Polymer matrix for CO2 sensing

Abstract

The present invention relates to a polymer matrix for optically sensing carbon dioxide and a method for manufacturing the polymer matrix. Also discussed are optical sensors for optically sensing carbon dioxide comprising the polymer matrix of the present invention and methods for manufacturing the optical sensors. The present invention further provides a method for measuring the carbon dioxide content of a sample using the sensor of the present invention. The optical sensor is useful for measuring the carbon dioxide content of a biological sample, such as blood.

Description

[Technical Field]

[0001] Technical field to which the invention belongs The present invention provides a polymer matrix for the optical detection of CO2 and a method for making the polymer matrix. The present invention also provides an optical sensor for the optical detection of CO2 comprising the polymer matrix of the present invention and a method for making the optical sensor. The present invention further provides a method for measuring the CO2 content of a sample using the sensor of the present invention. [Background technology]

[0002] Background of the Invention Detecting the presence or precise concentration of carbon dioxide in aqueous samples is important in many fields. Accurately measuring the pH of aqueous samples is also useful. When CO2 dissolves in an aqueous solution, some of it reacts with water to form carbonic acid (H2CO3), an acidic species, and these quantities can frequently be assessed in the same way. The presence of dissolved carbonic acid affects the pH of the solution. Therefore, measuring the pH of a solution not only provides the pH but also essentially the amount of dissolved CO2 (corrected for any other acidic or basic species present). Therefore, although pH measurements are affected by the amount of dissolved CO2, they do not provide a measure of CO2 unless corrected for the presence of other acidic or basic species. However, correcting for the presence of other acidic or basic species is very difficult, or even impossible, in practice. Therefore, specifically detecting CO2 in a sample is desirable. The detection of CO2 in aqueous solutions is most commonly performed with a Severinghaus electrode system. The Severinghaus electrode detects bicarbonate ions (HCO3 - The buffer solution is separated from the sample by a gas-permeable membrane. CO2 diffuses into the buffer, equilibrating with bicarbonate ions and changing the pH of the buffer. A pH electrode monitors the pH of the buffer, so changes in pH indicate changes in the amount of CO2 present in the sample. This system is effective for detecting CO2 in procedures such as fermentation. However, this electrode-based system has a major drawback when it comes to monitoring CO2 in biological systems such as blood. To obtain an accurate measurement, the Severinghaus electrode requires the sample to be equilibrated with the internal bicarbonate buffer. This requires time, typically 5 to 15 minutes. However, in biological systems such as blood, sudden changes in CO2 content can have catastrophic consequences for patients. Therefore, it is desirable to provide a CO2 detection system with a faster response time. Furthermore, it is desirable to provide a CO2 detection system that can continuously monitor samples and provide real-time information.

[0003] Another drawback of the Severinghaus electrode in blood gas sensing applications is that if the membrane separating the blood sample from the bicarbonate buffer is ruptured, bicarbonate can be released into the blood. Therefore, the Severinghaus electrode is not suitable for measuring the CO2 content of blood being returned to the patient. Previous researchers have attempted to provide sensors that overcome some of the above difficulties. Ge et al. have provided a so-called "naked optical CO2 sensor" (Ge et al., Biosensors & Bioelectronics, 18 (2003), pp857-865). This device utilizes the fluorescent dye pyranine, which has a pH-dependent fluorescence emission profile. Protonated pyranine has an emission peak at a different wavelength than deprotonated pyranine. Therefore, the ratio of the emission intensities at the two wavelengths can be used to determine the relative concentrations of the two forms of pyranine and, therefore, the CO2 content in the system.

[0004] To avoid the need for a bicarbonate buffer, the fluorescent dye is suspended in a polymer support along with a phase transfer agent. The phase transfer agent is usually a quaternary ammonium species that deprotonates the fluorescent dye, helping it to dissolve. CO2 diffuses into the polymer support and, together with the trapped water, generates carbonic acid, changing the pH within the polymer support and, consequently, the ratio of deprotonated to protonated fluorescent dye. However, fabricating this optical sensor poses considerable challenges. The sensing region must be created by preparing a polymer support and suspending the fluorescent dye and phase transfer agent within it. This suspending is achieved by providing a solution containing the fluorescent dye and phase transfer agent. The polymer support is saturated with this solution and then dried. However, the fluorescent dye, polymer, and phase transfer agent typically have very different solubilities. Therefore, it is difficult to provide a solution containing the fluorescent dye and phase transfer agent at the desired concentrations, and even more difficult to provide a solution that will penetrate the polymer support. Not only is it difficult to combine these necessary components, but it is also difficult to provide a polymer support / dye / phase transfer agent combination that can retain sufficient water to mediate a pH change in the presence of CO2. This can result in significant uncertainty in the amount of water, leading to poor uniformity between sensors and significant uncertainty in the concentrations of the dissolved dye and phase transfer agent. This makes it difficult to accurately determine the CO2 concentration. Careful selection of the polymer support, dye, and phase transfer agent is also required (Chu et al., Photonic Sensors, (2011) vol. 1, no. 3, pp. 234–250). The choice of polymer support is sometimes governed by solubility considerations rather than sensing suitability (e.g., gas permeability, ion impermeability, and other related properties). Furthermore, optimizing the amounts of fluorescent dye, phase transfer agent, and water is usually not possible. Therefore, there is a clear need to provide a means for accurate CO2 sensing that has a fast response time, can be used for continuous measurements, and overcomes the manufacturing difficulties mentioned above. Summary of the Invention

[0005] Summary of the Invention The present inventors have recognized that positively charged counterion moieties can be used to form ion pairs with fluorophores. These ion pairs can be disrupted by acid. Thus, the present inventors have recognized that prior art "phase transfer agents" can play an important role in optical CO2 and pH sensing, and as a result, have realized that the relative amounts of fluorophores and counterion moieties in a system are important and can be used to improve the accuracy of ion-pair and fluorophore-based optical sensors. Furthermore, the inventors have noted that the difficulty of providing an optical sensor containing the desired amount of both fluorophore and counterion moiety can be reduced by covalently attaching either the fluorophore or the counterion moiety to a polymer matrix. When either the fluorophore or the counterion moiety is attached to a polymer matrix, only one of the fluorophore and the counterion moiety needs to be provided within the matrix by other means (e.g., by being retained in the matrix). Thus, the need for the difficult task of providing a solution containing both the fluorophore and the oppositely charged "phase transfer moiety" at the desired concentrations is completely eliminated. One component may be retained in the polymer matrix, which is significantly easier than retaining two components with different solubilities. Therefore, as shown below, the relative amounts of fluorophore and counterion moiety can be more precisely controlled, resulting in a more accurate sensor. Thus, described herein is a polymer matrix for optical sensing of CO comprising a fluorophore, a positively charged counterion moiety, and a polymer support, where either the fluorophore or the counterion moiety is covalently attached to the polymer support by a polymer linkage moiety. Fluorophores or counterion moieties that are not covalently attached to the polymeric support may be provided within the polymeric support by any means, and typically, fluorophores or counterion moieties that are not covalently attached to the polymeric support by a polymer linkage moiety are retained within the polymeric support. Because the polymer matrix is ​​intended for CO2 sensing, it is generally configured to prevent contact of acids and bases (other than carbonic acid produced in the presence of CO2) with the fluorophore. For example, the polymer matrix may be a hydrophobic polymer or may include or consist of a hydrophobic polymer. Thus, the polymer matrix may be hydrophobic. This prevents aqueous acids from penetrating the matrix. Alternatively or additionally, the polymer matrix may be protected by a membrane that is gas-permeable but impermeable to liquids and hydrogen ions. This prevents other acids and bases from contacting the fluorophore. By "protected," we mean that the membrane is positioned to prevent liquids and hydrogen ions from entering the polymer matrix. It is particularly preferred that the polymer matrix be protected by the aforementioned gas-permeable membrane. This has the added benefit of maintaining a constant water concentration within the polymer matrix, improving accuracy in sensing applications.

[0006] The polymer matrix of the present invention can be prepared by any means. However, it is particularly convenient to provide a polymer support to which either a fluorophore or a counterion moiety is covalently attached, and then retain the other component (i.e., neither the fluorophore nor the counterion moiety is covalently attached to the polymer support) in the polymer support. The process of retaining either a fluorophore or a counterion moiety in the polymer support is significantly easier than prior art methods that require retaining both a fluorophore and an oppositely charged "phase transfer agent" in the polymer support, and provides a more accurate indication of the concentration of the species retained in the polymer support. Thus, a method for preparing a polymer matrix for optical sensing of CO2 is described herein, which method includes: (i) providing a polymeric support, wherein either the fluorophore or the counterion moiety is covalently attached to the polymeric support by a polymer linking moiety; and (ii) Retaining in the polymeric support either the fluorophore or the counterion moiety that is not covalently attached to the polymeric support via a polymer linkage moiety.

[0007] The polymer matrix of the present invention can be used to fabricate optical sensors with improved accuracy and improved manufacturability. Accordingly, an optical sensor for the optical detection of CO2 is also described, which comprises: a sensing region comprising a polymer matrix as described herein; and an optical waveguide arranged to guide light to said sensing region;

[0008] In a particularly preferred embodiment of the present invention, there is provided an optical sensor for the optical detection of CO2, comprising: a sensing region comprising a polymer matrix as described herein, wherein the polymer support comprises a hydrophilic polymer, and the sensing region further comprises a gas-permeable hydrophobic membrane that is impermeable to liquids and hydrogen ions, the sensor being configured to allow CO2 to enter the sensing region through the hydrophobic membrane; and an optical waveguide arranged to guide light to said sensing region;

[0009] This sensor configuration is highly desirable because the hydrophobic membrane prevents liquid water (and any acid or alkaline species therein) in an aqueous sample from entering the sensing region of the sensor. However, the hydrophobic membrane (which is generally gas-permeable but liquid-impermeable) allows gaseous CO2 in the sample to diffuse into the sensing region. Therefore, this sensor specifically detects CO2. The polymer matrix may be composed of a hydrophobic polymer or, alternatively, a hydrophobic polymer or polymers. When the polymer matrix is ​​composed of a hydrophobic polymer, the likelihood of interference from acids or bases is significantly reduced; however, care must be taken (when using the sensor) to ensure that some water is present in the polymer matrix so that CO2 can form carbonic acid. The polymer matrix of the present invention can be used to fabricate an optical sensor.

[0010] Thus, described herein is a method of making an optical sensor for the optical detection of CO, the method comprising: (i) providing an optical waveguide; (ii) disposing a polymer matrix as described herein over the optical waveguide; In a particularly advantageous embodiment, the method for fabricating an optical sensor further comprises disposing a hydrophobic membrane on the polymer matrix, which ensures that the sensor contains a known and consistent amount of water, and can be used for the specific detection of CO2, as described below.

[0011] The optical sensor of the present invention can be used to detect and / or quantify the amount of CO in a sample. Accordingly, the present invention provides a method for measuring the CO content of a sample, the method comprising: (i) contacting an optical sensor described herein with a sample; (ii) providing excitation light to the sensing region through an optical waveguide; and (iii) detecting the intensity I1 of light emitted from the fluorophore at a first wavelength λ1 through the optical waveguide; [Brief explanation of the drawings]

[0012] Brief description of the drawings [Figure 1] FIG. 1 is a schematic diagram of the sensing chemistry underlying the present invention, illustrated using 8-hydroxytrisodium 1,3,6-pyrenetrisulfonate ("HTPS") as the fluorophore and cetyltrimethylammonium hydroxide ("CTMAOH+") as the positively charged counterion moiety. [Figure 2] Figure 2 contains two graphs showing the emission spectra of the fluorophore HTPS under various conditions. Graph A shows the emission spectrum of HTPS when CTMAOH+ is present at a 50:1 molar ratio relative to HTPS, and graph B shows the emission spectrum of HTPS when CTMAOH+ is present at a 25:1 molar ratio relative to HTPS. In both cases, the spectrum in the presence of 0% CO2 (blue line; high intensity at 470.0 nm) and 100% CO2 (red line; low intensity at 470.0 nm) are shown. [Figure 3] FIG. 3 is a perspective view of the optical sensor. [Figure 4] FIG. 4 is a binding curve showing the binding of CO2 to the polymer matrix in a sensor of the present invention as a function of pCO2. [Figure 5] FIG. 5 shows the actual pCO2 and pCO2 values ​​measured by a sensor according to the invention over a 5-hour period. DETAILED DESCRIPTION OF THE INVENTION

[0013] Detailed Description of the Invention Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the relevant art. The present invention will now be described with reference to particular embodiments and drawings, but it is not intended that the present invention be limited to the particular embodiments or aspects of the following description. It should be noted that the following description of various aspects of the invention is applicable to each of the different embodiments of the invention, for example, the description of fluorophores relates to fluorophores present in the polymer matrices of the invention, and to fluorophores present in the optical sensors of the invention, as well as to fluorophores present in the various processes described herein. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein which equivalents are intended to be encompassed by the following claims.

[0014] definition As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the content clearly dictates otherwise. Thus, for example, reference to a "counterion moiety" includes two or more counterion moieties. In the present specification and claims, where the term "comprising" is used, it does not exclude other elements or steps. Furthermore, the terms first, second, third, etc. in the present specification and claims are used to distinguish between similar elements and not necessarily to describe a sequential or chronological order. The terms so used are interchangeable under appropriate circumstances, and it will be understood that the embodiments of the invention described herein are capable of operating in orders other than those described or illustrated herein. As used herein, "about" when referring to a measurable value such as an amount, temporal duration, etc. is intended to encompass a variation of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and even more preferably ±0.1% from the stated value, as appropriate for carrying out the disclosed methods. As used herein, "alkyl" refers to monovalent straight-chain and branched alkyl groups. Typically, the alkyl group is a straight-chain alkyl group. The alkyl group may have 1 to 30 carbon atoms (i.e., C 1-30 Typically, the alkyl group is a C 1-20 Alkyl group or C 1-10 Preferred alkyl groups include C 1-6 Alkyl groups, such as C 1-4 The alkyl group includes, for example, a methyl group, an ethyl group, a straight-chain or branched propyl group, a butyl group, and a pentyl group. Particularly, the alkyl group includes a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, a sec-butyl group, an isobutyl group, and a tert-butyl group. As used herein, "cycloalkyl" refers to a monovalent group derived from a saturated monocyclic hydrocarbon. A cycloalkyl group can have, for example, 3 to 12 carbon atoms, but "cycloalkyl" typically refers to a C 3-10 Refers to a cycloalkyl group. 3-7 Cycloalkyl groups are particularly preferred. Exemplary cycloalkyl groups include cyclopentyl and cyclohexyl groups. As used herein, "alkenyl" refers to a monovalent hydrocarbon moiety containing one or more carbon-carbon double bonds. Typically, an alkenyl group contains one carbon-carbon double bond. The hydrocarbon moiety can be straight-chain or branched; typically, the hydrocarbon moiety is straight-chain. An alkenyl group can have 2 to 30 carbon atoms (i.e., C 2-30 Typically, the alkenyl group is a C 2-20 , or C 2-10Alkenyl groups are preferred. 2-6 Alkenyl groups, such as C 2-4 Alkenyl groups are included. As used herein, "cycloalkenyl" refers to a non-aromatic monovalent group derived from a monocyclic hydrocarbon containing one or more carbon-carbon double bonds. Typically, a cycloalkenyl group contains one carbon-carbon double bond. A cycloalkenyl group may have 3 to 12 carbon atoms. "Cycloalkenyl" typically refers to a C 4-10 Refers to a cycloalkenyl group. 5-7 Cycloalkenyl groups are particularly preferred. As used herein, "alkynyl" refers to a monovalent hydrocarbon moiety containing one or more carbon-carbon triple bonds. Typically, an alkynyl group contains one carbon-carbon triple bond. The hydrocarbon moiety can be straight-chain or branched; typically, the hydrocarbon moiety is straight-chain. An alkynyl group can have from 2 to 30 carbon atoms (i.e., C 2-30 Typically, the alkynyl group is a C 2-20 , or C 2-10 Alkynyl groups are preferred. 2-6 Alkynyl groups, such as C 2-4 Alkynyl groups are included.

[0015] As used herein, "aryl" refers to a monovalent unsaturated aromatic carbocyclic group that may be monocyclic (e.g., a phenyl group) or polycyclic having multiple condensed rings (e.g., a naphthyl group). Aryl groups typically contain 6 to 14 carbon atoms. A preferred aryl group is phenyl. As used herein, "alkylaryl" refers to a monovalent group that includes alkylene and aryl species as described herein. The number of carbon atoms in the alkyl portion may be indicated by a prefix; for example, C 1-10 The alkylaryl moiety is C 1-10 It includes alkylene and aryl groups. The number of carbon atoms in the aryl moiety can also be expressed; for example, C 1-10 Alkyl C 6-14 The aryl species is C 1-10Alkylene and C 6-14 A preferred alkylaryl moiety is benzyl. As used herein, "heteroaryl" refers to a monovalent aromatic carbocyclic group having at least one heteroatom selected from oxygen, sulfur, and nitrogen. Heteroaryl groups typically contain one, two, or three heteroatoms independently selected from oxygen, sulfur, and nitrogen, and typically contain one such heteroatom. Heteroaryl groups may be monocyclic or polycyclic. Heteroaryl groups typically contain 5 to 14 carbon atoms. As used herein, "heterocycloalkyl" refers to a monovalent saturated monocyclic ring containing at least one heteroatom selected from oxygen, sulfur, and nitrogen. Heterocycloalkyl groups typically contain one, two, or three heteroatoms independently selected from oxygen, sulfur, and nitrogen, and typically contain one such heteroatom. Heterocycloalkyl rings may be monocyclic (e.g., piperidinyl) or polycyclic (e.g., decahydroquinoline). Heterocycloalkyl groups typically contain 5 to 14 carbon atoms. As used herein, "alkylene" refers to a divalent saturated hydrocarbon moiety that may be straight-chained or branched. Typically, an alkylene group is a straight-chain alkylene group. An alkylene group typically has 1 to 10 carbon atoms (i.e., C 1-10 However, preferred alkylene groups are C 1-6 Alkylene groups, such as C 1-4 Examples of alkyl groups include methylene (-CH2-) and ethylene (-CH2CH2-) groups. As used herein, "alkenylene" refers to a divalent hydrocarbon moiety containing one or more carbon-carbon double bonds. Typically, an alkenylene group contains one carbon-carbon double bond. The hydrocarbon moiety can be straight-chain or branched, and typically, the hydrocarbon moiety is straight-chain. An alkenylene group typically has 2 to 10 carbon atoms (i.e., C 2-10However, preferred alkenylene groups are C 2-6 Alkenylene groups, such as C 2-4 Contains alkenylene groups.

[0016] As used herein, "alkynylene" refers to a divalent hydrocarbon moiety containing one or more carbon-carbon triple bonds. Typically, an alkynylene group contains one carbon-carbon triple bond. The hydrocarbon moiety can be straight-chain or branched, and typically, the hydrocarbon moiety is straight-chain. An alkynylene group typically has 2 to 10 carbon atoms (i.e., C 2-10 However, preferred alkynylene groups are C 2-6 Alkynylene groups, such as C 2-4 Contains alkynylene groups. As used herein, the term "halogen" is intended to include fluorine, chlorine, bromine and iodine atoms, typically fluorine, chlorine or bromine. The term "oxo" refers to the group =0. The term "hydroxy" refers to the group -OH. Reference herein to a group in its protonated form should be considered to include a reference to any deprotonated forms that may be produced in solution. For example, a reference to "hydroxy" or "-COOH" will produce -O in solution. - or -COO - The term "aromatic group" should be considered to encompass such groups when deprotonated.

[0017] Sensing Chemistry The sensing chemistry employed in the present invention is shown in Figure 1. The polymer matrix of the present invention contains a fluorophore that is negatively charged when deprotonated and a counterion site that is positively charged. In Figure 1, deprotonated HTPS (hydroxypyrenetrisulfonic acid) and the quaternary ammonium cation CTMAOH are shown. + (cetyltrimethylammonium). These two oppositely charged ionic species interact to form an ion pair. When CO2 enters the sensing environment, it interacts with the water trapped in the polymer matrix. The reaction of H2O with CO2 produces carbonic acid, H2CO3. The carbonic acid interacts with the ion pair and protonates the fluorophore, thereby forming the bicarbonate anion, HCO3 - This leaves a residue that interacts with a positively charged counterion site to form a new ion pair. For example, in Figure 1, the deprotonated HTPS becomes protonated upon interaction with carbonic acid, forming HCO3 - and CTMAOH + A new ion pair containing This interaction changes the emission and / or absorption spectra of the system. A deprotonated fluorophore has a different fluorescence emission spectrum than a protonated fluorophore. Similarly, a deprotonated fluorophore has a different absorption spectrum than a protonated fluorophore. For example, deprotonated HTPS absorbs and emits most strongly around 465 nm, while protonated HTPS absorbs and emits most strongly around 405 nm. Thus, the polymer matrices of the present invention are useful in sensing methods for optical detection of CO2, where the sensing method involves disrupting the ion pair involving the fluorophore and the counterion moiety by protonation of the negatively charged moiety of the fluorophore. Therefore, monitoring the absorption or emission spectrum of the polymer matrix shows that as the CO2 concentration in the polymer matrix increases, the absorption or emission intensity of the spectral peak corresponding to the protonated form of the fluorophore increases. Correspondingly, the absorption or emission intensity of the spectral peak corresponding to the deprotonated form of the fluorophore decreases. Similarly, as the CO2 concentration in the polymer matrix decreases, the absorption or emission intensity of the spectral peak corresponding to the deprotonated form of the fluorophore is observed to increase. pH sensing relies on the deprotonation / protonation of the fluorophore. Therefore, pH sensing methods and applications do not require the presence of a positively charged counterion site. Therefore, although the polymer matrix of the present invention can be used for pH detection (if the acid is allowed to enter the matrix), the polymer matrix is ​​particularly suitable for CO2 detection. Therefore, optical sensors for CO2 detection and methods for detecting or quantifying CO2 content in a sample are preferred.

[0018] Note that in some cases, either the protonated or deprotonated form of a fluorophore may have a very weak absorption or emission spectrum. In such cases, you may not observe an increase or decrease in the absorption or emission intensity of the spectral peak corresponding to that form of the fluorophore in response to changes in its concentration. Rather, you may only observe an increase or decrease in the absorption or emission intensity of the spectral peak corresponding to the other form of the fluorophore. Positively charged counterion sites are generally used in excess. The more positively charged counterion sites there are, the greater the likelihood that each fluorophore site will form an ion pair with a counterion site in the absence of CO2. This increases the spectral change that occurs when CO2 enters the polymer matrix. This is because the fluorophore that becomes protonated (and the counterion site that binds to the carbonate ion) is more likely to have previously been part of an ion pair containing the fluorophore and the counterion. If the fluorophore does not form an ion pair with a counterion site in the absence of CO2, it is likely to remain protonated, and no protonation effect is observed when CO2 is added to the system. This is shown in Figure 2. Graph A in Figure 2 shows the + graph B is the emission spectrum of HTPS when CTMAOH is present in a molar ratio of 50:1 to HTPS; graph C is the emission spectrum of CTMAOH +Figure 1 shows the emission spectra of HTPS when present at a molar ratio of 25:1 to HTPS. In both cases, the spectrum in the presence of 0% CO2 (blue line; high intensity at 470.0 nm) and 100% CO2 (red line; low intensity at 470.0 nm) is shown. + It can be clearly seen that when the ratio of HTPS to CO2 is larger (50:1 compared to 25:1), a larger intensity change is observed upon CO2 addition. A larger intensity change indicates a higher sensitivity and better performance of the sensor. Therefore, the polymer matrix of the present invention is particularly advantageous because a large amount of counterion moieties can be easily incorporated into the polymer support, ensuring an excess of counterion moieties. Thus, in a preferred embodiment, the counterion moieties are covalently bound to the polymer support. It is also preferred that the counterion moieties are present in molar excess compared to the fluorophore. In a particularly preferred embodiment, the counterion moieties are covalently bound to the polymer support and present in molar excess compared to the fluorophore. For example, the polymer matrix may contain up to about 25% by weight of counterion sites, typically up to about 20% by weight, and preferably up to about 10% by weight. Thus, the polymer matrix may contain about 1-25% by weight of counterion sites, typically about 2-20% by weight, and preferably about 5-10% by weight. When counterion sites are incorporated into the polymer support, the polymer support may contain up to about 25% by weight of counterion sites, typically up to about 20% by weight, and preferably up to about 10% by weight. Thus, the polymer support may contain about 1-25% by weight of counterion sites, typically about 2-20% by weight, and preferably about 5-10% by weight.

[0019] In contrast, the polymer matrix of the present invention may contain up to about 10% by weight of fluorophore, typically up to about 7% by weight of fluorophore, and preferably up to about 5% by weight of fluorophore. Thus, the polymer matrix may contain about 0.1-10% by weight of fluorophore, preferably about 1-5% by weight of fluorophore, for example, about 2, 3, or 4% by weight of fluorophore. When fluorophores are incorporated into the polymer support, the polymer support may contain up to about 10% by weight of fluorophore, typically up to about 5% by weight of fluorophore. For example, the polymer support may contain about 0.1-10% by weight of fluorophore, preferably about 1-5% by weight of fluorophore, for example, about 2, 3, or 4% by weight of fluorophore. The molar ratio of counterion to fluorophore can be 2:1 or greater. Typically, the molar ratio of counterion to fluorophore is at least 20:1. Preferably, the molar ratio of counterion to fluorophore is at least 30:1, e.g., at least 40:1 or at least 50:1. Generally, the molar ratio of counterion to fluorophore is 20:1 to 1000:1, preferably 30:1 to 500:1 or 40:1 to 200:1. By way of example, the molar ratio of counterion to fluorophore can be about 50:1 or about 100:1.

[0020] Fluorophores I. Fluorescence and absorption properties A fluorophore is a moiety that can absorb light and emit fluorescence. Typically, the fluorophores described herein absorb light in the visible region of the electromagnetic spectrum. The fluorophores also typically emit light in the visible region of the electromagnetic spectrum. "Visible region of the electromagnetic spectrum" refers to electromagnetic radiation having wavelengths from about 400 nm to about 700 nm. Fluorophores may also absorb and / or emit radiation outside the visible region of the electromagnetic spectrum. Fluorophores can exist in deprotonated and protonated states. Protonation of a fluorophore affects its electronic structure. As a result, the protonated and deprotonated forms of a fluorophore typically absorb and emit light at different wavelengths. The protonated and deprotonated forms have different characteristic emission and absorption spectra. Therefore, measuring the absorption or emission spectrum of a fluorophore will indicate whether it is protonated, deprotonated, or a mixture of protonated and deprotonated forms. Of course, if a fluorophore has multiple different protonated and deprotonated forms (e.g., if the fluorophore has two or more labile protons), measuring the absorption or emission spectrum of the fluorophore can indicate which of the multiple different protonated and deprotonated forms are present. Thus, references to "a protonated form of a fluorophore" and "a deprotonated form of a fluorophore" should be understood to refer to "one of the protonated forms of the fluorophore" and "one of the deprotonated forms of the fluorophore" when the fluorophore has multiple protonated and deprotonated forms. Therefore, a fluorophore usually has at least one protonated form and at least one deprotonated form, and the fluorescence emission spectrum of the protonated form is different from the fluorescence emission spectrum of the deprotonated form.For example, the fluorescence emission spectrum of the protonated form may have a peak emission wavelength of λ1, and the fluorescence emission spectrum of the deprotonated form may have a peak emission wavelength of λ2, and λ1 is different from λ2.Usually, λ1 is different from λ2 by at least 5 nm, preferably λ1 is different from λ2 by at least 10 nm or at least 20 nm.The greater the difference between λ1 and λ2, the easier it is to distinguish the two peaks of the fluorescence emission spectrum. Similarly, in another preferred embodiment, the absorption spectrum of the protonated form is different from the absorption spectrum of the deprotonated form of the fluorophore.For example, the absorption spectrum of the protonated form may have an absorption peak wavelength of λ3, and the absorption spectrum of the deprotonated form may have an absorption peak wavelength of λ4, where λ3 is different from λ4.Typically, λ3 is different from λ4 by at least 5 nm, preferably λ3 is different from λ4 by at least 10 nm or at least 20 nm.The greater the difference between λ3 and λ4, the easier it is to distinguish the two peaks in the absorption spectrum.

[0021] Thus, the fluorophore has a pH-dependent absorption spectrum and / or a pH-dependent fluorescence emission spectrum. By "pH-dependent absorption spectrum" we mean that the absorption spectrum contains (a) an absorption peak corresponding to the absorption of the protonated form of the fluorophore; and / or (b) an absorption peak corresponding to the absorption of the deprotonated form of the fluorophore. By "pH-dependent fluorescence emission spectrum" we mean that the fluorescence emission spectrum contains (a) an emission peak corresponding to the emission of the protonated form of the fluorophore; and / or (b) an emission peak corresponding to the emission of the deprotonated form of the fluorophore. Preferably, the fluorophore has a pH-dependent absorption spectrum or a pH-dependent fluorescence emission spectrum that contains both (a) and (b), thereby allowing ratiometric measurement of the two forms. Increasing the concentration of acid (i.e., carbonate) in the polymer matrix increases the proportion of fluorophores that are in the protonated form, resulting in an increase in the intensity of the peaks in the absorption or emission spectra associated with the protonated fluorophores. Thus, the emission spectrum of a fluorophore typically includes (a) an emission peak corresponding to the fluorescence emission of the protonated form of the fluorophore, the intensity of which correlates with the concentration of the protonated fluorophore in the polymer matrix. Similarly, the emission spectrum of a fluorophore typically includes (b) an emission peak corresponding to the fluorescence emission of the deprotonated form of the fluorophore, the intensity of which correlates with the concentration of the deprotonated form of the fluorophore in the polymer matrix. Preferably, the emission spectrum includes (a) and (b). Similarly, the absorption spectrum of a fluorophore typically includes (a) an absorption peak corresponding to the absorption of the protonated form of the fluorophore, the intensity of which correlates with the concentration of the protonated fluorophore in the polymer matrix. Similarly, the absorption spectrum of a fluorophore typically includes (b) an absorption peak corresponding to the absorption of the deprotonated form of the fluorophore, the intensity of which correlates with the concentration of the deprotonated form of the fluorophore in the polymer matrix. Preferably, the absorption spectrum includes (a) and (b). When the intensity of an absorption or emission peak correlates with the concentration of the protonated or deprotonated form of a fluorophore, the concentration of that species (and thus the CO2 content of the system) can be calculated using only the intensity of that peak. However, a more accurate measurement is possible using a ratiometric approach. This is possible when the absorption or emission spectrum consists of two peaks corresponding to the protonated and deprotonated forms of the fluorophore. By calculating the intensity ratio of these two peaks and calculating the CO2 content of the system, calculation errors due to photobleaching of the fluorophore can be eliminated. Thus, in a preferred embodiment, the fluorophore is capable of undergoing fluorescence emission at a first wavelength λ1 with an intensity I1 and at a second wavelength λ2 with an intensity I2, the ratio of I1 to I2 varying depending on the pH. Similarly, in a preferred embodiment, the fluorophore is capable of absorbing light at a first wavelength λ3 with an intensity I3 and at a second wavelength λ4 with an intensity I4, the ratio of I3 to I4 varying depending on the pH. Generally, the fluorescence emission at λ1 is that of the protonated form of the fluorophore, and the fluorescence emission at λ2 is that of the deprotonated form of the fluorophore. Similarly, the absorption at λ3 is that of the protonated fluorophore, and the absorption at λ4 is that of the deprotonated fluorophore. These values ​​are merely labels, and of course the reverse is also possible.

[0022] II. Protonated, deprotonated, and salt forms of fluorophores Fluorophores have protonated and deprotonated forms and can gain or lose one or more protons upon interaction with water or water vapor. Systems containing HO and fluorophores tend toward an equilibrium in which the deprotonated and protonated forms of the fluorophore exist. The extent to which these forms exist (i.e., the position of the equilibrium) depends on the pH of the system and the pKa of the fluorophore moiety. If the system is strongly acidic, generally all fluorophores will be protonated; if the system is strongly alkaline, generally all fluorophores will be deprotonated. In biological systems, the pH is generally neither strongly acidic nor strongly alkaline. Fluorophores can be chosen with pKas appropriate for their application. Detection is most effective when the pKa of a fluorophore is the same as the pH of the environment in which it is used. If the pKa of a fluorophore is significantly different from the pH of the environment in which it is used, nearly all of the fluorophore will remain protonated or deprotonated as the pH of the environment changes. Therefore, the change in the absorption and / or emission properties of the system will be small, making it difficult to detect the difference using optical techniques. However, if the pKa of a fluorophore is close to the pH of the environment, a small change in the pH of the environment can cause a large change in the relative concentrations of the protonated and deprotonated forms of the fluorophore. Therefore, the change in the absorption and / or emission properties of the system will be larger, making it easier to detect via the system's absorption or emission spectrum. The polymer matrix is ​​useful for detecting carbonic acid (and thus CO2), which is a weak acid. Thus, typically, the fluorophore has a pKa of about 3.5 to 10. In preferred embodiments, the fluorophore has a pKa of about 4 to 9, e.g., 5 to 8 or 6 to 8. In some embodiments, the fluorophore comprises a weak acid or a salt of a weak acid. In the context of the present invention, "weak acid" means an acid with a pKa of 5 to 9, such as 5.5 to 8.5, preferably 6 to 8 or 6.5 to 8. Thus, the fluorophores described herein can be any fluorophores that carry a negatively charged moiety at near-neutral pH. Near-neutral pH is typically pH 5.5 to 8.5, particularly pH 6 to pH 8. The negatively charged moiety can be stabilized by a counterion moiety. However, fluorophores can also be protonated (e.g., in the presence of acid). Fluorophores with negatively charged moieties will typically exist in an equilibrium state containing the protonated fluorophore at near-neutral pH. Preferably, the equilibrium is in the deprotonated form of the fluorophore at near-neutral pH. For example, 50% or more of the fluorophore may be deprotonated (i.e., contain a negatively charged moiety) at near-neutral pH. Preferably, the fluorophore is -O at near-neutral pH. - group and / or -COO - Of course, the -O - group or -COO - Fluorophores containing groups include -O - group and / or -COO - may exist in equilibrium with a form of the fluorophore that is protonated. Fluorophores can also have multiple deprotonated forms, for example, fluorophores can have multiple charged sites that can accept protons. Reference to a fluorophore having a charge or charged site does not exclude the possibility that the fluorophore contains additional charged sites that can accept protons. Furthermore, reference to "fluorophore" in this specification should be considered to include both deprotonated and protonated forms of the fluorophore, unless otherwise indicated. When the fluorophore is in the form of a salt, for example, when the fluorophore comprises a salt of a weak acid, the fluorophore may comprise one or more positive ions. These positively charged ions are not covalently bound to the fluorophore or to the polymer matrix. Such ions are dissociated from the fluorophore when the fluorophore is in solution. The nature of the positive ions is not particularly limited. Suitable positive ions include cations of elements in Group I or II of the periodic table (e.g., Li + , Na + , K. + , Mg 2+ or Ca 2+ ) or ammonium salts (e.g., NH4 + , CH3NH3 + or CH3CH2NH3 + ) are listed. + and K. + is preferred. The overall charge of a fluorophore, even in its deprotonated form, is not necessarily negative, since the fluorophore may contain a positive ion. However, the positive ion need not necessarily be present; for example, washing after covalently attaching the fluorophore to a polymer support may remove the positive ion associated with the fluorophore. Thus, the overall charge of a fluorophore may be positive, neutral, or negative. Generally, the charge of a fluorophore is neutral or negative.

[0023] III. Fluorophore Structure Fluorophores exist in at least a protonated form and a deprotonated form. At near-neutral pH, fluorophores contain a negatively charged site capable of accepting a proton. The chemical structure of the fluorophore moiety is not otherwise particularly limited. Fluorophores may or may not be covalently attached to a polymeric support. For example, in one embodiment, a fluorophore may consist of a fluorophore moiety and, optionally, one or more associated ions, while in another embodiment, the fluorophore may be covalently attached to a polymeric support. Preferably, the fluorophore is not covalently attached to the polymeric support via a polymeric linkage moiety. The fluorophore is preferably soluble in a coating solution that can be added to a polymer matrix to retain the fluorophore in the polymer matrix. The coating solution can be comprised of any solvent, such as water and / or an organic solvent. An important feature of the fluorophore moiety is that it is typically stable in aqueous solution. It is also preferable that the fluorophore be resistant to photobleaching, as this allows the polymer matrix to be used repeatedly and / or for long periods of time. It is particularly preferable that the fluorophore be resistant to photobleaching over long periods of time, as this allows the polymer matrix to be used in sensing applications for continuous measurements over long periods of time. Thus, typically, the loss of the fluorophore due to photobleaching is less than 5%, preferably less than 2% or less than 1% per hour of visible light exposure. In particular, it is preferable that the loss of the fluorophore due to photobleaching is less than 5%, preferably less than 2% or less than 1% per 24 hours of visible light exposure. Preferably, the deprotonated form of the fluorophore is -O - Site or -COO -These species are preferred because the highly electronegative O atom can stabilize the negative charge of the deprotonated fluorophore. Therefore, when protonated, the fluorophore preferably contains an -OH or -COOH moiety. The deprotonated fluorophore is preferably an -O - It is particularly preferred that the aryl group contains a moiety that can be protonated to form an -OH moiety. Furthermore, fluorophores typically contain extended systems of delocalized electrons, as such systems typically give rise to the excited electronic states required to enable absorption and fluorescence in the visible region of the electromagnetic spectrum. Thus, in a preferred embodiment, the fluorophore comprises an optionally substituted polycyclic aryl or polycyclic heteroaryl species substituted with at least one -OH or -COOH group. Reference to an -OH or -COOH group refers to the fluorophore in its protonated form; the deprotonated form of the fluorophore is -O - Site or -COO - In particularly preferred embodiments, the fluorophore comprises an optionally substituted polycyclic aryl or polycyclic heteroaryl species substituted with at least one -OH group (again, references to -OH groups refer to the fluorophore in its protonated form).

[0024] In some embodiments, the fluorophore is pyranine or a derivative thereof. Pyranine (also called HTPS or 8-hydroxy-1,3,6-pyrenetrisulfonic acid) has the following structure: [ka] The OH moiety can be deprotonated to yield a deprotonated fluorophore having absorption and emission spectra that differ from those of the protonated form. Thus, in some embodiments, the fluorophore includes or is pyranine.

[0025] Modified forms of pyranine are also known to be useful as fluorophores in pH sensing. One example of such a compound is the modified DHDS (dihydroxypyrene disulfonic acid) shown below. [ka]

[0026] Thus, in some embodiments, the fluorophore comprises a species of formula (Fl-A). [ka]

[0027] Each R group can be H, halogen, cyano, nitro, hydroxy, or C 1-10 Alkyl, C 3-10 Cycloalkyl, C 4-10 Cycloalkenyl, aryl, alkylaryl, heteroaryl, heterocycloalkyl, C 2-10 alkenyl. 2-10 Alkynyl, -SR a , -OR a , -SO2R a , -SO3 - , -SOR a , -SO2NR a , -S(O)(NR a )R a , -NR a 2, -NR a COR a , -NR a CO2R a , -COR a , -CO2R a , -CONR a 2, or a covalent bond attaching the fluorophore to the polymer linkage site, or two adjacent R groups joining to form a carbocyclic or heterocyclic ring. In some embodiments, each R group is H, halogen, cyano, nitro, hydroxy, C 1-10 Alkyl, C 3-10 cycloalkyl. 4-10Cycloalkenyl, C 2-10 Alkenyl, C 2-10 Alkynyl, -SR a , -OR a , -SO2R a , -SO3 - , -SOR a , -SO2NR a , -S(O)(NR a )R a , -NR a 2, -NR a COR a , -NR a CO2R a , -COR a , -CO2R a , -CONR a 2. For example, each R is preferably H, hydroxy, C 1-6 Alkyl, C 2-6 Alkenyl, -OR a , -SO3 - , -COR a , and -CO2R a Particularly preferably, each R is independently selected from H, C 1-4 Alkyl, and -SO3 - are independently selected from Each R a The groups are H, C 1-10 Alkyl, C 3-10 Cycloalkyl, C 4-10 Cycloalkenyl, aryl, alkylaryl, heteroaryl, heterocycloalkyl, C 2-10 Alkenyl, and C 2-10 alkynyl. Preferably, each R a is H, C 1-10 Alkyl, C 3-6 Cycloalkyl, C 4-6 Cycloalkenyl, and C 2-10 alkenyl. Particularly preferably, each R a , H and C 1-4 alkyl. Any R group that can be substituted may be optionally substituted with one or more substituents. The one or more substituents may be present at any suitable position on the R group, and (if present) a The one or more substituents are typically halogen, oxo, cyano, nitro, hydroxy, C 1-10 Alkyl, C 3-10 Cycloalkyl, C 4-10 Cycloalkenyl, C 2-10 Alkenyl, C 2-10 Alkynyl, -OR b , -NR b 2, -COR b , -CO2R b , and -CONR b 2. Each R is usually substituted with 0, 1, 2 or 3 substituents, preferably 0 or 1 substituent. Preferred substituents include halogen, oxo, hydroxy, C 1-6 Alkyl, C 3-6 Cycloalkyl, C 2-6 Alkenyl, -OR b , -NR b 2, -COR b , and -CO2R b Particularly preferred substituents include halogen, hydroxy, C 1-4 Alkyl, -OR b , -NR b 2, -COR b , and -CO2R b is. R b is H, C 1-6 Alkyl, C 3-6 Cycloalkyl, C 4-6 Cycloalkenyl, and C 2-6 alkenyl. Preferably, R b is H, C 1-6 Alkyl, C 3-6 Cycloalkyl, and C 2-6 alkenyl. More preferably, R b , H and C 1-4 alkyl. Thus, in a preferred embodiment, each R group is selected from the group consisting of H, hydroxy, C 1-6 Alkyl, C 2-6 Alkenyl, -OR a , -SO3 - , -COR a , and -CO2R a are independently selected from a is H, C 1-10 Alkyl, C 3-6 Cycloalkyl, C 4-6 Cycloalkenyl, and C 2-10 In this embodiment, each R is independently selected from the group consisting of halogen, hydroxy, C 1-4 Alkyl, -OR b , -NR b 2, -COR b , and -CO2R b and optionally substituted with one substituent independently selected from b is H and C 1-4 alkyl. If the R group or a substituent thereon contains a hydroxy group or a -COOH group, then "hydroxy" or "-COOH" (i.e., -COR a , where R a and H) refer to the deprotonated forms of these groups, i.e., -O, respectively. - and -COO - It includes:

[0028] In preferred embodiments, the species of formula (Fl-A) has electron-withdrawing groups at specific positions on the heterocyclic backbone and no electron-withdrawing groups at other positions. Thus, in preferred embodiments, the fluorophore comprises a species of formula (Fl-B). [ka]

[0029] Each R 1 The groups are independently halogen, cyano, nitro, hydroxy, -OR a , -SO2R a , -SO3 -, -SOR a Selected from: -SO2NR a ,-S(O)(NR a )R a ,-NR a 2,-NR a COR a ,-NR a CO2R a ,-COR a ,-CO2R a ,-CONR a 2 and a covalent bond to the polymer linkage moiety. Typically, each R 1 The groups are halogen, cyano, nitro, hydroxy, -OR a , -SO2R a , -SO3 - , -SOR a , -SO2NR a , -S(O)(NR a )R a , -NR a 2, -NR a COR a , -NR a CO2R a , -COR a , -CO2R a , -CONR a 2. Preferably, each R 1 The groups are independently hydroxy, -OR a , -SO2R a , -SO3 - , -SOR a , -COR a , -CO2R a , and -CONR a Selected from 2. R 1 Particularly preferred examples of a , and -SO3 - is. Each R 2 The groups are H, C 1-10 Alkyl, C 3-10 Cycloalkyl, C 4-10 Cycloalkenyl, aryl, alkylaryl, heteroaryl, heterocycloalkyl, C 2-10 Alkenyl, C 2-10alkynyl, and a covalent bond to the polymer attachment site. Typically, each R 2 The groups are H, C 1-10 Alkyl, C 3-6 Cycloalkyl, C 4-6 Cycloalkenyl, and C 2-10 alkenyl. Preferably, each R 2 The groups are H and C 1-6 alkyl. Most preferably, each R 2 The group is H. Any R that can be substituted 1 or R 2 The group may be optionally substituted with one or more substituents. The one or more substituents may be R 1 or R 2 It may be present in any suitable position on the group, and (if present) R a The one or more substituents are typically halogen, oxo, cyano, nitro, hydroxy, C 1-10 Alkyl, C 3-10 Cycloalkyl, C 4-10 Cycloalkenyl, C 2-10 Alkenyl, C 2-10 Alkynyl, -OR b , -NR b 2, -COR b , -CO2R b , and -CONR b 2. Each R 1 or R 2 is usually substituted with 0, 1, 2 or 3 substituents, preferably 0 or 1 substituent. Preferred substituents include halogen, oxo, hydroxy, C 1-6 Alkyl, C 3-6 Cycloalkyl, C 2-6 Alkenyl, -OR b , -NR b 2, -COR b , and -CO2R b Particularly preferred substituents include halogen, hydroxy, C 1-4 Alkyl, -OR b , -NR b 2, -CORb , and -CO2R b is. R 1 , R 2 When a group or a substituent thereon contains a hydroxy group or a -COOH group, the designation "hydroxy" or "-COOH" refers to the deprotonated form of those groups, i.e., -O, respectively. - and -COO - It includes: R a and R b is as mentioned above. In a preferred embodiment, each R 1 is hydroxy, -OR a , -SO2R a , -SO3 - , -SOR a , -COR a , -CO2R a , and -CONR a 2 independently selected from each R 2 is H and C 1-6 R is independently selected from alkyl. a is H, C 1-10 Alkyl, C 3-6 Cycloalkyl, C 4-6 Cycloalkenyl, and C 2-10 In this embodiment, each R 1 and R 2 The group may be unsubstituted or may be selected from halogen, hydroxy, C 1-4 Alkyl, -OR b , -NR b 2, -COR b , and -CO2R b and R b is H and C 1-4 alkyl.

[0030] In some embodiments, the fluorophore is fluorescein or a derivative thereof. Fluorescein is a compound having the following structure: [ka]

[0031] However, structural variations on fluorescein are known to act as fluorophores. Thus, where the fluorophore is a derivative of or based on fluorescein, the fluorophore may include, for example, a species of the formula (Fl-C): [ka]

[0032] where R is as defined above and may be optionally substituted as explained above. For example, the fluorophore may comprise a species of formula (Fl-D): [ka]

[0033] R 2 is as defined above. R 3 is cyano, nitro, hydroxy, -OR a , -SO2R a , -SO3 - , -SOR a , -SO2NR a , -S(O)(NR a )R a , -NR a 2, -NR a COR a , -NR a CO2R a , -COR a , -CO2R a , -CONR a 2 and a covalent bond at the polymer linking site. 3 is hydroxy, -NR a 2, -NR a COR a and —COOH. Preferably, R 3 is hydroxy, -NR a 2, and -CO2R a; in particular selected from hydroxy. Any R that can be substituted 2 or R 3 The group may be optionally substituted with one or more substituents. The one or more substituents may be R 2 or R 3 It may be present in any suitable position on the group, and (if present) R a The one or more substituents are typically halogen, oxo, cyano, nitro, hydroxy, C 1-10 Alkyl, C 3-10 Cycloalkyl, C 4-10 Cycloalkenyl, C 2-10 Alkenyl, C 2-10 Alkynyl, -OR b , -NR b 2, -COR b , -CO2R b , and -CONR b 2. Each R 2 or R 3 is usually substituted with 0, 1, 2 or 3 substituents, preferably 0 or 1 substituent. Preferred substituents include halogen, oxo, hydroxy, C 1-6 Alkyl, C 3-6 Cycloalkyl, C 2-6 Alkenyl, -OR b , -NR b 2, -COR b , and -CO2R b Particularly preferred substituents include halogen, hydroxy, C 1-4 Alkyl, -OR b , -NR b 2, -COR b , and -CO2R b is. R a and R b is as mentioned above. In a preferred embodiment, each R 2 is H and C 1-6 alkyl; R 3 is hydroxy, -NR a 2, and -CO2R a Each Ra is H, C 1-10 Alkyl, C 3-6 Cycloalkyl, C 4-6 Cycloalkenyl, and C 2-10 In this embodiment, each R 2 and R 3 The group may be unsubstituted or may be selected from halogen, hydroxy, C 1-4 Alkyl, -OR b , -NR b 2, -COR b , and -CO2R b and R b is H and C 1-4 alkyl. R 1 and R 2 Similarly, R 3 If a group or a substituent thereon contains a hydroxy group or a -COOH group, then "hydroxy" or "-COOH" (i.e., -COR a where R a References to groups (H) include the deprotonated forms of those groups, i.e., -O, respectively. - and -COO - Includes:

[0034] Counterion site The key function of the counterion site is to form an ion pair with the deprotonated form of the fluorophore at near-neutral pH. Thus, the counterion site is positively charged at near-neutral pH. Of course, the counterion site may be negatively charged at more extreme pH values. In addition to carrying a positive charge at near-neutral pH, the counterion site is generally stable in aqueous solution. Many suitable water-stable positively charged cations are known to those skilled in the art. The counterion moiety may be retained in the polymer matrix of the present invention or may be covalently bound to the polymer support via a polymer linking moiety. Generally, it is preferred that the counterion moiety be covalently bound to the polymer support. This advantageously allows the counterion moiety to be present in large amounts, meaning that it can be provided in excess compared to the amount of fluorophore present without being limited by the solubility of the counterion. When the counterion moiety includes an organic moiety, a covalent bond from the polymer support to the counterion moiety is easily formed. Therefore, in a preferred embodiment, the counterion moiety includes an organic moiety.

[0035] Suitable organic cations include quaternary ammonium ions. Thus, in a preferred embodiment, the counterion site comprises a quaternary ammonium ion. For example, the counterion site may comprise a quaternary ammonium cation of formula (CI-A): [ka]

[0036] Each R 4 The group is H; or C 1-30 Alkyl, C 3-12 Cycloalkyl, C 4-12 Cycloalkenyl, aryl, alkylaryl, heteroaryl, heterocycloalkyl, C 2-30 Alkenyl, C 2-30 alkynyl, and a covalent bond to the polymer attachment site; or two R 4 The groups are bonded together to form C 3-16 Heterocycloalkyl group or C 4-16 A heterocycloalkenyl group may be formed. Preferably, each R 4 The groups are H, C 1-20 Alkyl, C 3-6 Cycloalkyl, C 2-20 alkenyl, and a covalent bond to the polymer attachment site. 4 Specific examples of 1-20 Alkyl, C 2-20alkenyl, and a covalent bond to the polymer attachment site. In an exemplary embodiment, one R 4 The R group is the covalent bond to the polymer linkage site, and the remaining three R 4 The groups are each independently H, C 1-20 Alkyl, C 3-10 Cycloalkyl, C 4-12 Cycloalkenyl, aryl, alkylarylheteroaryl, heterocycloalkyl, C 2-20 Alkenyl, C 2-20 alkynyl. Preferably, one R 4 The R group is the covalent bond to the polymer linkage site, and the remaining three R 4 The groups are independently H, C 1-20 Alkyl, and C 2-20 alkenyl. Any R that can be substituted 4 The group may be optionally substituted by one or more substituents, which are typically halogen, oxo, cyano, nitro, hydroxy, C 1-10 Alkyl, C 3-10 Cycloalkyl, C 4-10 Cycloalkenyl, C 2-10 Alkenyl, C 2-10 Alkynyl, -OR b , -NR b 2, -COR b , -CO2R b Each R 4 is usually substituted with 0, 1, 2 or 3 substituents, preferably 0 or 1 substituent. Preferred substituents include halogen, oxo, hydroxy, C 1-6 Alkyl, C 3-6 Cycloalkyl, C 2-6 Alkenyl, -OR b , -NR b 2, -COR b , and -CO2R b Particularly preferred substituents include halogen, hydroxy, C 1-4 Alkyl, -OR b , -NR b 2, -CORb , and -CO2R b is. R b , as defined above. In a preferred embodiment, one R 4 The R group is the covalent bond to the polymer linkage site, and the remaining three R 4 The groups are independently H, C 1-20 Alkyl, and C 2-20 alkenyl. Each substitutable R 4 The group may be unsubstituted or may be selected from halogen, hydroxy, C 1-4 Alkyl, -OR b , -NR b 2, -COR b , and -CO2R b and R b is H and C 1-4 alkyl. In some embodiments, the counterion moiety comprises a hexadecyltrimethylammonium ion or a derivative thereof. For example, the counterion moiety may be (C 16 H 33 )(CH3)3N + ; or (C 16 H 33 )(CH3)2R 4 N + , where R 4 may include being a covalent bond to the polymer linkage moiety. The counterion site may be associated with one or more negatively charged ions other than the negatively charged fluorophore. These negatively charged ions are not covalently bound to the counterion site or the polymer matrix. Such ions dissociate from the counterion site when the ions associated with the counterion site are contacted with water. Suitable negative ions include halide ions or hydroxide ions, preferably hydroxide ions. The associated ions may typically be present when the positively charged counterion site is introduced into the polymer matrix by retaining a dissolved salt containing the counterion site and one or more associated negative ions in the polymer matrix.

[0037] Polymer linking site The polymer matrix includes a polymer linkage moiety that forms a covalent bond between the polymer support and either the fluorophore or the counterion moiety. In some embodiments, the polymer linkage moiety is a covalent bond that directly connects the polymer support to the fluorophore or the counterion moiety. In other embodiments, the polymer linkage moiety may include one or more intervening atoms. Preferably, the linker is hydrolytically stable during storage. For example, the linker is preferably hydrolytically stable during storage for up to 1, 2, 3, 4, 5, or 6 months. By "hydrolytically stable" is meant that at least 90%, preferably at least 95%, of the linker does not undergo hydrolysis. The nature of the polymer linkage moiety is not particularly limited. The polymer linkage moiety can comprise one or more of a covalent bond, O, S, N, and C. Typically, the polymer linkage moiety is a covalent bond, -O-, -S-, -SO2-, -SO-, optionally substituted alkylene, optionally substituted alkenylene, optionally substituted alkynylene, and -NR 5 For example, the polymer linkage site can be a covalent bond, -O-, or -NR 5 -, -S-, -SO2-, -SO-, optionally substituted C 1-10 Alkylene, and optionally substituted C 2-10 It may contain one or more of the following: alkenylene. When the polymer linkage moiety comprises two or more of the aforementioned divalent species, the polymer linkage moiety is typically selected from the group consisting of -O-, -NR 5 -, -S-, -SO2-, -SO-, optionally substituted C 1-10 Alkylene, and optionally substituted C 2-10 It comprises two or three moieties independently selected from alkenylene, wherein at least two of the moieties are different. Preferably, the polymer linking moiety is a covalent bond, -O-, or -NR 5-, and optionally substituted C alkylene. Examples of polymer linkage moieties include a covalent bond, -O-, -NH-, -NR 5 -, and C 1-4 Alkylene is included. R 5 is typically H, C 1-10 Alkyl, C 3-10 Cycloalkyl, C 4-10 Cycloalkenyl, aryl, arylalkyl, heteroaryl, heterocycloalkyl, C 2-10 Alkenyl, C 2-10 Alkynyl, -COR a , -CO2R a , and -CONR a 2. Preferably, R 5 is H, C 1-6 Alkyl, C 3-6 Cycloalkyl, and C 2-10 Particularly preferably, R 5 is H and C 1-4 R is selected from alkyl. 5 Examples include hydrogen, methyl and ethyl groups. The polymer linkage site may optionally be substituted with one or more substituents. The one or more substituents, if present, are R a The alkylene, alkenylene, or alkynylene moiety of the polymer linkage moiety may be present at any suitable position on the polymer linkage moiety, including on the alkylene, alkenylene, or alkynylene group. In particular, the alkylene, alkenylene, or alkynylene moiety of the polymer linkage moiety may be optionally substituted. The one or more substituents are typically halogen, oxo, cyano, nitro, hydroxy, C 1-10 Alkyl, C 3-10 Cycloalkyl, C 4-10 Cycloalkenyl, C 2-10 Alkenyl, C 2-10 Alkynyl, -OR b , -NR b 2, -COR b , -CO2R b , and -CONR b2. The polymer linkage site is typically substituted with 0, 1, 2 or 3 substituents, preferably 0 or 1 substituent. Preferred substituents include halogen, oxo, hydroxy, C 1-6 Alkyl, C 3-6 Cycloalkyl, C 2-6 Alkenyl, -OR b , -NR b 2, -COR b , and -CO2R b Particularly preferred substituents include halogen, hydroxy, C 1-4 Alkyl, -OR b , -NR b 2, -COR b , and -CO2R b Includes: Preferably, the polymer linkage site is unsubstituted. R a and R b is as above. In a preferred embodiment, the polymer linkage moiety is a covalent bond, —O—, or —NR 5 -, and optionally substituted C alkylene. 5 is H and C 1-4 In this embodiment, the polymer linkage site is unsubstituted or is selected from halogen, hydroxy, C 1-4 Alkyl, -OR b , -NR b 2, -COR b , and -CO2R b and R b is H and C 1-4 Particularly preferred examples of polymer linkage moieties include a covalent bond, -O-, and -CH2-. For completeness, R or R 1 From R 3 is a covalent bond attaching the fluorophore to the polymer linkage moiety, and if the polymer linkage moiety is a covalent bond, the fluorophore may be attached to the respective R or R 1 From R3 Note that the polymer is attached to the support by a covalent bond at position R. 4 is a covalent bond linking the counterion to the polymer linkage moiety, and if the polymer linkage moiety is a covalent bond, then the counterion moiety is 4 The polymeric support is covalently attached at the position

[0038] Polymer Support The polymer matrix includes a polymer substrate, which is a polymer that may contain structural units derived from one type of monomer or from multiple different monomers. Generally, at least a majority of the structural units present in the polymer substrate are derived from one type of monomer. The polymer support is generally gas-permeable. When the polymer matrix is ​​gas-permeable, gases such as CO2 can diffuse into the matrix, allowing the polymer matrix to be used as a gas sensor. The polymer support may also be permeable to liquids such as water. In some preferred embodiments, the polymeric support comprises a hydrophilic polymer. For example, the polymeric support comprises or can comprise structural units derived from the polymerization of hydrophilic monomers. When the polymeric support comprises structural units derived from the polymerization of hydrophilic monomers, the polymeric support is typically itself hydrophilic. Suitable examples of hydrophilic polymers that can be included in the polymer support include hydrogels, cellulose derivatives, ethyl cellulose, sol-gels, or hydrophilic silicone-based polymers. Preferably, the polymer support includes a hydrogel. Suitable examples of hydrogels include polyacrylamide or polyhydroxyethyl methacrylate. In some embodiments, the polymer support comprises a hydrophobic polymer. For example, the polymer support may comprise or contain structural units derived from the polymerization of hydrophobic monomers. When the polymer support comprises structural units derived from the polymerization of hydrophobic polymers, the polymer support itself is typically hydrophobic. Hydrophobic polymer supports are particularly suitable for CO2 detection because they have low permeability or are impermeable to aqueous solutions that may contain acids or bases that may interfere with CO2 detection. For example, the polymeric support may comprise one or more of polystyrene, a hydrophobic silicone-based polymer, a hydrophobic cellulose derivative, or plasticized ethyl cellulose, of which polystyrene is preferred. The polymeric support can include structural units that include either fluorophores or counterion moieties. In a preferred embodiment, the polymeric support includes structural units that include counterion moieties. The inclusion of such structural units in the polymer matrix can be achieved in a variety of ways, including: (i) The polymeric support can be produced by copolymerizing (a) a monomer containing a fluorophore covalently attached to a polymerizable moiety by a polymer linkage moiety and (b) one or more other monomers. (ii) The polymeric support can be produced by copolymerizing (a) a monomer containing a counterion moiety covalently attached to a polymerizable moiety by a polymer linking moiety and (b) one or more other monomers. (iii) The polymeric support can be produced by copolymerizing (a) a monomer comprising a polymeric linkage moiety precursor and a polymerizable moiety with (b) one or more other monomers. A "polymeric linkage moiety precursor" is a species that can react with a fluorophore precursor or a counterion moiety precursor to form a fluorophore or counterion moiety covalently attached to the polymeric support via a polymeric linkage moiety. Thus, a polymeric linkage moiety precursor reacts with a fluorophore precursor or a counterion moiety precursor to produce a fluorophore or counterion moiety covalently attached to the polymeric support via a polymeric linkage moiety.

[0039] polymer matrix The polymer matrix comprises a fluorophore, a positively charged counterion site, and a polymer support. However, when the polymer matrix is ​​used for optical sensing as described herein, the polymer matrix is ​​used to transport H to and from the fluorophore. + The polymer matrix must contain water to mediate the transfer of ions. Thus, in some embodiments, the polymer matrix contains water. Typically, the molar ratio of water (HO) to fluorophore is 1:1 or greater. This ensures that there is sufficient HO to interact with each ion pair. For example, the molar ratio of water (HO) to fluorophore can be at least 2:1 or at least 5:1. Preferably, the polymer matrix contains a known amount of water, or at least a constant amount of water. This is particularly easy to achieve if a membrane is placed to prevent liquid and hydrogen ions from entering the polymer matrix. The membrane is preferably gas-permeable and impermeable to liquids, and more preferably impermeable to hydrogen ions. In such a case, the matrix and membrane can be exposed to an aqueous sample. The amount of water in the matrix may change slightly as water vapor migrates across the membrane while equilibrium is established. Once equilibrium is established, the amount of water in the sensor remains fixed while the matrix is ​​exposed to aqueous solutions during use as a sensor. This reduces signal fluctuations and improves accuracy. The molar amount of water present is typically in excess compared to the molar amount of fluorophore present. The molar amount of water present may also be in excess compared to the molar amount of counterion moieties. In some embodiments, the polymer matrix is ​​saturated with water.

[0040] Optical sensor The polymer matrix is ​​useful in an optical sensor for the optical detection of CO. The optical sensor generates an optical signal that varies with pH in the presence of CO. The optical signal can be generated by directing excitation light at the polymer matrix while the polymer matrix is ​​in contact (directly or indirectly) with a sample. Thus, described herein is an optical sensor 1 for the optical detection of CO, comprising: - a sensing region comprising a polymer matrix 5 as described herein; and - an optical waveguide 3 arranged to direct light towards the sensing area; An optical waveguide is a physical structure having a first end and a second end that is capable of guiding electromagnetic waves in the optical region of the spectrum between the first and second ends of the structure. The optical sensor 1 is suitable for detecting the CO content of a sample 9. The CO present in the sample is referred to herein as the analyte. The sample 9 can be any fluid of interest with a CO content. Exemplary samples include buffer solutions and biological samples such as saliva or blood. In a preferred embodiment, the sample 9 is a blood sample, e.g., a blood sample taken from a human patient. Thus, in a preferred embodiment, the optical sensor is a sensor for detecting the CO content of blood. When sample 9 is a biological sample, sample 9 is typically an ex vivo sample, i.e., sample 9 is usually outside the human or animal body. The design of an optical sensor 1 including a polymer matrix 5 of the present invention can be influenced by the nature of the polymer support, in particular whether the polymer support is hydrophobic or hydrophilic. If the polymer support is inherently hydrophobic, the polymer matrix 5 can be used to construct a so-called "naked sensor." If the polymer support is hydrophobic, the polymer matrix 5 can be placed in direct contact with an aqueous solution (e.g., a biological sample such as blood), and the aqueous solution either does not penetrate the polymer matrix 5 or penetrates the polymer matrix 5 slightly. Furthermore, if the polymer support is hydrophobic, it advantageously resists adsorption of hydrophilic proteins. In such cases, there is no need to provide an additional layer to prevent the aqueous solution or proteins from penetrating the polymer matrix 5. Bare sensors (which do not include a membrane positioned to prevent penetration of the aqueous sample into the polymer matrix 5) have several advantages. The membrane slows the passage of the analyte and acts as a reservoir for the analyte. This increases the response time of the sensor. Therefore, the response time of bare sensors is very fast.

[0041] Optical sensors constructed using hydrophilic polymer matrices also have particular advantages. When used as optical sensors, the polymer matrix 5 must contain water (to prevent the formation of carbonic acid during CO2 detection and H2O during acid detection). + If the water content is too low, not all of the CO2 can produce carbonic acid. Therefore, not all of the CO2 present is available to cause protonation of the fluorophore and change the absorption or emission properties of the polymer matrix. Therefore, an insufficient water content can lead to an optical sensor whose optical output does not accurately correspond to the CO2 content of the sample under test. When a hydrophobic polymer matrix 5 is used, it is difficult to ensure that the polymer matrix 5 contains enough water to enable the sensing chemistry. Therefore, it is advantageous to use a hydrophilic polymer matrix 5 in the optical sensor 1, as a hydrophilic polymer matrix 5 can easily become saturated with water. When the hydrophilic polymer matrix 5 comes into contact with water, it absorbs and retains a certain amount of water. The hydrophilic polymer matrix 5 (e.g., a hydrogel) can become saturated with water. When the hydrophilic polymer matrix 5 is placed in direct contact with an aqueous solution (e.g., a biological sample such as blood), the aqueous solution penetrates the hydrophilic polymer matrix 5. As shown in Figure 5, the optical sensor 1 typically also includes a hydrophobic membrane 7 positioned such that the sample 9 contacts the polymer matrix 5 through the hydrophobic membrane 7. Thus, the optical sensor 1 including the hydrophilic polymer matrix 5 allows the analyte to pass through the hydrophobic membrane 7 and into the polymer matrix 5. When an optical sensor includes a hydrophilic polymer matrix 5 and a gas-permeable hydrophobic membrane 7 and the optical sensor is placed in contact with an aqueous solution, gaseous water (water vapor) can pass across the gas-permeable hydrophobic membrane 7. Because equilibrium is maintained, the amount of water present in the polymer matrix 5 remains approximately constant. Therefore, optical sensors including a hydrophilic polymer matrix 5 are less sensitive to inaccuracies resulting from the presence of insufficient water or varying water content. Thus, in some embodiments, the optical sensor 1 comprises a membrane 7. Generally, the membrane 7 is gas-permeable. The membrane 7 may also be liquid-permeable. Typically, when the optical sensor 1 includes a membrane 7, the optical sensor 1 is configured to allow the analyte to enter the sensing region through the membrane 7. In such embodiments, the membrane 7 is typically hydrophobic, and preferably, the polymer matrix 5 is hydrophilic. In some embodiments, the polymer matrix 5 may penetrate the membrane 7. The membrane 7 may be disposed within or partially within the polymer matrix 5. Suitable membranes, particularly gas-permeable membranes, are known in the art. Such membranes include dialysis membranes and microporous membranes. Dialysis membranes are preferred.

[0042] The optical sensor 1 is specially configured for the detection of CO. Thus, in a particularly preferred embodiment, there is provided an optical sensor 1 for the optical detection of CO, comprising: - a sensing region comprising a polymer matrix 5 as described herein, further comprising a hydrophobic membrane 7, the sensor being configured to allow an analyte to enter the sensing region through the hydrophobic membrane 7; and - an optical waveguide 3 arranged to direct light towards the sensing area; The sample is CO2. In another particularly preferred embodiment, there is provided an optical sensor 1 for optically detecting CO2, comprising: - a sensing region comprising a polymer matrix 5 as described herein, wherein the polymer matrix is ​​a hydrophobic polymer matrix; the sensing area optionally further comprises a hydrophobic membrane 7, the sensor being configured to allow the analyte to enter the sensing area through the hydrophobic membrane 7; and An optical waveguide 3 arranged to direct light to the sensing area. During use, the hydrophobic membrane 7 is positioned between the sample under test 9 and the sensing area. The hydrophobic membrane 7 thus acts as a barrier to liquids, preventing liquids (especially water) in the sample under test 9 from entering the sensing area. Therefore, the hydrophobic membrane 7 is preferably impermeable to liquids. This prevents acids in the sample 9 from entering the sensor. However, because the hydrophobic membrane 7 is gas-permeable, gaseous or dissolved CO2 in the sample under test 9 can diffuse across the hydrophobic membrane 7 into the sensing area. Therefore, when the sensor includes the hydrophobic membrane 7, the optical sensor 1 specifically senses CO2. Furthermore, the sensor is not subject to interference from species contained in the liquid sample 9, such as acids or alkalis. Particularly preferably, the hydrophobic membrane 7 is impermeable to hydrogen ions. In this configuration, for the reasons explained above, it is preferred that the polymer matrix 5 be hydrophilic: a hydrophilic polymer matrix 5 can readily capture the water necessary to allow for the formation of carbonic acid and aid in the functioning of the sensor.

[0043] Thus, in a particularly preferred embodiment, the optical sensor 1 is an optical sensor 1 for the optical detection of CO2, comprising: - a sensing region comprising a polymer matrix 5 as described herein, wherein the polymer support comprises a hydrophilic polymer; the sensing region further comprises a gas-permeable hydrophobic membrane 7 that is impermeable to liquids and hydrogen ions, the sensor being configured to allow CO2 to enter the sensing region through the hydrophobic membrane 7; and - an optical waveguide 3 arranged to direct light towards the sensing area; Therefore, in this embodiment, the polymer matrix 5 is preferably hydrophilic. However, in this embodiment, the polymer matrix 5 may alternatively comprise a hydrophobic polymer and may be hydrophobic. A diagram of the optical sensor 1 in use is shown in FIG. 3. In the illustrated embodiment, the optical sensor 1 includes a gas-permeable membrane 7, although this is not required. An optical waveguide 3 is positioned to direct excitation light onto a sensing region comprising a polymer matrix 5. During use, the sensing region is in contact with a sample 9. The gas-permeable membrane 7 is positioned over the polymer matrix 5, allowing the analyte (CO2) present in the sample 9 to pass through the membrane 7 (if present) to contact the polymer matrix 5. The fluorophore can absorb the excitation light and emit light. The light emitted by the fluorophore passes through the optical waveguide 3. The absorption and emission spectra depend on the pH of the sample 9, which determines the degree of protonation of the fluorophore. The waveguide can be any optically transparent material. Typically, the waveguide will contain or include an optical fiber. Alternatively, a transparent optical window can be used. Optical fibers utilize total internal reflection to prevent loss of light from the fiber. This means that light is efficiently transported to and from the fluorophore, improving the signal and providing a higher quality, more reliable measurement. Optionally, the optical sensor 1 may include a reflector configured to reflect light emitted by the fluorophores into the optical waveguide 3. If present, the reflector increases the proportion of light emitted by the fluorophores that is collected by the waveguide and subsequently detected. The reflector may include a layer deposited on the polymer matrix 5, such as a film 7 disposed on the polymer matrix 5. Suitable materials that can be used as the reflector include polysulfone (PSU), polyethersulfone (PESU), and polyphenylsulfone (PPSU). Polysulfone is preferred. Other reflective compounds, such as silicon-containing titanium oxide or barium sulfate, could also be used. An optical sensor 1 comprising a sensing region and an optical waveguide 3, and possibly also a membrane 7 and / or a reflector, may be referred to as a sensor probe. The optical sensor 1 may further include a light source configured to provide excitation light to the fluorophore. The light source may be any light source capable of emitting light at the wavelength and intensity required to excite the fluorophore. For example, the light source may include a laser diode. The optical sensor 1 may further include a detector configured to detect light emitted by the fluorophore via the optical waveguide 3. The detector may be any device capable of generating a signal in response to receiving light of a wavelength emitted by the fluorophore. For example, the detector may include a charge-coupled device, an active pixel sensor, a photodiode, or a photoresistor. Part or all of the optical sensor 1 can be disposable. This is useful in clinical situations where the optical sensor 1 may come into contact with a biological sample inside or taken from a patient. In such cases, the portion of the optical sensor 1 that comes into contact with the biological sample must be sterile and cannot be reused between patients. For example, the sensor probe is disposable, but not the detector or light source, if present. In such cases, the optical sensor 1 may further include a connector interface configured to connect the optical waveguide 3 to the light source and detector. The optical sensor 1 may form part of an optical detection system further comprising a control system. The control system may be configured to cause the light source to emit light and to activate the detector as needed. The optical detection system may further comprise an analysis system. The analysis system may be configured to determine whether carbon dioxide is present in the sample 9 under test. In particular, the analysis system may be configured to determine the CO2 concentration of the sample 9 under test. The optical sensors 1 and optical sensing systems described herein can be used to provide rapid, real-time measurements of the CO2 content of a sample 9. This is particularly desirable in blood gas sensing. It is important to monitor the CO2 content of a patient's blood during procedures such as dialysis and surgery, especially cardiac bypass surgery, because CO2 content can change rapidly, potentially with dangerous consequences for the patient.

[0044] Thus, the optical sensing system may be an in-line blood gas sensing system comprising the optical sensor 1 described herein, where the in-line blood gas sensing system directs blood from the patient's body outside the body, and typically the in-line blood gas sensing system is further configured to return the blood to the patient's body. Generally, an in-line blood gas sensing system further includes an optical sensor 1 as described herein, a blood line, and a pump, the blood line configured to conduct blood from a patient's body out of the body and contact the blood sensing area of ​​the optical sensor 1, and return the blood to the patient's body. The pump is configured to pump blood along the blood line. In one example, the present invention provides a dialysis system including the in-line blood gas sensing system described herein. In another example, the present invention provides a cardiac bypass system comprising the in-line blood gas sensing system described herein.

[0045] Method for producing a polymer matrix The polymer matrix of the present invention can be produced by any suitable method. For example, it is possible to produce a polymer matrix in which the fluorophore or counterion moiety is already retained within the polymer matrix by carrying out a polymerization reaction in the presence of the fluorophore or counterion moiety to produce the polymer support. However, it is convenient to first produce a polymer support to which the fluorophore or counterion moiety is covalently attached, and then provide the fluorophore or counterion moiety (whichever is not attached to the polymer support) within the matrix. This method allows for the washing away of waste or remaining starting materials from the production of the polymer support.

[0046] Thus, provided herein is a method for producing a polymer matrix for optical sensing, the method comprising: (i) providing a polymeric support, wherein either the fluorophore or the counterion moiety is covalently attached to the polymeric support by a polymer linkage moiety; and (ii) Retaining in the polymeric support either the fluorophore or the counterion moiety that is not covalently attached to the polymeric support via a polymer linkage moiety. Generally, the optical sensing polymer matrix produced by the method of the present invention is a polymer matrix as described herein. Step (i) can involve covalently attaching a fluorophore or counterion moiety to a polymeric support. For example, step (i) can involve: - Producing a polymeric support by polymerizing the monomer; and - reacting the polymeric support with a fluorophore precursor comprising a fluorophore, or with a counterion precursor comprising a counterion moiety, to produce a fluorophore or counterion moiety covalently attached to the polymeric support by a polymer linkage moiety. Suitable monomers are any polymerizable moiety, typically a hydrophobic or hydrophilic polymerizable moiety. Examples include styrene or hydrogel monomers.

[0047] In another example, step (i) can include: - functionalization of the polymer support; and - reacting the functionalized polymeric support with a fluorophore precursor comprising a fluorophore, or with a counterion precursor comprising a counterion moiety, to produce a fluorophore or counterion moiety covalently attached to the polymeric support by a polymer linkage moiety. This method can be useful when an existing polymer, such as cellulose or its derivatives, is desired as the polymer support. Conveniently, the polymeric support can be directly manufactured with the fluorophore or counterion moiety incorporated. In such embodiments, step (i) comprises copolymerizing a fluorophore monomer or a counterion monomer with one or more other monomers. Preferably, step (i) comprises copolymerizing a counterion monomer with one or more other monomers. "Fluorophore monomer" means a polymerizable monomer comprising a fluorophore. Suitable "fluorophore monomers" are fluorophores described herein in which the covalent bond connecting the fluorophore to the polymer attachment site is replaced with a polymerizable moiety, typically an alkenyl or alkynyl group. "Counterionic monomer" means a polymerizable monomer comprising a counterionic moiety. Suitable "counterionic monomers" are those counterionic moieties described herein in which the covalent bond connecting the counterionic moiety to the polymer linkage moiety has been replaced with a polymerizable moiety, typically an alkenyl or alkynyl group.

[0048] An exemplary counterion monomer is a quaternary ammonium cation of the formula (CI-AM): [ka]

[0049] PL is a polymerizable moiety. Preferably, PL is an alkenyl or alkynyl group. For example, PL is C 2-10 Alkenyl group or C 2-10 It may be an alkynyl group. Particularly preferably, PL is C 2-6 It is an alkenyl group. Each R 6 The groups are independently selected from H or C 1-30 Alkyl, C 3-12 Cycloalkyl, C 4-12 Cycloalkenyl, aryl, alkylaryl, heteroaryl, heterocycloalkyl, C 2-30 Alkenyl, and C 2-30 alkynyl; or two R groups together form C 3-16 Heterocycloalkyl or C 4-16 A heterocycloalkenyl group may be formed. Preferably, each R 6 The groups are H, C 1-10 Alkyl, C 3-6 Cycloalkyl, and C 2-10 R may be independently selected from alkenyl. 6 The most preferred examples of 1-6 It is alkyl. PL and / or any replaceable R 6The group may be optionally substituted with one or more substituents. The one or more substituents may be present at any available position. The one or more substituents are typically halogen, oxo, cyano, nitro, hydroxy, C 1-10 Alkyl, C 3-10 Cycloalkyl, C 4-10 Cycloalkenyl, C 2-10 Alkenyl, C 2-10 Alkynyl, -OR b , -NR b 2, -COR b , -CO2R b , and -CONR b 2. PL and R 6 is typically substituted with 0, 1, 2 or 3 substituents, preferably 0 or 1 substituent. Preferred substituents include halogen, oxo, hydroxy, C 1-6 Alkyl, C 3-6 Cycloalkyl, C 2-6 Alkenyl, -OR b , -NR b 2, -COR b , and -CO2R b Particularly preferred substituents include halogen, hydroxy, C 1-4 Alkyl, -OR b , -NR b 2, -COR b , and -CO2R b Most preferably, P1 and R 6 is non-substituted. R b is as above. In a preferred embodiment, the PL is C 2-6 alkenyl group, and each R 6 The groups are H and C 1-6 P L and R are independently selected from alkyl. 6 are each unsubstituted or halogen, hydroxy, C 1-4 Alkyl, -OR b , -NR b 2, -COR b , and -CO2R b and R is substituted with one substituent independently selected fromb is H and C 1-4 alkyl. Intermediate steps may be performed between steps (i) and (ii), for example, the polymeric support having covalently attached fluorophores or counterion moieties thereto may be washed before step (ii) is performed. Step (ii) involves retaining the fluorophore or counterion moiety in the polymer matrix. For example, step (ii) can involve dissolving the fluorophore (e.g., a salt form of the fluorophore) in a coating solution such as water or an aqueous solution, and contacting the polymeric support produced in step (i) with the coating solution containing the dissolved fluorophore. Alternatively, step (ii) can involve dissolving the counterion moiety (e.g., a salt containing the counterion moiety, typically a hydroxide salt of the counterion moiety) in a coating solution such as water or an aqueous solution, and contacting the polymeric support produced in step (i) with the coating solution containing the dissolved counterion moiety. Further steps may be carried out after step (ii), for example, the method of producing the polymer matrix may further comprise drying the polymer matrix to remove excess water.

[0050] Optical sensor manufacturing method The polymer matrix can be used to provide an optical sensor. Accordingly, described herein is a method of making an optical sensor, the method comprising: (i) providing an optical waveguide; (ii) disposing a polymer matrix described herein on an optical waveguide; and (iii) optionally disposing a membrane on the polymer matrix; Generally, the optical sensor produced by this method is described herein as Optical Sensor 1. For example, the membrane is typically a gas-permeable membrane, and is preferably also hydrophobic. Disposing the polymer matrix over the optical waveguide does not necessarily require that the polymer matrix be in direct contact with the optical waveguide, for example, there may be an intervening coating present on the optical waveguide. Step (ii) may optionally include providing a polymer matrix as described herein that includes both a fluorophore and a counterion moiety (one of which is covalently attached to the polymer support), and then disposing the polymer matrix on the optical waveguide. The method of providing the polymer matrix may be as described herein. For example, a method of manufacturing an optical sensor may include: (i) providing an optical waveguide; (ii) providing a polymeric support having either a fluorophore or a counterion moiety covalently attached to the polymeric support by a polymer linkage moiety, and retaining in the polymeric support the fluorophore or the counterion moiety that is not covalently attached via a polymer linkage moiety to produce a polymeric matrix as described herein; and Disposing a polymer matrix over the optical waveguide. Alternatively, step (ii) may comprise forming in situ a polymer matrix as described herein on the optical waveguide. The method of forming the polymer matrix may be as described herein. For example, a method of manufacturing an optical sensor may include: (i) providing an optical waveguide; (ii)(a) disposing a polymeric support on an optical waveguide, wherein either the fluorophore or the counterion moiety is covalently attached to the polymeric support by a polymer linking moiety; and (b) Retaining in the polymeric support either the fluorophore or the counterion moiety that is not covalently attached to the polymeric support via a polymer linkage moiety.

[0051] In a typical example, a method for manufacturing an optical sensor may include: (i) providing an optical waveguide; (ii)(a) contacting the optical waveguide with a solution comprising a fluorophore monomer or a counterion monomer with one or more other monomers to initiate polymerization to produce a polymeric support disposed on the optical waveguide, wherein either the fluorophore or counterion moiety is covalently attached to the polymeric support by a polymer linkage moiety; and (b) retaining either the fluorophore or the counterion moiety (whichever is not covalently attached to the polymeric support via a polymer linkage moiety) in the polymeric support to produce a polymeric matrix as described herein. A washing step may be carried out between (ii)(a) and (ii)(b) to remove unreacted monomer and / or waste products. It may be desirable to incorporate the membrane into the optical sensor during its manufacture, which can be done after the polymer matrix of the present invention has been produced and placed on the sensor. For example, a method of manufacturing an optical sensor may include: (i) providing an optical waveguide; (ii) disposing a polymer matrix described herein onto an optical waveguide; and (iii) Subsequently, disposing the membrane on the polymer matrix. In other embodiments, the membrane can be incorporated earlier in the procedure, for example, before the polymer matrix is ​​created. For example, in some embodiments, a method of making an optical sensor can include: (i) providing an optical waveguide; (ii)(a) contacting the optical waveguide and membrane with a solution comprising a fluorophore monomer or counterion monomer with one or more other monomers to initiate polymerization to produce a polymeric support disposed on the optical waveguide, wherein the fluorophore or counterion moiety is covalently attached to the polymeric support by a polymer linking moiety; and (b) Retaining in the polymeric support either the fluorophore or the counterion moiety that is not covalently attached to the polymeric support via a polymer linkage moiety. A solution containing the fluorophore monomer or counterion monomer along with one or more other monomers can be passed through the membrane, e.g., the membrane can be immersed in the solution. The method for manufacturing an optical sensor may include one or more additional steps after (i) and (ii), as follows: These additional steps may be performed in any order. The method of manufacturing an optical sensor may further include contacting the optical sensor with water (in the form of water vapor and / or, more preferably, liquid water), thereby bringing the water content within the sensor into equilibrium with the external water, thereby minimizing water uptake or loss by the optical sensor when the sensor is in contact with a sample containing water (e.g., an aqueous sample such as a water solution, blood, or saliva).

[0052] The method for manufacturing an optical sensor may further include disposing a reflector on the optical sensor. For example, the method for manufacturing an optical sensor may include disposing a layer including one or more of polysulfone (PSU), polyethersulfone (PESU), and polyphenylsulfone (PPSU) on the polymer matrix. When a gas-permeable membrane is disposed on the polymer matrix, the method for manufacturing an optical sensor may include disposing a layer including one or more of polysulfone (PSU), polyethersulfone (PESU), and polyphenylsulfone (PPSU) on the gas-permeable membrane. Polysulfone is preferred. The method of manufacturing an optical sensor may further include incorporating a light source and / or a detector. The method may also include incorporating a control system and / or an analysis system.

[0053] Measurement method The sensors described herein can be used to detect CO2 in a sample. Optical sensors can further be used to determine the amount of CO2 in a sample. This can be achieved through a simple intensity measurement. For example, the optical sensor's light output (typically light emitted from a fluorophore) can be calibrated using one or more solutions of known CO2 content. Thus, the intensity of the fluorescent emission at wavelength λ1, I1, can be determined as a function of CO2 content. Alternatively or additionally, the absorbance at wavelength λ1', A1, can be determined as a function of CO2 content. λ1 and λ1' are the wavelengths at which the protonated form of the fluorophore emits / absorbs light, respectively. Calibration can also be performed on the deprotonated form. When excitation light is applied to an optical sensor in contact with a sample, the resulting emission (λ1) or absorbance (λ1') is measured and can be used to determine the CO2 content of the sample. Although the sensor can be operated in either absorption or emission mode, it is preferable to use the sensor to detect fluorescent emissions, since the path length of light through the sensing region of an optical sensor is generally too short to allow for strong absorption. Thus, the present invention provides a method for measuring the CO2 content of a sample, the method comprising: (i) contacting an optical sensor described herein with a sample; (ii) providing excitation light to the sensing region via an optical waveguide; and (iii) detecting the intensity I1 of light emitted from the fluorophore at a first wavelength λ1 via the optical waveguide. Contacting the sensor with the sample typically involves flowing the sample over the sensing area of ​​the sensor or immersing the sensing area of ​​the sensor in the sample.

[0054] Alternatively, the present invention provides a method for measuring the CO2 content of a sample using an absorption method, the method comprising: (i) contacting an optical sensor described herein with a sample; (ii) providing excitation light to the sensing region via an optical waveguide; (iii) detecting light returning from the sensing region via the optical waveguide; and (iv) determining the absorbance A1 of the fluorophore at a first wavelength λ1'; The method may include an initial step of calibrating the sensor. The method may include a subsequent calculation step involving comparing the detected emission intensity I1 or absorbance A1 to a calibration curve to determine the CO2 content of the sample. A single intensity measurement is advantageously simple. The accuracy of the results may be improved by taking one or more further measurements. Thus, in some embodiments, a method for measuring the CO content of a sample comprises: (i) contacting an optical sensor described herein with a sample; (ii) providing excitation light to the sensing region via an optical waveguide; (iii) detecting the intensity I1 of light emitted by the fluorophore at a first wavelength λ1 via the optical waveguide; and (iv) detecting the intensity I2 of light emitted by the fluorophore at a second wavelength λ2 via the optical waveguide. The method may further include, for example, determining the CO content based on each of I1 and I2, and then averaging (e.g., averaging) the two values ​​to arrive at an average CO content of the sample. Intensity measurements that rely on absolute intensity are subject to inaccuracies that arise when fluorophore moieties photobleach. Photobleaching of fluorophore moieties reduces the total intensity of fluorescent emission, inaccurately indicating changes in analyte concentration. Therefore, using ratiometric measurements to measure the CO2 content of samples is particularly preferred. Ratiometric measurements reduce or avoid errors caused by photobleaching of the optical sensor during the calibration process and measurement.

[0055] If the protonated and deprotonated forms of a fluorophore each undergo fluorescence emission at different wavelengths and / or if the protonated and deprotonated forms of a fluorophore absorb light at different wavelengths, a ratiometric method can be used. In the ratiometric method, the emission intensities (or absorbances) of the protonated and deprotonated forms are compared, and the ratio of these two emission intensities (or absorbances) is used to determine the relative amounts of the protonated and deprotonated fluorophore. Therefore, the CO2 content of the sample can be determined. Determining the CO2 content of the sample can be performed by calibrating the optical sensor (i.e., by determining the output of the sensor when contacted with one or more samples of known CO2 content), or the CO2 content of the sample can be determined based on a mathematical model of the system. Like simple intensity measurements, ratiometric measurements can be performed in absorption or emission mode, however, it is preferred to perform measurements in emission mode because the path length of light through the sensitive region of an optical sensor is generally too short in practice to allow for significant absorption. Thus, the present invention provides a method for measuring the CO2 content of a sample, the method comprising: (i) contacting an optical sensor described herein with a sample; (ii) providing excitation light to the sensing region via an optical waveguide; (iii) detecting the intensity I1 of light emitted by the fluorophore at a first wavelength λ1 via the optical waveguide; (iv) detecting the intensity I of light emitted by the fluorophore at a second wavelength λ via the optical waveguide; and (v) Comparison of I2 and I1. The method may include an initial step of calibrating the sensor. The method may include a subsequent calculation step involving comparing the detected emission intensity ratio I1:I2 to a calibration curve to determine the CO2 content of the sample. Each optical measurement is performed quickly, typically taking less than a second. Furthermore, the sample volume of the sensor's sensing area is very small and typically does not function as a sample reservoir. Therefore, the sensor's response time is very fast. Furthermore, optical sensors do not affect the sample except for removing very small amounts of analyte from it, so they do not substantially degrade or deplete the sample. Therefore, the sensor may be placed in contact with a biological sample (such as saliva or blood) within the body or within a patient's body, and typically is removed and returned from the patient's body. All these factors mean that the sensor is highly suitable for performing long-term continuous measurements on samples, particularly biological samples. Thus, in some embodiments, the method for measuring the CO content of a sample is a method for continuously measuring the CO content of a sample, wherein: - the optical sensor is continuously exposed to the sample for an exposure time of at least 10 minutes; - excitation light is provided to the sensing region via an optical waveguide either continuously or intermittently throughout the exposure period; and - the intensity I1 of the emission of the fluorophore at the first wavelength λ1 and optionally the intensity I2 of the emission of the fluorophore at the second wavelength λ2 are detected via the optical waveguide continuously or intermittently throughout the exposure period. The exposure time is preferably at least 1 hour. For example, the exposure period can be at least 100 hours, typically up to 144 hours (7 days). Therefore, this measurement method can be used in situations where long-term monitoring of biological samples (such as blood) is important, such as during dialysis or open heart surgery.

[0056] example Positively charged counterion sites covalently attached to the polymer support were generated as follows.

[0057] Process (a) Styrene (1.0 mass equivalent) and vinylbenzyl chloride (0.052 mass equivalent) were dissolved in toluene (55.7 volumes). The solution was stirred, and nitrogen was bubbled through the solution using a nitrogen dip tube for 30 minutes. The nitrogen dip tube was then removed, and the initiator AIBN (0.002 mass equivalent) was added. The solution was then heated at 60°C for 18 hours. The resulting polymer, poly(styrene-co-vinylbenzyl chloride), was then precipitated from the solution by the addition of methanol to give a crude material, which was twice dissolved in chloroform and precipitated with methanol. The resulting precipitate was then dried under high vacuum.

[0058] Process (b) The polymer support, poly(styrene-co-vinylbenzyl chloride) (1.0 equiv.) was dissolved in anhydrous DMF (20 vol.) and stirred under nitrogen until completely dissolved. The counterion site precursor, 45% aqueous trimethylamine (4.4 vol.), was added, and the resulting mixture was stirred overnight at room temperature. After stirring for 20 h, an IPC was obtained consistent with the formation of a new product. The solvent was removed under vacuum at 60 °C. The resulting product was poly(styrene-co-(vinylbenzyl)trimethylammonium hydroxide), containing 5 wt. % vinylbenzyltrimethylammonium hydroxide.

[0059] Process (c) Poly(styrene-co-(vinylbenzyl)trimethylammonium hydroxide) (500 mg) was dissolved in DCM (50 ml) and washed three times with NaOH (0.1 M, 50 ml), then the solvent was removed under vacuum.

[0060] The reaction scheme including steps (a) to (c) is shown below. [ka]

[0061] Next, a polymer matrix further containing the fluorophore moiety was prepared by dissolving poly(styrene-co-(vinylbenzyl)trimethylammonium hydroxide) (5 wt% vinylbenzyl)trimethylammonium hydroxide) (30 mg) in dichloromethane (600 μl). The fluorophore moiety HTPS (2 μl, 10 mg.ml in water) was added, and the mixture was shaken to extract the HTPS into the organic phase. The sensor was then prepared by immersing the tip of an optical waveguide (specifically an optical fiber) in the solution and drying the solution to form a polymer matrix at the tip of the optical fiber. The sensor was then tested in a CO2-containing environment. The bonding curves and performance data for these fibers are shown in Figures 4 and 5. Figure 4 shows the binding curves of CO2 to the polymer matrix in the sensor of the present invention as a function of pCO2. The percentage of CO2 that binds to the matrix agrees very well with the predicted value, indicating that the sensor effectively detects CO2. Figure 5 shows the actual pCO2 values ​​and the pCO2 values ​​measured by a sensor according to the present invention over a 5-hour period. Also shown in this figure is the temperature, which remained constant between 35 and 40°C over the test period. It is clear that the actual pCO2 values ​​(partial pressure of CO2 in the test sample), shown by the dotted line, closely match the values ​​detected using a sensor according to the present invention (shown by the solid line). The accuracy of the sensor according to the present invention is excellent. The sensor was found to be able to detect pCO2 values ​​within 3.67% accuracy over the test range (0.0-349.3 mmHg, 0-50%).

[0062] The following is further disclosed in relation to the present invention. [1] a CO molecule comprising a fluorophore, a positively charged counterion moiety, and a polymeric support, wherein either the fluorophore or the counterion moiety is covalently attached to the polymeric support by a polymer linkage moiety. 2 Polymer matrices for optical sensing of. [2] The polymer matrix according to [1], wherein the molar ratio of counterion to fluorophore is 2:1 or greater. [3] The fluorophore is preferably attached to a negatively charged site at near-neutral pH, preferably -O - group or -COO- The polymer matrix according to [1] or [2], which contains a group. [4] The polymer matrix according to any one of [1] to [3], wherein either the fluorophore or the counter ion moiety that is not covalently bound to the polymer support by a polymer linking moiety is retained in the polymer support. [5] The polymer matrix according to any one of [1] to [4], wherein the fluorophore has a pH-dependent fluorescence emission spectrum. [6] The fluorophore is irradiated at a first wavelength λ 1 Intensity I 1 and a second wavelength λ 2 Intensity I 2 can undergo fluorescence emission of I 1 vs. I 2 The polymer matrix according to any one of [1] to [5], wherein the ratio of changes depending on pH. [7] The polymer matrix according to any one of [1] to [6], wherein the fluorophore comprises a species of formula (FI-A):

change

change

[10] The polymer matrix according to any one of [1] to [9], wherein the counter ion site contains a quaternary ammonium ion.

[11] The polymer matrix according to any one of [1] to

[10] , wherein the counter ion site contains a quaternary ammonium cation of formula (CI-A).

change

[12] The polymer matrix according to any one of [1] to

[11] , wherein the counter ion site is a hexadecyltrimethylammonium ion or a derivative thereof.

[13] The polymer matrix according to any one of [1] to

[12] , wherein the polymer support is gas permeable.

[14] The polymer matrix according to any one of [1] to

[13] , wherein the polymer support comprises a hydrophilic polymer, preferably the polymer support comprises one or more of a hydrogel, a cellulose derivative, an ethyl cellulose, a sol-gel, or a hydrophilic silicone-based polymer, preferably a hydrogel selected from polyacrylamide or polyhydroxyethyl methacrylate.

[15] The polymer matrix according to any one of [1] to

[13] , wherein the polymer support comprises a hydrophobic polymer.

[16]

[15] The polymer matrix according to

[15] , wherein the polymer support comprises one or more of polystyrene, a hydrophobic silicone-based polymer, a hydrophobic cellulose derivative, or plasticized ethyl cellulose.

[17] The polymer matrix according to

[15] or

[16] , wherein the polymer support is hydrophobic.

[18] The polymer linking site is a covalent bond, -O-, -S-, or -SO 2 -, -SO-, optionally substituted alkylene, optionally substituted alkenylene, optionally substituted alkynylene, and -NR 5 including one or more of; R 5 is H, C 1-10 Alkyl, C 3-10 Cycloalkyl, C 4-10 Cycloalkenyl, aryl, arylalkyl, heteroaryl, heterocycloalkyl, C 2-10 Alkenyl, C 2-10 Alkynyl, -COR a , -CO 2 R a , and -CONR a 2 selected from; and R a The polymer matrix according to any one of [1] to

[17] , wherein is as defined in [7].

[19] The polymer matrix according to any one of [1] to

[18] , wherein the polymer matrix also contains water.

[20] CO 2 An optical sensor for optically detecting - a sensing region comprising the polymer matrix according to any one of [1] to

[19] ; ​​and an optical waveguide arranged to direct light onto the sensing area; The optical sensor.

[21] Blood CO 2

[20] The optical sensor according to

[20] , which is a sensor for detecting content.

[22] 20. The optical sensor of claim 21, wherein the sensing region further comprises a hydrophobic membrane, and the sensor is configured to allow an analyte to enter the sensing region through the hydrophobic membrane.

[23] The polymeric support comprises a hydrophilic polymer; the sensing region further comprises a gas-permeable hydrophobic membrane that is impermeable to liquids and hydrogen ions; and the sensor is 2 The optical sensor according to any one of

[20] to

[22] , wherein the optical sensor is configured so that the hydrophobic membrane can pass through the hydrophobic membrane and enter the sensing region.

[24] The polymeric support comprises a hydrophobic polymer; the sensing region further comprises a gas-permeable hydrophobic membrane that is impermeable to liquids and hydrogen ions; and the sensor is 2 The optical sensor according to any one of

[20] to

[22] , wherein the optical sensor is configured so that the hydrophobic membrane can pass through the hydrophobic membrane and enter the sensing region.

[25] CO 2 1. A method for producing a polymer matrix for optical detection of (i) providing a polymeric support, wherein either the fluorophore or the counterion moiety is covalently attached to the polymeric support by a polymer linkage moiety; and (ii) retaining in the polymeric support either the fluorophore or the counterion moiety that is not covalently attached to the polymeric support via a polymer linkage moiety; The method comprising:

[26] The method according to

[25] , wherein the polymer matrix for optical detection is as defined in any one of [1] to

[19] .

[27] CO 2 A method for manufacturing an optical sensor for detecting light comprising the steps of: (i) providing an optical waveguide; (ii) disposing a polymer matrix according to any one of [1] to

[19] on an optical waveguide; and (iii) optionally disposing a membrane on the polymer matrix; The method comprising:

[28] The method according to

[27] , wherein the optical sensor is as described in any one of

[20] to

[24] .

[29] Sample CO 2 A method for measuring the content, comprising: (i) contacting the optical sensor according to any one of

[20] to

[24] with a sample; (ii) providing excitation light to the sensing region via an optical waveguide; and (iii) transmitting a first wavelength λ via an optical waveguide; 1 is the intensity of light emitted from the fluorophore at I 1 Detecting The method comprising:

[30] moreover, (iv) a second wavelength λ 2 is the intensity of light emitted from the fluorophore at I 2 Detecting; and (v) In some cases, I 2 and I 1 To compare The method according to

[29] , comprising:

[31] Sample CO 2 The method according to

[29] or

[30] , which is a method for continuously measuring the content, comprising: - The optical sensor is in continuous contact with the sample for an exposure period of at least 10 minutes; - excitation light is provided to the sensing region via an optical waveguide either continuously or intermittently throughout the exposure period; and - first wavelength λ 1 Intensity of fluorophore emission at I 1 , and optionally a second wavelength λ 2 Intensity of fluorophore emission at I 2 is detected continuously or intermittently throughout the exposure period via the optical waveguide; The method.

Claims

1. a CO2 molecule comprising a fluorophore, a positively charged counterion moiety, and a polymeric support, wherein either the fluorophore or the counterion moiety is covalently attached to the polymeric support by a polymer linking moiety; ２ A polymer matrix for optical sensing of The polymer matrix as described above, wherein the molar ratio of counterion to fluorophore is 2:1 or greater.

2. The polymer matrix of claim 1 , wherein the fluorophore comprises a negatively charged moiety at near-neutral pH.

3. The polymer matrix according to claim 2, wherein the negatively charged site is an —O − group or a —COO − group.

4. 4. The polymer matrix of claim 1, wherein either the fluorophore or the counterion moiety that is not covalently attached to the polymer support by a polymer linkage moiety is retained in the polymer support.

5. The polymer matrix of any one of claims 1 to 4, wherein the fluorophore has a pH-dependent fluorescence emission spectrum.

6. The fluorophore is irradiated at a first wavelength λ １ Intensity I １ and a second wavelength λ ２ Intensity I ２ and I １ Against I ２ The polymer matrix according to any one of claims 1 to 5, wherein the ratio of

7. The polymer matrix of any one of claims 1 to 6, wherein the fluorophore comprises a species of formula (FI-A): 【Chemical FI-A】 where each R group is H, halogen, cyano, nitro, hydroxy, C １－１０ Alkyl, C ３－１０ Cycloalkyl, C ４－１０ Cycloalkenyl, aryl, alkylaryl, heteroaryl, heterocycloalkyl, C ２－１０ Alkenyl, C ２－１０ Alkynyl, -SR ａ , -OR ａ , -SO ２ R ａ , -SO ３ － , -SOR ａ , -SO ２ NR ａ , -S(O)(NR ａ ) R ａ , -NR ａ ２ , -NR ａ COR ａ , -NR ａ CO ２ R ａ , -COR ａ , -CO ２ R ａ , -CONR ａ ２ or a covalent bond attaching the fluorophore to the polymer linkage site; or two adjacent R groups joined to form a carbocyclic or heterocyclic ring; Each R ａ The groups are H, C １－１０ Alkyl, C ３－１０ Cycloalkyl, C ４－１０ Cycloalkenyl, aryl, arylalkyl, heteroaryl, heterocycloalkyl, C ２－１０ alkenyl, and C ２－１０ independently selected from alkynyl; The optional substitutable R groups are halogen, oxo, cyano, nitro, hydroxy, C １－１０ Alkyl, C ３－１０ Cycloalkyl, C ４－１０ Cycloalkenyl, C ２－１０ Alkenyl, C ２－１０ Alkynyl, -OR ｂ , -NR ｂ ２ , -COR ｂ , -CO ２ R ｂ , and -CONR ｂ ２ and optionally substituted by one or more substituents independently selected from: Here, R ｂ is H, C １－６ Alkyl, C ３－６ Cycloalkyl, C ４－６ cycloalkenyl, and C ２－６ alkenyl.

8. The polymer matrix of any one of claims 1 to 7, wherein the fluorophore comprises a species of formula (FI-B): 【Chemical FI-B】 Here, each R １ The groups include halogen, cyano, nitro, hydroxy, -OR ａ , -SO ２ R ａ , -SO ３ － , -SOR ａ , -SO ２ NR ａ , -S(O)(NR ａ ) R ａ , -NR ａ ２ , -NR ａ COR ａ , -NR ａ CO ２ R ａ , -COR ａ , -CO ２ R ａ , -CONR ａ ２ and a covalent bond to a polymer linkage moiety; Each R ２ The groups are H, C １－１０ Alkyl, C ３－１０ Cycloalkyl, C ４－１０ Cycloalkenyl, aryl, alkylaryl, heteroaryl, heterocycloalkyl, C ２－１０ Alkenyl, C ２－１０ independently selected from alkynyl, and a covalent bond to the polymer linkage moiety; Any substitutable R １ or R ２ The groups include halogen, oxo, cyano, nitro, hydroxy, C １－１０ Alkyl, C ３－１０ Cycloalkyl, C ４－１０ Cycloalkenyl, C ２－１０ Alkenyl, C ２－１０ Alkynyl, -OR ｂ , -NR ｂ ２ , -COR ｂ , -CO ２ R ｂ , and -CONR ｂ ２ and R ａ and R ｂ is as defined in claim 7.

9. The polymer matrix according to any one of claims 1 to 8, wherein the fluorophore is pyranine or a derivative thereof.

10. The polymer matrix of any one of claims 1 to 9, wherein the counter ionic moieties are covalently attached to the polymer support.

11. The polymer matrix of any one of claims 1 to 10, wherein the counterion sites comprise quaternary ammonium ions.

12. The polymer matrix of any one of claims 1 to 11, wherein the counterion sites comprise quaternary ammonium cations of formula (CI-A): 【Chemical CI-A】 Here, each R ４ The groups are independently H; or C １－３０ Alkyl, C ３－１２ Cycloalkyl, C ４－１２ Cycloalkenyl, aryl, alkylaryl, heteroaryl, heterocycloalkyl, C ２－３０ Alkenyl, C ２－３０ alkynyl, and a covalent bond to the polymer attachment site; or two R ４ The group is bonded to C ３－１６ Heterocycloalkyl or C ４－１６ may form a heterocycloalkenyl group; Any substitutable R ４ The groups include halogen, oxo, cyano, nitro, hydroxy, C １－１０ Alkyl, C ３－１０ Cycloalkyl, C ４－１０ Cycloalkenyl, C ２－１０ Alkenyl, C ２－１０ Alkynyl, -OR ｂ , -NR ｂ ２ , -COR ｂ , and —CO ２ R ｂ optionally substituted by one or more substituents independently selected from Here, R ｂ is as defined in claim 7.

13. The polymer matrix of any one of claims 1 to 12, wherein the counter ion moiety is a hexadecyltrimethylammonium ion or a derivative thereof.

14. One R ４ The R group is a covalent bond to the polymer linkage site, and the remaining three R ４ The groups are each independently H, C １－２０ Alkyl, and C ２－２０ The polymer matrix of claim 12, wherein the alkyl group is selected from alkenyl.

15. The polymer matrix of any one of claims 1 to 14, wherein the polymer support is gas permeable.

16. The polymer matrix of any one of claims 1 to 15, wherein the polymer support comprises a hydrophilic polymer.

17. The polymer matrix of claim 16, wherein the polymer support comprises a hydrogel selected from polyacrylamide or polyhydroxyethyl methacrylate.

18. The polymer matrix of any one of claims 1 to 15, wherein the polymer support comprises a hydrophobic polymer.

19. 20. The polymer matrix of claim 18, wherein the polymer support comprises one or more of polystyrene, a hydrophobic silicone-based polymer, a hydrophobic cellulose derivative, or plasticized ethyl cellulose.

20. 20. The polymer matrix of claim 18 or 19, wherein the polymer support is hydrophobic.

21. The polymer linking site is a covalent bond, —O—, —S—, or —SO ２ -, -SO-, alkylene, substituted alkylene, alkenylene, substituted alkenylene, alkynylene, substituted alkynylene, and -NR ５ including one or more of: R ５ is H, C １－１０ Alkyl, C ３－１０ Cycloalkyl, C ４－１０ Cycloalkenyl, aryl, arylalkyl, heteroaryl, heterocycloalkyl, C ２－１０ Alkenyl, C ２－１０ Alkynyl, —COR ａ , -CO ２ R ａ , and -CONR ａ ２ is selected from: R ａ A polymer matrix according to any one of claims 1 to 20, wherein is as defined in claim 7.

22. The polymer matrix of any one of claims 1 to 21, wherein the polymer matrix also comprises water.

23. CO ２ An optical sensor for optically detecting - a sensing area comprising a polymer matrix according to any one of claims 1 to 22; and - an optical waveguide arranged to direct light onto the sensing area; The optical sensor comprising:

24. CO in blood ２ 24. The optical sensor of claim 23, which is a sensor for detecting content.

25. 25. The optical sensor of claim 23 or claim 24, wherein the sensing region further comprises a hydrophobic membrane, the sensor configured to allow an analyte to enter the sensing region through the hydrophobic membrane.

26. The polymeric support comprises a hydrophilic polymer; the sensing region further comprises a gas-permeable hydrophobic membrane that is impermeable to liquids and hydrogen ions; and the sensor ２ The optical sensor according to any one of claims 23 to 25, wherein the optical sensor is configured so that the hydrophobic membrane can pass through the sensing region.

27. The polymeric support comprises a hydrophobic polymer; the sensing region further comprises a gas-permeable hydrophobic membrane that is impermeable to liquids and hydrogen ions; and the sensor ２ The optical sensor according to any one of claims 23 to 25, wherein the optical sensor is configured so that the hydrophobic membrane can pass through the sensing region.

28. CO ２ 1. A method for producing a polymer matrix for optical detection of (i) providing a polymeric support, wherein either the fluorophore or the counterion moiety is covalently attached to the polymeric support by a polymer linkage moiety; and (ii) retaining in the polymeric support either the fluorophore or the counterion moiety that is not covalently attached to the polymeric support via a polymer linkage moiety; The method comprising: The method, wherein the polymer matrix for optical sensing is as defined in any one of claims 1 to 22.

29. CO ２ A method for manufacturing an optical sensor for detecting light comprising the steps of: (i) providing an optical waveguide; and (ii) disposing a polymer matrix according to any one of claims 1 to 22 on an optical waveguide; The method comprising:

30. (iii) disposing a membrane on a polymer matrix; 30. The method of claim 29, further comprising:

31. The method according to claim 29 or 30, wherein the optical sensor is as defined in any one of claims 23 to 27.

32. Sample CO ２ A method for measuring the content, comprising: (i) contacting an optical sensor according to any one of claims 23 to 27 with a sample; (ii) providing excitation light to the sensing region via an optical waveguide; and (iii) transmitting a first wavelength λ via the optical waveguide; １ is the intensity of light emitted from the fluorophore at １ Detecting The method comprising:

33. moreover, (iv) transmitting a second wavelength λ via the optical waveguide; ２ is the intensity of light emitted from the fluorophore at ２ Detecting 33. The method of claim 32, comprising:

34. Furthermore, (v) comparing I 2 with I 1 ; 34. The method of claim 33, comprising:

35. CO of the sample ２ The method according to any one of claims 32 to 34, which is a method for continuously measuring the content, comprising: - the optical sensor is in continuous contact with the sample for an exposure period of at least 10 minutes; - excitation light is provided to the sensing area via an optical waveguide either continuously or intermittently throughout the exposure period; and - first wavelength λ １ Intensity I of the emission of the fluorophore at １ , and a second wavelength λ ２ Intensity I of the emission of the fluorophore at ２ is detected continuously or intermittently throughout the exposure period via the optical waveguide; The method.

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