Methods for detecting per- and poly-fluoroalkyl substances with naphthalene diimide-based fluorophores

Abstract

A method for detecting and discriminating between per- and poly-fluoroalkyl substances (PFAS) in an aqueous sample includes exciting the aqueous sample alone at a wavelength of between 330 - 390 nm, contacting the sample with a detection reagent comprising a naphthalene diimide-based cationic fluorophore, exciting the detection reagent and sample together at the excitation wavelength, and quantifying a change in a fluorescence emission intensity of the detection reagent while in contact with the sample relative to the fluorescence intensity of the detection reagent prior to being contacted with the sample.

Description

METHODS FOR DETECTING PER- AND POLY-FLUOROALKYL SUBSTANCES WITH NAPHTHALENE DIIMIDE-BASED FLUOROPHORESCROSS EFERENCE TO RELATED APPLICATION

[0001] This application claims priority under 35 U. S. C. §119(e) upon U. S. Provisional Patent Application No. 63 / 715,824, entitled "METHODS FOR DETECTING PER- AND POLYFLUOROALKYL SUBSTANCES WITH NAPHTHALENE DIIMIDE-BASED FLUOROPHORES" filed on November 4, 2024, by Ling Zang et al., the entire disclosure of which is incorporated herein by reference.FIELD OF THE DISCLOSURE

[0002] The present disclosure generally relates to methods for detecting per- and polyfluoroalkyl substances (PFAS) in a sample, and more particularly to methods for detecting PFAS with naphthalene diimide-based ratiometric fluorophores.B CKGROU D

[0003] Per- and poly-fluoroalkyl substances (PFAS), also known as "forever chemicals", are an emerging class of pollutants widely present in surface / ground waters and soils. These compounds have been used for over 60 years in hundreds of industrial applications and consumer products (e.g., carpet, apparel, upholstery, cookware, food wrappers, and aqueous fire-fighting foams [AFFFs)]). More than 9,000 PFAS have been identified, with perfluorooctanesulfonic acid (PFOS) and perfluorooctanoic acid (PFOA) being the most used and widely studied. The term "forever chemicals" reflects the high chemical stability of PFAS, which renders them highly resistant to hydrolysis, biodegradation, metabolism, photolysis (sunlight), and other degradation processes. The U. S. and European countries have begun to ban the manufacturing and use of many PFAS, but efforts to remediate the global spread of PFAS will likely take several decades.

[0004] Currently, the official health advisory level set by the U. S. Environmental Protection Agency (EPA) for PFOA and PFOS in drinking water is 70 parts per trillion (ppt), a level that may be markedly lowered to 0.004 ppt for PFOA and 0.02 ppt for PFOS according to an advisory set on June 15, 2022, pending the results of ongoing health-related studies. According to recent speculation, implementing new guidelines and regulations may result in water everywhere, including fresh rainwater, being labeled as contaminated and unfit forconsumption and use. To this point, new methods will be required that are highly sensitive for the detection of PFAS. These methods will also need to be highly selective for specific PFAS, such as PFOA and PFOS, so that appropriate remediation techniques can be used and the sources of contamination can be ascertained. Improved detection methods also may aid efforts to gauge the range and scope of the geographic distribution of PFAS contamination and assist in monitoring the efficiency of treatment.

[0005] Current PFAS detection methods are poorly suited for field deployment due in part to requirements such as derivatization prior to analysis, lengthy sample preparation, cost, specialized laboratories, trained personnel, and instrument maintenance. Predominant methods include mass spectrometry (MS)-based ex-situ laboratory techniques. Commonly used modes include analytical scale extraction and subsequent analysis by liquid chromatography tandem mass spectrometry (LC-MS / MS) and total oxidizable precursor (TOP). An alternative approach to MS involves the analysis of total fluorine by particle induced gamma ray emission (PIGE) spectroscopy. These methods are not portable, and, therefore, are poorly suited to use in the field. Moreover, the limit of detection (LOD)forthe most recent EPA-validated methods is 0.3 ppt for PFOS and PFOA, which falls short of the advisory level by nearly one hundred-fold.

[0006] Additionally, prior probes and sorbents for capture and detection of PFAS have exhibited poor uptake, slow kinetics, and / or poor selectivity. Some prior probes have been based on a general electronic affinity for electron rich hydrophobic, groups (e.g., perfluoroalkyl or similar species). While prior sensors can detect PFOS and PFOA in simple matrices (DI water and drinking water), this nonspecific binding limits both selectivity and sensitivity in practical matrices (e.g., for field deployment), due to the inability to screen out interferences with similar electronic properties and hydrophobicity.SUMMARY OF THE DSSCLOSURE

[0007] According to one aspect of the present disclosure, a method for detecting and discriminating between per- and poly-fluoroalkyl substances (PFAS) in an aqueous sample includes exciting the aqueous sample alone at a wavelength of between 330 - 390 nm, contacting the sample with a detection reagent comprising a naphthalene diimide-based cationic fluorophore, exciting the detection reagent and sample together at the excitation wavelength, and quantifying a change in a fluorescence emission intensity of the detectionreagent while in contact with the sample relative to the fluorescence intensity of the detection reagent prior to being contacted with the sample.

[0008] According to another aspect of the present disclosure, a fluorophore for detecting per- and poly-fluoroalky substances (PFAS) in an aqueous sample includes a fluorophore of Formula (I)

[0009] According to another aspect of the present disclosure, a fluorophore for detecting perand poly-fluoroalkyl substances (PFAS) in an aqueous sample includes a fluorophore of Formula (la)(la).

[0010] These and other features, advantages, and objects of the present disclosure will be further understood and appreciated by those skilled in the art by reference to the following specification, claims, and appended drawings.

[0011] In the drawings:

[0012] FIG. 1 illustrates an exemplary synthetic scheme for a precursor to naphthalene diimide (NDI), according to aspects of the present disclosure;

[0013] FIG. 2 illustrates schematic chemical structures of multiple naphthalene diimide (NDI)- based fluorophores synthesized according to various aspects of the present disclosure:

[0014] FIG. 3 illustrates a synthesis scheme for a naphthalene diimide (NDI-C1) fluorophore, according to aspects of the present disclosure;

[0015] FIG. 4 is an electrospray ionization mass spectrum of a naphthalene diimide (NDI-C1) fluorophore, according to aspects of the present disclosure;

[0016] FIG. 5 is a synthesis scheme for a naphthalene diimide (NDI-C3) fluorophore, according to aspects of the present disclosure;

[0017] FIG. 6 is an electrospray ionization mass spectrum of a naphthalene diimide (NDI-C3) fluorophore, according to aspects of the present disclosure;

[0018] FIG. 7 is a proton nuclear magnetic resonance (1H-NMR) spectrum of the NDI-C3 probe, according to aspects of the present disclosure;

[0019] FIG. 8 is a synthesis scheme for a naphthalene diimide (NDI-C4) fluorophore, according to aspects of the present disclosure;

[0020] FIG. 9 is an electrospray ionization mass spectrum of a naphthalene diimide (NDI-C4) fluorophore, according to aspects of the present disclosure;

[0021] FIG. 10 is a proton nuclear magnetic resonance (1H-NMR) spectrum of a naphthalene diimide (NDI-C4) fluorophore, according to aspects of the present disclosure;

[0022] FIG. 11 is a synthesis scheme for a naphthalene diimide (NDI-C6) fluorophore, according to aspects of the present disclosure;

[0023] FIG. 12 is an electrospray ionization mass spectrum of a naphthalene diimide (NDI-C6) fluorophore, according to aspects of the present disclosure;

[0024] FIG. 13 is a proton nuclear magnetic resonance (1H-NMR) spectrum of a naphthalene diimide (NDI-C6) fluorophore, according to aspects of the present disclosure;

[0025] FIG. 14 is a synthesis scheme for a naphthalene diimide (NDI-C8) probe, according to aspects of the present disclosure;

[0026] FIG. 15 is an electrospray ionization mass spectrum of a naphthalene diimide (NDI-C8) fluorophore, according to aspects of the present disclosure;

[0027] FIG. 16 is a proton nuclear magnetic resonance (1H-NMR) spectrum of a naphthalene diimide (NDI-C8) fluorophore, according to aspects of the present disclosure;

[0028] FIG. 17 is a synthesis scheme for a naphthalene diimide (NDI-C10) fluorophore, according to aspects of the present disclosure;

[0029] FIG. 18 is an electrospray ionization mass spectrum of a naphthalene diimide (NDI- C10) fluorophore, according to aspects of the present disclosure;

[0030] FIG. 19 is a synthesis scheme for a naphthalene diimide (NDI-C12) fluorophore, according to aspects of the present disclosure;

[0031] FIG. 20 is an electrospray ionization mass spectrum of a naphthalene diimide (NDI- C12) fluorophore, according to aspects of the present disclosure;

[0032] FIG. 21 is a proton nuclear magnetic resonance (1H-NMR) spectrum of a naphthalene diimide (NDI-C12) fluorophore, according to aspects of the present disclosure;

[0033] FIG. 22A is a graph illustrating a series of fluorescence emission spectra of a naphthalene diimide (NDI-C1) fluorophore in the presence of varying concentrations of PFOS in water, according to aspects of the present disclosure;

[0034] FIG. 22B is a graph illustrating a series of fluorescence emission spectra of a naphthalene diimide (NDI-C1) fluorophore in the presence of varying concentrations of perfluorohexanesulfonic acid (PFHxS) in water, according to aspects of the present disclosure;

[0035] FIG. 22C is a graph illustrating a series of fluorescence emission spectra of a naphthalene diimide (NDI-C1) fluorophore in the presence of varying concentrations of perfluorobutanesulfonic acid (PFBS) in water, according to aspects of the present disclosure;

[0036] FIG. 23A is a graph illustrating a series of fluorescence emission spectra of a naphthalene diimide (NDI-C8) fluorophore in the presence of varying concentrations of perfluorooctanesulfonic acid (PFOS) in water, according to aspects of the present disclosure;

[0037] FIG. 23B is a graph illustrating a series of fluorescence emission spectra of a naphthalene diimide (NDI-C8) fluorophore in the presence of varying concentrations of perfluorohexanesulfonic acid (PFHxS) in water, according to aspects of the present disclosure;

[0038] FIG. 23C is a graph illustrating a series of fluorescence emission spectra of a naphthalene diimide (NDI-C8) fluorophore in the presence of varying concentrations of perfluorobutanesulfonic acid (PFBS) in water, according to aspects of the present disclosure;

[0039] FIG. 24 is a bar graph showing the emissions intensity ratio of the measurement of two wavelengths in fluorescence emission spectra as a function of the alkyl chain length on a naphthalene diimide (NDl-based) cationic fluorophores showing the ratiometric change towards perfluorooctanesulfonic acid (PFOS), according to aspects of the present disclosure;

[0040] FIG. 25 is a bar graph showing the ratiometric fluorescence of a naphthalene diimide (NDI-C8) in the presence of various analytes, according to aspects of the present disclosure;

[0041] FIG. 26 is a graph showing the ratiometric fluorescence of a naphthalene diimide (NDI-C8) in varying concentrations of perfluorooctanesulfonic acid (PFOS) in pure water, according to aspects of the present disclosure;

[0042] FIG. 27 A is a principal component analysis plot generated from a naphthalene diimide (NDI-C8) fluorophore tested with varying concentrations of GenX, perfluorooctanoic acid (PFOA), and perfluorobutanesulfonic acid (PFBS), according to aspects of the present disclosure;

[0043] FIG. 27B is a principal component analysis plot generated from a naphthalene diimide (NDI-C8) fluorophore tested with varying concentrations of perfluorooctanesulfonic acid (PFOS), perfluorohexanesulfonic acid (PFHxS), and perfluorobutanesulfonic acid (PFBS), according to aspects of the present disclosure;

[0044] FIG. 28A is a bar graph showing the emissions intensity ratio of the measurement of two wavelengths in fluorescence emission spectra as a function of the alkyl chain length on a naphthalene diimide (NDI)-based cationic fluorophores showing the ratiometric change towards different per- and polyfluoroalkyl substances (PFASs), according to aspects of the present disclosure;

[0045] FIG. 28B is a heat map showing the emissions intensity ratio of the measurement of two wavelengths in fluorescence emission spectra as a function of the alkyl chain length on a naphthalene diimide (NDI)-based cationic fluorophores as dependent on different per- and polyfluoroalkyl substances (PFASs), according to aspects of the present disclosure;

[0046] FIG. 29A is a principal component analysis plot generated from a naphthalene diimide (NDI)-based fluorophores tested with varying concentrations of different per- and polyfluoroalkyl substances (PFASs), according to aspects of the present disclosure; and

[0047] FIG. 29B is a hierarchical clustering analysis generated from a naphthalene diimide (NDI)-based fluorophores tested with varying concentrations of different per- and polyfluoroalkyl substances (PFASs), according to aspects of the present disclosure.

[0048] The present illustrated embodiments reside primarily in combinations of method steps and apparatus components related to fluorescent sensors for detection of per- and poly-fluoroalkyl substances (PFAS) in a water sample. Accordingly, the apparatus components and method steps have been represented, where appropriate, by conventional symbols In the drawings, showing only those specific details that are pertinent to understanding theembodiments of the present disclosure so as not to obscure the disclosure with details that will be readily apparent to those of ordinary skill in the art having the benefit of the description herein. Further, like numerals in the description and drawings represent like elements.

[0049] For purposes of description herein, the terms "upper," "lower," "right," "left," "rear," "front," "vertical," "horizontal," and derivatives thereof, shall relate to the disclosure as oriented in FIG. 1. Unless stated otherwise, the term "front" shall refer to the surface of the device closer to an intended viewer of the device, and the term "rear" shall refer to the surface of the device further from the intended viewer of the device. However, it is to be understood that the disclosure may assume various alternative orientations, except where expressly specified to the contrary. It is also to be understood that the specific devices and processes illustrated in the attached drawings, and described in the following specification are simply exemplary embodiments of the inventive concepts defined in the appended claims. Hence, specific dimensions and other physical characteristics relating to the embodiments disclosed herein are not to be considered as limiting, unless the claims expressly state otherwise.

[0050] The terms "including," "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. An element preceded by "comprises a.. does not, without more constraints, preclude the existence of additional identical elements in the process, method, article, or apparatus that comprises the element.

[0051] The present disclosure relates to a fluorescence-based detection method for detecting and discriminating between per- and poly-fluoroalky! substances (PFAS) in a water sample using naphthalene diimide (NDI)-based cationic fluorophores. The target PFAS substances include, but are not limited to, perfluorooctanesulfonic acid (PFOS), perfluorohexanesulfonic acid (PFHxS), perfluorobutanesulfonic acid (PFBS), pefluorononanoic acid (PFNA), perfluorooctanoic acid (PFOA), and the ammonium salt of hexafluoropropylene oxide dimer acid (GenX™). In some implementations, the NDI molecule is modified with cationic side groups at both imide (carbonyl-nitrogen-carbonyl) positions. Further, the ND! molecule may be modified with alkyl side chains. In some implementations, the ND! molecule is modifiedwith cationic functional groups at both imide positions and then further modified with alkyl side chains. These methods of detection are both highly sensitive (having limits of detection of between about 1 and about 50 ppb) and highly selective, enabling sensitive detecting and discrimination of a variety of PFAS analogues. In some examples, the ND! -based fluorophore is specifically a water-soluble cationic napthalene diimide derivative such as NDI-C1, NDI-C3, NDI-C4, NDI-C6, NDI-C8, NDI-C10 and NDI-C12, which are based on a napthalene diimide (NDI) structure modified with quaternary amine functional groups and terminal alkyl side groups having 1-12 carbon atoms. In some aspects, a limit of detection (LOD) for NDI-C8 can reach as low as 8.2 nM (4.1 ppb).

[0052] Definitions of specific functional groups and chemical terms are described in more detail below.

[0053] The term "alkyl," as used herein, means a straight or branched, saturated hydrocarbon chain. The term "lower alkyl" or "C1-6alkyl" means a straight or branched chain hydrocarbon containing from 1 to 6 carbon atoms. The term "C1-4alkyl" means a straight or branched chain hydrocarbon containing from 1 to 4 carbon atoms. Representative examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tertbutyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methyl hexyl, 2,2-dimethylpentyl, 2,3- dimethylpentyl, n-heptyl, n-octyl, n-nonyl, and n-decyl.

[0054] The term "alkoxy," as used herein, refers to a group -O-alkyl. Representative examples of alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy and tert-butoxy.

[0055] The term "alkenyl," as used herein, means a straight or branched, hydrocarbon chain containing at least one carbon-carbon double bond.

[0056] The term "alkylene" as used herein, refers to a bivalent saturated aliphatic radical, such as ethylene (-CH2-CH2-), which bridges two other groups in a molecule.

[0057] The term "alkoxyalkyl," as used herein, refers to an alkoxy group, as defined herein, appended to the parent molecular moiety through an alkyl group, as defined herein.

[0058] The term "alkylamino," as used herein, means at least one alkyl group, as defined herein, is appended to the parent molecular moiety through an amino group, as defined herein.

[0059] The term "amide," as used herein, means -C(O)NR- or -NRC(O)-, wherein R may be hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, heterocycle, alkenyl, or heteroalkyl.

[0060] The term "aminoalkyl" as used herein, means at least one amino group, as defined herein, is appended to the parent molecular moiety through an alkylene group, as defined herein.

[0061] The term "amino," as used herein, means -NRxRy, wherein Rx and Ry may independently be hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, heterocycle, alkenyl, or heteroalkyl. In the case of an aminoalkyl group or any other moiety where amino appends together two other moieties, amino may be -NRx-, wherein Rx may be hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, heterocycle, alkenyl, or heteroalkyl.

[0062] The term "aryl," as used herein, refers to a phenyl or a phenyl appended to the parent molecular moiety and fused to a cycloalkane group (e.g., the aryl may be indan-4-yl), fused to a 6-membered arene group (i.e., the aryl is naphthyl), or fused to a non-aromatic heterocycle (e.g., the aryl may be benzo[d][l,3]dioxol-5-y!). The term "phenyl” is used when referring to a substituent and the term 6-membered arene is used when referring to a fused ring. The 6- membered arene is monocyclic (e.g., benzene or benzo). The aryl may be monocyclic (phenyl) or bicyclic e.g., a 9- to 12-membered fused bicyclic system).

[0063] The term "cyanoalkyl," as used herein, means at least one -CN group, is appended to the parent molecular moiety through an alkylene group, as defined herein.

[0064] The term "cycloalkoxy," as used herein, refers to a cycloalkyl group, as defined herein, appended to the parent molecular moiety through an oxygen atom.

[0065] The term "cycloalkyl" or "cycloalkane," as used herein, refers to a saturated ring system containing all carbon atoms as ring members and zero double bonds. The term "cycloalkyl" is used herein to refer to a cycloalkane when present as a substituent. A cycloalkyl may be a monocyclic cycloalkyl (e.g., cyclopropyl), a fused bicyclic cycloalkyl (e.g., decahydronaphthalenyl), or a bridged cycloalkyl in which two non-adjacent atoms of a ring are linked by an alkylene bridge of 1, 2, 3, or 4 carbon atoms (e.g., bicyclo[2.2.1]heptanyl). Representative examples of cycloalkyl Include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, adamantyl, and bicyclo[l.l.l]pentanyl.

[0066] The term "cycloalkenyl" or "cycloalkene," as used herein, means a non-aromatic monocyclic or multicyclic ring system containing all carbon atoms as ring members and at least one carbon-carbon double bond and preferably having from 5-10 carbon atoms per ring. The term "cycloalkenyl" is used herein to refer to a cycloalkene when present as a substituent.A cycloalkenyl may be a monocyclic cycloalkenyl (e.g., cyclopentenyl), a fused bicyclic cycloalkenyl (e.g., octahydronaphthalenyl), or a bridged cycloalkenyl in which two non- adjacent atoms of a ring are linked by an alkylene bridge of 1, 2, 3, or 4 carbon atoms (e.g., bicyclo[2.2.1]heptenyl). Exemplary monocyclic cycloalkenyl rings Include cyclopentenyl, cyclohexenyl or cycloheptenyl. Exemplary monocyclic cycloalkenyl rings include cyclopentenyl, cyclohexenyl or cycloheptenyl.

[0067] The term "carbocyclyl" means a "cycloalkyl" or a "cycloalkenyl." The term "carbocycle" means a "cycloalkane" or a "cycloalkene." The term "carbocyclyl" refers to a "carbocycle" when present as a substituent.

[0068] The terms "cycloalkylene" and "heterocyclylene" refer to divalent groups derived from the base ring, i.e., cycloalkane, heterocycle. For illustration, an example cycloalkylenemay be cyclohexene orand a heterocyclylene may be. Cycloalkylene and heterocyclylene include a geminal divalent groups such as 1,1-C3-5cycloalkylene. A further example is 1,1-cyclopropylene.

[0069] The term "halogen" or "halo," as used herein, means Cl, Br, I, or F.

[0070] The term "haloal kyl," as used herein, means an alkyl group, as defined herein, in which one, two, three, four, five, six, seven or eight hydrogen atoms are replaced by a halogen.

[0071] The term "haloalkoxy," as used herein, means at least one haloalkyl group, as defined herein, is appended to the parent molecular moiety through an oxygen atom.

[0072] The term "halocycloalkyl," as used herein, means a cycloalkyl group, as defined herein, in which one or more hydrogen atoms are replaced by a halogen.

[0073] The term "heteroalkyl," as used herein, means an alkyl group, as defined herein, in which one or more of the carbon atoms has been replaced by a heteroatom selected from S, O, P and N. Representative examples of heteroalkyls include, but are not limited to, alkyl ethers, secondary and tertiary alkyl amines, amides, and alkyl sulfides.

[0074] The term "heteroaryl," as used herein, refers to an aromatic monocyclic heteroatomcontaining ring (monocyclic heteroaryl) or a bicyclic ring system containing at least one monocyclic heteroaromatic ring (bicyclic heteroaryl). The term "heteroaryl" is used herein to refer to a heteroarene when present as a substituent. The monocyclic heteroaryl are five or six membered rings containing at least one heteroatom independently selected from thegroup consisting of N, 0 and S (e.g., 1, 2, 3, or 4 heteroatoms independently selected from 0, S, and N). The five membered aromatic monocyclic rings have two double bonds, and the six membered aromatic monocyclic rings have three double bonds. The bicyclic heteroaryl is an 8- to 12-membered ring system and includes a fused bicyclic heteroaromatic ring system (i.e., lOn electron system) such as a monocyclic heteroaryl ring fused to a 6-membered arene (e.g., quinolin-4-yl, indol-l-yl), a monocyclic heteroaryl ring fused to a monocyclic heteroarene (e.g., naphthyridinyl), and a phenyl fused to a monocyclic heteroarene (e.g., quinolin-5-yl, indol-4-yl). A bicyclic heteroaryl / heteroarene group includes a 9-membered fused bicyclic heteroaromatic ring system having four double bonds and at least one heteroatom contributing a lone electron pair to a fully aromatic 10n electron system, such as ring systems with a nitrogen atom at the ring junction (e.g., imidazopyridine) or a benzoxadiazolyl. A bicyclic heteroaryl also includes a fused bicyclic ring system composed of one heteroaromatic ring and one non-aromatic ring such as a monocyclic heteroaryl ring fused to a monocyclic carbocyclic ring (e.g., 6,7-dihydro-5H-cyclopenta[b]pyridinyl), or a monocyclic heteroaryl ring fused to a monocyclic heterocycle (e.g., 2,3-dihydrofuro[3,2-b]pyridinyl). The bicyclic heteroaryl is attached to the parent molecular moiety at an aromatic ring atom. Other representative examples of heteroaryl include, but are not limited to, indolyl (e.g., indol-l-yl, indol-2-yl, indol-4-yl), pyridinyl (including pyridi n-2-yl, pyridin-3-yl, pyridin-4-yl), pyrimidinyl, pyrazinyl, pyridazinyl, pyrazolyl (e.g., pyrazol-4-yl), pyrrolyl, benzopyrazolyl, 1,2,3-triazolyl (e.g., triazol-4-yl), 1,3,4-thiadiazolyl, 1,2,4-thiadiazolyl, 1,3,4-oxadiazolyl, 1,2,4-oxadiazolyl, imidazolyl, thiazolyl (e.g., thiazol-4-yl), isothiazolyl, thienyl, benzimidazolyl (e.g., benzimidazol-5-yl), benzothiazolyl, benzoxazolyl, benzoxadiazolyl, benzothienyl, benzofuranyl, isobenzofuranyl, furanyl, oxazolyl, isoxazolyl, purinyl, isoindolyl, quinoxalinyl, indazolyl (e.g., indazol-4-yl, indazol-5-yl), quinazolinyl, 1,2,4-triazinyl, 1,3,5-triazinyl, isoquinolinyl, quinolinyl, imidazo[l,2-a]pyridinyl (e.g., imidazo[l,2-a]pyridin-6-yl), naphthyridinyl, pyridoimidazolyl, thiazolo[5,4-b]pyridin-2-yl, and thiazolo[5,4-d]pyrimidin-2-yl.The term "heterocycle" or "heterocyclic," as used herein, means a monocyclic heterocycle, a bicyclic heterocycle, or a tricyclic heterocycle. The term "heterocyclyl" is used herein to refer to a heterocycle when present as a substituent. The monocyclic heterocycle is a three-, four-, five-, six-, seven-, or eight-membered ring containing at least one heteroatom independently selected from the group consisting of 0, N, and S. The three- or four-membered ring contains zero or one double bond, and one heteroatom selected from the group consisting of 0, N, and S. The five-membered ring contains zero or one double bond and one, two or three heteroatoms selected from the group consisting of 0, N and S. The sixmembered ring contains zero, one or two double bonds and one, two, or three heteroatoms selected from the group consisting of 0, N, and S. The seven- and eight-membered rings contains zero, one, two, or three double bonds and one, two, or three heteroatoms selected from the group consisting of 0, N, and S. Representative examples of monocyclic heterocyclyls include, but are not limited to, azetidinyl, azepanyl, aziridinyl, diazepanyl, 1,3-dioxanyl, 1,3-dioxolanyl, 1,3-dithiolanyl, 1,3-dithianyl, imidazolinyl, imidazolidinyl, isothiazolinyl, isothiazolidinyl, isoxazolinyl, isoxazolidinyl, morpholinyl, 2-oxo-3-piperidinyl, 2-oxoazepan-3-yl, oxadiazolinyl, oxadiazolidinyl, oxazolinyl, oxazolidinyl, oxetanyl, oxepanyl, oxocanyl, piperazinyl, piperidinyl, pyranyl, pyrazolinyl, pyrazolidinyl, pyrrolinyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahydrothienyl, thiadiazolinyl, thiadiazolidinyl, 1,2-thiazinanyl, 1,3-thiazinanyl, thiazolinyl, thiazolidinyl, thiomorpholinyl, 1,1-dioxidothiomorpholinyl (thiomorpholine sulfone), thiopyranyl, and trithianyl. The bicyclic heterocycle is a monocyclic heterocycle fused to a 6-membered arene, or a monocyclic heterocycle fused to a monocyclic cycloalkane, or a monocyclic heterocycle fused to a monocyclic cycloalkene, or a monocyclic heterocycle fused to a monocyclic heterocycle, or a monocyclic heterocycle fused to a monocyclic heteroarene, or a spiro heterocycle group, or a bridged monocyclic heterocycle ring system in which two non-adjacent atoms of the ring are linked by an alkylene bridge of 1, 2, 3, or 4 carbon atoms, or an alkenylene bridge of two, three, or four carbon atoms. The bicyclic heterocyclyl is attached to the parent molecular moiety at a non-aromatic ring atom (e.g., indolin-l-yl). Representative examples of bicyclic heterocyclyls include, but are not limited to, chroman-4-yi, 2,3-dihydrobenzofuran-2-yl, 2,3-dihydrobenzothien-2-yl, l,2,3,4-tetrahydroisoquinolin-2-yl, 2-azaspiro[3.3]heptan-2-yl, 2-oxa-6-azaspiro[3.3]heptan-6-yl, azabicyclo[2.2.1]heptyl (including 2-azabicyclo[2.2.1]hept-2-yl), azabicyclo[3.1.0]hexanyl (including 3-azabicyclo[3.1.0]hexan-3-yl), 2,3-dihydro-lH-indol-1-yl, isoindolin-2-yl, octahydrocyclopenta[c]pyrrolyl, octahydropyrrolopyridinyl, tetrahydroisoquinolinyl, 7-oxabicyclo[2.2.1jheptanyl, hexahydro-2H-cyclopenta[b]furanyl, 2-oxaspiro[3.3]heptanyl, 3-oxaspiro[5.5)undecanyl, 6-oxaspiro[2.5]octan-l-yl, and 3-oxabicydo[3.1.0]hexan-6-yl. Tricyclic heterocycles are exemplified by a bicyclic heterocycle fused to a 6-membered arene, or a bicyclic heterocycle fused to a monocyclic cycloalkane, ora bicyclic heterocycle fused to a monocyclic cycloalkene, or a bicyclic heterocycle fused to a monocyclic heterocycle, or a bicyclic heterocycle in which two non-adjacent atoms of the bicyclic ring are linked by an alkylene bridge of 1, 2, 3, or 4 carbon atoms, or an alkenylene bridge of two, three, or four carbon atoms. Examples of tricyclic heterocycles include, but are not limited to, octahydro-2, 5-epoxypentalene, hexahydro-2H-2,5- methanocyclopenta[b)furan, hexahydro-lH-l,4-methanocyclopenta[c]furan, azaadamantane (l-azatricyclo[3.3.1.13,7]decane), and oxa-adamantane (2- oxatricyclo[3.3.1.13,7]decane). The monocyclic, bicyclic, and tricyclic heterocyclyls are connected to the parent molecular moiety at a non-aromatic ring atom.

[0076] The term "hydroxyl" or "hydroxy," as used herein, means an -OH group.

[0077] The term "hydroxyalkyl," as used herein, means at least one -OH group, is appended to the parent molecular moiety through an alkylene group, as defined herein.

[0078] Terms such as “alkyl," "cycloalkyl,” "alkylene," etc. may be preceded by a designation indicating the number of atoms present in the group in a particular instance (e.g., "Cualkyl,” "Cs bCycloalkyl,” "Ci^alkylene”). These designations are used as generally understood by those skilled in the art. For example, the representation “C" followed by a subscripted number indicates the number of carbon atoms present in the group that follows. Thus, "Csalkyl" is an alkyl group with three carbon atoms (i.e., n-propyl, isopropyl). Where a range is given, as in "Ci-4," the members of the group that follows may have any number of carbon atoms falling within the recited range. A "Cwalkyl," for example, is an alkyl group having from 1 to 4 carbon atoms, however arranged (i.e., straight chain or branched).

[0079] The term "substituted" refers to a group that may be further substituted with one or more non-hydrogen substituent groups. Substituent groups include, but are not limited to, halogen, --O (oxo), --S (thioxo), cyano, nitro, fluoroalkyl, alkoxyfluoroalkyl, fluoroalkoxy, alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy, heteroalkyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocycle, cycloalkylalkyl, heteroarylalkyl, arylalkyl, hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, alkylene, aryloxy, phenoxy, benzyloxy, amino, alkylamino, acylamino, aminoalkyl, arylamino, sulfonylamino, sulfinylamino, sulfonyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl, sulfinyl, -COOH, ketone, amide, carbamate, and acyl.

[0080] "Fluorescence" as used herein is a cyclical process where a luminescence is generated by certain molecules in which the molecular absorption of a photon triggers the emission of another photon with a longer wavelength. Certain molecules are capable of being excited, viaabsorption of light energy, to a higher energy state, also called an excited state. The energy of this short-lived excited state decays (or decreases), resulting in the emission of light energy. The emission of light via this process is "fluorescence." Molecules that emit light in this manner are said to "fluoresce" and are generally referred to as "fluorophores" or "fluorescent dyes."0081] A "fluorophore," "fluorescent probe," or "fluorescent dye," as used herein, is a molecule that is capable of fluorescing. In its ground state, the fluorophore molecule is in a relatively low-energy, stable configuration, and it does not fluoresce. When light from an external source of one or more particular wavelengths contacts a fluorophore, the fluorophore can absorb the light energy. If the fluorophore absorbs sufficient energy, the fluorophore is excited to an excited state (high energy); this process is known as excitation. There may be multiple excited states or high energy levels that a fluorophore can attain, depending on the wavelength and energy of the external light source. Since a fluorophore is unstable at high-energy configurations, it eventually decays to the lowest-energy excited state, which is semi-stable. The excited lifetime (the length of time that a fluorophore is in an excited state) is very short; the fluorophore then decays from the semi-stable excited state back to the ground state, and at least a portion of the excess energy released by this decay may be emitted as light. The emitted light is of a lower energy, and a longer wavelength, than the absorbed light, and thus the color of the light that is emitted is different from the color of the light that has been absorbed. Upon reaching the ground state, a fluorophore can again absorb light energy to enter an excited state.

[0082] A fluorophore or fluorescent dye absorbs light over a range of wavelengths, and every dye has a characteristic range of excitation wavelengths. This range of excitation wavelengths is referred to as the fluorescence "excitation spectrum," "absorption spectrum” and / or "absorbance spectrum" and reflects the range of possible excited states that the dye can achieve. Certain wavelengths within this range are more effective for excitation than other wavelengths. A fluorophore is excited most efficiently by light of a particular wavelength. This wavelength is the excitation maximum for the fluorophore. As used herein, "excitation maximum" refers to the specific wavelength for each fluorescent dye that most effectively induces fluorescence. Less efficient excitation can occur at wavelengths near the excitation maximum; however, the intensity of the emitted fluorescence is reduced. Although illumination at the excitation maximum of the fluorophore produces the greatestfluorescence output, illumination at lower or higher wavelengths affects only the intensity of the emitted light; the range and overall shape of the emission profile are unchanged.

[0083] As used herein, "excitation" refers to the process where a photon of energy supplied by an external source, such as a laser or a lamp, is absorbed by the fluorophore, creating an excited electronic singlet state (Si') from the So ground state. The excited state exists for a finite time during which the fluorophore undergoes conformational changes and is also subject to a multitude of possible interactions with its molecular environment. These processes have two important consequences. One of these consequences is that the energy of Si' is partially dissipated, yielding a relaxed singlet excited state (Si) from which fluorescence emission originates. Molecules in an excited state (Si') can relax by various competing pathways. They can undergo 'non-radiative relaxation' in which the excitation energy is dissipated as heat (vibrations) to the solvent. Excited organic molecules can also relax via conversion to a triplet state, which may subsequently relax via phosphorescence or by a secondary non-radiative relaxation step. The term "relax" as used herein refers to the energy loss of an excited molecule. Not all the molecules initially excited by absorption return to the ground state (So) by fluorescence emission. Relaxation of an Si' state can also occur through interaction with a second molecule through fluorescence quenching. Other processes, such as, but not limited to, collisional quenching or fluorescent resonance energy transfer (FRET), may also depopulate Si.

[0084] FRET is a radiationless process in which energy is transferred from an excited donor molecule to an acceptor molecule. Radiationless energy transfer is the quantum-mechanical process by which the energy of the excited state of one fluorophore is transferred without actual photon emission to a second fluorophore or molecule. Briefly, a fluorophore absorbs light energy at a characteristic wavelength. The first fluorophore is generally termed the donor ("D”) and may have an excited state of higher energy than that of the second fluorophore, termed the acceptor ("A").

[0085] An essential feature of FRET is that the emission spectrum of the donor overlap with the excitation spectrum of the acceptor, and that the donor and acceptor be sufficiently close. In addition, the distance between "D" and "A" must be sufficiently small to allow the radiationless transfer of energy between the fluorophores. Because the rate of energy transfer is inversely proportional to the sixth power of the distance between the donor and acceptor, the energy transfer efficiency is extremely sensitive to distance changes. Energytransfer is said to occur with detectable efficiency in the 1-10 nm distance range but is typically 4-6 nm for optimal results. The distance range over which radiationless energy transfer is effective depends on many other factors as well, including the fluorescence quantum efficiency of the donor, the extinction coefficient of the acceptor, the degree of overlap of their respective spectra, the refractive index of the medium, and the relative orientation of the transition moments of the two fluorophores.

[0086] As used herein, the term "quencher" means a substance, which reduces or quenches the emission of fluorescence from a fluorophore. As used herein, "fluorescence quenching" may be achieved by any mechanism, typically by FRET between a fluorophore and a non- fluorescent quenching moiety or by collisional (i.e., contact) quenching.

[0087] Fluorophore molecules, when excited, emit over a range of wavelengths. This range of wavelengths is referred to as the fluorescence "emission spectrum." There is a spectrum of energy changes associated with these emission events. The emission maximum is the wavelength where the population of molecules fluoresces most intensely. The emission maximum for a given fluorophore is always at a longer wavelength (lower energy) than the excitation maximum. This difference between the excitation and emission maxima is called the Stokes shift. The magnitude of the Stokes shift is determined by the electronic structure of the fluorophore and is characteristic of the fluorophore molecule. The Stokes shift occurs because some of the energy of the excited fluorophore is lost through molecular vibrations that occur during the brief lifetime of the molecule's excited state, which is dissipated as heat to surrounding solvent molecules as they collide with the excited fluorophore. Remaining energy that is emitted as light fluorescence is thus less than the amount of energy required for excitation.

[0088] Fluorescence requires a source of excitation energy. There are many light source options for fluorescence. Selecting the appropriate light source, and filters for both excitation and emission, can increase the sensitivity of signal detection. Several types of light sources are used to excite fluorescent dyes. The most common sources used are broadband sources, such as, for example, mercury-arc and tungsten-halogen lamps. These lamps produce white light that has peaks of varying intensity across the spectrum. When using broadband white light sources, it is necessary to filter the desired wavelengths needed for excitation; this is most often done using optical filters. Optical filters selectively allow light of certainwavelengths to pass while blocking out undesirable wavelengths. A bandpass excitation filter transmits a narrow range of wavelengths and may be used for selective excitation.

[0089] Laser excitation sources provide wavelength peaks that are well-defined, selective, and of high intensity, allowing more selective illumination of the sample. The best performance Is achieved when a dye's peak excitation wavelength is close to the wavelength of the laser. Several lasers commonly used include, for example, the compact violet 405 nm laser, 488 nm blue-green argon-ion laser, 543 nm helium-neon green laser, and 633 nm helium-neon red laser. Mixed-gas lasers such as, for example, the krypton-argon laser, can output multiple laser lines which may require optical filters to achieve selective excitation. High-output light-emitting diodes (LEDs) provide selective wavelengths, low cost and energy consumption, and long lifetime. Single-color LEDs are ideal for low-cost instrumentation where they can be combined with simple long-pass filters that block the LED excitation and allow the transmission of the dye signal. However, the range of wavelengths emitted from each LED is still relatively broad and may also require the use of a filter to narrow the bandwidth.

[0090] Filters are important for selecting excitation wavelengths and for isolating the fluorescence emission emanating from the dye of interest. Stray light arising from sources other than the emitting fluorophores (for example, from the excitation source) interferes with the detection of the fluorescence emission. Stray light, therefore, must be contained to ensure only the fluorescence of the sample registers with the instrument's light-sensitive detectors. When a single fluorophore is used, a long pass emission filter that selectively blocks out the excitation light to reduce background noise may be used to maximize collection of a signal. If multiple fluorophores are used in the sample, a band pass emission filter can be used to isolate the emission from each dye.

[0091] Ratiometric fluorescence (RF) is a method that uses intensities of two or more wavelengths of an excitation or emission spectrum that are then measured to detect changes to a localized environment. RF probes can be used that are specifically sensitive to changes in an environment, such as ion concentration, analyte concentration, pH, viscosity, or polarity. RF sensors utilize emission intensities at two wavelengths, independent of interfering factors, that may enable more accurate detection through self-calibration of dual emission. For instance, a single dye molecule exhibiting two well-separated fluorescence emission bands (e.g., blue and green) serves as a reporter in a molecular sensor construction. An analytebinding to the RF sensor may be detected by the switch in emission intensity from one band to the other. This response can be expressed as the bound analyte concentration as a function of intensity ratio. The ratio of the intensities at two wavelengths from these peaks can directly correlate with a concentration of the bound sensor-analyte. The use of RF sensors provides an internal calibration at a molecular level, independent of instrumental factors, sensor element size, or sensor molecule density. The internal calibration allows for the reduction of noise within data collected, which may improve measurement accuracy and precision. Ratiometric detection may also enhance sensitivity of the analyte by improving measurement reproducibility, thus allowing for a lower detection limit than traditional sensing methods targeting a single wavelength.

[0092] Generally, the methods of the present disclosure include the use of naphthalene diimide (NDI)-based fluorophores to selectively detect, quantify, and distinguish between PFAS analogues in an aqueous sample. These methods include contacting a sample with the NDl-based fluorophores disclosed herein. If PFAS is present in the sample, then the PFAS interacts with the NDl- based fluorophores in a manner that induces a change in ability of the NDl-based fluorophore to fluoresce at a particular emission wavelength(s) while simultaneously increasing the fluorescence at a different particular emission wavelength(s). The ratio of the intensities of these peaks of the NDl-based fluorophores when they come into contact with a sample can thus be used to detect and / or quantify the amount of PFAS in the sample. Thus, the NDl-based fluorophores disclosed herein can be used as ratiometric detection reagents. Precise discrimination may be made among PFAS with similar functional groups, even at low concentrations. Low concentrations of PFAS that can be discriminated according to aspects of the present disclosure may include concentrations in a range of greater than 0 nM to approximately 5 nM or more.

[0093] The NDl-based fluorophores of the present disclosure are configured to selectively interact with various PFAS analogues in a highly sensitive manner that significantly changes their emission intensities at particular emission wavelengths. According to a method of the present disclosure, synthesizing the fluorophores to use for detection first may involve the synthesis of naphthalene diimide (NDI) from a precursor or intermediate molecule as illustrated in FIG. 1. This NDI may then be used as the basis for all fluorophores with varying alkyl chain lengths. The salt form of exemplary fluorophores synthesized from NDI are illustrated in FIG. 2. The core of each of the exemplary fluorophores are identical with respectto the NDI moiety. However, the fluorophores may differ with respect to the terminal alkyl group, which may range from a 1-carbon chain (Ci) to a 12-carbon chain (C12).

[0094] According to another method of the present disclosure, the interaction between the NDl-based fluorophores of the present disclosure and PFAS causes a decrease in the fluorescence of the fluorophore at one or more emission wavelengths (e.g., PFAS functions as a fluorescence quencher to the NDl-based fluorophores disclosed herein) and an increase in the fluorescence of the fluorophore at a different one or more emission wavelengths. These changes in fluorescence of the fluorophore allows for use of the fluorophore to detect and / or quantify PFOS in a sample with limits of detection between at least about 1 and about 50 parts per billion (ppb). In some aspects, the limit of detection (LOD) may be as low as 5-10 ppb, as low as 4 ppb, as low as 3 ppb, as low as 2 ppb, or even as low as 1 ppb. To detect and / or quantify PFOS in a sample at these limits of detection, the detection reagent may be excited at an excitation wavelength of about 360 and the fluorescence intensity may then be quantified at an emission wavelength at about 493 and about 394 nm. When the NDl-based fluorophore contains alkyl chains from either ends of the molecule, as illustrated In FIG. 2, a ratiometric fluorescence emission change can appear. Similar ratiometric changes were observed with other NDl-based fluorescent probes featuring different alkyl chain lengths when contacted with a sample containing a PFAS analogue. Utilizing the fluorescence emission intensities at two wavelengths allows for independent measurements through selfcalibration of dual emission. The ratio of the emission intensity of the NDl-based fluorophore probe at the two different wavelengths, 493 and 394 nm in this example, directly correlates with the concentration of the PFAS bound NDl-based probe. The ratiometric approach of the present disclosure involves measuring a ratio between the changes in emission intensity from the NDl-based cationic fluorophores, rather than relying on a single absolute value. Accordingly, selectivity is improved by canceling out the effects of environmental factors and other common interferences. Environmental factors may include, but are not limited to, changes in the aqueous solvent sample or presence of other molecular solutes. Changes in the aqueous solvent sample may include differences in ion concentration, pH, turbidity, viscosity, etc. Other molecule solutes may include organic compounds commonly found in water, nonlimiting examples include acetic acid, phenol, and nitro benzene.

[0095] Again, the present disclosure utilizes the NDl-based fluorophores for a method of detecting and discriminating between PFAS analogues in aqueous samples. The aqueoussample is first excited at a wavelength of 360 and the fluorescence emissions is measured at 493 and 394 nm. The aqueous sample is then put into contact with one or more of the NDI- based fluorophores. The aqueous sample in combination with the NDl-based fluorophore is then excited at a wavelength of 360 and the fluorescence emissions may be measured at 493 and 394 nm. The concentration of PFAS bound to the NDl-base fluorophore is a function of the ratio of intensity of the fluorescence emissions. For example, a bound [PFAS]+[fluorophore] complex exhibits a different or unique fluorescence emission than that of a free fluorophore (e.q., the fluorophore without PFAS bound thereto) when excited by the same wavelength of light. As detection {e.g., emitted fluorescence) may be possible prior to all of the free fluorophores bind to PFAS molecule(s), the fluorescence emission may be quantified by an average fluorescence emission of the bound [PFAS]+[fluorophore] complex over a predetermined period of time, such as during the excited lifetime. By comparing the average fluorescence emission of the bound [PFAS]+[fluorophore] complex relative to the free fluorophore, the increase of the average fluorescence emission of the [PFAS]+[f!uorophore] complex with respect to the free fluorophore represents the ratio intensity of the fluorescence emissions. In some aspects, the ratiometric changes promote a color change of the light that is emitted (from blue to green) from the fluorophore that may be visualized with the naked eye. The visualization via naked eye, may allow for the detection of PFAS without the need for expensive instruments and enables real-time monitoring without the need for sample processing and length analysis. Further, measuring the ratiometric responses of two or more of the NDl-based fluorophores for various PFAS analogues may further allow for the discrimination among PFAS analogues with similar molecular structures with varying alkyl chain lengths.

[0096] Without wishing to be bound by theory, the formation of a supramolecular complex (e.g., a binding event) between PFAS and the cationic fluorophores, facilitated by the synergistic interplay of electrostatic, hydrophobic, and n-n stacking interactions, enables a rapid fluorometric sensing response for the detection of PFAS analogues in aqueous systems. Stated another way, the supramolecular complex may be in the form of a non-covalent complex between the PFAS and the NDl-based fluorophore of the present disclosure. The cationic head groups on the fluorophores facilitate the electrostatic interactions with anionic groups present within PFAS molecules. The varying alkyl chain lengths on the NDl-base fluorophores are designed to control sensitivity and selectivity towards different molecularsizes of PFAS. The formation of the analyte and the NDl-based probe complex in the selfassembly processes results in a change in fluorescence emission from blue to green, indicative of the ratiometric dual emission behavior. PFAS binding to NDl-based fluorophores results in the stacking of the NDI backbones in the fluorophore and triggers quenching of the original H-H electronic emission, while generating a new emission intrinsic to an aggregate state. It has been discovered that the NDl-based fluorophore sensitivity towards PFOS, specifically, increases with increasing alkyl chain length, reaching an optimum condition at Cg before decreasing with further chain length increments (C10-C12). Once the alkyl chain becomes too long, the NDl-based fluorophore may become poorly soluble in an aqueous solution. A detection limit for PFOS using methods of the present disclosure was found to be as low as 8.2 nM (4.1 ppb) for the NDl-based probe having an alkyl chain length of 8, showcasing the high sensitivity of the sensor for PFOS detection.

[0097] According to another method of the present disclosure, in order to overcome a true positive (e.g. targeted PFAS) and a false positive (e.g. interfering substance) of traditional sensors which use single emission intensity, an array-based sensor system with ratiometric detection is used. The array utilizes a systematic approach to contact the sample with multiple NDl-based fluorophores of the present disclosure to elicit varying responses to different PFAS analogues. In other words, the aqueous PFAS samples can be divided into a plurality of samples and separately contacted with different fluorophores (serving as detection agents). When used with a single NDl-based fluorophore, the system is capable of selectively detecting multiple perfluoroalkyl sulfonic acids with similar functional groups (e.g., PFOS, PFHxS, and PFBS) which are chemically similar in structure with varying chain lengths. The array-based sensor system relies on the combined responses from multiple tests on individual samples using an array of NDl-based fluorophores rather than a single NDl-based fluorophore. The fluorescence changes can vary for each combination of detection agent and PFAS tested, thereby enabling discrimination based on these differences. It is possible to selectively detect multiple PFAS molecules using the array based on the ratiometric response of the different fluorophores with carrying chain lengths. For example, a first example PFAS molecule may induce a particular fluorescence response when in contact with a particular fluorophore while a second example PFAS molecule may induce a different fluorescence response when in contact with the same fluorophore. The length of the alkyl chain may have an impact on the way a particular fluorophore is able to fluoresce in different supramolecular complexes. In afurther example, the interaction between the first example PFAS molecule and the particular fluorophore may induce a response that includes the change of color of the aqueous sample while the second example PFAS molecule may induce a minor change of color or no change of color at all.

[0099] Specifically, each NDl-based fluorophore exhibits a distinct ratiometric fluorescence emission response to PFAS analogues with the same functional group (e.g. sulfate, SO3-) while including varying chain lengths for each. The NDl-based sensor system involves individual molecular sensors that the sample is introduced to that each contain an NDl-based fluorophore with a different length of alkyl chain. Analyzing the ratiometric response patterns across the entire array, the system may not only detect the presence of PFAS but also differentiate between various PFAS analogues. This multisensory approach also offers robustness and redundancy, as even if one sensor experiences interference, the remaining sensors can still provide valuable data.

[0099] The disclosed method further utilizes a dataset generated by the sensor / array system, containing the ratiometric response patterns from each NDl-based sensor molecule, to be further analyzed using Principal Component Analysis (PCA). PCA methods extract the most meaningful information from this data by identifying underlying patterns and trends. This analysis allows for visualization of data trends, reduction of data complexity, and may be more precise in the differentiation between PFAS analogues. PCA is a linear method for reducing the dimensionality of data, commonly used In data analysis. PCA works by transforming the data into a new coordinate system, where the axes (principal components) align with the directions that capture the greatest variation in the dataset. In this way, the data may be visualized in terms of the selectivity of desired analytes.

[0100] In one aspect of the present disclosure, a method for detecting PFAS in a sample includes contacting the sample with a detection reagent including a compound of formula (I), or salt thereof:

[0101] in another example, LI of formula (I), at each occurrence, is Ci-ealkylene and R, at each occurrence, is Ci-izalkyi.

[0102] In yet another example, the compound of formula (I), or a salt thereof, is, more specifically, the compound of formula (la), or a salt thereof:(la),

[0103] wherein R, at each occurrence, is Ci izaikyl.

[0104] In some aspects of the present disclosure the compound of formula (la), or a salt thereof, may be selected from the group consisting of:

[0105] The NDl-based fiuorophores of the present disclosure may exist as salts when not dissolved in a solvent, and as ionic compounds with at least one counterion that stabilizes their ionic charge when in solution dissolved in a solvent. Examples of counterions include, but are not limited to, iodide, chloride, bromide, and combinations thereof.EXAMPLES

[0106] Exemplary Synthesis of D -based fluorophores[00107 Naphthalene diimide (100 mg, 0.22 mM) was dispersed in 15 ml of ethanol. Then 10 equivalents of an alkyl iodide (where the number of carbons on the alkyl chain are in the range of 1-12) was added into the solution and the mixture was refluxed overnight. The precipitate was filtered and washed with a large excess of ethanol and dried in an air oven. The resulting product was a naphthalene diimide (ND!) based probe represented as NDI-CX, where X is the number of carbons in the alkyl iodide used. Molecules were verified with mass spectrometry (MS) and / or1H-NMR.

[0108] FIG. 3 shows an exemplary synthetic scheme 200 for synthesizing an NDI-C1 fluorophore or fluorescent probe. This probe is the result when the alkyl iodide contains an alkyl chain including one carbon. The resulting yield was 87%. It is understood that NDI-C1 may be synthesized according to other processes. It is also understood that scheme 200, may include additional, or alternative, steps without deviating from the scope of the present disclosure. FIG. 4 shows the electrospray ionization mass spectrometry (ESI-MS) spectrum of NDI-C1. This spectrum verifies successful synthesis of the NDI-C1 fluorophore.

[0109] FIG. 5 shows an exemplary synthetic scheme 210 for synthesizing an NDI-C3 fluorophore or fluorescent probe. This probe is the result when the alkyl iodide contains an alkyl chain including three carbons. In this way, synthesis of NDl-based probes of the present disclosure may include using a base amount of ND! and then adding an equivalent amounts of alkyl iodide. The resulting yield was 83%. It will be understood that NDI-C3 may be synthesized according to other processes. It is also understood that scheme 210, may include additional, or alternative, steps without deviating from the scope of the present disclosure. FIG. 6 shows an electrospray ionization mass spectrometry (ESI-MS) spectrum of NDI-C3. FIG.7 shows the1H-NMR spectrum of NDI-C3. These spectra verify successful synthesis of the NDI-C3 fluorophore.

[0110] FIG. 8 shows an exemplary synthetic scheme 220 for synthesizing an NDI-C4. This probe is the result when the alkyl iodide used contains an alkyl chain including four carbons. The resulting yield was 85%. It will be understood that NDI-C4 may be synthesized according to other processes. It is also understood that scheme 220, may include additional, or alternative, steps without deviating from the scope of the present disclosure. FIG. 9 shows the electrospray ionization mass spectrometry (ESI-MS) spectrum of NDI-C4. FIG. 10 showsthe1H-NMR spectrum of NDI-C4. These spectra verify successful synthesis of the NDI-C4 fluorophore.[00111 FIG. 11 shows an exemplary synthetic scheme 230 for synthesizing an NDI-C6. This probe is the result when the alkyl iodide used in the synthesis contains an alkyi chain including six carbons. The resulting yield was 80%. it is understood that NDI-C6 may be synthesized according to other processes. It is also understood that scheme 230, may include additional, or alternative, steps without deviating from the scope of the present disclosure. FIG. 12 shows the electrospray ionization mass spectrometry (ESI-MS) spectrum of NDI-C6. FIG. 13 shows the1H-NMR spectrum of NDI-C6. These spectra verify successful synthesis of the NDI-C6 fluorophore.

[0112] FIG. 14 shows an exemplary synthetic scheme 240 for synthesizing an NDI-C8. This probe is the result when the alkyl iodide used contains an alkyl chain including eight carbons. The resulting yield was 83%. It is understood that NDI-C8 may be synthesized according to other processes. It is also understood that scheme 240, may include additional, or alternative, steps without deviating from the scope of the present disclosure. FIG. 15 shows the electrospray ionization mass spectrometry (ESI-MS) spectrum of NDI-C8. FIG. 16 shows the1H-NMR spectrum of NDI-C8. These spectra verify successful synthesis of the NDI-C8 fluorophore.

[0113] FIG. 17 shows an exemplary synthetic scheme 250 for synthesizing an NDI-C10. This probe is the result when the alkyl iodide used contains an alkyl chain including ten carbons. The resulting yield was 73%. It is understood that NDI-C10 may be synthesized according to other processes. It is also understood that scheme 250, may include additional, or alternative, steps without deviating from the scope of the present disclosure. FIG. 18 shows the electrospray ionization mass spectrometry (ESI-MS) spectrum of NDI-C10. These spectra verify successful synthesis of the NDI-C10 fluorophore.

[0114] FIG. 19 shows an exemplary synthetic scheme 260 for synthesizing an NDI-C12. This probe is the result when the alkyl iodide used contains an alkyl chain including twelve carbons. The resulting yield was 78%. It is understood that NDI-C12 may be synthesized according to other processes, it is also understood that scheme 260, may include additional, or alternative, steps without deviating from the scope of the present disclosure. FIG. 20 shows the electrospray ionization mass spectrometry (ESI-MS) spectrum of NDI-C12. FIG. 21 shows the1H-NMR spectrum of NDI-C12. These spectra verify successful synthesis of the NDI-C12 fluorophore.[00115 Fluorescence Spectroscopic Studies of NDl-based Probes[00116 FIG. 22A illustrates a series of fluorescence emission spectra of an example NDI-C1 in the presence of varying concentrations of PFOS (0 pM - 5. M). In this example, a 2. M NDI- C1 solution in water was excited at an excitation wavelength of 360 nm and fluorescence peaks were observed between 375 and 450 nm. With the addition of perfluorooctance sulfonic acid (PFOS) having concentrations ranging from 0 to 5 p to the solution, there was very little change observed in emission intensity. The minor change in emission intensity with an increase in PFOS concentration highlights the key role of the longer alkyl chains in NDI- based probes.

[0117] FIG. 22B and 22C illustrate a series of fluorescence emission spectra of an example NDI-C1 in the presence of varying concentrations of perfluorohexanesulfonic acid (PFHxS) (0 pM - 5. M) and perfluorobutanesulfonic acid (PFBS) respectively (0 pM - 5 pIVI). In this example, a 2. M NDI-C1 solution in water was excited at an excitation wavelength of 360 nm and fluorescence peaks were observed between 375 and 450 nm. With the addition of the analyte from 0 to 5 pM to the solution, there was no change in emission intensity. There was little to no distinct change in the emission spectrum of NDI-C1 after 5. M of other analytes were added, thereby illustrating that the selectivity of NDl-based probes towards PFOS, even in less desirably performing versions such as NDI-C1.

[0118] FIG. 23A illustrates a series of fluorescence emission spectra of an example NDI-C8 in the presence of varying concentrations of PFOS (0 pM - 5 pM). In this example, a 2 M NDI- C8 solution in water was excited at an absorbance wavelength of 360 nm and fluorescence peaks were observed between 375 and 450 nm. With the addition of perfluorooctance sulfonic acid (PFOS) from 0 to 5 pM, there was a decrease in the peak intensity from 375 to 450 nm and increase in a new peak in the range of 450 to 600 nm. The increase in concentration added of PFOS into an NDI-C8 solution was also accompanied by a color change from blue to green. The decrease of the initial peak with the immergence of a new peak, at two well-separated fluorescence emission bands, highlight the NDl-based probe system's ability to use NDI-C8 as a ratiometric fluorescence sensor.

[0119] FIGS. 23B and 23C illustrate a series of fluorescence emission spectra of an example NDI-C8 in the presence of varying concentrations of perfluorohexanesulfonic acid (PFHxS) andperfluorobutanesulfonic acid ( PFBS) respectively (0 pM - 5 jiM). In this example, a 2 pM NDI- C8 solution in water was excited at an absorbance wavelength of 360 nm and fluorescence peaks were observed between 375 and 450 nm. With the addition of PFHxS from 0 to 5 pM, there a decrease in the peak intensity from 375 to 450 nm and small increase in a new peak in the range of 450 to 600 nm. It should be noted that the increase in the new peak in the range of 450 to 600 nm in FIG. 23B is not as large as the increase in the new peak in the range of 450 to 600 nm in FIG. 23A with the inclusion of PFOS. The addition of PFBS from 0 to 5 pM, there was a decrease in the peak intensity from 375 to 450 nm and no new peak in the range of 450 to 600 nm was observed. FIGS. 23B and 23C, when analyzed in view of FIG. 23A, highlight the potential selectivity of the NDI-C8 probe towards PFOS compared to the different PFAS chemicals PFHxS and PFBS.

[0120] FIG. 24 illustrates the dependence of the ratiometric change for an NDI based cationic probe towards PFOS on the terminal alkyl chain length of the probe. A fluorescence emissions spectrum similar to FIG. 23A was obtained for each of the NDl-based probes: NDI-C1, NDI-C4, NDI-C6, NDI-C8, NDI-C10, and NDI-C12. The intensity of the fluorescence emission was measured at 493 and 394 nm, and the ratio of those intensities (I493 / I394) was plotted against the terminal alkyl chain length of the NDI probe. FIG. 24 shows that the largest ratiometric change towards PFOS, which was observed with NDI-C8 making it a better candidate for PFOS detection compared to the other tested NDI probes.

[0121] Selectivity Studies of NDI-Based Probes

[0122] FIG. 25 shows the selectivity of the NDl-based method towards PFOS. The analytes that were studied were the top six targeted PFAS chemicals by the EPA: PFOS, perfluorohexanesulphonic acid (PFHxS), perfluorobutanesulfonic acid (PFBS), perfluorooctanoic acid (PFOA), perfluorononanoic add (PFNA), and GenX as well as common salts and organic contaminants found in water: sodium dodecyl sulfate (SDS), cetrimonium bromide (CTAB), acetic acid, octanoic acid, p-toluene sulfonic acid, ethylenediaminetetraacetic acid (EDTA), phenol, nitro benzene, sodium chloride (NaCI), potassium chloride (KCI), calcium chloride (CaCI?), and magnesium sulfate (MgSC ). Each study was performed similar to the procedure used with respect to FIG. 23A. The ratiometric change of the fluorescence of NDI-C8 towards each of the analytes was determined by the ratio of the intensities of the fluorescence emission measured at 493 and 394 nm (I493 / I394).The bar plot of FIG. 25 illustrates the selectivity of the NDI-C8 probe towards PFOS over other analytes.[00123 FIG. 26 illustrates the linear regression of the percentage of emission ratios (I493 / I394) with respect to PFOS concentration in pure water. To obtain the limit of detection (LOD) of PFOS using NDI-C8, a linear regression of the percentage change of fluorescence intensity of NDI-C8 at 493 and 394 in the presence of varying amounts of PFOS (0-50 nM) was calculated. The LOD was calculated for NDI-C8 to be 8.2 nM (4.1 ppb).

[0124] FIGS. 27A and 27B illustrate a principal component analysis (PCA) generated from the NDI-C8 probe tested with seven different concentrations of PFAS. PCA was conducted based on the data obtained for the NDI-C8 probe. PCA is a statistical technique that is able to summarize data sets into smaller principal components. In this way, the data can be visualized in terms of the selectivity of the desired analytes. FIG. 27A demonstrates that the current ratiometric sensor can selectively detect PFOS over GenX, PFOA, and PFBS. Further, FIG. 27B illustrates the NDI-C8 probe's ability to discriminate among the three PFOS analogues (PFOS, PFHxS, and PFBS).

[0125] FIGS. 28A and 28B show the ratiometric fluorescence responses towards different NDI fluorophores, by varying the alkyl chain, and different PFAS chemicals, PFBS, PFHxS, PFOS, PFOA, PFNA, and GenX. Each PFAS sample was tested with each NDI fluorophore independently. The ratiometric change of each NDl-based probe towards each of the analytes was determined by the ratio of the intensities of the fluorescence emission measured at 493 and 394 nm (I493 / I394). These results are summarized in the bar plot as shown in FIG. 28A and the heat map as shown in FIG. 28B. As seen in both figures, the NDI-C8 probe was the best at both detecting PFOS in the sample and discriminating PFOS against the other PFAS chemicals.

[0126] FIGS. 29A and 29B show an overall fluorescence data matrix "6 fluorophores x 6 PFAS x 5 replicates" as transformed using a PCA (FIG. 29A) and a hierarchical clustering analysis (HCA). HCA can be used to understand the structure of data. Both PCA and HCA allow the data to be visualized in terms of the selectivity of the desired analytes. Both analyses confirm the selective separation of PFOS from other PFAS used when measured at 5 pM for all analytes.

[0127] According to one aspect of the present disclosure, a method for detecting and discriminating between per- and poly-fluoroalkyl substances (PFAS) in an aqueous sample includes exciting the aqueous sample alone at a wavelength of approximately 360 nm, contacting the aqueous sample with a reagent comprising a naphthalene diimide-basedcationic fluorophore, exciting the detection reagent and aqueous sample combination at the wavelength of approximately 360 nm, and quantifying a change in a fluorescence emission intensity of the detection reagent while in contact with the sample relative to the fluorescence intensity of the detection reagent prior to being contacted with the sample.[00128 According to another aspect of the present disclosure, a method for detecting and discriminating between per- and poly-fluoroalkyl substances (PFAS) in an aqueous sample includes exciting the aqueous sample alone at a wavelength of between 330 - 390 nm, contacting the sample with a detection reagent comprising a naphthalene diimide-based cationic fluorophore, exciting the detection reagent and sample together at the excitation wavelength, and quantifying a change in a fluorescence emission intensity of the detection reagent while in contact with the sample relative to the fluorescence intensity of the detection reagent prior to being contacted with the sample.

[0129] According to another aspect of the present disclosure, a method for detecting and discriminating between per- and poly-fluoroalkyl substances (PFAS) in an aqueous sample includes the excitation wavelength is approximately 360 nm.[00130 According to another aspect of the present disclosure, a method for detecting and discriminating between per- and poly-fluoroalkyl substances (PFAS) in an aqueous sample includes quantifying a change in the fluorescence emission intensity of the detection reagent is performed by measuring a ratio between the change in the fluorescence emission intensity of the detection reagent while in contact with the sample relative to the fluorescence intensity of the detection reagent prior to being contacted with the sample at a minimum of two different wavelengths.

[0131] According to another aspect of the present disclosure, a method for detecting and discriminating between per- and poly-fluoroalkyl substances (PFAS) in an aqueous sample includes dividing the aqueous sample into a first sample and a second sample, contacting the first sample by a first detection reagent, contacting the second sample by a second detection reagent, each detection reagent including an alkyl chain length different than the other.

[0132] According to another aspect of the present disclosure, a method for detecting and discriminating between per- and poly-fluoroalkyl substances (PFAS) in an aqueous sample includes quantifying a change in the fluorescence emission intensity of the detection reagent is performed by measuring a ratio between the change in the fluorescence emission intensity of the detection reagent while in contact with the sample relative to the fluorescenceemission intensity of the detection reagent prior to being contacted with the sample, each measured at a first wavelength and a second wavelength, different from the first wavelength.[00133 According to another aspect of the present disclosure, a method for detecting and discriminating between per- and poly-fluoroalkyl substances (PFAS) in an aqueous sample includes the two unique wavelengths are between 350 and 550 nm.[00134 According to another aspect of the present disclosure, a method for detecting and discriminating between per- and poly-fluoroalkyl substances (PFAS) in an aqueous sample includes the alkyl chain length comprises between 1 to 12 carbon atoms.

[0135] According to yet another aspect of the present disclosure, a method for detecting and discriminating between per- and poly-fluoroalkyl substances (PFAS) in an aqueous sample includes conducting a principal component analysis of the first and second samples in contact with the first and second detection reagents.

[0136] According to yet another aspect of the present disclosure, a method for detecting and discriminating between per- and poly-fluoroalkyl substances (PFAS) in an aqueous sample includes conducting a hierarchical clustering analysis (HCA) of the first and second samples in contact with the at least first and second detection reagents.

[0137] According to yet another aspect of the present disclosure, a method for detecting and discriminating between per- and poly-fluoroalkyl substances (PFAS) in an aqueous sample includes a limit of detection for at least one of the PFAS is in the range of 5-15 nM.

[0138] According to yet another aspect of the present disclosure, a method for detecting and discriminating between per- and poly-fluoroalkyl substances (PFAS) in an aqueous sample includes said per- and poly-fluoroalkyl substances is at least one of perfluorooctanesulfonic acid, perfluorohexanesulphonic acid, perfluorobutanesulfonic acid, perfluorooctanoic acid, perfluorononanoic acid, and the ammonium salt of hexafluropropylene oxide dimer acid (GenX).

[0139] According to yet another aspect of the present disclosure, a method for detecting and discriminating between per- and poly-fluoroalkyl substances (PFAS) in an aqueous sample includes the limit of detection is independent from environmental factors.[00140 According to yet another aspect of the present disclosure, a method for detecting and discriminating between per- and poly-fluoroalkyl substances (PFAS) in an aqueous sample includes environmental factors comprise at least one of ion concentration or solution pH.

[0141] According to yet another aspect of the present disclosure, a method for detecting and discriminating between per- and poly-fluoroalkyl substances (PFAS) in an aqueous sample includes at least one of the first sample and second sample changes color when the first detection reagent or second detection reagent, respectively, contacts the PFAS molecule.

[0142] According to yet another aspect of the present disclosure, a method for detecting and discriminating between per- and poly-fluoroalkyl substances (PFAS) in an aqueous sample includes the naphthalene diimide-based cationic fluorophore is selected from:

[0144] According to yet another aspect of the present disclosure, a method for detecting and discriminating between per- and poly-fluoroalkyl substances (PFAS) in an aqueous sample includes the contacting the aqueous sample with a detection reagent comprising a naphthalene diimide-based cationic fluorophore comprises a binding event between the detection reagent and said PFAS in a non-covalent manner.

[0145] According to another aspect of the present disclosure, a fluorophore for detecting PFAS in an aqueous sample includes a compound of formula (I):

[0146] (!)•

[0147] According to another aspect of the present disclosure, a fluorophore for detecting PFAS in an aqueous sample includes R, at each occurrence, is Ci-nalkyl.

[0148] According to another aspect of the present disclosure, a fluorophore for detecting PFAS in an aqueous sample includes a compound of formula (I), or a salt thereof:

[0149] wherein: LI, at each occurrence, is Ci-galkylene; and R, at each occurrence, is Ci-ualkyl.

[0150] According to yet another aspect of the present disclosure, R, at each occurrence, is Ci- salkyl.

[0151] According to another aspect of the present disclosure, a fluorophore for detecting PFAS in an aqueous sample includes R at each occurrence, is Csalkyl.According to another aspect of the present disclosure, a fluorophore for detecting PFAS in an aqueous sample includes said fluorophore is selected frorr

[0152] According to another aspect of the present disclosure, a fluorophore for detecting PFAS in an aqueous sample includes said PFAS is at least one of perfluorooctanesulfonic acid, perfluorohexanesulphonic acid, perfluorobutanesulfonic acid, perfluorooctanoic acid, perfluorononanoic acid, or the ammonium salt of hexafluropropylene oxide dimer acid (GenX).

[0153] According to another aspect of the present disclosure, a fluorophore for detecting PFAS in an aqueous sample includes said PFAS can form a non-covalent supramolecular complex in solution with said fluorophore.

[0154] According to another aspect of the present disclosure, a fluorophore for detecting PFAS in an aqueous sample includes a limit of detection for at least one of said PFAS is in the range of 5-15 nM.

[0155] According to another aspect of the present disclosure, a fluorophore for detecting per- and poly-fluoroalkyl substances (PFAS) in an aqueous sample of Formula (la):(la).00156 According to another aspect of the present disclosure, a fluorophore for detecting PFAS in an aqueous sample includes R, at each occurrence, is Ci-nalkyl.

[0157] According to another aspect of the present disclosure, a fluorophore for detecting per- and poly-fluoroalkyl substances (PFAS) in an aqueous sample of Formula (la):(la);

[0158] wherein R, at each occurrence, is Ci-ualkyl.

[0159] According to yet another aspect of the present disclosure, the fluorophore for detecting PFAS in an aqueous sample, R at each occurrence is Ci-nalkyl.

[0160] According to another aspect of the present disclosure, a fluorophore for detecting PFAS in an aqueous sample includes when the fluorophore is in the form of a salt.[00161 According to another aspect of the present disclosure, a fluorophore for detecting PFAS in an aqueous sample includes a counterion for the salt is one of iodide, chloride, and bromide.

[0162] According to another aspect of the present disclosure, a fluorophore for detecting PFAS in an aqueous sample includes said fluorophore binds noncovalently to said PFAS in solution.

[0163] It will be understood by one having ordinary skill in the art that construction of the described disclosure and other components is not limited to any specific material. Other exemplary embodiments of the disclosure disclosed herein may be formed from a wide variety of materials, unless described otherwise herein.

[0164] For purposes of this disclosure, the term "coupled” (in all of its forms, couple, coupling, coupled, etc.) generally means the joining of two components (electrical or mechanical) directly or indirectly to one another. Such joining may be stationary in nature or movable in nature. Such joining may be achieved with the two components (electrical or mechanical) and any additional intermediate members being integrally formed as a single unitary body with one another or with the two components. Such joining may be permanent in nature or may be removable or releasable in nature unless otherwise stated.

[0165] It is also important to note that the construction and arrangement of the elements of the disclosure, as shown in the exemplary embodiments, is illustrative only. Although only a few embodiments of the present innovations have been described in detail in this disclosure, those skilled in the art who review this disclosure will readily appreciate that many modifications are possible (e.g., variations in sizes, dimensions, structures, shapes and proportions of the various elements, values of parameters, mounting arrangements, use of materials, colors, orientations, etc.) without materially departing from the novel teachings and advantages of the subject matter recited. For example, elements shown as integrally formed may be constructed of multiple parts, or elements shown as multiple parts may be integrally formed, the operation of the interfaces may be reversed or otherwise varied, the length or width of the structures and / or members or connector or other elements of the system may be varied, the nature or number of adjustment positions provided between the elements may be varied. It should be noted that the elements and / or assemblies of thesystem may be constructed from any of a wide variety of materials that provide sufficient strength or durability, in any of a wide variety of colors, textures, and combinations. Accordingly, all such modifications are intended to be included within the scope of the present innovations. Other substitutions, modifications, changes, and omissions may be made in the design, operating conditions, and arrangement of the desired and other exemplary embodiments without departing from the spirit of the present innovations.

[0166] It will be understood that any described processes or steps within described processes may be combined with other disclosed processes or steps to form structures within the scope of the present disclosure. The exemplary structures and processes disclosed herein are for illustrative purposes and are not to be construed as limiting.

[0167] It is also to be understood that variations and modifications can be made on the aforementioned structures and methods without departing from the concepts of the present disclosure, and further it is to be understood that such concepts are intended to be covered by the following claims unless these claims by their language expressly state otherwise.

Claims

What is claimed is:

1. A method for detecting and discriminating between per- and poly-fluoroalkyl substances (PFAS) in an aqueous sample, the method comprising:exciting the aqueous sample alone at an excitation wavelength of between 330 - 390 nm;contacting the aqueous sample with a detection reagent comprising a naphthalene diimide-based cationic fluorophore;exciting the detection reagent and aqueous sample together at the excitation wavelength; andquantifying a change in a fluorescence emission intensity of the detection reagent while in contact with the sample relative to the fluorescence emission intensity of the detection reagent prior to being contacted with the sample.

2. The method for detecting and discriminating between per- and poly-fluoroalkyl substances (PFAS) in an aqueous sample of claim 1, wherein the excitation wavelength is approximately 360 nm.

3. The method for detecting and discriminating between per- and poly-fluoroalkyl substances (PFAS) in an aqueous sample of either of claims 1 or 2, wherein quantifying a change in the fluorescence emission intensity of the detection reagent is performed by measuring a ratio between the change in the fluorescence emission intensity of the detection reagent while in contact with the sample relative to the fluorescence emission intensity of the detection reagent prior to being contacted with the sample, each measured at a first wavelength and a second wavelength, different from the first wavelength.

4. The method for detecting and discriminating between per- and poly-fluoroalkyl substances (PFAS) in an aqueous sample of claim 1, wherein the aqueous sample is divided into at least a first sample and a second sample, the first sample contacted by a first detection reagent and the second sample contacted by the second detection reagent, wherein the first and second detection reagents include an alkyl chain length different than the other.

5. The method for detecting and discriminating between per- and poly-fluoroalkyl substances (PFAS) in an aqueous sample of claim 4, wherein the first and second detection reagents includes a naphthalene diimide core.

6. The method for detecting and discriminating between per- and poly-fluoroalkyl substances (PFAS) in an aqueous sample either of claims 4 or 5, further comprising:quantifying a ratio of fluorescence intensity of the first and second detection reagents at two unique wavelengths alone relative to the ratio of fluorescence intensity of the one or more detection reagents while in contact with the sample at the two unique wavelengths.

7. The method for detecting and discriminating between per- and poly-fluoroalkyl substances (PFAS) in an aqueous sample of claim 6, wherein the two unique wavelengths are between 350 and 550 nm.

8. The method of detecting and discriminating between per- and poly-fluoroalkyl substances (PFAS) in an aqueous sample of any one of claims 4-7, wherein the alkyl chain length comprises between 1 to 12 carbon atoms.

9. The method for detecting and discriminating between per- and poly-fluoroalkyl substances (PFAS) in an aqueous sample of any one of claims 4-8, further comprising:conducting a principal component analysis of the first and second samples after contact with the first and second detection reagents.

10. The method for detecting and discriminating between per- and poly-fluoroalkyl substances (PFAS) in an aqueous sample of any one of claims 4-9, further comprising:conducting a hierarchical clustering analysis (HCA) of the first and second samples in contact with the at least first and second detection reagents.

11. The method for detecting and discriminating between per- and poly-fluoroalkyl substances (PFAS) in an aqueous sample of any one of claims 1-10, wherein a limit of detection for at least one of the PFAS is in a range of 5-15 nM.

12. The method for detecting and discriminating between per- and poly-fluoroalkyl substances (PFAS) in an aqueous sample of any one of claims 1-11, wherein said per- and poly-fluoroalkyl substances is at least one of perfluorooctanesulfonic acid, perfluorohexanesulphonic acid, perfluorobutanesulfonic acid, perfluorooctanoic acid, perfluorononanoic acid, and the ammonium salt of hexafluropropylene oxide dimer acid (GenX).

13. The method for detecting and discriminating between per- and poly-fluoroalkyl substances (PFAS) in an aqueous sample of any one of claims 11-12, wherein the limit of detection is independent from environmental factors.

14. The method for detecting and discriminating between per- and poly-fluoroalkyl substances (PFAS) in an aqueous sample of any one of claims 11-13, wherein environmental factors comprise at least one of ion concentration or solution pH.

15. The method for detecting and discriminating between per- and poly-fluoroalkyl substances (PFAS) in an aqueous sample of any one of claims 4-14, wherein at least one of the first sample and second sample changes color when the first detection reagent or second detection reagent, respectively, contacts the PFAS molecule.

16. The method for detecting and discriminating between per- and poly-fluoroalkyl substances (PFAS) in an aqueous sample of any one of claims 1-15, wherein the naphthalene diimide-based cationic fluorophore is selected from:

17. The method for detecting and discriminating between per- and poly-fluoroaikyl substances (PFAS) in an aqueous sampie of any one of ciaims 1-15, wherein the contacting the aqueous sample with a detection reagent comprises a binding event between the detection reagent and said PFAS in a non-covalent manner.

18. A fluorophore for detecting per- and poly-fluoroalkyl substances (PFAS) in an aqueous sample of Formula (I):

19. The fluorophore for detecting PFAS in an aqueous sample of claim 18, wherein R, at each occurrence, is Crualkyl.

20. The fluorophore for detecting PFAS in an aqueous sample of either one of claims 18 or 19, wherein LI, at each occurrence, is Ci-salkylene.

21. The fluorophore for detecting PFAS in an aqueous sample of any one of claims 18-20- 19, wherein R at each occurrence, is Cgalkyl.

22. The fluorophore for detecting PFAS in an aqueous sample of any one of claims 18-21, wherein said fluorophore is selected from:

23. The fluorophore for detecting PFAS in an aqueous sample of any one of claims 18-22, wherein said PFAS is at least one of perfluorooctanesulfonic acid, perfluorohexanesulphonic acid, perfluorobutanesulfonic acid, perfluorooctanoic acid, perfluorononanoic acid, or the ammonium salt of hexafluropropylene oxide dimer acid (GenX).

24. The fluorophore for detecting PFAS in an aqueous sample of any one of the claims 18- 23, wherein said PFAS can form a non-covalent supramolecular complex in solution with said fluorophore.

25. The fluorophore for detecting PFAS in an aqueous sample of any one of the claims 18- 24, where a limit of detection for at least one of said PFAS is in a range of 5-15 nM.

26. A fluorophore for detecting per- and poly-fluoroalkyl substances (PFAS) in an aqueous sample of Formula (la):(la).

27. The fluorophore for detecting PFAS in an aqueous sample of claim 26, wherein R, at each occurrence, is CM? alkyl.

28. The fluorophore for detecting PFAS in an aqueous sample of claim 26, wherein R at each occurrence, is Ci-s alkyl.

29. The fluorophore for detecting PFAS in an aqueous sample of either one of claims 26 or 28, wherein the fluorophore is in a form of a salt.

30. The fluorophore for detecting PFAS in an aqueous sample of claim 29, wherein a counterion for the salt is one of iodide, chloride, and bromide.

31. The fluorophore for detecting PFAS in an aqueous sample of any one of claims 26-30, wherein said fluorophore binds noncovalently to said PFAS in solution.

Images