhimorium (Anti-Salm, 50 μg / ml, 10 mM borate buffer, pH 8), the achieved levels of bound antibody were 180, 102, or 98 ng/cm² according to the SPR. After rinsing with borate buffer, the brush was deactivated by (2-aminoethoxy)acetic acid or glycine or deactivation buffer (10 mM sodium borate + 10 mM imidazole + 10 mM NaCl, pH 8, 40 minutes) and then incubated with undiluted blood plasma as described in Comparative example 1. Plasma deposits which remained on the surface after rinsing with PBS, and deposits after further rinsing with a high ionic strength solution (PBS* ¹, PBS containing 0.75 M NaCl, pH 7.4) were determined by SPR measurement as in Comparative example 1.

Table 2: Deposition from undiluted human blood plasma and undiluted human cerebrospinal fluid on the polyCBAA brush functionalized by antibody against carcinoembryonic antigen (anti- CEA) or NH₂-DNA oligonucleotide probes for detection of microRNA (NH₂-DNA-ON) and subsequently deactivated by deactivation reagents

PBS, 0.75 M NaCl, pH 7.4, 5 minutes

^*2 (2-aminoethoxy)acetic acid, AEAA, 1M, pH 7, 30 minutes

glycine, 1M, pH 7, 30 minutes

ethanolamine 1M, pH 8, 30 minutes

deactivation buffer (10 mM sodium borate + 10 mM imidazole + 10 mM NaCl, pH 8) 40 minutes anti-CEA, 125 ng/cm²

LOD deposit was below the SPR detection limit of 0.03 ng/cm² The effect of the reaction of functionalized polyCBAA brush with AEAA solution was compared with the effect of traditional reaction of activated carboxyl groups with glycine solution of the same concentration and with regeneration of carboxyl groups by spontaneous hydrolysis of active esters in a deactivation buffer.

Blood plasma deposit on polyCBAA with bound anti-CEA and deactivated by AEAA was under the SPR detection limit. On the same brush deactivated by glycine, 7.5 ng/cm² of blood plasma was deposited. Only slightly more, namely 10.8 ng/cm², was deposited on the brush deactivated by spontaneous hydrolysis of active esters in the deactivation buffer.

The effect of the reaction of polyCBAA brush functionalized with 125 ng/cm² of anti-CEA with traditional deactivating agents was evaluated in a separate experiment. Deactivation by glycine did not lead to significantly higher blood plasma deposition than deactivation by ethanolamine or deactivation buffer.

After binding of small molecules of NH₂-DNA-ON, which, unlike large antibodies, can penetrate to active esters of side groups of polymers inside the brush, the deactivation of a small number of residual active esters by deactivating agents has only a little effect on the regeneration of the brush structure.

This example demonstrates that the extraordinarily high efficiency of deactivation by AEAA (1M, pH 7, 30 minutes) approaches the complete recovery of the original ability of the polyCBAA surface to withstand non-specific deposition from biological media even after binding of different types of bioactive substances, such as antibodies or NH₂-oligonucleotide probes, at high surface concentrations by SPR biosensor coated with polyCBAA brush with bound anti-CEA (198 ng/cm²) and deactivated by AEAA was detected with CEA (10 μg/mL) added to the undiluted blood plasma in the detection channel. The signal of the detection channel was compensated by a signal from the reference channel containing plasma without CEA (see Fig. 2).

Table 3 : Deposition from homogenized hamburger, undiluted blood plasma, and cerebrospinal fluid on the polyCBAA brush functionalized by anti-Salmonella (anti-Salm) binding followed by deactivation by deactivating agents

Functionalization anti-Salm anti-Salm anti-Salm

180 ± 0.9 ng/cm² 102 ± 5.5 ng/cm² 98 ng/cm²

Deposits [ng/cm²]

Hamburger Blood plasma Cerebrospinal fluid

Rinsing deposits with PBS PBS^{* 1} PBS PBS^{* 1} PBS PBS^{* 1} Before activation 0.5 0.3 5.2 ± 0.5 1.3 ± 1.1 0.5 < LOD

Functionalization/deactivation 10.9 < LOD 24.9 ± 2.9 4.1 ± 0.9 1.8 < LOD by AEAA *²

Activation/deactivation by 18.7 10.4 32.3 ± 1.3 1 1.1 ± 0.8 6.5 0.3 glycine ³

PBS, 0.75 M NaCl, pH 7.4, 5 minutes

^*2 (2-aminoethoxy)acetic acid, AEAA, 1M, pH 7, 30 minutes

3 glycine, 1M, pH 7, 30 minutes

< LOD deposit was below the SPR detection limit of 0.03 ng/cm²

The effect of the reaction of the functionalized polyCBAA brush with AEAA solution was compared with that of a known reaction of activated carboxyl groups with a glycine solution of the same concentration.

On polyCBAA with bound anti-Salm, the deactivation by AEAA led, compared to deactivation by glycine, to hamburger and cerebrospinal fluid deposition under the SPR detection limit and 2.7 times lower plasma deposition. Anti-Salm has been tested here as a typical receptor for the detection of pathogenic bacteria in food and as a model antibody to obtain information on purely non-specific blood plasma and cerebrospinal fluid deposition not affected by specific antigen bond. Salmonella is present neither in plasma nor cerebrospinal fluid.

Using an SPR biosensor coated with polyCBAA brush with bound anti-Salm (175 ng/cm²) and deactivated by reaction with AEAA, Salmonella (10⁷ CFU/mL) was detected, which was added to the cucumber extract in the detection channel. The detection channel signal was compensated by the signal from the reference channel containing Salmonella-free extract. Fig. 3 shows reference- compensated data obtained by subtracting the SPR response from the reference channel with anti- Salm incubated in a homogenized cucumber solution from the measuring channel with anti-Salm incubated in Salmonella-added homogenized cucumber solution (1 ^χ 10⁷ CFU/mL).

Comparative Example 3: Reaction of functionalized poly(HPMAA-co-CBMAA/ 15 mol%) brushes with (2-aminoethoxy)acetic acid increases their resistance to biological deposition

The golden surface of the SPR chip was covered with a copolymer brush containing 15 mol% CBMAA (poly(HPMAA-co-CBMAA/15 mol%) with a thickness ranging from 19 nm to 31 nm according to the protocol described in PCTCZ2016050011, PV 2015-313. The brush was activated, functionalized by covalent bonding of an antibody to Staphylococcal enterotoxin B (anti-SEB) or NH₂-DNA oligonucleotide probes, deactivated by AEAA or glycine by the procedure described in Comparative example 2. Table 4: Deposition from non-diluted blood plasma on poly(HPMAA-co-CBMAA/15 mol%) brushes functionalized by binding of an antibody to Staphylococcal enterotoxin B (anti-SEB) or NH₂-DNA-oligonucleotide probes (NH₂-DNA-ON) for detection of microRNA and subsequently deactivated by deactivating agents.

PBS, 0.75 M NaCl, pH 7.4, 5 minutes

*² (2-aminoethoxy)acetic acid, AEAA 1M, pH 7, 30 minutes

3 glycine, 1M, pH 7, 30 minutes Also in this example, the effect of AEAA deactivation on blood plasma deposits was more significant than with glycine deactivation. In addition, the example shows that as in the case of polyCBAA, in poly(HPMAA-co-CBMAA/15 mol%), after activation, binding of bioactive substances and deactivation by AEAA (1 M, pH 7, 30 minutes), even total regeneration of original surface resistance to biological deposition may take place.

Comparative Example 4: Resistance of carboxybetaine polymer brushes to biological deposition from undiluted blood plasma and food samples after EDC/NHS activation and after reaction with deactivating agents at different concentrations

The golden surface of the chip for measuring surface plasmon resonance (SPR), a glass plate coated with 50 nm thick, gold film, was coated using SI ATRP method with a polyCBMAA brush or a copolymer brush containing 15 mol% CBMAA (poly(HPMAA-co-CBMAA/15 mol%) with a thickness ranging from 19 nm to 31 nm, according to the protocol described in PV 2015-313. Subsequently, the chip was rinsed with water and mounted in the SPR sensor chamber with six flow microfluidic channels. Carboxybetaine groups of the brush were activated in the 2^nd to 6^th channel by reaction with an aqueous solution of N-hydroxysuccinimide (NHS, 0.1 M) and 1-ethyl- 3-(3-dimethylaminopropyl)carbodiimide (EDC, 0.5 M) for 20 minutes at 20 °C (step (a) in Example 1). After rinsing with water for 5 minutes and with PBS and water pH 8 (10 mM Na₂HP0₄, 1.8 mM KH₂P0₄, 137 mM NaCl, 2.7 mM KC1, pH 7.4) for 10 minutes, the activated brush was exposed to glycine solution (1 M, water pH 8) for 30 minutes in the 3^rd channel, (2- aminoethoxyacetic) acid solution (0.1 M, water pH 8) for 30 minutes in the 4^th channel, ethanolamine solution (1M, water, pH 8) for 30 minutes in the 5^th channel, and deactivation buffer (10 mM sodium borate + 10 mM imidazole + 10 mM NaCl, pH 8) for 40 minutes in the 6^th channel at 20 °C (step (c) in Example 1). In this way, the SPR sensor was prepared in which the 1^st channel contained an inactivated brush, the 2^nd channel brush after activation, the 3 ^rd channel brush after deactivation by glycine, the 4^th channel brush after deactivation by (2-aminoethoxy)acetic acid, the 5^th channel brush after deactivation by ethanolamine, and the 6^th channel brush after deactivation by deactivation buffer. Always after the brush modification carried out in the respective channel, the brush was rinsed with water for 5 minutes and with PBS for 10 minutes. Then the SPR resonance wavelength (λ^ was measured, the surface was incubated with undiluted blood plasma for 10 minutes, rinsed with PBS and again λ₂ was measured. The size of the biological deposit in ng/cm² was calculated from the difference λ₂ -

In the same way, the biological deposits were measured on other chips from the complex food samples prepared by homogenization according to the protocol described in CSN ISO 7251 and CSN EN ISO 6579. The results are shown in Tables 5 and 6.

Table 5: Deposition from complex biological solutions on the poly(HPMAA-co-CBMAA/15 mol%) brush activated by EDC/NHS and deactivated by deactivating agents

¹ 1 M glycine, pH 8, 30 minutes

100 mM, (2-aminoethoxy)acetic acid, pH 5, 30 minutes

70 mM, [2-(2-aminoethoxy) ethoxy]acetic acid, pH 9, 30 minutes ^*4 1 M ethanolamine, pH 8, 30 minutes

^*5 10 mM sodium borate + 10 mM imidazole + 10 mM NaCl, pH 8, 40 minutes

Table 6: Deposition from complex biological solutions on the polyCBMAA brush activated by EDC/NHS and deactivated by deactivating agents

1 M glycine, pH 8, 30 minutes

100 mM (2-aminoethoxy)acetic acid, pH 5, 30 minutes

1 M ethanolamine, pH 8, 30 minutes

10 mM sodium borate + 10 mM imidazole + 10 mM NaCl, pH 8, 40 minutes

Tables 5 and 6 present deposition from all tested biological samples for both types of carboxybetaine polymer brushes after deactivation by incubation with a solution of (2- aminoethoxy)acetic acid. This is comparable or slightly lower than deposition after deactivation by incubation with 10 times more concentrated glycine solution and significantly lower compared with traditional deactivation by hydrolysis in deactivation buffer or by ethanolamine binding. This example demonstrates that even at much lower concentrations of (2-aminoethoxy)acetic acid compared to glycine, deactivation by AEAA is comparable or even more effective than deactivation by glycine.

Claims

C LA I M S

1. A method of preparation of a substrate surface containing carboxybetaine groups, with increased resistance to undesirable deposition of biological media components on the substrate surface, comprising the following steps:

a) chemical activation of carboxybetaine groups on the surface of the substrate by converting the carboxybetaine carboxyl group into an active ester;

characterized in that following the step b), step c) is carried out in which the product of step b) reacts with an acid of general formula (I)

NH₂-(CH₂-CH₂-0)_n-CH₂-COOH (I),

2. The method according to claim 1, characterized in that the acid of general formula (I) is (2-aminoethoxy)acetic acid.

3. The method according to claims 1 or 2, characterized in that the concentration of the acid of general formula (I) in step c) is in the range of from 0.5 M to 2 M.

4. The method according to claims 1, 2 or 3, characterized in that step c) is carried out by first rinsing the product of step b) with a buffer, which was used as a solvent in step b), followed by rinsing with a buffer, which will be used in the next step for incubation, then the product is incubated with a solution of aminoethoxyacetic acid of general formula (I) in a buffer, and then it is rinsed with the buffer previously used for incubation, and then rinsed with a solution into which it is then deposited for storage or with water and dried.

5. The method according to any one of the preceding claims, characterized in that the carboxybetaine groups are contained in a polymeric layer on the substrate surface, selected from a group comprising a polymer brush, prepared by surface polymerization or grafting of polymers to the substrate surface, or a polymer coating, whereas preferably the polymeric layer has a thickness ranging from 5 nm to 5 μιη.

6. The method according to claim 5, characterized in that the polymeric layer contains at least one polymer comprising carboxybetaine groups.

7. The method according to claim 5, characterized in that the polymeric layer comprising carboxybetaine groups is a polymer brush of poly(carboxybetaine methacrylamide), poly(carboxybetaine methacrylate), poly(carboxybetaine acrylamide) or copolymer poly (A-co-B), wherein A is a monomeric unit from the group of N-(2-hydroxypropyl) methacrylamide, 2- hydroxyethyl methacrylate, 3-hydroxypropyl methacrylate and oligo(hydroxy ethylene glycol) methacrylate, and B is a monomeric unit from the group of carboxybetaine methacrylamide, carboxybetaine methacrylate and carboxybetaine acrylamide at a concentration of from 1 to 99 mol%.

8. The method according to any one of the preceding claims, characterized in that the substrate is selected from the group comprising particles with preferable size of from 5 nm to 1 mm, porous membranes, cell carriers, and biosensors.

9. The method according to claim 8, characterized in that the substrate is a biosensor detection surface for direct detection or multistage detection of analytes in complex biological media.