OBJECT OF THE INVENTION The present invention refers to the preparation of new polymer materials that behave like fluorescent sensors for anions and, in particular, for chlorides, in aqueous media, such as for example industrial waters, human consumption waters and/or sweat.

BACKGROUND OF THE INVENTION There are currently a large number of rules and/or directives that apply to the field of control of chemical species in industrial waters and human consumption waters (Royal Decree 140/2003, of 7 February). Among said chemical species, various anions, including chloride anions, are measured on a weekly basis, since they may vary greatly and rapidly. Chlorine salts are commonly used to prevent the growth of microorganisms in human consumption waters. A defect in the levels of these chlorine salts can favour the growth of microorganisms; however, in excess they can trigger the exponential growth of concentrations of chloride anions, with all the health risks it entails.

The most widely used methods for sensing chlorides in waters include, namely, the chloride ion-selective electrode. It is basically a device that translates the concentration of chlorides into an electric signal that is subsequently analysed and interpreted. Obviously, the equipment requires an electrical outlet to operate, in addition to calibration prior to measurement.

There are also methods based on the precipitation of insoluble chloride salts, such as silver chloride. In this case, the technique requires handling various chemical reagents, some of which reach very high prices, which is also a limitation for performing routine assays. The methods used to sense chlorides in sweat include, namely, the so-called "sweat test" or "Gibson and Cooke Method" (G. Mastella, G. Di Cesare, A. Borruso, L. Menin, L. Zanolla, Acta Pediatr., 2000, 89, 933-937), which is applied particularly in young patients as a simple method for the pre-diagnosis of cystic fibrosis and whose procedure is amply detailed in the bibliography (V. A. LeGrys, J. R. Yankaskas, L. M. Quittell, B. C. Marshall, P. J. Mogayze, J. Pediatr., 2007, 151 , 1 , 85-89). A high concentration (above 60 millimoles per litre) of chlorides in sweat, is one of the most representative symptoms of cystic fibrosis. There are currently various examples of marketed kits for performing this assay. They are all based on three steps: (1 ) the stimulation of sweat glands by iontophoresis (J. Guodemar, P. Garcia, E. M. Rodriguez, Revista de la Facultad de Ciencias de la Salud, 2004, 2, "Iontophoresis, doses and treatments") and with the help of pilocarpine (I. Largo-Garcia, Rev. Ped. Elec, 2009, 6, 1 -18); (2) sample collection; and (3) analysis.

In addition to cystic fibrosis, there are other diseases related to a high concentration of chlorides in sweat, such as for example: Untreated adrenal failure, ectodermal dysplasia, familial cholestasis syndrome, hypothyroidism, malnutrition, Mauriac syndrome, glycogen- storage disease type I, mucopolysaccharidosis, insipid nephrogenic diabetes, fucosidosis, nephrosis and glucose-6-phosphate deficiency.

Various efforts have been made to develop sensors to detect anions in a concentration dependent manner and which are stable and of simple use. In this sense, the use of fluorescent immobilized dyes in a substrate as a method to detect anions in a solution is known, however, the loss of the dye from the substrate, called "dye leaching" has been a drawback in their development.

Several substrates have been proposed to immobilize fluorescent dyes, among which glass substrates (S1O2) and polymeric membranes. Although said glass substrates have been proposed for being less prone to bacterial development, the error on the anion concentration measured with those is sometimes too large. In fact, Geddes et al. (Meas. Sci. Technol. 12 (2001 ) R53-R88) report that glass substrates in which acridinium, or quinolinium, dyes are embedded provide an error in chloride determination of about 5%, due to dye leaching, in a solution with a concentration of 100 mM which the normal concentration of chloride in blood.

Geddes et al. also describe (Sensors and actuators B 72 (2001 ); 188-195, J. of Appl. Pol. Sci., vol. 76 (2000); 603-6015 and Meas. Sci. Technol. 12 (2001 ) R53-R88) the use of polymeric membranes in which fluorescent dyes are attached covalently to avoid errors due to dye leaching. Those sensor membranes proposed by Geddes et al. include fluorescent dyes attached by means of an ester bond to a polymeric chain. Although a clear reduction in dye leaching is obviously obtained when compared to the loosely embedded dyes in glass or membrane support, Geddes et al. (Sensors and actuators B 72 (2001 ); 188-195) admit that, in order to correct the decrease in fluorescence intensity due to dye degradation or leaching, the use a reference sensor film is always needed. It is important to note that Geddes et al. perform all "dye bleaching" measurements at pH 10 (see J. of Appl. Pol. Sci., vol. 76 (2000); 603-6015 and Meas. Sci. Technol. 12 (2001 ) R53-R88), a higher pH than the one of drinking water or sweat. The same authors indicate (see Meas. Sci. Technol. 12 (2001 ) R53-R88) that lowering the pH results in dye leaching, due to the hydrolysis of the ester bond and, therefore, they conclude that meticulous studies of dye attachment should be carried out, especially if the sensors are to be used in living systems.

Ester hydrolysis may occur in a higher or smaller degree depending on pH or, on the presence of nucleophilic species in the solution. In sweat, several components as ammonia, bicarbonate and lactate may influence the pH taking it to values as low as 4.5 where hydrolysis of esters may be significant. Moreover, some of the sweat components such as ammonia are nucleophiles, which may contribute even more to hydrolysis.

In this context, the development of polymers and copolymers based on monomers with different functional groups and in the form of gel-like hydrophilic membranes, and that detect different chemical species, both in solution and in the gas phase, is a subject of great scientific interest (S. Vallejos, A. Mufioz, S. Ibeas, F. Serna, F. Garcia, J. M. Garcia, J. Mater. Chem. A.

2013, 1 , 15435-15441 , S. Vallejos, A. Mufioz, S. Ibeas, F. Serna, F. Garcia, J. M. Garcia, J.

Hazard. Mater. 2014, 276, 52-57, S. Vallejos, A. Mufioz, S. Ibeas, F. Serna, F. Garcia, J. M.

Garcia, ACS Appl. Mater. Interfaces. 2015, 7, 921-928, J. L. Pablos, S. Vallejos, A. Mufioz, M. J. Rojo, F. Serna, F. C. Garcia, J. M. Garcia, Chem. Eur. J. 2015, 21 , 8733-8736, S. Vallejos,

A. Mufioz, F. Garcia, R. Colleoni, R. Biesuz, G. Alberti, J. M. Garcia, Sens. Actuators B. 2016,

233, 120-126).

BRIEF DESCRIPTION OF THE INVENTION The present invention relates to a copolymer comprising a salt of a group of formula (I):

Pi wherein one of the R3, R ₄ , R5, R6, R7, Rs or R9 groups is an OR10 group and the rest of the groups R3, R ₄ , R5, R6, R7, Rs and R9 are each selected independently from H, OH, halogen, sulphonyl, N R11 R12, COOR10, CON R11 R12, alkyl, cycloalkyl, aryl, non-aromatic heterocyclic and hetaryl groups; wherein R10 is selected from alkyl, cycloalkyl, aryl, non-aromatic heterocyclic or hetaryl groups;

wherein Rn and R12 are each selected independently from H, alkyl, cycloalkyl, aryl, non-aromatic heterocyclic and hetaryl groups; and

- wherein said group of formula (I) is covalently linked to the copolymer chain by a hydrocarbon group selected from the group consisting of aryl, cycloalkyl or alkyl.

Another aspect of the present invention relates to a method to obtain a copolymer comprising a salt of a group of formula (I):

wherein said method comprises the steps of:

(a) obtaining a copolymer through the polymerization of at least two monomers, wherein at least one of said two monomers is a monomer Z includes a R2 group, wherein:

said group R2 is independently selected from a group consisting of aryl, cycloalkyl or alkyl, and

- said group R2 is also substituted with at least one halogen group, and where said polymerization is carried out by direct reaction of polymerizable groups present in each of the monomers;

(b) reaction of the copolymer obtained in step (a) with a compound of formula (la):

wherein one of the R3, R ₄ , R5, 6, R7, Rs or R9 groups is an OR10 group and the rest of said groups R3, R ₄ , R5, R6, R7, Rs and R9 are each selected independently from H, OH, halogen, sulphonyl, N R11 R12, COOR10, CON R11 R12, alkyl, cycloalkyl, aryl, non-aromatic heterocyclic and hetaryl groups; wherein R10 is selected from alkyl, cycloalkyl, aryl, non-aromatic heterocyclic or hetaryl groups; and wherein Rn and R12 are each selected independently from H, alkyl, cycloalkyl, aryl, non-aromatic heterocyclic and hetaryl groups.

The present invention also refers to a copolymer comprising a salt of a group of formula (I), obtainable through the method described herein.

Another aspect of the present invention relates to the use of a copolymer comprising a salt of a group of formula (I), obtainable by the method of the present invention, for sensing and/or quantifying anions and, in particular, chlorides.

An additional aspect of the present invention relates to the use of a copolymer comprising a salt of a group of formula (I), obtainable by the method of the present invention, for detecting a disease selected independently from cystic fibrosis, untreated adrenal failure, ectodermal dysplasia, familial cholestasis syndrome, hypothyroidism, malnutrition, Mauriac syndrome, glycogen-storage disease type I, mucopolysaccharidosis, insipid nephrogenic diabetes, fucosidosis, nephrosis and glucose-6-phosphate deficiency. A embodiment refers to the use of a copolymer comprising a salt of a group of formula (I), of the present invention, for detecting cystic fibrosis. Another aspect of the present invention relates to a dressing comprising a copolymer comprising in turn, a salt of a group of formula (I), obtainable by the method of the present invention, to detect a disease selected independently from cystic fibrosis, untreated adrenal failure, ectodermal dysplasia, familial cholestasis syndrome, hypothyroidism, malnutrition, Mauriac syndrome, glycogen-storage disease type I, mucopolysaccharidosis, insipid nephrogenic diabetes, fucosidosis, nephrosis and glucose-6-phosphate deficiency. A particular embodiment refers to a dressing comprising a copolymer comprising in turn, a salt of a group of formula (I), of the present invention, to detect cystic fibrosis.

DESCRIPTION

Present invention refers to a method for obtaining a copolymer comprising a salt of a group of formula (I):

I wherein said method comprises the steps of:

(a) obtaining a copolymer through the polymerisation of at least two monomers, wherein at least one of said two monomers is a monomer Z comprising a group R2, wherein:

said group R2 is independently selected from a group consisting of aryl, cycloalkyl and alkyl, and

said group R2 is also substituted with at least one halogen group; and where said polymerisation is carried out by direct reaction of polymerizable groups present in each of the monomers;

(b) reaction of the copolymer obtained in step (a) with a compound of formula (la):

wherein one of the R3, R ₄ , R5, R6, R7, Rs or R9 groups is an OR10 group and the rest of said groups R3, R ₄ , R5, R6, R7, Rs and R9 are each selected independently from H, OH, halogen, sulphonyl, NR11R12, COOR10, CONR11R12, alkyl, cycloalkyl, aryl, non-aromatic heterocyclic and hetaryl groups; wherein R10 is selected from alkyl, cycloalkyl, aryl, non-aromatic heterocyclic or hetaryl groups; and wherein Rn and R12 are each selected independently from H, alkyl, cycloalkyl, aryl, non-aromatic heterocyclic and hetaryl groups. The present invention therefore also relates to a copolymer comprising a salt of a of formula (I):

wherein one of the R3, R ₄ , R5, R6, R7, Rs or R ₉ groups is an OR10 group and the rest of the groups R3, R ₄ , R5, R6, R7, Rs and R9 are each selected independently from H, OH, halogen, sulphonyl, N R11 R12, COOR10, CON R11 R12, alkyl, cycloalkyl, aryl, non-aromatic heterocyclic and hetaryl groups; wherein R10 is selected from alkyl, cycloalkyl, aryl, non-aromatic heterocyclic or hetaryl groups;

- wherein Rn and R12 are each selected independently from H, alkyl, cycloalkyl, aryl, non- aromatic heterocyclic and hetaryl groups; and

wherein said group of formula (I) is covalently linked to the copolymer chain by a hydrocarbon group selected from the group consisting of aryl, cycloalkyl or alkyl.

The present invention also refers to a copolymer comprising a salt of a group of formula (I), obtainable through the method described above herein.

For the purposes of the present invention, examples of salts of groups of formula (I) comprise salts of an organic or inorganic acid. In one embodiment, said salt is selected independently from a fluoride, chloride, bromide, iodide, sulphate, sulphite, phosphate, carbonate, bicarbonate, nitrate, hydroxide nitrite, acetate, lactate, citrate and oxalate salt. In general, in one embodiment of the invention, said salt comprises any anion present in industrial waters, human consumption waters or in sweat. In one embodiment of the invention, the salt is a fluoride, chloride, bromide or iodide salt, more preferably a bromide salt.

For the purposes of the present invention, the term "alkyl" relates to a linear or branched hydrocarbon aliphatic chain of 1 to 20 carbons that may or may not have different replacements. In a preferred embodiment, said alkyl is selected independently from methyl, ethyl or propyl. For the purposes of the present invention, the term "cycloalkyl" relates to a hydrocarbon cyclical group that may or may not have different replacements. In a preferred embodiment, said cycloalkyl is selected independently from cyclopropyl, cyclopentyl or cyclohexyl.

For the purposes of the present invention, the term "alcoxy" relates to a hydrocarbon group bonded to an oxygen atom such as ORio. In a preferred embodiment, said alcoxy group is selected from methoxy, ethoxy, butoxy, propoxy, isopropoxy, tert-butoxy and phenoxy. In a more preferred embodiment, said alcoxy group is methoxy.

For the purposes of the present invention, the term "non-aromatic heterocyclic group" relates to an aliphatic hydrocarbon cycle, having at least one heteroatom, and which may or may not have different replacements. In a preferred embodiment, said non-aromatic heterocyclic group is selected independently from piperidyl, pyrrolidine, tetrahydropyran, tetrahydrofuran and tetrahydrothiophene.

For the purposes of the present invention, the term "aryl" relates to a group that may comprise one or more hydrocarbon aromatic rings replaced or not replaced by other functional groups. In a preferred embodiment, said aryl group is selected independently between phenyl and naphthyl.

For the purposes of the present invention, the term "hetaryl" relates to a group that may comprise one or more aromatic rings comprising one or more heteroatoms. In a preferred embodiment, said hetaryl group is selected independently from pyridyl, pyrazole, pyran, furan and thiophene

Said group of formula (I) interacts with anions and, in particular, with chlorides, causing a change in fluorescence of the copolymer and, therefore, allowing the sensing and/or quantification of said anions. Specifically, the fluorescence of the material is reduced, i.e. an ON-OFF fluorescence process takes place. This process can be observed in a fluorometer (fluorescence spectrophotometer). In the event of sensing chloride anions, the fluorescence band centred at around 440 nm (observed when the sample is excited at 321 nm) begins to decrease until practically disappearing as the concentration of chlorides increases in the medium. The change in fluorescence is so important that, in addition to a fluorometer, the change can also be observed qualitatively with the naked eye. Additionally, the use of the copolymers of the present invention is completely reversible, such that, if said copolymer is washed with water once used to sense an anion, it becomes fully operational once again.

The copolymers of the present invention are, therefore, applicable in the sensing and/or quantification of anions and, in particular, chlorides. One embodiment relates to the use of the copolymers that comprise a salt of a group of formula (I) for sensing and/or quantifying anions, and chlorides in particular, in industrial waters and in human consumption waters.

Additionally, the copolymers of the present invention are biocompatible for application in the sensing and/or quantification of chlorides in sweat. This chloride sensing and/or quantification property by immersing the copolymer of the invention in an aqueous medium, or simply by contact, in the case of sweat, allows said copolymers to be used as continuous chloride sensors (in the case of industrial waters or human consumption waters) and as a dressing (in the case of sweat) that contains them.

Therefore, another embodiment relates to the use of the copolymers of the invention for sensing and/or quantifying chlorides in sweat.

The use of the copolymers of present invention as sensors of chlorides in sweat, can be applied in the diagnosis of diseases related to a high concentration of chlorides in sweat, such as cystic fibrosis, untreated adrenal failure, ectodermal dysplasia, familial cholestasis syndrome, hypothyroidism, malnutrition, Mauriac syndrome, glycogen-storage disease type I, mucopolysaccharidosis, insipid nephrogenic diabetes, fucosidosis, nephrosis and glucose-6- phosphate deficiency.

Therefore, one embodiment relates to the use of the copolymers of the invention in the detection of a disease related to a high concentration of chlorides in sweat. Preferably, said disease is selected independently from cystic fibrosis, untreated adrenal failure, ectodermal dysplasia, familial cholestasis syndrome, hypothyroidism, malnutrition, Mauriac syndrome, glycogen-storage disease type I, mucopolysaccharidosis, insipid nephrogenic diabetes, fucosidosis, nephrosis and glucose-6-phosphate deficiency. More preferably, said disease is cystic fibrosis.

The method for sensing and/or quantifying chlorides described herein is therefore based on the capacity of said chloride anions to eliminate the fluorescence from the group of formula (I) present in the copolymers of the present invention. Said group of formula (I), present in the copolymers of the present invention, is a quinoline-derived group covalently bonded to the polymer chain by the nitrogen atom of the quinoline, and where said nitrogen atom is a positively charged quaternary nitrogen.

The copolymers of the present invention, comprising at least one salt of a group of formula (I), may be linear or cross-linked copolymers. The term copolymer relates to a molecule comprising one or more successively repeated structural units. Said units are called monomers. The polymers are obtained from the repeated bonding of monomers by reaction of reagent groups (or polymerizable groups) present in each of the monomers, in a process called polymerisation. A copolymer therefore comprises more than one different monomer and is obtained, therefore, by polymerisation of at least two or more different monomers.

Cross-linked copolymer refers to a copolymer that gives rise to a network formed by the bonding of different copolymer chains. The formation of said network by different polymer chains is called cross-linking.

The copolymers comprising a salt of a group of formula (I), described herein, are presented in the form of films or solid membranes. Said solid membranes may be dense membranes or porous membranes. The present invention relates to said dense membranes, but also to the porous membranes obtained by means of chemical and/or physical foaming processes carried out in the previously described dense membranes.

The copolymers that comprise a salt of a group of formula (I), described herein, present changes in fluorescence in the presence of anions. For the purposes of the present invention, chloride [Ch], bromide [Br], iodide [I-], lactate (CH3CHOHCOO ^" ) and acetate [CH3COO-] are non-limiting examples of anions. In a preferred embodiment, said change in fluorescence occurs when there are chloride anions present in the medium.

For the purposes of the present invention, the term copolymer is used equivalently to the term membrane, due to the membrane structure of the copolymers described herein. Furthermore, the linear or cross-linked copolymers comprising a salt of a group of formula (I), described herein, are indistinctly called sensor membrane, sensor copolymer or fluorogenic sensors due to their properties, described herein.

In a preferred embodiment, one of the R3, R ₄ , R5, 6, R7, Rs or R9 groups is a methoxy group and the rest of said groups R3, R ₄ , R5, R6, R7, Rs and R9 are H. In an even more preferred embodiment, R5 is an OR10 group and the rest of said groups R3, R ₄ , R5, R6, R7, Re and R9 are H; wherein R10 is selected from alkyl, cycloalkyi, aryl, non-aromatic heterocyclic and hetaryl groups. More preferably, R10 is an alkyl group; even more preferably, R10 is selected from methyl, ethyl, propyl, isopropyl, butyl and tert-butyl. In an even more preferred embodiment, R ₅ is a methoxy group and the other groups are H.

In another preferred embodiment, one of the R3, R ₄ , R5, R6, R7, Rs or R ₉ groups is an OR10 group and the rest of said groups R3, R ₄ , R5, R6, R7, Rs and R9 are each selected independently between H or CH3; wherein R10 is selected from alkyl, cycloalkyi, aryl, non-aromatic heterocyclic and hetaryl groups. More preferably, R10 is an alkyl group; even more preferably, R10 is selected from methyl, ethyl, propyl, isopropyl, butyl and tert-butyl. More preferably, R10 is methyl.

In a more preferred embodiment, R5 is an OR10 group and the rest of the groups R3, R ₄ , R5, R6, R7, Rs and R9 are each selected independently between H and CH3; where R10 is selected from alkyl, cycloalkyi, aryl, non-aromatic heterocyclic and hetaryl groups. More preferably, R10 is an alkyl group; even more preferably, R10 is selected from methyl, ethyl, propyl, isopropyl, butyl and tert-butyl. More preferably, R10 is methyl.

In another preferred embodiment, one of the R3, R ₄ , R5, R6, R7, Rs or R9 groups is an OR10 group and the rest of said groups R3, R ₄ , R5, R6, R7, Rs and R9 are each selected independently between H and sulphonyl; wherein R10 is selected from alkyl, cycloalkyi, aryl, non-aromatic heterocyclic and hetaryl groups. More preferably, R10 is an alkyl group; even more preferably, R10 is selected from methyl, ethyl, propyl, isopropyl, butyl and tert-butyl. More preferably, R10 is methyl.

In a more preferred embodiment, R ₅ is an OR10 group and the rest of the groups R3, R ₄ , R5, R ₆ , R7, Rs and Rg are each selected independently between H and sulphonyl; where R10 is selected from alkyl, cycloalkyi, aryl, non-aromatic heterocyclic and hetaryl groups. More preferably, R10 is an alkyl group; even more preferably, R10 is selected from methyl, ethyl, propyl, isopropyl, butyl and tert-butyl. More preferably, R10 is methyl.

The monomers used in step (a) described herein may be both commercial monomers and synthesis monomers. In a preferred embodiment of the present invention, the reaction of the step (b) between the copolymer obtained in step (a) and a compound of formula (la) comprises: i. immersing the copolymer obtained in step (a) in said compound of formula (la) or in a solution thereof, at a temperature between 0°C and 150°C for at least 5 hours, ii. immersing the copolymer obtained in step (i) in an organic solvent and repeating this process to completely remove of the compound of formula (la) that did not react, iii. immersing the copolymer obtained in step (ii) in distilled water.

In one embodiment of the invention, step (b) comprises (i) immersing the copolymer obtained in step (a) in a compound of formula (la), or in a solution thereof, at a temperature between 50°C and 100°C for at least 5 hours.

In one embodiment of the invention, step (b) comprises (i) immersing the copolymer obtained in step (a) in a compound of formula (la), or in a solution thereof, and heating at a temperature between 50°C and 150°C for at least 12 hours.

In one embodiment of the invention, the step (b) comprises (ii) immersing the copolymer obtained in step (i) in an organic solvent and repeating this process at least five times. A preferred embodiment of step (b) of the present invention comprises (ii) immersing the copolymer obtained in step (i) in an organic solvent and repeating this process at least five times, each of said immersions having a duration of 5 minutes.

In a preferred embodiment of the invention, step (b) comprises (ii) immersing the copolymer obtained in step (i) in an organic solvent and repeating this process to completely remove the compound of formula (la) that did not react, where said organic solvent is an organic solvent miscible with water.

For the purposes of the present invention, the term "organic solvent miscible with water" relates to any organic solvent that can be mixed in any proportion of water, being the resulting mixture a homogeneous mixture or solution.

In a more preferred embodiment, said organic solvent is selected from dimethylformamide, acetonitrile, dimetylacetamide, dimethylsulphoxide, N-methylpyrrolidone, methanol, ethanol, isopropanol and THF. In an even more preferred embodiment, said organic solvent is acetone.

For the purposes of the present invention, the copolymers resulting from step (a) are obtained by means of copolymerization, where said polymerization is carried out by bonding or direct reaction between monomers, where said monomers comprise polymerizable groups responsible for the polymerization reaction that gives rise to the polymer.

For the purposes of the present invention, non-limiting examples of monomers with polymerizable groups are the vinyl, methacrylate, acrylate, methacrylamide and acrylamide.

For the purposes of the present invention, carrying out an immersion implies that the copolymer is completely immersed in the solution used.

In a preferred embodiment, the copolymer obtained in step (a) is obtained in the presence of a thermal or photochemical initiator.

The group R2 of the monomer Z used in step (a) of the method of the present invention is called, for the purposes of the present description, "anchor group R2", since said group is responsible for the subsequent bonding of the groups of formula (I), which act as fluorogenic sensor groups for anions, preferably chlorides.

For the purposes of the present invention, the term "anchor monomer" refers to the monomer Z used in step (a) of the method of the present invention, where said "anchor monomer Z" comprises an "anchor group R2" and a polymerizable group.

In a preferred embodiment, the anchor monomer Z is a monomer of formula (II):

wherein Ri is selected independently between H and CH3; and R2 is independently selected from the group consisting of aryl, cycloalkyl and alkyl, and said group R2 is also substituted with at least one halogen group

In a preferred embodiment the group R2 is an alkyl group substituted with at least one halogen.

In a more preferred embodiment, the monomer of formula (II) is 5-bromo-1 -pentene or 4- bromo-1-butene. In an even more preferred embodiment, the monomer of formula (II) is 5- bromo-1-pentene.

Similarly, for the purposes of the present invention, the copolymer obtained in step (a) is called anchor copolymer or anchor membrane, since it comprises R2 anchor groups, which enable, in accordance with step (b) of the process of the present invention, the subsequent bonding of rests of formula (I). Step (b) of the process of the present invention is therefore called, for the purposes of the present invention, a process of anchoring the groups of formula (I).

The process of anchoring the groups of formula (I) in step (b) of the process of the present invention, which occurs when a compound of formula (la) reacts with the anchor group R2 (by halogen nucleophilic substitution), forming a copolymer comprising a salt of a rest of formula (I), of the present invention, which can be represented in accordance with Diagram 1 below:

Hal

Diagram 1 wherein the halogen (Hal) is covalently linked to a carbon of an aromatic ring, non-aromatic ring or aliphatic chain of the anchor group R2 and, wherein one of the R3, R ₄ , R5, R6, R7, Rs or R9 groups is an OR10 and the rest of said groups R3, R ₄ , R5, R6, R7, Rs and R9 are each selected independently from H, OH, halogen, sulphonyl, N R11 R12, COOR10, CON R11 R12, alkyl, cycloalkyl, aryl, non-aromatic heterocyclic and hetaryl groups; wherein R10 is selected from alkyl, cycloalkyl, aryl, non-aromatic heterocyclic and hetaryl groups; and where Rn and R12 are each selected independently from H, alkyl, cycloalkyl, aryl, non-aromatic heterocyclic and hetaryl groups.

Said group R2 is independently selected from the group consisting of aryl, cycloalkyl or alkyl group, substituted with at least one halogen group (Hal), and forms part of an anchor copolymer obtained according to step (a) described previously. Said (Hal) halogen that substitutes the R2 group is selected independently from Fl, CI, Br and I.

In a preferred embodiment of the method of the invention, step (a) includes obtaining a copolymer with monomers Z and also with monomers X and Y, where the proportion of Z represents between 0.1 % and 10% of the total number of monomers, and wherein the proportion of X with respect to Y is between 1 :9 and 9:1. In a preferred embodiment, the proportion of X with respect to Y is 3:1 . In a preferred embodiment, Z represents 1 % of the total number of monomers.

In another preferred embodiment, X represents from 70% to 80%, Y represents from 19% to 29% and Z represents 1 % of the total number of monomers. In a preferred embodiment of the invention, the monomers X and Y used in step (a) are each selected independently from N-vinylpyrrolidone, methyl methacrylate, butyl acrylate, butyl methacrylate, methyl acrylate, styrene, 2-hydroxyethyl acrylate, 2-hydroxyethyl methacrylate, 2-ethoxyethyl methacrylate, lauryl acrylate, lauryl methacrylate, vinyl acetate, methacrylic acid, methacrylic anhydride, acrylic acid and 2-N,N-dimethylaminoethyl methacrylate. In a preferred embodiment, the monomers X and Y used in step (a) are each selected independently from N-vinylpyrrolidone, 2-hydroxyethyl acrylate, 2-hydroxyethyl methacrylate and methyl methacrylate.

In a preferred embodiment of the invention, one of the monomers used in step (a) is N- vinylpyrrolidone. In a preferred embodiment of the present invention, one of the monomers used in step (a) is 2-hydroxyethyl acrylate.

In a preferred embodiment of the method of present invention, step (a) includes obtaining a copolymer including monomers Z, and also including monomers X and Y, where the monomers X and Y are N-vinylpyrrolidone and 2-hydroxyethyl acrylate. In a more preferred embodiment, the Z monomer is 5-bromo-1 -pentene or 4-bromo-1 -butene. In an even more preferred embodiment, the Z monomer is 5-bromo-1 -pentene.

In a preferred embodiment of the invention, the polymerization described in step (a) is carried out in solution or block.

For the purposes of the present invention, the terms "block polymerization" or "mass polymerization" refers to a polymerization technique where only the monomers and the initiator are present in the reaction medium. In the event that the polymerization is carried out by thermal initiation without need for an initiator, only the monomers are present in the reaction medium. For the purposes of the present invention, the term "solution polymerization" refers to the polymerization technique wherein a solvent is present in the reaction medium in addition to the monomers and initiator.

In a preferred embodiment step (b) of the method of present invention, includes the compound of formula (la),

wherein one of the R3, R ₄ , R5, 6, R7, Rs or R9 groups is a methoxy group and the rest of said groups R3, R ₄ , R5, R6, R7, Rs and R9 are H. In another preferred embodiment, one of the R3, R ₄ , R5, R6, R7, Rs or R9 groups is a methoxy group and the rest of the groups R3, R ₄ , R5, R6, R ₇ , Rs and R9 is selected independently between H and CH3. In another preferred embodiment, one of the R3, R ₄ , R5, R6, R7, Rs or R ₉ groups is a methoxy group and the rest of the groups R3, R ₄ , R5, R6, R7, Rs and R9 are each selected independently between H and sulfonyl.

In another preferred embodiment, R ₅ is an OR10 group and the rest of the groups R3, R ₄ , R5, R ₆ , R7, Rs and Rg is H; where R10 is selected from alkyl, cycloalkyl, aryl, non-aromatic heterocyclic and hetaryl groups. More preferably, R10 is an alkyl group; even more preferably, R10 is selected from methyl, ethyl, propyl, isopropyl, butyl and tert-butyl. More preferably, R10 is methyl.

In another preferred embodiment, R5 is an OR10 group and the rest of the groups R3, R ₄ , R5, R6, R7, Rs and Rg are each selected independently between H and CH3; wherein R10 is selected from alkyl, cycloalkyl, aryl, non-aromatic heterocyclic and hetaryl groups. More preferably, R10 is an alkyl group; even more preferably, R10 is selected from methyl, ethyl, propyl, isopropyl, butyl and tert-butyl. More preferably, R10 is methyl.

In a more preferred embodiment, R5 is an OR10 group and the rest of the groups R3, R ₄ , R5, R6, R7, Rs and Rg are each selected independently between H and sulphonyl; where R10 is selected from alkyl, cycloalkyl, aryl, non-aromatic heterocyclic and hetaryl groups. More preferably, R10 is an alkyl group; even more preferably, R10 is selected from methyl, ethyl, propyl, isopropyl, butyl and tert-butyl. More preferably, R10 is methyl.

In a preferred embodiment of step (b) of the present invention, the compound of formula (la) is 6-methoxyquinoline. The synthesis of the copolymers comprising a salt of a group of formula (I), described herein, cannot be carried out by conventional methods in organic chemistry, i.e. (i) synthesising a monomer having a group of formula (I), derived from quinoline, and, (ii) performing a subsequent copolymerization with other monomers, as described in literature (S. Vallejos, A. Mufioz, S. Ibeas, F. Serna, F. Garcia, J. M. Garcia, J. Mater. Actuators, B, 201 1 , 157, 686- 690). Said conventional process is not possible due to the fact that species with formal charges, such as the ion structure of formula (I), inhibit radical polymerisation processes (A. A. Yassin, J. Polym. Sci., Part A: Polym. Chem., 1978, 16, 1475-1485; A. A. Yassin, N. A. Rizk, POLYMER, 1978, 19, 57-62) and therefore, it would be impossible to obtain the copolymers comprising a salt of a group of formula (I) of the invention by said conventional methods. However, upon firstly performing the synthesis of an anchor copolymer, in step (a) of the method of the present invention, the group of formula (I), with formal charges, is only formed once the polymerization is performed and the sensor copolymer can easily be obtained in accordance with step (b) of the method of the present invention. To this end, the use of an anchor monomer Z comprising a group R2, responsible for the reaction described in step (b) of the method of the present invention, is required.

In one embodiment of the invention, the polymerisation of step (a) of the method of the invention comprises a monomer Z which is a monomer of formula (II), a monomer X which is a monomer of formula (III) and a monomer Y which is a monomer of formula (IV):

wherein each Ri is selected independently between H and CH3, wherein R2 is independently selected from the group consisting of aryl, cycloalkyi or alkyl, substituted with at least one halogen group. In a preferred embodiment, the monomer of formula (II) represents between 0.1 % and 10% of the total number of monomers and the proportion of monomer of formula (III) with respect to that of monomer of formula (IV) is from 1 :9 to 9:1 . In a more preferred embodiment, the proportion of monomer of formula (III) with respect to that of monomer of formula (IV) is 3:1. In a more preferred embodiment, monomer of formula (II) represents 1 % of the total number of monomers. One embodiment of the method to obtain a copolymer comprising a salt of a group of formula (I), of the present invention, comprises:

(a) obtaining a copolymer through the polymerization of at least two monomers, wherein at least one of said two monomers is a monomer of formula (II) and also comprises monomers of formula (III) and monomers of formula (IV), described previously, and where said polymerization is carried out by direct reaction of polymerizable groups present in each of the monomers;

(b) reacting the copolymer obtained in step (a) with a compound of formula (la) described previously:

In a preferred embodiment, the compound of formula (la), is 6-methoxyquinoline. In a preferred embodiment, monomer (II) represents between 0.1 % and 10% of the total number of monomers and the proportion of monomer (III) with respect to monomer (IV) is 1 :9 to 9:1 . In a more preferred embodiment, the proportion of monomer (III) with respect to monomer (IV) is 3:1. In an even more preferred embodiment, monomer (II) represents 1 % of the total number of monomers.

In a preferred embodiment, the polymerization of step (a) of the method of the invention comprises a monomer Z which is 5-bromo-1-pentene, a monomer X which is N- vinylpyrrolidone and a monomer Y which is 2-hydroxyethyl acrylate:

In a preferred embodiment, 5-bromo-1-pentene represents between 0.1 % and 10% of the total number of monomers and the proportion of N-vinylpyrrolidone with respect to that of 2- hydroxyethyl acrylate is 1 :9 to 9:1 . One embodiment of the method to obtain a copolymer comprising a salt of a rest of formula (I), of the present invention, comprises:

(a) obtaining a copolymer through the polymerization of at least two monomers, where at least one of said two monomers is 5-bromo-1 -pentene and also comprises N- vinylpyrrolidone and 2-hydroxyethyl acrylate, and where said polymerization is carried out by direct reaction of polymerizable groups present in each of the monomers;

(b) reacting the copolymer obtained in step (a) with a compound of formula (la) described previously.

In a preferred embodiment, the compound of formula (la), is 6-methoxyquinoline. In a preferred embodiment, 5-bromo-1-pentene represents between 0.1 % and 10% of the total number of monomers and the proportion of N-vinylpyrrolidone with respect to that of 2- hydroxyethyl acrylate is from 1 :9 to 9:1 . In a more preferred embodiment, the proportion of N- vinylpyrrolidone with respect to that of 2-hydroxyethyl acrylate is 3:1 . In an even more preferred embodiment, 5-bromo-pentene represents 1 % of the total number of monomers. In general, the polymerization of the monomers of step (a) described herein, both comprising the use of commercial vinyl monomers or not, can be carried out by any of the methods described in literature for the polymerisation of multiple links.

The anchor monomers Z, according to present description, can be obtained commercially.

In a preferred embodiment of the present invention, the anchor monomer used in step (a) is 5- bromo-1-pentene, which can be obtained commercially.

The copolymers of the invention have a three-dimensional structure that enables permeation when placed in contact with or immersed in water or other solvents. This property is the basis for enabling the sensing and/or quantification of dissolved anions, such as for example chlorides. Said permeation can be controlled by varying the proportion between the different monomers used to prepare the copolymers.

Furthermore, the copolymers of the present invention, membranes, films, coatings and materials in solid state obtained therefrom, are characterised by an ideal combination of mechanical properties, both dry and expanded, i.e. with water within the polymer network. This converts the polymers of the present invention into adequate materials for preparing dense membranes which can be used, among other applications, for the detection and/or quantification of anions, such as chloride, in water and/or in aqueous media such as sweat. That is, the copolymers of the invention are fluorescent sensors for anions in water and/or in aqueous media such as sweat.

The change in fluorescence due to the presence of anions, and particularly chlorides, can be observed by immersing the membranes or copolymers comprising rests of formula (I), of the present invention, in the different media without any type of previous sample treatment. Therefore, the copolymers of the present invention can be used as sensors for the qualitative or quantitative sensing of the anions in question.

Therefore, one embodiment of the present invention relates to the use of a copolymer obtained in the method described herein, or of a copolymer comprising a salt of a rest of formula (I), for the sensing and/or quantification of anions, preferably for the sensing and/or quantification of chlorides.

One embodiment of the present invention relates to the use of a copolymer obtained using the method described herein comprising a salt of a rest of formula (I), for the sensing and/or quantification of anions using at least one method selected independently between: - detection with the naked eye; and

use of spectroscopic techniques based on fluorescence.

A more preferred embodiment relates to the detection and/or quantification of chlorides in industrial or human consumption waters. More preferably, the detection and/or quantification of chlorides is performed in sweat. Another embodiment relates to the use of a copolymer obtained using the method described herein, or of a copolymer comprising a salt of a group of formula (I), to detect a disease related to a high concentration of chlorides in sweat; preferably a disease selected independently from cystic fibrosis, untreated adrenal failure, ectodermal dysplasia, familial cholestasis syndrome, hypothyroidism, malnutrition, Mauriac syndrome, glycogen-storage disease type I, mucopolysaccharidosis, insipid nephrogenic diabetes, fucosidosis, nephrosis and glucose-6- phosphate deficiency. More preferably, the disease is cystic fibrosis.

One embodiment of the present invention relates to the use of a dressing comprising a copolymer obtained using the method described herein, or of a copolymer comprising a salt of a group of formula (I), to detect a disease related to a high concentration of chlorides in sweat; preferably a disease selected independently from cystic fibrosis, untreated adrenal failure, ectodermal dysplasia, familial cholestasis syndrome, hypothyroidism, malnutrition, Mauriac syndrome, glycogen-storage disease type I, mucopolysaccharidosis, insipid nephrogenic diabetes, fucosidosis, nephrosis and glucose-6-phosphate deficiency; more preferably for the detection of cystic fibrosis.

One embodiment of the invention also relates to the porous membranes obtained by means of chemical and/or physical foaming processes carried out using the previously described solid membranes. Examples of physical foaming processes include dissolved high-pressure gas (CO2 and/or N2) and chemical foaming processes include some non-limiting examples such as leaching using polymer salts or mixtures or the use of endo- or exothermal chemical foaming agents produced by the cell structure by heating and releasing the gas and, in general, any foaming process that gives rise to a porous structure inside the solid membrane.

DESCRIPTION OF THE FIGURES

Figure 1. Disappearance of the fluorescence band of a copolymer of the invention prepared according to Example 2 and immersed in Milli-Q water. Different quantities of NaCI were added and the effect analysed was the disappearance of a band in the fluorescence spectrum. Figure 1A shows the decrease in the fluorescent band centred at 440 nm, which is obtained upon exciting the sample at 321 nm, as the concentration of chlorides increases in the medium and; figure 1 B shows the ratio between the concentration mM of added chloride and the fluorescence intensity normalised as [1-(lntensity / Initial intensity)] of the aforementioned band at 440 nm, and figure 1 C shows the ratio between the logarithm of the concentration of added chloride and fluorescence intensity normalised as [1-(lntensity / Initial intensity)] of the aforementioned band at 440 nm. In this manner the fluorescence intensity values can be more easily observed for each of the assayed chloride concentrations.

Figure 2. Disappearance of the fluorescence band of a copolymer of the invention prepared according to Example 2 and immersed in a solution simulating human sweat (SS). Increasing amounts of NaCI/KCI were added (within a molar range 8.5/1.5), from the minimum concentration to the maximum possible concentration present in human sweat (between 6.17 mM and 100.16 mM). The graph shows the ratio between the concentration of added chloride and the fluorescence intensity normalised as [1 -(Intensity / Initial intensity)] at 440 nm, which is obtained upon exciting the sample at 321 nm.

Figure 3. Reusability of the copolymers of the invention. The graph shows the use and wash cycles, which translate into the decrease and increase, respectively, of the fluorescence band centred at 440 nm, and which is obtained upon exciting the sample at 321 nm. EXAMPLES

The following illustrative examples are not intended to be limiting and describe:

(1 ) Example 1 : Synthesis of an anchor copolymer comprising a vinyl-type anchor monomer Z in accordance with step (a) of the method of the present invention;

(2) Example 2: Synthesis of a sensor copolymer according to the method of the present invention departing from the anchor copolymer obtained in Example 1 ;

(3) Example 4: Fluorescent sensor-like behaviour of the sensor copolymer obtained in Example 2 in the presence of chloride in water;

(4) Example 5: Fluorescent sensor-like behaviour of the sensor copolymer obtained in Example 2 in the presence of chloride in a solution simulating human sweat (SS) prepared in

Example 3;

(5) Example 6: Reusability of the sensor copolymer obtained in Example 2.

Example 1 . Synthesis of an anchor copolymer in accordance with step (a) of the method of the present invention. A membrane having the composition indicated below was prepared by means of block copolymerization. Monomers: N-vinylpyrrolidone, 2-hydroxyethyl methacrylate and 5-bromo- 1-pentene, having a molar ratio of 74.25:24.75:1 , respectively. 2,2-Dimethoxy-2- phenylacetophenone as a photochemical initiator, with a percentage by weight of 1 %. The resulting copolymer solution was injected in a 100 μηη thick silanised crystal mould, in the absence of oxygen, and placed under a UV lamp for a whole night, obtaining said copolymer in the form of a membrane.

Example 2. Synthesis of a sensor copolymer comprising a salt of a group of formula (I) in accordance with the invention.

To prepare the sensor copolymer, a piece of the membrane prepared in Example 1 was immersed in a test tube with the necessary amount of 6-methoxyquinoline such that the piece of membrane was completely covered by the liquid reagent. The test tube was covered with a piece of parafilm and was kept thermostated at 70°C for 12 hours. Once this time had elapsed, the liquid reagent was transferred to a vial in order to be reused and the piece of membrane was washed repeatedly with acetone. Lastly, the membrane was immersed in distilled water, being ready for use. Example 3. Preparation of a solution simulating human sweat (SS).

In order to prepare a solution simulating human sweat, the corresponding quantities of the substances specified in Table 1 were weighed.

Table 1 includes a composition of an aqueous solution that simulates human sweat, which we will refer to with the abbreviation "SS". Concentration data of the 12 water-soluble components most abundant in sweat are shown, in addition to the corresponding quantities of salts required to prepare the solution and the resulting concentration of each in the mixture.

Table 1 Once weighed, the components were dissolved in distilled water and the solution was made up to a final volume of 1 L.

Example 4. Fluorescent sensor-like behaviour in the presence of chlorides in distilled water of the sensor copolymer of the invention obtained in Example 2. This example illustrates the fluorescent sensor-like behaviour of a copolymer comprising a salt of a group of formula (I), whose synthesis is illustrated in Example 2, in the presence of chloride anions in an aqueous medium. The immersion of the membrane prepared in Example 2 in distilled water gave rise to a fluorescence spectrum with a band from 350 to 600 nm, centred at 440 nm, upon exciting the sample at 321 nm. The addition of increasing amounts of sodium chloride between 1.25E-15 and 0.1 17 M/L caused the disappearance of this fluorescence band as observed in Figure 1A. Furthermore, Figure 1 B graphically shows the normalised fluorescence intensity values with respect to the concentration of chloride, which appear in Table 2. The normalised fluorescence intensity value is shown as 1 -(Measured intensity/Initial intensity) indicated as 1 -(l/l o) in Table 2 and in Figure 1 B:

Table 2

The detection limit obtained was 8.43- 10 ^"16 moles per litre and the limit of quantification was 2.55- 10 ^"15 moles per litre. The of detection limit (LOD) and quantification limit (LOQ) were estimated using the following equations: LOD= 3.3xSD/p and LOQ=10xSD/p, where SD is standard deviation and p is the slope of the calibration curve in a low-chloride-content zone.

Example 5. Fluorescent sensor-like behaviour of the sensor copolymer obtained in Example 2 in the presence of chlorides in a human sweat simulation (SS) prepared according to Example 3,

This example illustrates the fluorescent sensor-like behaviour of a copolymer comprising a salt of a group of formula (I), the synthesis of which is illustrated in Example 2, in the presence of chlorides in a medium simulating human sweat (SS), being the preparation of said solution illustrated in Example 3. The immersion of the membrane prepared in Example 2, in the solution prepared in Example 3, gave rise to a fluorescence spectrum with a band from 350 to 600 nm, centred at 440 nm, upon exciting the sample at 321 nm. The addition of increasing quantities of between 6.17 mM and 100 mM of sodium chloride and potassium chloride, within a molar range of 8.5/1 .5, respectively, caused the disappearance of this fluorescence band as observed in Figure 2, figure which reproduces the normalised fluorescence intensity values with respect to the concentration of chloride, appearing in Table 3. The normalised fluorescence intensity value is shown as 1-(Measured intensity/Initial intensity) indicated as 1- (l/lo) in Table 3 and in Figure 2:

Table 3

Example 6. Reusability of a copolymer comprising a salt of a group of formula (I) for detecting chlorides.

The behaviour of the membrane prepared in Example 2 was studied over various cycles of use as a chloride sensor in water with a subsequent wash.

A dense membrane prepared according to Example 2 was die-cut in forms of 10 mm diameter discs and immersed in 2 ml of distilled water. NaCI was added up to a concentration of 100 mM, and the effect on the disappearance of a band in the fluorescence spectrum was measured. Subsequently, the membrane was washed with distilled water and, upon removing the chlorides from the system, the initial fluorescence of the material was recovered. This use wash cycle was repeated six times. Figure 3 shows the use wash cycles, which translate into the decrease and increase, respectively, of the fluorescence band centred at 440 nm, and which is obtained upon exciting the sample at 321 nm.