WO2010061032A1 - Preparation of functionalized surfaces of polystyrene substrates - Google Patents

Abstract

The present invention describes the preparation of polystyrene surfaces chemically modified with a wide variety of functional groups. This involves a wet modification performed in two steps. The first step is the activation of the surface on creating chlorosulphonyl groups by immersing the polystyrene substrates in a chlorosulphonic acid solution at low temperatures. After washing, the modified substrate is immersed in an aqueous solution that contains a bifunctional compound, one of the functional groups being a primary aliphatic amine that serves as anchorage to the surface by means of a sulphonamide covalent bond. The second functional group is available on the modified surface and can be used to immobilize biomolecules when a covalent connection is created or to adjust surface parameters such as wettability or adhesion. The optical quality of the samples is maintained unaltered during the modification, which allows the use of the modified surfaces as an ideal tool for diagnostic assays of the ELISA or DNA chip type.

Description

 PREPARATION OF FUNCTIONALIZED SURFACES OF POLYSTYRENE SUBSTRATES

SECTOR OF THE TECHNIQUE This invention describes a method for producing in a simple and economical way activated polystyrene surfaces and the subsequent specific preparation of surfaces with a wide variety of functional groups such as amine groups (primary, secondary and tertiary), carboxylic groups, groups sulfonic, sulfonazides or methyl esters with excellent optical quality and their application in diagnostic tests in the biomedical and pharmacological sectors.

STATE OF THE TECHNIQUE:

In the last decades the scientific community of the biological, clinical and pharmaceutical areas have recognized that microassays are very useful tools for high yields in the determination of a variety of biological and biochemical interactions and functions. Microassays on supports or other formats, for example, can help reduce the consumption of expensive or limited agents, used for biological or biochemical analysis. It is widely accepted that the microassay format will remain a key tool in the foreseeable future. Applications for microassay technology will continue to expand to the areas of drug discovery and development, chemical detection, diagnosis and basic research. For these reasons, the development of a solid phase material that guarantees optimum bioactivity without loss of biomolecules is a common objective of scientists, clinical laboratories and manufacturers of diagnostic kits. Until now, several methods for covalent anchoring of molecules in previously activated solid supports have been described in the literature. The polymers have also become an attractive support material because they can be readily linked chemically or physically with biomolecules such as enzymes, antibodies and phospholipids to form biologically functional systems. A particular advantage in using polymers is that microassays can be carried out economically and in large numbers, which makes their use in commercial applications especially attractive.

Generally, polymeric surfaces are hydrophobic and quite unreactive. To immobilize biomolecules on solid polymeric surfaces through covalent or ionic bonds, the surface must be chemically modified by introducing reactive groups. The selection of the polymer solid phase is frequently influenced by the availability of compatible instrumentation and automation systems. Polystyrene is one of the most commonly used polymeric materials in the biomedical sector because it has excellent optical clarity, is easily moldable and relatively inexpensive. Polystyrene plates and multi-well plates have an increasing acceptance, partly because the processes of pipetting, washing and detecting the signal are easy to automate. Other advantages include the possibility of analyzing multiple samples simultaneously and the compatibility with a large number of different detection systems (for example by calorimetry, fluorescence and chemiluminescence). Already in the 1970s microplates of this polymer were used as reaction vessels for ELISA (Enzyme-Linked Immunosorbent Assays) assays that require surfaces capable of immobilizing proteins and other biomolecules.

However, Polystyrene also has a great drawback: it is a very hydrophobic polymer with low wettability and the anchoring of cells and many other biomolecules is difficult. Fortunately, the surface of the polystyrene can be easily modified. By different methods of chemical and physical treatments it is possible to anchor a wide variety of chemical groups such as hydroxy, ketone, aldehyde, carboxy and amine groups to the polymer that allow the covalent anchoring of different reactive groups for the subsequent covalent immobilization of biomolecules. Typical treatments of the surface of the polystyrene employ corona discharges and deposition of chemical vapors as well as the ozoneation in gas phase and in the presence of irradiation with UV light. These methods have been widely used to improve the wettability and adhesion properties of polymer surfaces. Another conventional method to modify polystyrene surfaces with amine groups is the plasma polymerization of allylamine or plasma treatments in the presence of N ₂ or NH ₃ . The PS surfaces modified in this way are used with bifunctional crosslinkers (eg glutaraldehyde, carbodiimide) to couple biomolecules covalently and thus achieve their immobilization.

Using a technology for photochemical modification [Producing low binding hydrophobic surfaces by treating with a low HLB number non-ionic surfactant. Bookbinder Dana Craig [US]; Fewkes Jr. Edgard John [US]; Griffin James Arthur [US]; French Smith M [US]; Tennent David, Patent number: US6093559], the Corning Group was able to preactivate multi-well polystyrene plates for the specific and covalent immobilization of biomolecules. This anchoring of reactive groups produces the following stable surfaces:

- the surface with N-oxysuccinimide groups (NOS) that is capable of reacting with amine groups, - the surface with maleimide groups for the covalent coupling of biomolecules bearing SH groups,

- the surface with hydrazide groups that is reactive towards carbohydrates activated with periodates, - aminated surfaces for covalent anchoring of biomolecules carrying COOH groups.

J. D. Page et.al. [J.D. Page, R. Durango, A.E. Huang, Chemical modification of PS surface and its effect on immobilized antibodies, Colloids and Surfaces A: Physicochemical and Engineering Aspects VoI.132, 193,

(1998)] on the other hand he developed methods of chemical modification in wetting of PS particles in order to obtain particles of amine and macroporous copolymers based on styrene and divinylbenzene. In this work, the PS cross-linked in the first step is subjected to a classical aromatic nitration. In a second step the nitro groups are reduced using SnCl2 / HCI to form primary aromatic amine groups.

Another wet method for multi-well amination was proposed by Zammatteo [N. Zammatteo, C. Girardeaux, D. Delforge, Amination of PS microwells: application to the covalent grafting of DNA Probes for Hybridization Assays. Analytical Biochemistry VoI 236, 85, (1996)]. In this work the polymeric substrate is oxidized in a first step using potassium permanganate, creating carboxylic groups that react in a second step with an aliphatic diamine, creating an aminated surface. The amount of amine groups formed was of the order of magnitude of picomoles / cm ² .

The present invention has several advantages in comparison with the procedures described above for the preparation of functionalized substrates based on PS. On the one hand it allows to obtain homogeneous surfaces with a controllable number of functional groups that is between one and two orders of magnitude greater than that conventionally achieved by plasma or UV treatments. On the other hand, the surfaces obtained have excellent optical quality with a high degree of transparency. Finally, the wet chemical treatment in two The steps described in this patent selectively and reproducibly produce only the preselected functional group.

DESCRIPTION OF THE INVENTION: BRIEF DESCRIPTION

An aspect of the present invention is the process for obtaining polystyrene modified with chlorosulfonyl groups comprising the following steps: a) cooling a solution of pure chlorosulfonic acid in an inert atmosphere at low temperature. b) introduction into solution a) of a polystyrene substrate between 0 and 3 hours, preferably 30 minutes c) extraction of the substrate and subsequent washing in a concentrated acid bath for a time between 0 and 10 min. d) washing of the modified substrate in a water / ice bath between 0 and 5 min. preferably 30 seconds and subsequent drying

In which the temperature of stage a) is between -2O ⁰ C and

2O ⁰ C, and the acid used in stage c) is sulfuric acid The temperature of the acid bath washing of stage c) is between -2O ⁰ C and

1 O ⁰ C,

A preferred aspect of the present invention is that with the process of obtaining the invention, polystyrene modified with chlorosulfonyl groups is obtained that preserves the optical transparency properties of the transparent starting polystyrene.

Another preferred aspect of the present invention is the use of transparent polystyrene modified with chlorosulfonyl groups as an intermediate in reactions for obtaining functionalized polystyrene. Another more preferred aspect of the present invention is the use of polystyrene modified with chlorosulfonyl groups that reacts with disubstituted alkanes containing a primary aliphatic amine group to prepare functionalized polystyrene derivatives through sulfonamide bonds according to formula (III). where R is a functional group selected from the group comprising an amino (NH2), a Bromide (Br), a carboxylic (COOH), an alkyl ester (COOCH3), a morpholine group or a hydrogen (H); and x takes a value between O and 12

Another more preferred aspect of the present invention is the use of polystyrene modified with chlorosulfonyl groups, in the synthesis of polystyrene functionalized with sulfonic groups according to formula (IV).

Another more preferred aspect of the present invention is the use of polystyrene modified with chlorosulfonyl groups, in the synthesis of polystyrene functionalized with sulfonazide groups according to formula (V).

Another more preferred aspect is that the functionalized polystyrene substrates (III), (IV) and (V) obtained retain their optical transparency properties.

Another preferred aspect of the present invention is the use of polystyrene substrates modified according to formulas (III), (IV) and (V) for the coupling of biomolecules.

DETAILED DESCRIPTION

The main objective of the present invention is to propose a new method for manufacturing solid polystyrene-based substrates with controlled functionalization surfaces. This objective can be achieved with a two step reaction. In the first, the surface of the substrate is modified with chlorosulfonyl groups and in the second, these functional groups are reacted with bifunctional molecules that must contain a primary aliphatic amino group (for anchoring to the pre-activated surface in the first step) and a second functional group that will determine the functionality of the surface. The length of the spacer between the two functional groups determines the accessibility and reactivity of the functionality of the surface.

Chlorosulfonation of polystyrene is a well known reaction for grafting reactive groups in this polymer and has been described in different works using PS in the form of crosslinked beads or fibers. However, no use of the reaction on transparent surfaces is described. The particular value of the present invention is to have found the experimental conditions that allow the application of said reaction to solid polystyrene substrates without causing degradation of the surface or damaging the optical properties, such as its transparency.

Thus, one aspect of the present invention is the process for obtaining polystyrene modified with chlorosulfonyl groups comprising the following steps: a) cooling a solution of pure chlorosulfonic acid in an inert atmosphere at low temperature. b) introduction into solution a) of a polystyrene substrate between 0 and 3 hours, preferably 30 minutes c) extraction of the substrate and subsequent washing in a concentrated acid bath for a time between 0 and 10 min. d) washing of the modified substrate in a water / ice bath between 0 and 5 min. preferably 30 seconds and subsequent drying In which the temperature of stage a) is between -2O ⁰ C and 2O ⁰ C, and the acid used in stage c) is sulfuric acid The temperature of the acid bath washing of stage c) is between -2O ⁰ C and 1O ⁰ C,

A preferred aspect is that the temperature of stage a) is -1O ⁰ C Another preferred aspect is that the temperature of stage c) is -1O ⁰ C

Another preferred aspect of the present invention is that the process for obtaining polystyrene modified with chlorosulfonyl groups is carried out on polystyrene substrates introduced in stage b) and that are transparent.

A preferred aspect of the present invention is that with the process of obtaining the invention, polystyrene modified with chlorosulfonyl groups is obtained that preserves the optical transparency properties of the transparent starting polystyrene.

By varying the reaction parameters (time, temperature, chlorosulfonic acid purity), surfaces with a variable amount of chlorosulfonyl groups that can be easily detected and quantified by FTIR-ATR spectroscopy can be achieved in a controllable manner as observed in Figure 1.0

The objective of the second step of the modification is to benefit from the high reactivity of the chlorosulfonyl groups on the surface to create new functional groups in a controlled manner.

In this way, another preferred aspect of the present invention is the use of transparent polystyrene modified with chlorosulfonyl groups as an intermediate in reactions for obtaining functionalized polystyrene.

A more preferred aspect of the present invention is the use of polystyrene modified with chlorosulfonyl groups that reacts with disubstituted alkanes containing a primary aliphatic amine group for prepare functionalized polystyrene derivatives through sulfonamide bonds according to formula

donde R es un grupo funcional seleccionado del grupo que comprende un amino (NH₂), un Bromuro (Br), un carboxílico (COOH), un áster alquílico (COOCH₃), un grupo morfolina ó un hidrógeno (H); y x toma un valor de entre O y 123,

where R is a functional group selected from the group comprising an amino (NH ₂ ), a Bromide (Br), a carboxylic (COOH), an alkyl ester (COOCH ₃ ), a morpholine group or a hydrogen (H); and x takes a value between O and 12

This objective is achieved by submerging the modified PS substrates in aqueous solutions of disubstituted alkanes containing a primary aliphatic amino group and a second functional group to choose R. The amine groups are anchored through sulfonamide bonds to the surface. The second functional group of the chosen molecule is free and available for future anchoring of some biomolecule.

Another more preferred aspect of the present invention is the use of polystyrene modified with chlorosulfonyl groups, in the synthesis of polystyrene functionalized with sulfonic groups (IV) by hydrolysis reaction under suitable conditions

(IV)

Another more preferred aspect of the present invention is the use of polystyrene modified with chlorosulfonyl groups, in the synthesis of polystyrene functionalized with sulfonazide groups (V) by reaction of the activated surface with sodium azide.

(V)

Another preferred aspect is that the functionalized polystyrene substrates (III), (IV) and (V) retain their optical transparency properties. As it is a modification of the polymeric surface and not a coating, the surface obtained is consistent and stable over time. By varying the reaction parameters (time, temperature, concentration of the modifying compound aqueous solution, number of alkyl groups of the disubstituted alkanes), surfaces with a variable amount of functional groups can be achieved. In addition, in the new materials, the distance between the surface and the functional groups created through the length of the aliphatic spacer used can be adjusted. That allows excellent accessibility for biomolecules. In the case of an amination of the surface, values of between 1 and 50 nmol / cm ² are achieved for the number of functional groups that are anchored to the surface, giving this method values that are of an order of magnitude between 1 and 2 greater than Ia that is obtained with conventional methods. Another preferred aspect of the present invention is the use of polystyrene substrates modified according to formulas (III), (IV) and (V) for the coupling of biomolecules.

A more preferred aspect of the present invention is the use of polystyrene substrates modified according to formulas (III), (IV) and (V) in which the coupled biomolecules can be antibodies and enzymes.

DESCRIPTION OF THE FIGURES:

Figure 1: Raman spectrum of PS modified with chlorosulfonyl groups. X axis is the wave number, the y axis is the aborbance in arbitrary units. Chlorosulfonyl groups are characterized by two strong bands at 1373 cm-1 and 1171 cm-1 that correspond to the valence bands of S = O.

EXAMPLES OF REALIZATION

1) Preparation of a PS substrate modified with chlorosulfonyl groups: A reactor with magnetic stirring is filled with pure Chlorosulfonic Acid, passes N ₂ through the solution and cools the liquid to a temperature of - 1O ⁰ C. A rectangular film of PS with dimensions 75mm ^* 25mm ^* 2mm in the reactor for example 20 minutes. After from the reaction the sample is taken out of the reactor and washed in a bath of concentrated sulfuric acid at -1O ⁰ C for 30 seconds. Finally, the modified film is washed in a water / ice bath for 30 seconds and dried. The quantity of the chlorosulfonyl groups can be analyzed qualitatively and quantitatively by FTIR-ATR where characteristic bands come out at 1170 and 1370 cm ^"1. Their quantification is carried out by means of a calibration curve with samples of chlorosulfonated PS in powder whose sulfur content It is determined by elementary analysis.

2) Preparation of a PS substrate modified with primary amine groups a) distance from the polymer chain of three aliquyl groups:

A chlorosulfonated film is immersed for 15 minutes in a solution of 1,3-diaminopropane in water at a concentration of c = 8 mol / l (20 ml diamine, 10 ml H ₂ O) at 4O ⁰ C. After washing with Water at room temperature dries the film.

b) distance from the polymer chain of six aliquyl groups: A chlorosulfonated film is immersed for 240 minutes in a solution of 1,6-diamino (hexane) in water at a concentration of c = 6 mol / l (30 ml diamine, 8.4 mi H ₂ O) at 4O ⁰ C. After a wash with water at room temperature the film dries.

c) distance of the polymer chain from ten alkyl groups:

A chlorosulfonated film is immersed for 240 minutes in a solution of 1, 10-diamino (decane) in water at a concentration of c = 2 mol / l (7 ml diamine, 9.4 ml H ₂ O) at 6O ⁰ C. After washing with water at room temperature the film dries.

d) Characterization and quantification of amine groups The formation of free primary amine groups on the surface can be qualitatively checked by FTIR-ATR: bands at 3369 and 3278 cm -1 are observed that correspond to the associated and free NH bond. The anchoring of the reagent to the surface by means of the formation of a sulfonamide group is verified by the unequivocal displacement of the bands at 1170 and 1370 cm ^"1 of the chlorosulfonyl groups towards 1156 and 1312 cm ^{" 1} .

For the evaluation of the number of free amine groups on the surface, a colorimetric study is carried out using Orange Il [Hamerli P., Weigel Th., Groth Th., Paul D. Biomaterials 24 (2003) 3989-3999]. This reagent binds to primary amines via an acid-base reaction and absorbs at 485 nm.

Method:

1. The surface is immersed in Orange Il 0.5μM in aqueous solution pH3.

2. It is left in contact overnight at room temperature and mechanical agitation 30rpm / mi.

3. Rinse 5 times with water at pH3

4. Extract, under mechanical stirring, with 10 ml 0.1 M NaOH for 30 minutes at room temperature in your case use 5h at 45 ⁰ C. I absorbance at 485 nm is measured. Application examples e1) Adhesion of antibodies Adhesion of antibodies to aminated PMMA surfaces. Method:

1. The surfaces are immersed in a 10% glutaraldehyde solution in water with Λ / - (3-Dimethylaminopropyl) -Λ / '- ethylcarbodiimide hydrochloride, Λ / -Hydroxysulfosuccinimide sodium salt and triethylamine

(5: 1.5: 10), for 1h at room temperature.

2. Rinse with water four times.

3. Streptavidin 0.1 mg / ml is put in 1x PBS overnight at 4 ⁰ C.

4. Rinse 4 times in 1 x 5 PBS. The surface is immersed in a 1% BSA solution in water for 30 minutes.

6. Rinse with water 4 times.

7. The surface in contact with the 0.04mg / ml biotinylated antibody is incubated at room temperature for 1 hour in 1x PBS. 8. Rinse with PBS 4 times.

e2) enzyme-linked immunosorbent assay ELISA

ELISA tests have been performed on synthesized aminated surfaces. In addition to the aminated surfaces, three different procedures have been performed for anchoring the anti-IL-6 antibody to the surface (Step 1).

Methods:

1. Functionalization of the surface with the anti-IL-6 antibody A. Following the protocol established by the manufacturer: a. Dilution 1/250 in "coating buffer". 48ul of Ac in 12ml buffer. b. Pour 100ul / well C. Leave overnight at 4 ⁰ C d. Remove the liquid and wash 5 times with wash buffer pouring more than 250ul / well. Let stir 1 minute each wash. Wash buffer: PBSIx + Tween 20 0.05% (Sigma

P3563) e. Dilute 1 part of Assay diluent in 4 parts of distilled water and add 200ul / well. Leave 1 hour RT. F. Vacuum and wash 5 times as in point d.

by glutaraldheido crosslinker a. 10% Glutaraldehyde for 20 'and rinse 4 times with water. b. Dilution 1/250 in "coating buffer". 48ul of Ac in 12ml buffer. C. Pour 100ul / well d. Leave overnight at 4 ⁰ C e. Remove the liquid and wash 5 times with wash buffer pouring more than 250ul / well. Let stir 1 minute each wash.

Wash buffer: PBSIx + Tween 20 0.05% (Sigma P3563) f. Dilute 1 part of Assay diluent in 4 parts of distilled water and add 200ul / well. Leave 1 hour RT. g. Vacuum and wash 5 times as in point d. C. Through EDAC crosslinker a. Dilution 1/250 in "coating buffer". 12ul of Ac + 0.03ul of triethylamine + 0.12mg of EDAC + 7.5ug of NH-sulfo in 3ml of buffer. b. Pour 100ul / well c. Leave overnight at 4 ⁰ C d. Remove the liquid and wash 5 times with wash buffer pouring more than 250ul / well. Let stir 1 minute each wash. Wash buffer: PBSIx + Tween 20 0.05% (Sigma

P3563) e. Dilute 1 part of Assay diluent in 4 parts of distilled water and add 200ul / well. Leave 1 hour RT. F. Vacuum and wash 5 times as in point d.

3a) Preparation of a PS substrate modified with carboxy groups:

A chlorosulfonated film is immersed for 60 minutes in an aqueous solution of the β-alanine methyl ester at a concentration of c = 6g / 40ml at 4O ⁰ C. The modified film is washed with water at room temperature and subsequently immersed for 15 minutes in an aqueous solution of KOH at 4O ⁰ C. The film is washed first in concentrated HCI and then in water and dried.

The anchoring of the β-alanine methyl ester to the surface is checked on the one hand by the complete formation of sulfonamide groups (see also 2d) and on the other hand by the formation of a band at 1737cm ^"1 corresponding to the carbonyl group of an ester It is also possible to transform the surface with carboxylic groups reversibly in the salt corresponding to immersing the film in an aqueous solution of NaOH. When subsequently exposed to an acid solution (pH = 1), the carboxylic groups are completely recovered.

b) Determination of carboxy groups

For the evaluation of the degree of carboxylation obtained with each process, a colorimetric study is performed using toluidine blue O (TBO) [Goddard J. M., Talbert J. N., and Hotchkiss J. H. Journal of Food Science, vol 12, Nr 1, 2007]. This reagent binds to carboxylic groups and absorbs 697 nm.

Method:

1. The surface is immersed in 0.5mM TBO in pH10 aqueous solution.

2. It is left in contact overnight at room temperature and mechanical agitation 30rpm / mi. 3. Rinse 5 times with water at pH10.

4. Extract, under mechanical stirring, with 1OmI of 37% HCI for 30 minutes at room temperature.

5. Absorbance is measured at 697nm (the peak of the TBO is at 633nm in 50% acetic acid when changing the extraction medium has changed the maximum adsorption peak) c) Applications: enzyme-linked immunosorbent assay (Elisa)

ELISA assays have been performed on synthesized carboxylated surfaces. The procedure established by the manufacturer has been followed. 1. Capture of antigen g. Dilute the standard (IL-6) 5ul in 25ml in Assay diluent 1x so it would be 200pg / ml, so make the required dilutions and pour 100dl / well. Cover and close by incubating overnight at 4 ⁰ C. h. Vacuum and wash as in 1.d.

2. Antigen detection a. Dilute the detection antibody (biotin anti IL-6) 1/250, that is, 48ul per 12ml of Assay diluent 1x. Pour 100ul / well. Cover and incubate 1 h RT. b. Vacuum and wash 5 times as in 1d

3. Immobilization of the enzyme a. Dilute the enzyme with avidin (avidin-HRP) 1/250, that is, 48ul per 12ml of Assay diluent 1x. Pour 100ul / well. Cover and incubate 30 minutes at RT. b. Aspirate and wash 7 times as in 1d, leaving 1-2 minutes in each wash.

4. Enzymatic activity a. Add 100ul / well of undiluted substrate. Incubate RT 15 minutes. b. Add 50ul / well stop solution (2N (1M) H2SO4)

5. Reading and data analysis a. Measure the plate at 450nm.

4) Preparation of a PS substrate modified with sulfonic groups: A chlorosulfonated film is immersed for 20 minutes in an aqueous solution of potassium carbonate at a concentration of c = 1g / 20ml at 5O ⁰ C. After 2 hours the reaction is complete. The modified film is washed with water at room temperature and the film is dried. The formation of sulfonic groups is verified by observing the complete disappearance of the 1370 cm ^{"1 band} of the chlorosulfonyl groups and the formation of new characteristic bands of the sulfonic group at 1131, 1040 and 1010 cm ^{" 1} . It is also possible to transform the surface with sulfonic groups reversibly in the corresponding salt by immersing the film in an aqueous NaOH solution. When subsequently exposed to an acid solution (pH = 1), the sulfonic groups are completely recovered.

5) Preparation of a PS substrate modified with sulfonazide groups:

A chlorosulfonated film is immersed for 3 hours in an aqueous solution of sodium azide at a concentration of c = 10g / 20ml at 4O ⁰ C. After 24 hours the reaction is complete. The modified film is washed with water at room temperature and the film is dried. The formation of sulfonazide groups is verified by observing the complete disappearance of the 1370 cm ^{"1 band} of the chlorosulfonyl groups and the formation of new characteristic bands of the azide group at 2133 cm ^{" 1}

Claims

1- Process for obtaining polystyrene modified with chlorosulfonyl groups comprising the following steps: a) cooling a solution of pure chlorosulfonic acid in an inert atmosphere at low temperature. b) introduction into solution a) of a polystyrene substrate between 0 and 3 hours, preferably 30 minutes c) extraction of the substrate and subsequent washing in a concentrated acid bath for a time between 0 and 10 min. d) washing of the modified substrate in a water / ice bath between 0 and 5 min., preferably 30 seconds and subsequent drying and is characterized in that: • in stage a) the temperature is between -2O ⁰ C and 2O ⁰ C, • in stage c) the acid used is sulfuric acid and • in stage c) the acid wash temperature is between -20 and 1O ⁰ C 2- Procedure for obtaining polystyrene modified with chlorosulfonyl groups according to claim 1 wherein the temperature of stage a) is -1O ⁰ C 3- Procedure for obtaining polystyrene modified with chlorosulfonyl groups according to claim 1 wherein the temperature of stage c) is -1O ⁰ C 4- Procedure for obtaining polystyrene modified with chlorosulfonyl groups according to claim 1 characterized in that the polystyrene substrate introduced in step b) is transparent 5- Polystyrene modified with chlorosulfonyl groups according to claim 1 to 4, characterized in that it retains the optical properties of the transparent starting polystyrene. 6- Use of polystyrene modified with chlorosulfonyl groups according to claim 5, as an intermediate in reactions for obtaining functionalized polystyrene 7- Use of the polystyrene modified with chlorosulfonyl groups according to claim 6 that reacts with disubstituted alkanes containing a primary aliphatic amine group for the preparation of functionalized polystyrene derivatives through sulfonamide bonds according to formula (III), wherein • -R is a functional group selected from the group comprising an amino (NH ₂ ), a Bromide (Br), a carboxylic (COOH) an alkyl ester (COOCH ₃ ), a morpholine group or a hydrogen (H); • -y x takes a value between O and 12 3,

-H x = 0-12 (III) 8- Use of the polystyrene modified with chlorosulfonyl groups according to claim 6 wherein the polystyrene is functionalized with sulfonic groups according to formula (IV)

- Use of the polystyrene modified with chlorosulfonyl groups according to claim 6 wherein the polystyrene is functionalized with sulfonazide groups according to formula (V)

0-Modified substrates according to claims 7, 8 and 9 characterized in that the modified polystyrenes according to formulas (IV) and (V) retain their optical transparency properties 1 -Use of modified polystyrene substrates according to formula (III), (IV) and (V) for the coupling of biomolecules. 2-Use of the modified polystyrene substrates according to claim 11 wherein the biomolecules are antibodies. 3-Use of the modified polystyrene substrates according to claim 11 wherein the biomolecules are enzymes