RU2309180C2 - Method for preparing photoreactive polymers for immobilization biomolecules on them - Google Patents

Abstract

FIELD: chemistry of polymers, biochemistry.

SUBSTANCE: invention relates to a rapid method for preparing photoreactive polymers and immobilization of biomolecules on indicated polymers. Invention describes a method for preparing photoreactive polymers followed by immobilization of molecules on them that involves the following steps: (a) effect of microwave radiation on mixture of photolinker and solvent; (b) washing out polymer with a corresponding solvent followed by drying at the environment temperature to obtain photoreactive polymer; (c) addition of biomolecule dissolved in corresponding buffer on photoreactive polymer; (d) treatment of mixture by the photoenergy source for period from 2 min to 2 h for immobilization of molecule on indicated polymer, and (e) washing out indicated polymer having immobilized molecule in corresponding buffer and the following drying indicated polymer at the environment temperature to obtain photoreactive polymer with molecules immobilized on its.

EFFECT: improved preparing method.

22 cl, 2 tbl, 18 ex

Description

Field of Invention

This invention relates to a quick method for producing photoreactive polymers and immobilizing biomolecules on these polymers. Photoreactive polymers are polymers that, when exposed to light of a specific wavelength, become highly reactive and form a covalent bond with biomolecules. This method of immobilization is soft, simple and does not depend on pH and temperature. An advantage of the invention is that the photoimmobilization technology is easily controllable and controllable.

BACKGROUND OF THE INVENTION

The need for immobilization of biomolecules on organic and inorganic polymers has increased significantly in recent years, especially in the post-genomic era. Immobilized molecules are widely used in clinical laboratories, biosensors, membrane bioreactors, diagnostics, genomics, proteomics, drug screening, affinity chromatography and many other related fields. There are many ways to immobilize biomolecules on an inert plane, such as (i) activating the polymer plane, (ii) activating the biomolecules, or (iii) activating both the polymer plane and the biomolecule before immobilization. From a practical point of view, method (i) is the most successful, since in most cases the biomolecules are very sensitive to chemicals and may be lost during the activation process.

Biomolecules immobilized on an inert surface are widely used in diagnostics, biosensors, membrane bioreactors, genomics, proteomics, drug screening, affinity chromatography, and many other related fields. [Krysteva, et. al., Covalent binding of enzymes to synthetic membrane containing acrylamide units, using formaldehyde, Biotechnol Appl. Biochem. 13: 106-111 (1991); Pandey, et. al., Amperometric enzyme sensor for glucose based on graphite paste-modified electrodes, Appl. Biochem. Biotechnol. 33: 139-144 (1992)].

The need for a simple and quick way to immobilize biomolecules on an inert surface continues to grow in the post-genomic era.

There are various methods for immobilizing biomolecules on an inert surface of a polymer. Covalent immobilization can improve biomolecular activity, reduce non-specific adsorption and increase stability. [Tiller, J. et. al., A novel efficient enzyme-immobilization reaction on NH ₂ polymers by means of L-ascorbic acid, Biotechnol. Appl. Biochem. 30, 155-162 (1992)].

There are many methods for covalent immobilization of biomolecules on various polymer substrates. Glucose oxidase, uricase, xanthine oxidase, and penicillin acylase were immobilized on alkylamine glass or silica gel by the glutaraldehyde coupling method. [Guo, et. al., Immobilization of glucose oxidase and peroxidase and their application in flow injection analysis for glucose in serum, Appl. Biochem. Biotechnol. 23 (1): 15-24 (1990); Nakamura, et. al., Immobilization of uricase on protamine bound to glass beads and its application to determination of uric acid, Anal. Biochem. 152 (2): 386-390 (1986); Tawa, et. al., Immobilization of xanthine oxidase to controlled pore-glass. Application to high-performance liquid chromatography, Nucleic Acids Symp. Ser. 12: 107-110 (1983)]. Glucose oxidase, cholesterol oxidase, and uricase immobilized on glass with a controlled pore size were used to determine serum glucose, cholesterol, and urea, respectively.

Thionyl chloride-activated porous glass with succinamidopropyl-controlled pore size was used to covalently immobilize anti-bull IgG. [Stabel, et. al., Anti-IgG immobilized controlled pore glass. Thionyl chloride-activated succinamidopropyl glass as a covalent immobilization matrix, Appl. Biotechnol. 36 (2): 87-96 (1992)]. Enzymes such as acetyl coenzyme A synthetase were immobilized on CNBr-activated porous glass. It has been reported that the activation of inert organic polymer surfaces, such as polystyrene, polypropylene or polyethylene, occurs by radiation graft polymerization or gas plasma techniques [deQueiroz et. al., Surface studies of albumin immobilized onto PE and PVC films, J. Biomater. Sci. Polym. 8: 667-681 (1997); Siephia et. al., Immobilization of enzymes on polypropylene bead surfaces by anhydrous ammonia gaseous plasma technique. J Biomed. Mater. Res. 22 (5): 417-22 (1988)].

However, all of these methods have one or more disadvantages, for example (i) they are labor intensive, (ii) they are time consuming, (iii) they use hazardous chemicals or (iv) harsh conditions are required for the reaction.

Photolinker-mediated technology provides covalent immobilization of biomolecules on a solid surface under mild reaction conditions. This method is based on the presence of at least two functional groups in the photolinker, one of which, as a rule, is a photoactivated group.

There are many methods suitable for immobilizing biomolecules via photolinker. Elsner et. al., U.S. Pat. 5427799, a method for activating an inert plane by means of a double ring heterocyclic photolinker, such as psoralen, by a photochemical reaction is described.

Jacobsen et al. U.S. Pat. 6033784, a method for photochemical activation of a polymer surface using quinone is disclosed. Using quinone derivatives containing a primary amine, amino groups are applied to the polystyrene surface. However, these groups are not readily bound to protein without the addition of one or more activating reagents. The preparation of such plates and photolinker is a lengthy and cumbersome process.

Recently, Nahar et. al. and Bora, et. al [Nahar, et. al., Light-induced activation of an inert surface for covalent immobilization of a protein ligand, Anal. Biochem. 294: 148-153 (2001); Nahar P. pending U.S. Patent Application, Method for photochemical activation of polymer surface and immobilization of biomolecules onto activated surface; Bora, et. al., Covalent immobilization of proteins onto photoactivated polystyrene microtiter plates for enzyme-linked immunosorbent assay procedures, J. Immunol. Methods 268: 171-177 (2002)] and later Naqvi et. al. [Naqvi, et. al., Introduction of functional groups onto polypropylene and polyethylene surfaces for immobilization of enzymes, Anal. Biochem. 306: 74-78 (2002)] published a simple, quick, and mild method of induced photoactivation of an inert polymer, such as polystyrene, polypropylene, and polyethylene, using 1-fluoro-2-nitro-4-azidobenzene.

However, in many of these photolinker-mediated methods, surfaces were activated by a photochemical reaction, while biomolecules were immobilized by a thermochemical reaction. Therefore, biomolecules must have one or more functional groups for reaction with an activated surface, creating a covalent bond. Whereas in the photochemical method of immobilization, biomolecules can be immobilized regardless of their functional group. There are only a few reports of photochemical immobilization of biomolecules.

Sigrist H. et al. immobilized the biomolecule on an inert surface using a photolinker polymer obtained by the reaction between BSA and 3- (triethylamine) -3- (m-isothiocyanophenyldiazirine). The photolinker polymer was then used to bind streptavidin, while the polystyrene surface was simultaneously exposed to light. [Sigrist et. al., Light dependent, Covalent immobilization of biomolecules on inert surface, Biotechnology 10: 1026-1028 (1992)]. However, the preparation of such a photolinker is a multi-stage, lengthy procedure.

Guire U.S. Pat. 3959078, a method for immobilizing an enzyme using a 1-fluoro-2-nitro-4-azidobenzene photolinker is disclosed. In this method, the amino-containing matrix is activated with 1-fluoro-2-nitro-4-azidobenzene during a thermochemical reaction at 37 ° C for 20 hours. In the next stage, the enzyme is immobilized for 16 hours on an activated surface by means of a photochemical reaction. The imperfection of this method consists in the duration of the reaction at both stages. None of these methods allows you to simply and quickly prepare a photoreactive surface and quickly immobilize a biomolecule on this photoreactive surface. The table below shows the disadvantages of the prior art.

Table I State of the art disadvantages one The C-H carrier matrix was activated photochemically using 1-fluoro-2-nitro-4-azidobenzene, followed by addition of the enzyme via a thermochemical reaction (Nahar et al., 2001) The method provided a limiting choice of biomolecule, because selected biomolecules must have an amino group or other nucleophilic groups to interact with the activated surface. 2 Jorg Tiller (Tiller et al.) Describes a method for activating NH ₂ polymer films by (i) immersing the film in a concentrated ascorbic acid solution until the swelling becomes visible to the naked eye. Then the enzymes were immobilized on an activated film for 16 hours,
(ii) the polymer film was allowed to swell in dimethylacetamide, followed by placing it in 10 ml of ice-cold 0.5M HCl solution, and 1 ml of 1M NaNO ₂ solution was added dropwise thereto _. The enzyme was then immobilized for 12 hours at a temperature of 4 ° C. The procedure for activating an amino polymer film is cumbersome. The stage of immobilization is time consuming. 3 The biomolecule was immobilized on an inert surface using a photolinker polymer obtained by the reaction of BSA and 3- (triethyleneamine) -3- (m-isothiocyanophenyl diazirin). Further, the polymer-photolinker was used for simultaneous binding under the influence of light with both streptavidin and a polystyrene surface. Sirgis H. et al., (1992) The process of preparing a photolinker is time-consuming and time-consuming. four Guir et al., US Patent No. 500,258 describes a method for modifying an alcohol-containing polymer by keeping it in the dark at room temperature for 16 hours using a photolinker. The activation procedure is time consuming. 5 US Pat. No. 3,959,078 describes a procedure for modifying an amine-containing polymer by the action of a photolinker containing an azido group for 20 hours. Immobilization on this polymer was carried out at 0 ° C-150 ° C for 16 hours with a photochemical reaction using a 40 watt tungsten lamp. Both stages, activation and immobilization, are time consuming.

The purpose of the invention

The main objective of the invention is to provide a simple and quick method for preparing a photoreactive polymer and the rapid immobilization of biomolecules on such polymers.

Another objective of the invention is the provision of a photoreactive polymer as a result of a microwave-mediated reaction of 1-fluoro-2-nitro-4-azidobenzene with the polymer. Another objective of the present invention is the provision of a simple, soft and quick way to immobilize biomolecules, independent of their functional groups, through a photochemical reaction.

Another objective of this invention is the creation of a photoreactive polymer surface for efficient and rapid immobilization of a biomolecule through ultraviolet (UV) light.

Another objective of this invention is the creation of a photoreactive polymer surface suitable for the light-induced immobilization of a biomolecule required for use in the chemical industry, pharmacological industry, diagnostics, separation technology and in many other related fields.

The novelty of the present invention is a quick method for producing a photoreactive polymer, which takes from one to ten minutes, depending on the particular polymer.

SUMMARY OF THE INVENTION

In order to achieve the claimed in the present invention and to circumvent the disadvantages of the methods of the prior art, a quick, simple and effective method has been developed to obtain a microwave-mediated photoreactive polymer and to quickly immobilize the biomolecule under the influence of light, regardless of the presence of functional groups on the polymer.

Accordingly, the present invention provides a method for producing photoreactive polymers with molecules immobilized on them, and this method includes:

(a) the interaction of a polymer containing a nucleophilic group with a photolinker molecule dissolved in an appropriate solution,

(b) washing the polymer in a suitable solution, followed by drying the washed polymer at ambient temperature, to obtain a photoreactive polymer,

(c) attaching a molecule dissolved in an appropriate buffer to a photoreactive polymer,

d) exposing the mixture to a photovoltaic source for a period of from 2 minutes to 2 hours to immobilize the molecule on the above photoreactive polymer, and

(e) washing said polymer having an immobilized molecule with an appropriate buffer and then drying at ambient temperature to obtain photoreactive polymers with molecules immobilized on them.

In one embodiment of the present invention, the photolinker has a thermochemical group selected from the group capable of forming a covalent bond with the nucleophilic group of the polymer.

In a further embodiment of the present invention, the thermochemical group of the photolinker is selected from the group consisting of aldehyde, carbonyl, isothiocyanate, halide and isocyanate.

In another embodiment of the present invention, the photolinker has a photochemical group that is photoreactive and capable of forming a covalent bond with a molecule as a result of a photochemical reaction, without the use of any other reagents.

In a further embodiment of the present invention, the photochemical group of the photolinker is selected from the group consisting of carbene precursors, nitrene precursors and an oxygen radical.

In yet another embodiment of the present invention, the photolinker is 1-fluoro-2-nitro-4-azidobenzene (FNAB).

In another embodiment of the present invention, the polymer includes any polymer having an amino, hydroxyl, thiol or any other reactive nucleophilic group, such as, for example, silica gel, porous glass with long chain alkylamino-controlled pore size, polyvinyl alcohol, aminopolystyrene, and alkylaminosilica gel.

In another embodiment of the present invention, the reaction in step (a) is carried out by exposing the mixture of photolinker, polymer and solvent to microwave irradiation or by thermal incubation at 37 ° C, microwave irradiation is most effective at a frequency of from 2000 to 2600 Hz and an applied power in the range of 100 to 1000 watts over a period of 10 seconds to 20 minutes.

In another embodiment of the present invention, the solvent used to wash the polymer obtained in step (a) has the ability to dissolve the photolinker and its dissolution products without causing degradation of the polymer, such as methanol and ethanol.

In another embodiment of the present invention, the solvent used in step (a) is selected from the group consisting of dimethylformamide and toluene.

Preferably, the reaction in step (a) is carried out in the presence of a catalyst, such as 30% KOH.

In yet another embodiment of the present invention, the polymer is selected in the form of a bead, hemisphere, sheet, powder, rod, plate, strip or tube.

In another embodiment of the present invention, a molecule immobilized on a polymer surface is selected from the group consisting of an organic molecule, an organic polymer, a biomolecule, and any molecule with a C-H bond.

In a further embodiment of the present invention, the biomolecule is selected from the group consisting of protein, nucleic acids, hydrocarbon, oligonucleotide, enzyme, antigen, antibody and peptide.

The enzyme is preferably horseradish peroxidase.

In another embodiment of the present invention, the photovoltaic source is selected from the group consisting of ultraviolet (UV) light, sunlight, light flux, and a laser beam.

In a further embodiment of the present invention, the ultraviolet (UV) light used as the photo-energy source has a wavelength in the range of 300 to 400 nm, and the process of immobilizing the molecule is carried out in the range of 2 minutes to 2 hours.

The reaction between the nucleophilic group of the polymer and the photolinker in step (a) is preferably carried out in a microwave oven in the presence of a solvent.

The invention also relates to the use of a photoreactive polymer with a molecule immobilized on it for diagnostics, affinity chromatography, proteomics, genomics and drug screening.

A brief description of the accompanying drawings.

Figure 1. Optimization of the amount of FNAB to obtain photoreactive silica gel (Example 1)

The amount of FNAB needed to produce photoreactive silica gel was optimized by adding different amounts of FNAB (0, 6.25, 12.5, 50, 75 and 100 mg) to each reactive sample containing 50 mg of alkylaminosilica gel, followed by microwave irradiation in within 60 seconds.

The photoreactive alkylamino silica gel thus obtained in each reactive sample was tested by immobilizing HRP (μg) on a support (50 mg) as a result of a photochemical reaction. Absorption was measured after colorimetric analysis of the immobilized enzyme.

Figure 2. Optimization of the amount of FNAB to obtain photoreactive LCAA-CPG (Example 2)

The optimized amount of FNAB required to produce photoreactive LCAA-CPG was determined by varying the concentration of FNAB (0, 6.25, 12.5, 25, and 50 mg) in each reactive sample containing 50 mg of LCAA-CPG, followed by microwave irradiation for 60 seconds. The LCAA-CPG thus obtained in each reactive sample was checked by photo-irradiation of HRP (1 μg) with 50 mg of the substrate and colorimetric analysis of the immobilized enzyme.

Figure 3. Optimization of the amount of FNAB to obtain photoreactive polyvinyl acetate (PVA) (Example 3)

The optimized amount of FNAB needed to produce photoreactive PVA was determined by adding various amounts of FNAB (12.5, 25, 50, and 75 mg) to the reaction mixture consisting of 50 mg of PVA, 6.5 μl of 30% KOH and 5 ml of toluene. The reaction mixtures were kept at room temperature for one hour. The PVA thus obtained in each reaction mixture was tested by HRP irradiation (16 μg) from a 50 mg support. Absorption was measured after addition of the substrate to the immobilized enzyme.

Figure 4. Optimization of the exposure time of microwave irradiation to obtain photoreactive alkylamino-silica gel (Example 4)

The exposure time of microwave irradiation in the process of producing photoreactive alkylamino-silica gel was optimized by varying the exposure time of microwave irradiation (30, 50, 60, and 70 seconds) in a reaction mixture of 50 mg of alkylamino-silica gel with 50 mg of FNAB. The photoreactive surface thus obtained in each reaction mixture was tested by irradiating 1 μg HRP with 50 mg of the substrate and the immobilized enzyme was colorimetrically analyzed.

Figure 5. Optimization of the exposure time of microwave irradiation for the activation of aminopolystyrene (Example 5)

The exposure time of microwave irradiation to obtain photoreactive aminopolystyrene was optimized by varying the exposure time (40, 50 and 60 seconds) in each reaction mixture of 20 mg of aminopolystyrene with 20 mg of FNAB. The photoreactive surface thus obtained was checked by irradiating HRP (35 μg) with 20 mg of the substrate. Absorption was measured after addition of the substrate to the immobilized enzyme.

6. Optimization of the concentration of KOH upon receipt of photoreactive PVA (Example 6)

The concentration of KOH in obtaining photoreactive PVA was optimized by adding various amounts of a 30% KOH solution (1.62, 3.25, 6.5, 13, and 20 μl) to the reaction mixture containing 50 mg PVA and 50 mg FNAB in 5 ml toluene. The photoreactive surface thus obtained in each reaction mixture was tested by irradiating HRP (16 μg) with 50 mg of the substrate and colorimetric analysis of the immobilized enzyme.

7. Optimization of PVA activation time at room temperature (Example 7)

The activation time for obtaining photoreactive PVA was optimized by using different incubation periods (5, 10, 60, 180 and 300 minutes) for each reaction mixture of 50 mg PVA, 50 mg FNAB in 5 ml of toluene and 6.5 μl of 30% KOH . The photoreactive surface thus obtained in each reaction mixture was tested by irradiating HRP (16 μg) with 50 mg of the substrate and colorimetric analysis of the immobilized enzyme.

Fig. 8. Optimization of irradiation time for immobilization of the enzyme on the surface of alkylamino silica gel (Example 8)

The immobilization time was optimized by changing the time of ultraviolet (UV) light irradiation of the HRP mixture (1 μg) with 50 mg of photoreactive alkylaminosilica gel (2, 5, 10, 20, 40, 60, and 80 minutes) for each reaction mixture. Absorption was measured after addition of the substrate to the immobilized enzyme.

Fig.9. Optimization of exposure time for immobilization of the enzyme on the photoreactive surface of LCAA-CPG (Example 9)

The immobilization time was optimized by changing the time of exposure to ultraviolet (UV) light of a mixture of 50 mg of photoreactive LCAA-CPG with HRP (1 μg) (2, 10, 20, 40 and 60) for each reaction mixture. The immobilized enzyme was colorimetrically tested after addition of the substrate.

Figure 10. Optimization of the time of exposure to ultraviolet (UV) light to immobilize the enzyme on the photoreactive surface of PVA (Example 10)

The immobilization time was optimized by changing the time of exposure to ultraviolet (UV) light of a mixture of 50 mg photoreactive PVA with HRP (16 μg) (5, 20, 30, 60, 120, and 180 minutes) for each reaction mixture. Absorption was measured after adding a substrate dye buffer to the immobilized enzyme.

11. Optimization of the concentration of the enzyme for its immobilization on photoreactive PVA (Example 11)

HRP concentration was optimized using different amounts of the enzyme (0.25, 0.5, 1.0, 2.0, 4.0, 8.0 and 16.0 μg) per 50 mg of photoreactive PVA in each reaction mixture, followed by ultraviolet irradiation for 30 minutes. The immobilized enzyme was colorimetrically tested after addition of the substrate.

Fig. 12. Optimization of the concentration of the enzyme for its immobilization on the microwave-activated photoreactive surface of the LCAA-CPG (Example 12)

HRP concentration was optimized using various amounts of the enzyme (0.2, 0.5, 1, 2, and 4 μg) for 50 mg of photoreactive LCAA-CPG, followed by ultraviolet irradiation for 20 minutes. Absorption was measured after addition of substrate staining buffer to the immobilized enzyme.

Fig.13. Optimization of the concentration of the enzyme for its immobilization on microwave-activated photoreactive surface of alkylamino-silica gel (Example 13)

HRP concentration was optimized for 50 mg of photoreactive alkylaminosilica gel using different amounts of the enzyme (0.5, 1.0, 2.0, 4.0, and 6.0 μg) for each reaction mixture, followed by ultraviolet irradiation for 20 minutes. The immobilized enzyme was colorimetrically tested after addition of the substrate.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is a quick and effective method of modifying a polymer surface to introduce a latent reactive group into said polymer surface and immobilizing biomolecules on said modified polymer surface by means of light energy. More specifically, the surface of a polymer with a latent photoreactive group is quickly obtained by microwave-mediated reaction of 1-fluoro-2-nitro-4-azidobenzene with a polymer containing a reactive nucleophilic group. The modified polymer (photoreactive polymer) obtained in this way has a photoreactive azido group which, under the influence of ultraviolet light (UV), creates a highly reactive nitrene capable of binding to a biomolecule.

The concentration of photolinker plays an important role in obtaining a photoreactive polymer. The best result was obtained when the ratio of photolinker (FNAB) and polymer 1: 1 (wt./wt.). A subsequent increase in the concentration of FNAB does not lead to an increase in photoreactive groups in the polymer. The effectiveness of the photoreactive surface was determined by immobilizing HRP on the surface and then testing the immobilized enzyme. When using an untreated surface (FNAB: 0 mg), immobilized biomolecules were practically not found on it (Figure 1, Figure 2 and Figure 3).

Microwaves have been found to be an excellent tool for creating a photoreactive surface. Thus, the photoreactive LCAA-CPG obtained by microwave irradiation 70, as it turned out, gives better results than the photoreactive surface obtained by the prior art technology, i.e. at 37 ° C for 20 hours (table 1).

It was also found that microwave irradiation for 60 seconds gives a photoreactive aminated silica surface with good results (Figure 4). To obtain photoreactive amino polystyrene, a 60 second microwave irradiation was sufficient for the reaction of the polymer and FNAB (Figure 5). In contrast to the amino polymer, the hydroxyl group containing the polymer needs a basic catalyst to react with FNAB. The optimization of the amount of KOH (catalyst) was determined by the reaction of PVA and FNAB with stirring at room temperature for one hour in the presence of toluene and various amounts of KOH in an aqueous solution. The optimal amount of KOH for the reaction of PVA and FNAB (50 mg each) in the presence of 5 ml of toluene was 6.5 μl of 30% KOH (Figure 6). The inventors have also optimized the time required to produce photoreactive PVA using KOH at room temperature. The optimal time was 60 minutes (Fig.7). However, with a 10-minute microwave exposure, comparable results were obtained.

In the photoactivation procedure, PVA was activated by exposure to ultraviolet light on a PVA coated FNAB. Photoactivated PVA was then used to immobilize horseradish peroxidase at 37 ° C for 1 hour. In the method according to the invention, photoreactive PVA was obtained by the reaction of PVA and FNAB under the influence of microwave radiation (or by thermal incubation at 37 ° C). Immobilization of horseradish peroxidase on the specified photoreactive PVA was then carried out under the influence of ultraviolet light. Table 2 shows that a photoreactive polymer (polymer surface is thermally activated) gives better results on the immobilization of biomolecules on it than a thermoset polymer (polymer surface is activated by exposure to light). Therefore, the photochemical immobilization procedure (thermochemical activation and photochemical immobilization), as described in the method according to the invention, is a better option compared to thermochemical immobilization (photochemical activation and thermochemical immobilization).

The inventors have optimized the concentration of the FNAB linker and the incubation time with the polymer (PVA) upon receipt of the photoreactive polymer. In the second stage, the enzyme was immobilized (HRP) on the polymer by means of its photoreactive group under the influence of light energy. Thus, the enzyme was immobilized on the surface of alkylamino silica gel under the influence of ultraviolet light on a UV stratalinker unit for various time intervals. It was found that the optimal time for immobilization of the enzyme on photoreactive alkylaminosilica gel is 10 minutes. However, an increase in the time of ultraviolet irradiation over 10 minutes did not lead to an increase in the degree of immobilization (Fig. 8). The immobilization of HRP on the photoreactive surface of the LCAA-CPG is optimal for a period of time of 20 minutes (Figure 9). However, photoreactive PVA requires 60 minutes of ultraviolet radiation for optimal HRP immobilization. A further increase in the time of ultraviolet irradiation reduces absorption, which can occur due to the fact that it is possible to inactivate some immobilized enzymes (Figure 10). Enzyme concentration is also an important factor for immobilization. The concentration of 2 μg HRP / 50 mg was optimal for immobilization on photoreactive PVA, and a further increase in concentration did not lead to a noticeable increase in the immobilization of the enzyme (Figure 11). However, the optimal amount of HRP was 4 μg / 50 mg of the substrate for immobilization on photoreactive LCAA-CPG (Figure 12) and alkylaminosilica gel (Figure 13). It was found that the total activity of the immobilized enzyme per 20 mg of LCAA-CPG and 20 mg of alkylamino-silica gel is respectively 160 units. and 120 units.

Thus, the method for producing photoreactive substrates according to the invention is simple, quick and avoids the disadvantages of published methods. And, in addition, the immobilization of biomolecules on photoreactive surfaces is quick and easy.

Table II presents the advantages of the method according to the invention.

Table II
THE PRESENT INVENTION AND ITS ADVANTAGES The method according to the invention Advantage one The method according to the invention provides a fast microwave-mediated method for producing photoreactive polymers capable of forming covalent bonds with biomolecules, regardless of the presence of a functional group in the biomolecule. The method according to the invention allows the activation of aminopolymers within 50 seconds, and alcohol-containing polymers within 10 minutes by microwave irradiation. Therefore, it allows to accelerate the production of photoreactive polymer. 2 The method according to the invention is also a fast technology that allows you to vary the time for immobilization of biomolecules on a photoreactive polymer in the range from 10 to 70 minutes. In the method according to the invention
1) The optimal time for immobilization of biomolecules is 10-70 minutes, which is significantly less than (12-16 hours) when using the same photolinker as in previous developments
2) The potentially activated surface is used in chromatographic separators, genomics, proteomics, where immobilized molecules are used.
3) 1-fluoro-2-nitro-4-azidobenzene used as a photolinker can be obtained in a simpler way.

Horseradish peroxidase, porous glass with long chain alkyl amine-Controlled pore glass (normal diameter 500 E, mesh size: 80-120), o-phenylenediamine were purchased from the manufacturer Sigma, USA. Silica gel LR (100-200 mesh) was purchased from the manufacturer SD fine chemicals. H ₂ O ₂ , methanol, dimethylformamide, toluene and 3-aminopropyltriethoxysilane, sodium chloride, monosubstituted sodium orthophosphate, disubstituted sodium orthophosphate, and analytical grade citric acid were purchased from the manufacturer Merck, India. FNAB was obtained from 4-fluoro-3-nitroaniline by the diazotization reaction, as previously published [15]. The microwave irradiated reaction was carried out in a BPL Sanyo microwave oven operating at a frequency of 2450 Hz at a power of 700 watts. Photoimmobilization was performed at a wavelength of 365 nm using a UV stratalinker; Model 2400, (Stratagene, USA), consisting of five 15-watt tubes. All solutions were prepared in triplicate distilled water immediately before use. A phosphate buffered saline (PBS) was prepared by mixing 0.85% NaCl with 0.01 M phosphate buffer (pH 7.2). A wash buffer was prepared by adding 0.1% Tween 20 in PBS. Freshly prepared substrate staining buffer contained 12 ml of citrate buffer (0.025 M citric acid and 0.5 M Na ₂ HPO ₄ · 2H ₂ O, pH 5), 5 μl H ₂ O ₂ (30% w / v) and 4 mg o- phenylenediamine.

Example 1. Optimization of the amount of 1-fluoro-2-nitro-4-azidobenzene to obtain photoreactive silica gel (Figure 1)

5 g of dry (moisture-free) silica gel mixed with 10 ml of 3-aminopropyltriethoxysilane and 80 ml of toluene was stirred at 28 ° C for 3 hours. Then the mixture was filtered and washed with methanol, water, a mixture of methanol: water (1: 1) and methanol, respectively. Then the substrate was dried and checked for the presence of an amino group using ninhydrin. A positive ninhydride test confirms the binding of the amino group to silica gel. A conical flask containing 50 mg of alkylaminosilica gel, 6.25 mg of FNAB and 10 ml of DMF was microwaved for 60 s. After that, the substrate extracted from the conical flask was washed with methanol, dried and placed in a Petri dish. The FNAB concentration was optimized by conducting the same experiment with different amounts of FNAB (12.5, 25, 50, 75 and 100 mg, respectively). Next, HRP (1 μg / 80 μl PBS) was added to 50 mg of silica gel and irradiated using a UV stratalinker at 365 nm for 20 minutes. The substrate was washed in washing buffer, followed by the addition of 300 μl of substrate staining buffer. The stained solution was transferred to the corresponding polystyrene wells of a microtiter plate and absorbance was measured at 490 nm using a reader. A control experiment in the same manner was performed using untreated alkylamino silica gel (FNAB: 0 mg).

Example 2. Optimization of the concentration of 1-fluoro-2-nitro-4-azidobenzene to obtain photoreactive porous glass with long chain alkylamino-controlled pore size (LCAA-CPG) (Figure 2)

A mixture of 50 mg LCAA-CPG, 6.25 mg FNAB and 10 ml DMF was placed in a conical flask, irradiated with microwaves for 60 s. The concentration of FNAB was optimized by conducting a similar experiment using different amounts of FNAB (12.5, 25 and 50 mg, respectively).

Enzyme immobilization and testing was carried out analogously to Example 1. A control experiment was performed using untreated LCAA-CPG (FNAB: 0 mg).

Example 3. Optimization of the concentration of 1-fluoro-2-nitro-4-azidobenzene to obtain photoreactive PVA (Figure 3)

A conical flask containing 50 mg of PVA, 50 mg of FNAB, 6.5 μl of 30% KOH and 5 ml of toluene was kept in the dark with stirring at room temperature (28 ° C) for 1 hour. A similar procedure was carried out with three more conical flasks, but the reaction mixture was prepared with an FNAB concentration of 12.5, 25 and 75 mg, respectively. After an hour, the conical flask substrates were washed with methanol, dried and placed in Petri dishes. HRP (16 μg / 80 μl PBS) was then added to each photoreactive PVA (50 mg) and irradiated using a UV stratalinker at 365 nm for 30 minutes. The enzyme was immobilized and tested in the same way as in Example 1. A control experiment was performed in the same way using untreated bead-shaped PVA.

Example 4. Optimization of the exposure time of microwave irradiation for the activation of alkylamino silica gel (Figure 4)

Four conical flasks containing 50 mg of alkylamino-silica gel, 50 mg of FNAB and 10 ml of DMF each were microwave irradiated for 30, 50, 60 and 70 seconds, respectively. After the appointed time, the substrates from each conical flask were separately washed with methanol, dried and placed in four Petri dishes. The HRP was immobilized and carried out as described in Example 1. A control experiment was carried out in the same way using untreated alkylamino silica beads.

Example 5. Optimization of the exposure time of microwave irradiation for the activation of aminopolystyrene (Figure 5)

20 mg of amino polystyrene, 20 mg of FNAB and 10 ml of ethanol were placed in a 50 ml conical flask and subjected to microwave irradiation for 40 seconds. The other two experiments were carried out in a similar way, but with a microwave exposure time of 50 and 60 seconds, respectively. After the appointed time, the conical flask support was washed with ethanol, dried and placed in Petri dishes. Then HRP (35 μg / 80 μl PBS) was added to each photoreactive PVA (50 mg) and irradiated using a UV stratalinker at 365 nm for 20 minutes. The enzyme was tested in the same manner as in Example 1. A control experiment was performed in the same manner using untreated aminopolystyrene.

Example 6. Optimization of the concentration of KOH during thermal activation of PVA (Figure 6)

A conical flask containing 50 mg of PVA, 50 mg of FNAB, 1.62 μl of 30% KOH and 5 ml of toluene was kept in the dark with constant stirring at room temperature (28 ° C) for 1 hour. The other four experiments were carried out in a similar manner, but with solutions containing different concentrations of 30% KOH (3.25, 6.5, 13 and 20 μl, respectively). After an hour, the substrates from the conical flask were washed separately with methanol, dried and placed in Petri dishes. HRP (16 μg / 80 μl PBS) was then added to each photoreactive PVA (50 mg) and irradiated with a UV stratalinker at 365 nm for 30 minutes. The substrates were washed in washing buffer, followed by the addition of 300 μl of substrate staining buffer. The stained solution was transferred to the corresponding polystyrene wells of a microtiter plate and absorbance was measured at 490 nm using a reader. A control experiment was performed in the same manner using untreated bead-shaped PVA.

Example 7. Optimization of time for thermal activation of PVA (Fig.7)

Four conical flasks containing 50 mg of PVA, 50 mg of FNAB, 6.5 μl of 30% KOH and 5 ml of toluene were each kept in the dark with constant stirring at room temperature (28 ° C) for 5, 10, 60, 180 and 300 minutes, respectively. Immobilization of HRP on photoreactive PVA and its subsequent testing was carried out as described in example 6.

Example 8. Optimization of the time of ultraviolet radiation to immobilize the enzyme on the surface of photoreactive alkylamino-silica gel activated by microwave irradiation (Fig. 8)

300 mg of alkylamino silica gel was activated by microwave irradiation of a mixture of 300 mg of FNAB in 10 ml of DMF for 70 seconds. Six Petri dishes containing 50 mg of photoreactive alkylaminosilica gel and HRP (1 μg / 80 μl PBS) each were irradiated using a UV stratalinker at 365 nm for 2, 5, 10, 20, 40, 60 and 180 minutes, respectively. The substrates were washed in washing buffer followed by the addition of 300 μl of substrate staining buffer. The stained solution was transferred to the corresponding polystyrene wells of a microtiter plate and absorbance was measured at 490 nm using a reader. A control experiment was performed in the same manner using untreated alkylaminosilica gel.

Example 9. Optimization of ultraviolet irradiation time for immobilizing an enzyme on the surface of photoreactive LCAA-CPG activated by microwave irradiation (Figure 9)

300 mg of LCAA-CPG was activated by microwave irradiation of a mixture of 300 mg of FNAB in 10 ml of DMF for 70 seconds. Six Petri dishes containing 50 mg of photoreactive LCAA-CPG and HRP (1 μg / 80 μl PBS) each were irradiated using a UV stratalinker at 365 nm for 2, 10, 20, 40 and 60 minutes, respectively. The immobilized enzyme was tested in the same manner as described in Example 8. A control experiment was performed in the same way using untreated LCAA-CPG.

Example 10. Optimization of the time of ultraviolet irradiation to immobilize the enzyme on the surface of photoreactive PVA activated by microwave irradiation (Figure 10)

300 mg of PVA was activated by microwave irradiation of 300 mg of FNAB dissolved in 10 ml of DMF for 10 seconds. Six Petri dishes containing 50 mg of photoreactive PVA mixed with HRP (16 μg / 80 μl PBS) each were irradiated using a UV stratalinker at 365 nm for 5, 20, 30, 60, 120 and 180 minutes, respectively. The substrates were washed in washing buffer, followed by the addition of 300 μl of substrate staining buffer. The stained solution was transferred to the corresponding polystyrene wells of a microtiter plate and absorbance was measured at 490 nm using a reader. A control experiment was performed in the same way using untreated PVA.

Example 11. Optimization of the concentration of the enzyme for its immobilization on photoreactive PVA (Figure 11)

A stock solution was prepared in a proportion of 1 mg HRP to 100 ml PBS. Seven Petri dishes containing 50 mg photoreactive PVA and HRP (16 μg / 80 μl PBS) in a variable concentration, i.e. 2.5, 5, 10, 20, 40, 80 and 160 μl, corresponding to 0.25, 0.5, 1.0, 2.0, 4.0, 8.0 and 16.0 μl of HRP, respectively, irradiated using a UV stratalinker at 365 nm for 30 minutes. The substrate was washed in washing buffer, followed by 300 μl of substrate staining buffer. The immobilized enzyme was tested in the same manner as described in Example 1. A control experiment was performed in the same way using untreated PVA.

Example 12. Optimization of the concentration of the enzyme for immobilization on the surface of the LCAA-CPG activated by microwave irradiation (Fig.12)

Five Petri dishes containing 50 mg of photoreactive LCAA-CPG and 10, 25, 50, 100 or 200 μl HRP solution corresponding to 0.2, 0.5, 1.0, 2.0 or 4.0 μg HRP, respectively irradiated using a UV stratalinker at 365 nm for 20 minutes. The enzyme immobilized on photoreactive LCAA-CPG was further tested as described in Example 8. A control experiment was performed in the same manner using untreated LCAA-CPG.

Example 13. Optimization of the concentration of the enzyme for its immobilization on the surface of photoreactive alkylamino-silica gel, activated by microwave irradiation (Fig.13)

Five Petri dishes containing 50 mg of microwave-activated photoreactive alkylamino silica gel and 0.5, 1.0, 2.0, 4.0, or 6.0 μg HRP were irradiated using a UV stratalinker at 365 nm for 20 minutes. The immobilized enzyme was further tested as described in Example 8. A control experiment was carried out in the same manner using untreated alkylamino silica gel.

Example 14. Optimization of the exposure time of microwave irradiation for binding of the photolinker to LCAA-CPG (table 1)

Four conical flasks containing 50 mg LCAA-CPG, 50 mg FNAB and 10 ml DMF each were microwave irradiated for 30, 50, 60 and 70 seconds, respectively. After the appointed time, the conical flask substrate was separately washed with ethanol, dried and placed in four Petri dishes. HRP was then immobilized and tested as described in Example 1. A control experiment was performed in the same manner using untreated bead-shaped LCAA-CPG.

Example 15. Optimization of the activation time of LCAA-CPG at a temperature of 37 ° C (table 1)

Four conical flasks containing 50 mg LCAA-CPG, 50 mg FNAB and 10 ml DMF each were mixed for 5, 10, 15 and 20 hours, respectively, at a temperature of 37 ° C. After the appointed time, the substrate from each of the conical flasks was separately washed with ethanol, dried and placed in four Petri dishes. HRP was then immobilized and tested as described in Example 1. A control experiment was performed in the same manner using untreated bead-shaped LCAA-CPG.

Example 16. Comparison of photochemical and thermochemical activation methods (table 2)

Photochemical activation of PVA: 250 mg of PVA and 250 mg of FNAB were dissolved in 2500 μl of methanol, stirring on a Petri dish. After evaporation of methanol, the reaction mixture was irradiated using a UV stratalinker at 365 nm for 60 minutes. After that, the substrate was washed with methanol and dried. In four Petri dishes, 50 mg of thermosetting PVA and 2, 4, 8, and 16 μg HRP, respectively, were mixed and incubated at 37 ° C for 60 minutes. The enzyme was checked as in example 1. A control experiment was performed in the same way using untreated PVA.

Thermochemical activation of PVA: thermal activation of PVA and the immobilization of HRP on photoreactive PVA was carried out as described above and in accordance with the procedure described in example 3.

Example 17. Determination of the activity of HRP immobilized on photoreactive LCM-CPG and alkylaminosilicon

HRP was immobilized on photoreactive LCM-CPG by irradiating the mixture (1 μg HRP / 50 μl PBS and 50 mg photoreactive LCAA-CPG) using a UV stratalinker for 20 minutes. The activity of immobilized HRP was determined by measuring the degree of staining at 490 nm, using dianisidine as a hydrogen donor and hydrogen peroxide as a substrate. The standard curve was constructed using the following HRP values (0.5, 1, 2, 4, 8, 16, 32, and 64 ng). Absorption was measured once per minute for 10 minutes and a per-minute degree of change in absorption was obtained. The activity of immobilized HRP on a photoreactive surface was determined as described above.

Example 18. Comparison of microwave and thermal activity of PVA

PVA (50 mg) was mixed with FNAB (50 mg), toluene (5 ml) and 6.5 μl of 30% KOH and stirred at room temperature for 1 hour. The same reaction mixture was microwaved for 10 minutes. After the reaction, the substrate was washed with methanol and dried. 50 mg of photoreactive alkylamino silica gel obtained at room temperature was mixed in Petri dishes with 1 μl of HRP and irradiated using a UV stratalinker at 365 nm for 30 minutes. The same experiment was carried out with photoreactive PVA obtained by microwave irradiation. Immobilized HRP was tested as described above. A control experiment was performed in the same way using untreated PVA.

Table 1 Time (seconds / hours) Activation method + FNAB -FNAB 30 seconds microwave 0.823 0.215 50 seconds microwave 1,021 0.311 60 seconds microwave 1,722 0.320 70 seconds microwave 1,960 0.415 5 o'clock 37 ° C 0.885 0.337 10 hours 37 ° C 0.966 0.321 15 hours 37 ° C 0,998 0.331 20 hours 37 ° C 1,368 0.320

table 2 HRP
(mcg) PVA activation method Thermal activation Photo activation + L -L + L -L 2 0,992 0.177 0.359 0.181 four 1,447 0.221 0.482 0.211 8 1,742 0.335 0.831 0., 320 16 1,791 0.411 1,317 0.359

Table 1. The results of studies of photoreactive LCAA-CPG obtained by microwave irradiation and incubation at 37 ° C for various time intervals (Example 14, 15)

Photoreactive LCAA-CPG was obtained at different exposure times of microwave irradiation (30, 50, 60 and 70 seconds) and at different incubation times at 37 ° C. The photoreactive surface was checked by photo irradiation of a mixture of 1 μg HRP with 50 mg of the substrate and colorimetric testing of the immobilized enzyme.

Table 2. Comparison of the effectiveness of immobilization on thermochemically and photochemically activated PVA (Example 16)

Photoreactive and thermosetting PVA were obtained by photochemical and thermochemical reactions, respectively. The effectiveness of their immobilization was compared by immobilizing various amounts of HRP (2, 4, 8, and 16 μg) on them and subsequent analysis of the immobilized enzyme.

Benefits

1. Invented a method for simple, fast and efficient production of a photoreactive polymer and the rapid immobilization of a biomolecule on such a polymer by ultraviolet light.

2. According to the invention, any polymer having a reactive nucleophilic group can be activated.

3. According to the invention, the polymer activation process lasts 50 seconds for amine-containing polymers and 10 minutes for the hydroxyl group of alcohol-containing polymers.

4. According to the invention, activation is carried out by microwave irradiation.

5. The photoreactive surface according to the invention can potentially be used to immobilize the molecules used in diagnostics, genomics, proteomics, affinity chromatography, drug screening, and in many other related fields.

Information sources

1. Krysteva et al., "Covalent binding of enzymes to synthetic membranes containing acrylamide units, using formaldehyde", Biotechnol. Appl. Biochem. 13, 106-111 (1991).

2. Pandey et al., "Amperometric enzyme sensor for glucose based on graphite paste-modified electrodes", Appl. Biochem. Biotechnol. 33, 139-144 (1992).

3. Tiller J. et al., "A novel efficient enzyme-immobilization reaction on NH ₂ polymers by means of L-ascorbic acid", Appl. Biochem. 30, 155-162 (1992).

4. Guo et al., "Immobilization of glucose oxidase and peroxidase and their application in their application in flow injection analysis for glucose in serum", Appl. Biochem. Biotechnol. 23 (1), 15-24 (1990).

5. Nakamura et al., "Immobilization of uricase on protamine bound to glass beads and its application to determination of uric acid", Anal. Biochem. 152 (2), 386-390 (1986).

6. Tawa et al., "Immobilization of xanthine oxidase to controlled pore glass. Application to high performance liquid chromatography", Nucleic Acids Symp. Ser. 12, 107-110 (1983).

7. Stabel et al., "Anti-IgG immobilized controlled-pore glass. Thionyl chloride-activated succinamidopropyl-glass as a covalent immobilization matrix", Appl. Biotechnol. 36 (2), 87-96 (1992).

8. de Queiroz et al., "Surface studies of albumin immobilized onto PE and PVC films", J. Biomater. Sci. Polym. 8, 667-681 (1997).

9. Siephia et al., "Immobilization of enzymes on polypropylene bead surfaces by anhydrous ammonia gaseous plasma technique", J. Biomed. Mater. Res. 22 (5), 417-22 (1988).

10. Nahar et al., "Light-induced activation of an inert surface for covalent immobilization of a protein ligand", Anal. Biochem. 294, 148-153 (2001).

11. Naqvi et al., "Introduction of functional groups onto polypropylene and polyethylene surfaces for immobilization of enzymes", Anal. Biochem. 30, 74-78 (2002).

12. Bora et al., "Covalent immobilization of proteins onto photoactivated polystyrene microtiter plates for enzyme-linked immunosorbent assay procedures", J. Immunol. Methods 268, 171 (2002).

13. Nahar P. A method for photochemical activation of polymer surface and immobilization of biomolecules onto the activated surface. U.S. Patent Pending.

14. Sigrist et al., "Light dependent, Covalent immobilization of biomolecules on 'inert' surface", Biotechnology 10, 1026-1028 (1992).

Other references: US Patent Documents

5,427,799 to Elsner, et al., 1995;

6033784 Jacobsen, et al., 2000;

3,959,078 Guire, 1976;

5002582 Guire, et al., 1991.

Claims (22)

1. The method of producing photoreactive polymers with subsequent immobilization of molecules on them, including (a) a reaction of a polymer containing a nucleophilic group with a photolinker molecule dissolved in an appropriate solvent, which is performed by exposure to it by microwave irradiation, (b) washing the polymer with an appropriate solvent, followed by drying at ambient temperature, to obtain a photoreactive polymer, (c) adding a biomolecule dissolved in an appropriate buffer to the photoreactive polymer, (d) treating the mixture with a photovoltaic source for a period of 2 minutes to 2 hours to immobilize the molecule on said polymer, and (e) washing said polymer having an immobilized molecule in an appropriate buffer, followed by drying said polymer at ambient temperature to obtain a photoreactive polymer with molecules immobilized thereon. 2. The method according to claim 1, in which the photolinker has a thermochemical group selected from the group capable of forming a covalent bond with the nucleophilic group of the polymer. 3. The method according to claim 2, in which the thermochemical group of the photolinker is selected from the group consisting of aldehyde, carbonyl, isothiocyanate, halide and isocyanate. 4. The method according to claim 1, in which the photolinker has a photochemical group that is photoreactive and capable of forming a covalent bond in the photochemical reaction with a biomolecule without the addition of any other reagent. 5. The method according to claim 4, in which the photochemical group of the photolinker is selected from the group consisting of carbene precursors, nitrene precursors and an oxygen radical. 6. The method according to claim 1, in which the photolinker is 1-fluoro-2-nitro-4-azidobenzene (FNAB). 7. The method according to claim 1, in which the polymer includes any polymer having an amino, hydroxyl, thiol or any other nucleophilic group. 8. The method according to claim 1, in which the polymer is selected from the group consisting of silica gel, glass with an adjustable pore size containing alkylamines, polyvinyl alcohol, aminopolystyrene and alkylamino-silica gel. 9. The method according to claim 1, in which the solvent used in stage (a) is selected from the group consisting of dimethylformamide and toluene. 10. The method according to claim 1, in which stage (a) is performed in the presence of a catalyst for polymers having a hydroxyl group. 11. The method according to claim 10, in which the catalyst contains 30% KOH. 12. The method according to claim 1, in which the microwave irradiation is carried out at a frequency of from 2000 Hz to 2600 Hz with an applied power in the range from 100 V to 1000 V for 10 s to 20 min. 13. The method according to claim 1, in which the solvent used to wash the polymer obtained in stage (a) includes a solvent capable of dissolving the photolinker and its degradation products without destroying the polymer. 14. The method according to item 13, in which the solvent used is selected from methanol and ethanol. 15. The method according to claim 1, wherein the polymer is selected from the polymer in the form of a bead, hole, sheet, powder, rod, plate, strip or tube. 16. The method according to claim 1, in which the biomolecule immobilized on a photoreactive polymer is selected from the group consisting of an organic molecule, an organic polymer, and any biomolecule molecule with a C-H bond. 17. The method according to clause 16, in which the biomolecule is selected from the group consisting of protein, nucleic acids, hydrocarbon, oligonucleotides, enzyme, antigen, antibodies and peptides. 18. The method according to 17, in which the enzyme is horseradish peroxidase. 19. The method according to claim 1, wherein the photo-energy source is selected from the group consisting of UV light, sunlight, light flash and a laser beam. 20. The method according to claim 1, in which the UV light used as a source of photovoltaic energy has a wavelength in the range from 300 nm to 400 nm, and processing to immobilize the molecule is carried out for a period in the range from 2 minutes to 2 hours . 21. The method according to claim 1, in which the reaction between the nucleophilic group of the polymer surface and the photolinker is carried out in a microwave oven in the presence of a solvent. 22. The method according to claim 1, in which the photoreactive polymer is used to immobilize the biomolecule necessary for diagnosis, affinity chromatography, proteomics, genomics and screening of drugs.

Images