Methods for detecting per- or poly-fluoroalkyl substances with fluorophores

Abstract

Described herein are compounds which experience fluorescence activation or fluorescence quenching in the presence of per- and / or poly-fluoroalkyl substances (PFAS). Also described herein are methods for selectively or non-selectively detecting PFAS with the disclosed fluorophores covalently attached or non-covalently adhered to a surface.

Description

[0001] 026389-0053-W001

[0002] METHODS FOR DETECTING PER- OR POLY-FLUOROALKYL SUBSTANCES WITH FLUOROPHORES

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] This application claims priority to U.S. Provisional Patent Application No. 63 / 717,453 filed on November 7, 2024, which is incorporated by reference herein in its entirety.

[0005] FEDERALLY SPONSORED RESEARCH

[0006] This invention was made with government support under R43 ES035349 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0007] BACKGROUND

[0008] Per- and poly-fluoroalkyl substances (PFAS), also known “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)]. Exposure to PFAS through drinking water and other processes can cause serious health problems, especially when accumulated in blood and tissue. More than 9,000 PFAS have been identified, with the perfluorooctanesulfonic acid (PFOS) and perfluorooctanoic acid (PFOA) being the most used and widely studied. Recent research has found direct links between these two compounds and chronic kidney disease, thyroid disfunction, and some forms of cancer. While now banned in the U.S. and many other countries, PFOS and PFOA were commonly used in AFFFs, fluoropolymer production, and the water / stain proofing of leather and fabrics.

[0009] The presence of these forever chemicals carries severe health risks due to their trace level accumulation in drinking water, foods, and other consumer products. 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.

[0010] Currently, the official health advisory level set by the U.S. Environment Protection Agency for PFOA and PFOS in drinking water is 70 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 026389-0053-W001 guidelines and regulations may make water everywhere, including fresh rainwater, being labeled as contaminated and unfit for consumption and use. Local, state, and federal governments may be imminently faced with many challenges to ensuring that drinking water is safe. To this point, new methods will be required that are highly sensitive for the detection of PFAS. Moreover, these methods may 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 gage the range and scope of the geographic distribution of PFAS contamination and assist in monitoring the efficiency of treatment.

[0011] Current PFAS detection methods are poorly suited for field deployment due in part to requirements such as of derivatization prior to analyses, lengthy sample preparation, cost, 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 analyses of total fluorine by particle induced gamma ray emission (PIGE) spectroscopy. However, these methods are poorly suited to use in the field. Moreover, the limits of detection (LOD) for the 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.

[0012] Additionally, prior probes and sorbents for PFAS capture and detection 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 were able detect PFOS and PFOA in simple matrices (DI water and drinking water), the nonspecific binding limited both selectivity and sensitivity in practical matrices, due to the inability to screen out interferences with similar electronic properties and hydrophobicity.

[0013] For at least these reasons, there is a need for compounds that can detect and quantify various PFAS, such as PFOS and PFOA.

[0014] SUMMARY

[0015] The present application discloses methods for detecting one or more per- or polyfluoroalkyl substances (PFAS) in a sample.

[0016] In some embodiments, the methods comprise: 026389-0053-W001 contacting the sample with a detection reagent comprising a compound of formula (I), or a salt thereof: wherein

[0017] R1is hydrogen, halogen, cyano, Ci^alkyl, Ci-ehaloalkyl, -OR1a, -SR1a, -CC>21a, -C(O)R1a, -SO2R1a, -N(R1a)2, -CO2N(R1a)2, or -NO2, where:

[0018] R1a, at each occurrence, is independently, hydrogen, Ci-ealkyl, or Ci-2haloalkyl;

[0019] R2, at each occurrence, is independently hydrogen, halogen Ci-6alkyl, Ci-ehaloalkyl, -OR1b, -SR1b, -CO2R1b, -C(O)R1b, -SO2R1b, -N(R1b)2, -CO2N(R1 b)2, or -NO2, where:

[0020] R1b, at each occurrence, is independently, hydrogen, Ci-ealkyl, or Ci.2haloalkyl;

[0021] G1, at each occurrence, is independently a 6- to 12-membered aryl or a 5- to 6-membered heteroaryl, wherein G1is optionally substituted with 1-5 substituents independently selected from the group consisting of Ci-4alkyl, Ci.2haloalkyl, halogen, cyano, -OCi-4alkyl, and -OCi.2haloalkyl;

[0022] G2, at each occurrence, is independently a 5- to 12-membered heteroarene or -N(GX)-, where:

[0023] Gx, at each occurrence, is a 6- to 12-membered aryl optionally substituted with 1-5 substituents independently selected from the group consisting of Ci-4alkyl, Ci- 2haloalkyl, halogen, cyano, -OCi-4alkyl, and -OCi-2haloalkyl;

[0024] G3is a 5- to 6-membered heteroaryl or a 6- to 12-membered aryl, wherein G3is optionally substituted with 1-5 substituents independently selected from the group consisting of Ci-4alkyl, Ci.2haloalkyl, halogen, cyano, -OCi-4alkyl, and -OCi.2haloalkyl; m is 0-1 ; and n is 0-2; and quantifying a change in fluorescence intensity of the detection reagent being contacted with the sample, relative to a baseline fluorescence intensity of the detection reagent prior to contact with the sample.

[0025] In some embodiments, the methods comprise: 026389-0053-W001 contacting the sample with a detection reagent comprising a compound of formula (II), or a salt thereof: wherein

[0026] X is O and n is 0, or X is N and n is 1 ;

[0027] R10is hydrogen, halogen, cyano, Ci-6alkyl, Ci-ehaloalkyl, or -OR10a, -SR10a, -CC>210a, -C(O)R10a, -SO2R10a, -N(R10a)2, -CO2N(R10a)2, or -NO2, where:

[0028] R10a, at each occurrence, is independently, hydrogen, Ci-ealkyl, or Ci-2haloalkyl;

[0029] R20is hydrogen, halogen Ci-ealkyl, Ci-ehaloalkyl, or -OR10b, -SR10b, -CO2R10b, -C(O)R10b, -SO2R10b, -N(R10b)2, -CO2N(R10b)2, or -NO2, wherein:

[0030] R10b, at each occurrence, is independently, hydrogen, Ci-ealkyl, or Ci-2haloalkyl;

[0031] R30, at each occurrence is hydrogen or Ci-ealkyl;

[0032] G10is a 4- to 12-membered heterocyclylene, a Cs- carbocyclylene, a 6- to 12-membered arene, or a 5- to 12-membered heteroarene, wherein G10is optionally substituted with 1-6 substituents, each independently selected from the group consisting of Ci-4alkyl, Ci-2haloalkyl, halogen, cyano, -OCi-4alkyl, and -OCi-2haloalkyl;

[0033] G20, at each occurrence, is independently a 5- to 12-membered heteroarene, wherein G20is optionally substituted with 1-5 substituents, each independently selected from the group consisting of Ci-4alkyl, Ci.2haloalkyl, halogen, cyano, -OCi-4alkyl, and -OCi-2haloalkyl;

[0034] G30, at each occurrence, is independently a 5- to 12-membered heteroaryl or a 6- to 12- membered aryl, wherein G30is optionally substituted with 1-5 substituents, each independently selected from the group consisting of Ci-4alkyl, Ci-2haloalkyl, halogen, cyano, -OCi-4alkyl, and -OCi-2haloalkyl; w is 0-1 ; x is 1-2; n is 0-1 ; and 026389-0053-W001 quantifying a change in fluorescence intensity of the detection reagent being contacted with the sample, relative to a baseline fluorescence intensity of the detection reagent prior to being contacted with the sample.

[0035] In some embodiments, the methods comprise: contacting the sample with a detection reagent comprising a compound of formula (III), or a salt thereof, wherein

[0036] Z is a solid support;

[0037] L10is Ci-ioalkylene; Ci- alkylene-C(O)-, or -O-Ci-ioalkylene-C(O)-; , where: m is 20-60;

[0038] R10is hydrogen, halogen, cyano, Ci-ealkyl, Ci-ehaloalkyl, -OR10a, -SR10a, -CC>210a, -C(O)R10a, -SO2R10a, -N(R10a)2, -CO2N(R10a)2, or -NO2, where:

[0039] R10a, at each occurrence, is independently, hydrogen, Ci-ealkyl, or Ci-2haloalkyl;

[0040] R20is hydrogen, halogen Ci-ealkyl, Ci-ehaloalkyl, -OR10b, -SR10b, -CO2R10b, -C(O)R10b, -SO2R10b, -N(R10b)2, -CO2N(R10b)2, or -NO2, where:

[0041] R10b, at each occurrence, is independently, hydrogen, Ci-ealkyl, or Ci-2haloalkyl;

[0042] G10is a 4- to 12-membered heterocyclylene, a Cs- carbocyclylene, a 6- to 12-membered, aryl, or a 5- to 12-membered heteroaryl, wherein G10is optionally substituted with 1-6 substituents independently selected from the group consisting of Ci-4alkyl, Ci. 2haloalkyl, halogen, cyano, -OCi.4alkyl, and -OCi.2haloalkyl;

[0043] G20, at each occurrence, is independently a 5- to 12-membered heteroaryl, wherein G20is optionally substituted with 1-5 substituents independently selected from the group consisting of Ci.4alkyl, Ci.2haloalkyl, halogen, cyano, -OCi.4alkyl, and -OCi. 2haloalkyl;

[0044] G30, at each occurrence, is independently a 5- to 12-membered heteroaryl or a 6- to 12- membered aryl, wherein G30is optionally substituted with 1-5 substituents 026389-0053-W001 independently selected from the group consisting of Ci-4alkyl, Ci-shaloalkyl, halogen, cyano, -OC- alkyl, and -OCi-2haloalkyl; n is 0-1 ; w is 0-1 ; and x is 1-2; and quantifying a change in fluorescence intensity of the detection reagent while being contacted with the sample, relative to a baseline fluorescence intensity of the detection reagent prior to being contacted with the sample.

[0045] In some instances, the methods comprise: contacting the sample with a detection reagent comprising a compound of formula (IV), or a salt thereof: wherein

[0046] X100is S, N(R110), or O;

[0047] L100is Co-3alkylene-C=C-Co-3alkylene or Ci-6alkylene;

[0048] R100is -N(GY)2, -N(R100a)2, -O-(GY)2, -O-(R100a), or -O-(GY) where:

[0049] GY, at each occurrence, is independently a 6- to 12-membered aryl or a 5- to 12- membered heteroaryl, wherein GYis optionally substituted with 1-5 substituents independently selected from the group consisting of hydrogen, halogen, Ci-4alkyl , Ci-2haloalkyl, cyano, -OCi-4alkyl, or -OCi-2haloalkyl; and

[0050] R100a, at each occurrence, is independently hydrogen or Ci-ealkyl;

[0051] R110is hydrogen, Ci.6alkyl, or Ci.2haloalkyl;

[0052] R120is hydrogen, halogen, Ci.4alkyl, Ci.2haloalkyl, cyano, -OCi.4alkyl, or-OCi.2haloalkyl; and

[0053] R130is hydrogen, halogen, Ci-4alkyl, Ci.2haloalkyl, cyano, -OCi^alkyl, or-OCi-2haloalkyl; and

[0054] X- is a counterion; and quantifying a change in fluorescence intensity of the detection reagent being contacted with the sample relative to a baseline fluorescence intensity of the detection reagent prior to being contacted with the sample.

[0055] In some instances, the methods comprise: 026389-0053-W001 contacting the sample with a detection reagent comprising a compound of formula (V), or a salt thereof: wherein

[0056] R200is halogen, hydrogen, Ci-ealkyl, Ci-2haloalkyl, or -OCi-2haloalkyl;

[0057] G200is a 4- to 12-membered heterocyclylene or a Cs- carbocyclylene, wherein G200is optionally substituted with 1-4 substituents, each independently selected from the group consisting of Ci-4alkyl , Ci.2haloalkyl, halogen, cyano, -OCi-4alkyl, and -OC1. 2haloalkyl;

[0058] G210is a 5- to 12-membered heteroaryl or a 6- to 12-membered aryl, wherein G210is optionally substituted with 1-4 substituents, each independently selected from the group consisting of Ci-4alkyl, Ci -2haloal kyl, halogen, cyano, -OCi-4alkyl, and -OCi. 2haloalkyl; and quantifying a change in fluorescence intensity of the detection reagent while being contacted with the sample relative to a baseline fluorescence intensity of the detection reagent prior to being contacted with the sample.

[0059] DESCRIPTION OF DRAWINGS

[0060] FIG. 1 shows fluorescence emission spectra at an emission wavelength of 460 nm (when excited at 350 nm) in response to 2 pM IPY in the presence 0 to 150 pM of PFOA.

[0061] FIG. 2A is a linear plot of fluorescence intensity at an emission wavelength of 460 nm (when excited at 350 nm) in response to 2 pM IPY as a function of PFOA concentration from 0 to 140 nM. From the linear fitting, a limit of detection of 40.4 nM (16.7 ppb) was calculated for IPY detection of PFOA. FIG. 2B displays a Stern-Volmer plot of I0 / I at an emission wavelength of 460 nm (when excited at 350 nm) in response to 2 pM IPY as a function of PFOA concentration (0 pM to 0.15 pM), where Zois the fluorescence intensity of IPY in the absence of PFOA and I is the fluorescence intensity of IPY in the presence of PFOA. From the linear fitting, a Stern-Volmer plot quenching constant (Ksv) of 1.9 x 105M-1was calculated using the equation: loll = KSV[Q] + 1 , where [Q] is the concentration of PFOA.

[0062] FIG. 3 shows the absorption spectra of 10 pM IPY upon addition of different concentration of PFOA with isosbestic point at 226 nm and 372 nm. 026389-0053-W001

[0063] FIG. 4 shows a Benesi-Hildebrand plot with Amax-Ao / Ax-Ao in response to 10 pM IPY as a function of 1 / [PFOA] for the formation of IPY + PFOA complex. The Benesi-Hildebrand plot was used to calculate the binding constant (Ka) for the binding of PFOA to IPY. A Kavalue of 2.94 x 104M’1was calculated from the plot.

[0064] FIG. 5 displays a bar graph showing the fluorescence quenching efficiency of 150 pM of various analytes on the fluorescence of 2 pM IPY at 460 nm (when excited at 350 nm).

[0065] FIG. 6 displays a bar graph showing the competition studies of 2 pM IPY, 150 pM PFOA, and 300 pM of various potential interferants.

[0066] FIG. 7 displays the structures of various PFAS and interferants.

[0067] FIG. 8 is a plot of the fluorescence emission intensity of 2 pM of IPY at an emission wavelength of 460 nm (when excited at wavelength of 350 nm) in the absence of 150 pM PFOA and in the presence of 150 pM PFOA as a function of time.

[0068] FIG. 9 is a plot of the fluorescence emission intensity of 2 pM IPY at an emission wavelength of 460 nm (when excited at an absorbance wavelength of 350 nm) in the absence of PFOA and in the presence of 150 pM PFOA as a function of ionic strength (i.e. , 0-10 mM NaCI).

[0069] FIG. 10 is a plot of the fluorescence emission intensity of 2 pM IPY at an emission wavelength of 460 nm (when excited at an absorbance wavelength of 350 nm) in the absence of PFOA and in the presence of 150 pM PFOA as a function of temperature (i.e., 25-70 °C).

[0070] FIG. 11 displays a Job’s plot of fluorescence intensity as a function of the mole fraction of IPY. Job’s plot shows peak fluorescence intensity at a mole fraction of 0.5 IPY and 0.5 PFOA, indicating a 1 :1 ratio of IPY:PFOA in the IPY + PFOA complex.

[0071] FIG. 12 shows fluorescence spectra at an emission wavelength of 525 nm (when excited at 400 nm) in response to 3.3 pM NA-3N in the presence of 0 to 4 pM PFOA in acetonitrile.

[0072] FIG. 13 shows a linear plot of the Benesi-Hildebrand equation with fluorescence data for / max- / o / / x- / o of NA-3N as a function of 1 / [concentration of PFOA], The complex NA-3N + PFOA complex is assumed to be 1 :1. The high-quality linear fitting of the fluorescence data confirms the 1 :1 complex between NA-3N and PFOA. From the Benesi-Hildebrand equation, a limit of detection (LOD) of 15.7 nM (6.5 ppb) was calculated for NA-3N detection of PFAS.

[0073] FIG. 14 shows a schematic illustration showing the mechanism of fluorescence activation of NA-3N by PFOA. Upon complexation with PFOA (shown on the right), the rotational dynamics between the electron donor group (here the pyridyl piperazine) and the naphthalic fluorophore (acting as an electron acceptor) gets restricted, thus blocking the electron transfer to certain extent depending on the complexation strength, resulting in fluorescence activation. 026389-0053-W001

[0074] FIG. 15 shows the UV-vis absorption spectra of 50 pM NA-3N in acetonitrile with 0 pM to 100 pM of PFOA with two isosbestic points, indicating quantitative conversion from the free molecules of NA-3N to the complexed state.

[0075] FIG. 16 shows a Benesi-Hildebrand plot with Amax-Ao / Ax-Ao of 50 pM NA-3N as a function of 1 / [concentration of PFOA] for the formation of NA-3N + PFOA complex at 385 nm.

[0076] FIG. 17 is a bar chart showing interference testing that compares the ability of 4 pM PFOA to quench the fluorescence emission of 3.3 pM NA-3N at 525 nm (when excited at 400 nm) in water in the presence of various other analytes, each at a concentration of 40 pM, thus showing the selectivity of NA-3N towards PFOA.

[0077] FIG. 18 displays a Job’s plot of fluorescence intensity as a function of PFOA. Job’s plot shows peak fluorescence intensity at a mole fraction of 0.5 PFOA (50 pM) and 0.5 NA-3N (50 pM), indicating a 1:1 ratio of NA-3N: PFOA in the NA-3N + PFOA complex.

[0078] FIG. 19 shows the fluorescence spectra at an emission wavelength of 480 nm (when excited at 370 nm) in response to 0.5 pM DIPY in the presence of 0 to 5 pM of PFOA.

[0079] FIG. 20 displays a linear plot of fluorescence intensity of 0.5 pM pM DIPY as a function of PFOA concentration from 0 to 140 nM measured an emission wavelength of 480 nm (when excited at 370 nm). From the linear fitting, a limit of detection (LOD) of 28.5 nM (11.8 ppb) was calculated for DIPY detection of PFOA.

[0080] FIG. 21 shows UV-visible absorption spectra of 5 pM DIPY with different concentrations of PFOA (0 to 100 pM). The spectra show an isosbestic point at 393 nm, indicating the formation of the PFOA and DIPY complex.

[0081] FIG. 22 shows a Benesi-Hildebrand plot with Amax-Ao / Ax-Ao of 5 pM DIPY as a function of 1 / [concentration of PFOA] for the formation of DIPY + PFOA complex at an absorbance of 420 nm. The Benesi-Hildebrand plot was used to calculate the binding constant (Ka) for the binding of PFOA to DIPY. A Kavalue of 1.80 x 104M’1was calculated from the plot.

[0082] FIG. 23 is a bar graph showing the fluorescence quenching efficiency of 10 pM of various analytes on the fluorescence of 2 pM DIPY at 480 nm (when excited at 370 nm). The fluorescence quenching efficiency calculated in the experiment was the % decrease in intensity of the DIPY as measured at an emission wavelength of 480 nm (when excited at a wavelength of 370 nm) in the presence of the various other analytes relative to the intensity of the fluorescence intensity of the DIPY in the absence of the analyte.

[0083] FIG. 24 is a bar chart showing interference testing that compares the ability of 2 pM PFOA to quench the fluorescence emission of 2 pM DIPY at 480 nm (when excited at 370 nm) in water 026389-0053-W001 in the presence of various other analytes, each at a concentration of 20 pM, thus showing the selectivity of DIPY towards PFOA.

[0084] FIG. 25 is a plot of the fluorescence emission intensity of 2 pM DIPY at an emission wavelength of 480 nm (when excited at 370 nm) in the absence of PFOA and in the presence of 10 pM PFOA as a function of ionic strength (i.e., 0-1.4 mM NaCI).

[0085] FIG. 26 is a plot of the fluorescence emission intensity of 2 pM DIPY at an emission wavelength of 480 nm (when excited at 370 nm) in the absence of PFOA and in the presence of 10 pM PFOA as a function of temperature (i.e., 25-70 °C).

[0086] FIG. 27 displays a Job’s plot of fluorescence intensity of DIPY as a function of PFOA at an emission wavelength of 480 nm (when excited at 370 nm). Job’s plot shows peak fluorescence intensity at a mole fraction of 0.5 PFOA (2 pM) and 0.5 DIPY (2 pM), indicating a 1 :1 ratio of DIPY: PFOA in the DIPY + PFOA complex.

[0087] FIG. 28 shows the fluorescence spectra at an emission wavelength of 480 nm (when excited at 350 nm) in response to 2 pM TPIPY in the presence of 0 to 10 pM of PFOA.

[0088] FIG. 29 shows a linear plot of fluorescence intensity of 2 pM TPIPY as a function of PFOA concentration (0 to 1.8 pM) measured at an emission wavelength of 480 nm (when excited at 350 nm). From the linear fitting, a limit of detection (LOD) of 20.5 nM (8.50 ppb) was calculated for TPIPY detection of PFOA.

[0089] FIG. 30 displays a Stern-Volmer plot of I0 / I of TPIPY as a function of PFOA concentration, where / 0is the fluorescence intensity of TPIPY in the absence of PFOA and I is the fluorescence intensity of TPIPY in the presence of PFOA. Using the Stern-Volmer plot, a Stern-Volmer plot quenching constant (Ksv) of 3.54 x 105M-1was calculated using the equation: Zo / / =SV[Q]+1 , where [Q] is the concentration of PFOA.

[0090] FIG. 31 shows UV-visible absorption spectra of 2 pM TPIPY with different concentrations of PFOA (0 to 50 pM). The spectra show an isosbestic point at 388 nm, indicating the formation of the PFOA and TPIPY complex.

[0091] FIG. 32 FIG. 22 shows a Benesi-Hildebrand plot with Amax-Ao / A<-Ao of 10 pM TPIPY as a function of 1 / [PFOA] for the formation of TPIPY + PFOA complex at an absorbance of 435 nm. The Benesi-Hildebrand plot was used to calculate the binding constant (Ka) for the binding of PFOA to TPIPY. A Ka value of 2.15 105IVT1was calculated from the plot.

[0092] FIG. 33 is a bar graph showing the fluorescence quenching efficiency of 10 pM of various analytes on the fluorescence of 2 pM TPIPY at 480 nm (when excited at 350 nm). The fluorescence quenching efficiency calculated in the experiment was the % decrease in intensity of the TPIPY as measured at an emission wavelength of 480 nm (when excited at a wavelength 026389-0053-W001 of 350 nm) in the presence of the various other analytes relative to the intensity of the fluorescence intensity of the DIPY in the absence of the analyte.

[0093] FIG. 34 is a bar chart showing interference testing that compares the ability of 10 pM PFOA to quench the fluorescence emission of 2 pM TPIPY at 480 nm (when excited at 350 nm) in water in the presence of various other analytes, each at a concentration of 20 pM, thus showing the selectivity of DIPY towards PFOA.

[0094] FIG. 35 is a plot of the fluorescence emission intensity of 2 pM TPIPY at an emission wavelength of 480 nm (when excited at 350 nm) in the absence of PFOA and in the presence of 10 pM PFOA as a function of ionic strength (i.e., 0-30 mM NaCI).

[0095] FIG. 36 is a plot of the fluorescence emission intensity of 2 pM TPIPY at an emission wavelength of 480 nm (when excited at 350 nm) in the absence of PFOA and in the presence of 10 pM PFOA as a function of temperature (i.e., 25-70 °C).

[0096] FIG. 37 displays a Job’s plot of fluorescence intensity of TPIPY as a function of PFOA at an emission wavelength of 480 nm (when excited at 350 nm). Job’s plot shows peak fluorescence intensity at a mole fraction of 0.5 TPIPY (2 pM) and 0.5 PFOA (2 pM), indicating a 1 :1 ratio of TPIPY: PFOA in the TPIPY + PFOA complex.

[0097] FIG. 38 shows the fluorescence spectra at an emission wavelength of 620 nm (when excited at 430 nm) in response to 2 pM BTD-3N in the presence of 0 to 20 pM of PFOA.

[0098] FIG. 39 shows a linear plot of fluorescence intensity of 2 pM BTD-3N as a function of PFOA concentration (0 to 6 pM) measured at an emission wavelength of 620 nm (when excited at 430 nm). From the linear fitting, a limit of detection (LOD) of 12.7 nM (5.26 ppb) was calculated for BTD-3N detection of PFOA.

[0099] FIG. 40 shows UV-visible absorption spectra of 10 pM BTD-3N with different concentration of PFOA (0 to 100 pM).

[0100] FIG. 41 is a bar graph showing the increase in fluorescence intensity of 2 pM BTD-3N at an emission wavelength of 620 nm (when excited at 430 nm) in response 10 pM of various analytes on the fluorescence.

[0101] FIG. 42 is a bar chart showing interference testing that compares the ability of 10 pM PFOA to quench the fluorescence emission of 2 pM BTD-3N at an emission wavelength of 620 nm (when excited at 430 nm) in water in the presence of various other analytes, each at a concentration of 20 pM, thus showing the selectivity of BTD-3N towards PFOA.

[0102] FIG. 43 is a plot of the fluorescence emission intensity of 2 pM BTD-3N at an emission wavelength of 620 nm (when excited at 430 nm) in the absence of PFOA and in the presence of 10 pM PFOA as a function of ionic strength (i.e., 0-30 mM NaCI). 026389-0053-W001

[0103] FIG. 44 is a plot of the fluorescence emission intensity of 2 pM BTD-3N at an emission wavelength of 480 nm (when excited at 430 nm) in the absence of PFOA and in the presence of 10 pM PFOA as a function of temperature (i.e., 25-70 °C).

[0104] FIG. 45 shows the fluorescence spectra at an emission wavelength of 590 nm (when excited at 520 nm) in response to 2 pM Benz-1 + in the presence of 0 to 10 pM of PFOS.

[0105] FIG. 46 is a linear plot of fluorescence intensity of 5 pM Benz-1 + as a function of PFOS concentration (0 to 2 pM) measured at an emission wavelength of 590 nm (when excited at 520 nm). From the linear fitting, a limit of detection (LOD) of 48.0 nM (25.8 ppb) was calculated for Benz-1 + detection of PFOS.

[0106] FIG. 47 shows UV-visible absorption spectra of 5 pM Benz-1 + with different concentration of PFOS (0 to 10 pM). The spectra show isosbestic points at 441 nm and 578 nm, indicating the formation of the PFOS + Benz-1 + complex.

[0107] FIG. 48 is a bar graph showing the fluorescence quenching efficiency of 10 pM of various analytes on the fluorescence of 2 pM Benz-1 + at and emission wavelength of 590 nm (when excited at 520 nm). The fluorescence quenching efficiency calculated in the experiment was the % decrease in intensity of the Benz-1 + as measured at an emission wavelength of 590 nm (when excited at a wavelength of 520 nm) in the presence of the various other analytes relative to the intensity of the fluorescence intensity of the Benz-1+ in the absence of the analyte.

[0108] FIG. 49 is a bar chart showing interference testing that compares the ability of 10 pM PFOS to quench the fluorescence emission of 5 pM Benz-1+ at an emission wavelength of 590 nm (when excited at 520 nm) in water in the presence of various other analytes, each at a concentration of 20 pM, thus showing the selectivity of Benz-1+ for PFOS.

[0109] FIG. 50 is a plot of fluorescence emission intensity of 2 pM Benz-1 + at an emission wavelength of 590 nm (when excited at 520 nm) in the absence of PFOS and in the presence of 10 pM PFOS as a function of ionic strength (i.e., 0-70 mM NaCI).

[0110] FIG. 51 shows the fluorescence spectra at an emission wavelength of 670 nm (when excited at 520 nm) in response to 5 pM TP-1+ in the presence of 0 to 10 pM of PFOS.

[0111] FIG. 52 is a linear plot of fluorescence intensity of 5 pM TP-1+ as a function of PFOS concentration (0 to 10 pM) measured at an emission wavelength of 670 nm (when excited at 520 nm). From the linear fitting, a limit of detection (LOD) of 7.2 nM (3.9 ppb) was calculated for TP- 1+ detection of PFOS.

[0112] FIG. 53 shows UV-visible absorption spectra of 5 pM TP-1 + with different concentration of PFOS (0 to 10 pM). The spectra show isosbestic points at 407 nm and 554 nm, indicating the formation of the PFOS + TP-1+ complex. 026389-0053-W001

[0113] FIG. 54 is a bar graph showing the fluorescence quenching efficiency of 10 pM of various analytes on the fluorescence of 5 pM TP-1+ at and emission wavelength of 670 nm (when excited at 520 nm). The fluorescence quenching efficiency calculated in the experiment was the % decrease in intensity of the TP-1+ as measured at an emission wavelength of 670 nm (when excited at a wavelength of 520 nm) in the presence of the various other analytes relative to the intensity of the fluorescence intensity of the TP-1+ in the absence of the analyte.

[0114] FIG. 55 is a bar chart showing interference testing that compares the ability of 10 pM PFOS to quench the fluorescence emission of 5 pM TP-1+ at an emission wavelength of 670 nm (when excited at 520 nm) in water in the presence of various other analytes, each at a concentration of 10 pM, thus showing the selectivity of TP-1+ for PFOS.

[0115] FIG. 56 is a plot of the fluorescence emission intensity of 5 pM TP-1+ at an emission wavelength of 670 nm (when excited at 520 nm) in the absence of PFOA and in the presence of 5 pM PFOS as a function of ionic strength (i.e., 0-40 mM NaCI).

[0116] FIG. 57 is a plot of the fluorescence emission intensity of 5 pM TP-1+ at an emission wavelength of 670 nm (when excited at 520 nm) in the absence of PFOS and in the presence of 10 pM PFOS as a function of pH (i.e., 1-11).

[0117] FIG. 58 shows the fluorescence spectra at an emission wavelength of 670 nm (when excited at 520 nm) for TP-1+ in 0% to 90% glycerol with weak fluorescence at 0% glycerol and increasing fluorescence with an increasing amount of glycerol.

[0118] FIG. 59 is a plot of fluorescence intensity at an emission wavelength of 670 nm (when excited at 520 nm) for TP-1 +. Experiments were performed twice as shown by the graph with the first experiment (circles) and second experiment (squares).

[0119] FIG. 60A shows the fluorescence spectra at an emission wavelength of 435 nm (when excited at 350 nm) in response to 2 pM NI-TPY in the presence of 0 to 10 pM of PFOA. FIG. 60B is a linear plot of the fluorescence intensity of the NI-TPY measured at 435 nm in the presence of 0 to 100 pM PFOA. FIG. 60C is a linear regression plot of the fluorescence intensity measure at 435 nm. From the linear regression fitting of the data, the limit of detection (LOD) of 23.3 nM (9.70 ppb) was calculated for NI-TPY detection of PFOA.

[0120] FIG. 61 shows a Stern-Volmer plot of lo / l of NI-TPY as a function of PFOA concentration, where / o is the fluorescence intensity of NI-TPY in the absence of PFOA and I is the fluorescence intensity of NI-TPY in the presence of PFOA. From the linear fitting of the data, a Stern-Volmer plot quenching constant ( / <sv) of 6.20 x 105M-1was calculated using the equation: Zo / / =SV[Q]+1 , where [Q] is the concentration of PFOA. 026389-0053-W001

[0121] FIG. 62A shows UV-visible absorption spectra of 10 pM NI-TPY with different concentration of PFOA (0 to 100 pM). The spectra show an isosbestic point at 293 nm, indicating the formation of the PFOA and NI-TPY complex. FIG. 62B is a Benesi-Hildebrand plot with Amax-Ao / Ax-Ao of 10 pM NI-IPY as a function of 1 / [PFOA] for the formation of NI-IPY + PFOA complex at an absorbance of 350 nm. The Benesi-Hildebrand plot was used to calculate the binding constant (a) for the binding of PFOA to TPIPY. A Kavalue of 7.1 x 104M’1was calculated from the plot.

[0122] FIG. 63 shows is a bar chart showing the quenching efficiency of 2 pM NI-TPY with various other analytes, each at a concentration of 100 pM. The fluorescence quenching efficiency calculated in the experiment was the % decrease in intensity of the NI-TPY as measured at an emission wavelength of 435 nm (when excited at a wavelength of 350 nm) in the presence of the various other analytes relative to the intensity of the fluorescence intensity of the NI-TPY in the absence of the analyte.

[0123] FIG. 64 is a bar chart showing interference testing that compares the ability of 100 pM PFOA to quench the fluorescence emission of 2 pM NI-TPY at an emission wavelength of 435 nm (when excited at 350 nm) in water in the presence of various other analytes, each at a concentration of 200 pM, thus showing the selectivity of NI-TPY towards PFOA.

[0124] FIG. 65 is a plot of the fluorescence emission intensity of 2 pM NI-TPY at an emission wavelength of 435 nm (when excited at 350 nm) in the absence of PFOA and in the presence of 100 pM PFOA as a function of response time.

[0125] FIG. 66 is a plot of the fluorescence emission intensity of 2 pM NI-TPY at an emission wavelength of 435 nm (when excited at 350 nm) in the absence of PFOA and in the presence of 100 pM PFOA as a function of ionic strength (i.e. , 0-30 mM NaCI).

[0126] FIG. 67 is a plot of the fluorescence emission intensity of 2 pM NI-TPY at an emission wavelength of 435 nm (when excited at 350 nm) in the absence of PFOA and in the presence of 100 pM PFOA as a function of temperature (i.e., 25-70 °C).

[0127] FIG. 68 displays a Job’s plot of fluorescence intensity of NI-TPY as a function of PFOA at an emission wavelength of 435 nm (when excited at 350 nm). Job’s plot shows peak fluorescence intensity at a mole fraction of 0.5 NI-TPY (2 pM) and 0.5 PFOA (2 pM), indicating a 1 :1 ratio of NI- TPY: PFOA in the NI-TPY + PFOA complex.

[0128] FIG. 69 shows the fluorescence spectra of 1 mg / mL Si@EPTES in the absence or presence of Fluorescamine (F.A.) (50 pM or 100 pM) measured at an emission wavelength of 475 nm (when excited at 380 nm). 026389-0053-W001

[0129] FIG. 70 shows the fluorescence spectra of 1 mg / mL Si@EPTES+PEG in the absence or presence of Fluorescamine (F.A.) (0-130 pM) measured at an emission wavelength of 475 nm (when excited at 380 nm).

[0130] FIG. 71 is a linear plot of fluorescence intensity as a function of fluorescamine (F.A.) concentration.

[0131] FIG. 72 shows the fluorescence spectra at an emission wavelength of 524 nm (when excited at 400 nm) in response to 1 mg / mL 5pSG-PEG44-NI-3N (5:1) in the presence of 0 to 800 pM of PFOA.

[0132] FIG. 73 is a linear plot of the fluorescence intensity at an emission wavelength of 524 nm (when excited at 400 nm) in response to 1 mg / mL 5pSG-PEG44-NI-3N (5:1) in the presence of 0 to 800 pM of PFOA.

[0133] FIG. 74 is a linear regression plot of the fluorescence intensity of 1 mg / mL 5pSG-PEG44- NI-3N (5:1) measured at 435 nm (when excited with 400 nm) in the presence of PFOA (0 to 400 pM). From the linear regression fitting of the data, the limit of detection (LOD) of 26 pM (10.7 ppm) was calculated for 5pSG-PEG44-NI-3N (5:1) detection of PFOA.

[0134] FIG. 75 shows the fluorescence spectra at an emission wavelength of 524 nm (when excited at 400 nm) in response to 1 mg / mL pM 5pSG-PEG44-NI-3N (20:1) in the presence of 0 to 700 pM of PFOA.

[0135] FIG. 76 is a plot of fluorescence intensity of 1 mg / mL 5pSG-PEG44-NI-3N (20:1) measured at 524 nm (when excited at 400 nm) as a function of PFOA concentration (0 to 700 pM).

[0136] FIG. 77 is a linear regression plot of the fluorescence intensity of 1 mg / mL 5pSG-PEG44- NI-3N (20:1) measured at 435 nm (when excited with 380 nm) in the presence of PFOA (0 to 400 pM). From the linear regression fitting of the data, the limit of detection (LOD) of 31 pM (12.9 ppm) was calculated for 5pSG-PEG44-NI-3N (20:1) detection of PFOA.

[0137] FIG. 78 shows the fluorescence spectra at an emission wavelength of 524 nm (when excited at 400 nm) in response to 1 mg / mL 5pSG-PEG44-NI-3N (50:1) in the presence of 0 to 700 pM of PFOA.

[0138] FIG. 79 is a plot of fluorescence intensity of 1 mg / mL 5pSG-PEG44-NI-3N (50:1) measured at 524 nm (when excited at 400 nm) as a function of PFOA concentration (0 to 700 pM).

[0139] FIG. 80 is a linear regression plot of the fluorescence intensity of 1 mg / mL 5pSG-PEG44- NI-3N (50:1) measured at 435 nm (when excited with 400 nm) in the presence of PFOA (0 to 400 026389-0053-W001 pM). From the linear regression fitting of the data, the limit of detection (LOD) of 34.6 pM (14.32 ppm) was calculated for 5pSG-PEG44-NI-3N (50:1) detection of PFOA.

[0140] FIG. 81 the fluorescence spectra at an emission wavelength of 524 nm (when excited at 400 nm) in response to 1 mg / mL non-porous 5pSG-PEG44-NI-3N (5:1) in the presence of 0 to 750 pM of PFOA.

[0141] FIG. 82 is a linear regression plot of the fluorescence intensity of 1 mg / mL non-porous 5pSG-PEG44-NI-3N (5:1) measured at 435 nm (when excited with 400 nm) in the presence of PFOA (0 to 400 pM). From the linear regression fitting of the data, the limit of detection (LOD) of 35 pM (15 ppm) was calculated for 5pSG-PEG44-NI-3N (5:1) detection of PFOA.

[0142] DETAILED DESCRIPTION

[0143] Described herein are methods for detecting one or more per- or poly-fluoroalkyl substance (PFAS) fluorophores. 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 of various PFAS.

[0144] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For example, any nomenclatures used in connection with, and techniques of chemistry described herein are well known and commonly used in the art. In case of conflict, the present disclosure, including definitions, will control. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the embodiments and aspects described herein.

[0145] As used herein, terms such as “include,” “including,” “contain,” “containing,” “having,” and the like mean “comprising.” The present disclosure also contemplates other embodiments “comprising,” “consisting essentially of,” and “consisting of” the embodiments or elements presented herein, whether explicitly set forth or not. As used herein, “comprising,” is an “open- ended” term that does not exclude additional, unrecited elements or method steps. As used herein, “consisting essentially of” limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristics of the claimed invention. As used herein, “consisting of” excludes any element, step, or ingredient not specified in the claim.

[0146] As used herein, the term “a,” “an,” “the” and similar terms used in the context of the disclosure (especially in the context of the claims) are to be construed to cover both the singular and plural unless otherwise indicated herein or clearly contradicted by the context. In addition, “a,” “an,” or “the” means “one or more” unless otherwise specified. 026389-0053-W001

[0147] As used herein, the term “or” can be conjunctive or disjunctive.

[0148] As used herein, the term “and / or” refers to both the conjunctive and disjunctive.

[0149] As used herein, the term “substantially” means to a great or significant extent, but not completely.

[0150] As used herein, the term “about” or “approximately” as applied to one or more values of interest, refers to a value that is similar to a stated reference value, or within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, such as the limitations of the measurement system. In one aspect, the term “about” refers to any values, including both integers and fractional components that are within a variation of up to ± 10% of the value modified by the term “about.” Alternatively, “about” can mean within 3 or more standard deviations, per the practice in the art. Alternatively, such as with respect to biological systems or processes, the term “about” can mean within an order of magnitude, in some embodiments within 5-fold, and in some embodiments within 2-fold, of a value. As used herein, the symbol means “about” or “approximately.”

[0151] All ranges disclosed herein include both end points as discrete values as well as all integers and fractions specified within the range. For example, a range of 0.1-2.0 includes 0.1 , 0.2, 0.3, 0.4 . . . 2.0. If the end points are modified by the term “about,” the range specified is expanded by a variation of up to ±10% of any value within the range or within 3 or more standard deviations, including the end points, or as described above in the definition of “about.”

[0152] As used herein, the terms “room temperature,” “RT,” or “ambient temperature” refer to the typical temperature in an indoor laboratory setting. In one aspect, the laboratory setting is climate controlled to maintain the temperature at a substantially uniform temperature or with a specific range of temperatures. In one aspect, “room temperature” refers a temperature of about 15-30 °C, including all integers and endpoints within the specified range. In another aspect, “room temperature” refers a temperature of about 15-30 °C; about 20-30 °C; about 22-30 °C; about 25-30 °C; about 27-30 °C; about 15-22 °C; about 15-25 °C; about 15-27 °C; about 20-22 °C; about 20-25 °C; about 20-27 °C; about 22-25 °C; about 22-27 °C; about 25-27 °C; about 15 °C ± 10%; about 20 °C ± 10%; about 22 °C ± 10%; about 25 °C ± 10%; about 27 °C ± 10%; ~20 °C, ~22 °C, ~25 °C, or ~27 °C, at standard atmospheric pressure.

[0153] As used herein, the terms “control,” or “reference” are used herein interchangeably. A “reference” or “control” level may be a predetermined value or range, which is employed as a baseline or benchmark against which to assess a measured result. “Control” also refers to control experiments or control cells. 026389-0053-W001

[0154] Definitions of specific functional groups and chemical terms are described in more detail below. For purposes of this disclosure, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th ed., inside cover, and specific functional groups are generally defined as described therein. Additionally, general principles of organic chemistry, as well as specific functional moieties and reactivity, are described in Organic Chemistry, Thomas Sorrell, University Science Books, Sausalito, 1999; Smith and March March’s Advanced Organic Chemistry, 5thed., John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989; Carruthers, Some Modern Methods of Organic Synthesis, 3rded. Cambridge University Press, Cambridge, 1987; the entire contents of each of which are incorporated herein by reference.

[0155] 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 tertbutoxy.

[0156] The term “alkyl,” as used herein, means a straight or branched, saturated hydrocarbon chain. The term “lower alkyl” or “Ci-ealkyl” means a straight or branched chain hydrocarbon containing from 1 to 6 carbon atoms. The term “Ci-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, / so-propyl, n-butyl, sec-butyl, / so-butyl, tert-butyl, n- pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n- heptyl, n-octyl, n-nonyl, and rj-decyl.

[0157] The term “alkenyl,” as used herein, means a straight or branched, hydrocarbon chain containing at least one carbon-carbon double bond.

[0158] 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.

[0159] 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.

[0160] 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.

[0161] 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.

[0162] The term “amino,” as used herein, means -NRXR wherein Rxand Ry may 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 - 026389-0053-W001

[0163] NRX-, wherein Rxmay be hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, heterocycle, alkenyl, or heteroalkyl.

[0164] 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][1 ,3]dioxol-5-yl). 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).

[0165] 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.

[0166] The term “cycloalkoxy,” as used herein, refers to a cycloalkyl group, as defined herein, appended to the parent molecular moiety through an oxygen atom.

[0167] 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[1 .1 ,1]pentanyl.

[0168] 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.

[0169] 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. 026389-0053-W001

[0170] The terms “cycloalkylene” and “heterocyclylene” refer to divalent or trivalent groups derived from the base ring, i.e., cycloalkane, heterocycle. For illustration, an example cycloalkylene may be cyclohexene or and a heterocyclylene may be Cycloalkylene and heterocyclylene include a geminal divalent groups such as 1 ,1-C3-6cycloalkylene. A further example is 1 ,1 -cyclopropylene.

[0171] The term “halogen” or “halo,” as used herein, means Cl, Br, I, or F.

[0172] The term “haloalkyl,” 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.

[0173] 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.

[0174] 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.

[0175] 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.

[0176] 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 the group consisting of N, O and S (e.g., 1, 2, 3, or 4 heteroatoms independently selected from O, 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., 10TT electron system) such as a monocyclic heteroaryl ring fused to a 6-membered arene (e.g., quinolin-4-yl, indol-1-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 10TT 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 026389-0053-W001 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-1-yl, indol-2-yl, indol-4-yl), pyridinyl (including pyridin-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[1 ,2-a]pyridinyl (e.g., imidazo[1 ,2-a]pyridin-6-yl), naphthyridinyl, pyridoimidazolyl, thiazolo[5,4-b]pyridin-2-yl, and thiazolo[5,4-d]pyrimidin-2-yl.

[0177] 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 O, N, and S. The three- or four-membered ring contains zero or one double bond, and one heteroatom selected from the group consisting of O, N, and S. The fivemembered ring contains zero or one double bond and one, two or three heteroatoms selected from the group consisting of O, N and S. The six-membered ring contains zero, one or two double bonds and one, two, or three heteroatoms selected from the group consisting of O, 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 O, 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 026389-0053-W001 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-1-yl). Representative examples of bicyclic heterocyclyls include, but are not limited to, chroman-4-yl, 2,3-dihydrobenzofuran-2-yl, 2,3-dihydrobenzothien-2-yl, 1 , 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-1 H-indol-1-yl, isoindolin-2-yl, octahydrocyclopenta[c]pyrrolyl, octahydropyrrolopyridinyl, tetrahydroisoquinolinyl, 7- oxabicyclo[2.2.1]heptanyl, hexahydro-2H-cyclopenta[b]furanyl, 2-oxaspiro[3.3]heptanyl, 3- oxaspiro[5.5]undecanyl, 6-oxaspiro[2.5]octan-1-yl, and 3-oxabicyclo[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, or a 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-2 H-2, 5- methanocyclopenta[b]furan, hexahydro-1 H-1 ,4-methanocyclopenta[c]furan, aza-adamantane (1- 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.

[0178] The term “hydroxyl” or “hydroxy,” as used herein, means an -OH group.

[0179] 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.

[0180] 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., “Ci.4alkyl,” “C3- ecycloalkyl,” “Ci-4alkylene”). 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 “C1.4,” the members of the group that follows may have any number of carbon atoms falling within the recited 026389-0053-W001 range. A “Ci^alkyl,” for example, is an alkyl group having from 1 to 4 carbon atoms, however arranged (i.e., straight chain or branched).

[0181] 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, =0 (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.

[0182] As used herein, the term “fluorescence” refers to the physical phenomenon in which a substance absorbs light or other electromagnetic radiation at a first wavelength (typically in the ultraviolet or visible range) and subsequently emits light at a second, longer wavelength. Fluorescence occurs when electrons in a molecule are excited to a higher energy state and then return to their ground state, releasing energy in the form of emitted photons. The intensity, wavelength, and duration of the emitted fluorescence can vary depending on the chemical structure of the fluorescent molecule and its surrounding environment. In the context of the present disclosure, fluorescence is used as a measurable signal to detect the presence or concentration of per- or poly-fluoroalkyl substances (PFAS) or other analytes. Changes in fluorescence intensity, emission wavelength, or fluorescence lifetime may be indicative of specific interactions between a detection reagent and the target analyte.

[0183] As used herein, the term “detection reagent” refers to any chemical substance, composition, or formulation that is capable of producing a detectable signal (e.g., a change in fluorescence, absorbance, luminescence, or color) upon interaction with a target analyte, including per- or poly-fluoroalkyl substances (PFAS). A detection reagent may include one or more active components such as fluorophores, chromophores, binding ligands, or molecular recognition elements, and may be used alone or in combination with other reagents, solvents, or buffers to facilitate detection under defined assay conditions. Examples of detection reagents include the compounds described herein, e.g., compounds of formula (l)-(V).

[0184] As used herein, the term “fluorescence intensity” refers to the measurable magnitude of light emitted by a fluorescent substance following excitation by an external light source, typically at a specific wavelength. Fluorescence intensity is commonly expressed in relative fluorescence units (RFU) or other quantitative metrics depending on the detection system employed, and may be influenced by factors such as fluorophore concentration, excitation energy, environmental 026389-0053-W001 conditions, and interactions with analytes. In the context of the present disclosure, fluorescence intensity is used as a quantifiable signal to assess the presence or concentration of per- or polyfluoroalkyl substances (PFAS). A change in fluorescence intensity — relative to a baseline fluorescence intensity — may indicate binding, quenching, enhancement, or other interactions between a detection reagent and PFAS compounds.

[0185] As used herein, the term “baseline fluorescence intensity”, e.g., of a detection reagent, refers to the measurable fluorescence output of a detection reagent, such as a fluorophore described herein, under defined conditions prior to exposure to a test sample. The baseline fluorescence intensity serves as a reference value against which changes in fluorescence (e.g., resulting from interaction with per- or poly-fluoroalkyl substances (PFAS) or other analytes) can be quantified. The baseline may be determined in the absence of PFAS, in a control solution, or under standardized buffer conditions, and may be expressed in absolute or relative fluorescence units depending on the detection system employed.

[0186] As used herein, the terms “fluorophore” and “compound” may be used interchangeably. A fluorophore or fluorescent dye is a molecule capable of emitting light through fluorescence, either inherently or upon interaction with a per- or poly-fluoroalkyl substance (PFAS) or other analyte. In its ground state, the fluorophore is in a low-energy, stable configuration and does not fluoresce. Upon exposure to light of specific wavelengths, the fluorophore absorbs energy and transitions to an excited state. It then returns to the ground state, releasing part of the absorbed energy as emitted light at a longer wavelength. This excitation-emission cycle may repeat as long as the fluorophore continues to absorb light energy.

[0187] As used herein, “excitation” refers to the process by which a photon from an external light source (e.g., laser or lamp) is absorbed by a fluorophore, promoting it from the ground electronic singlet state (So) to an excited singlet state (S1'). The excited state exists briefly and may undergo conformational changes or interact with the surrounding molecular environment. These interactions may dissipate energy, resulting in a relaxed excited state (S1), from which fluorescence emission originates. Competing relaxation pathways include non-radiative decay, intersystem crossing to a triplet state, and fluorescence quenching.

[0188] As used herein, “excitation maximum” refers to the specific wavelength at which a fluorophore is most efficiently excited. Each fluorophore has a characteristic excitation spectrum, representing the range of wavelengths it can absorb. While excitation may occur at wavelengths near the excitation maximum, the resulting fluorescence intensity is reduced. Illumination at the excitation maximum produces the strongest fluorescence signal without altering the shape or range of the emission spectrum. 026389-0053-W001

[0189] As used herein, the term “quencher” refers to a molecule or chemical entity that reduces, suppresses, or eliminates the fluorescence emitted by a fluorophore. Quenching may occur through one or more mechanisms, including but not limited to:

[0190] • Collisional quenching, wherein the quencher physically interacts with the excited fluorophore, dissipating its energy without photon emission;

[0191] • Static quenching, wherein the quencher forms a non-fluorescent complex with the fluorophore prior to excitation;

[0192] • Forster resonance energy transfer (FRET), wherein energy from the excited fluorophore is transferred to a nearby quencher molecule that does not emit light;

[0193] • Photoinduced electron transfer (PET), wherein the quencher facilitates electron transfer to or from the excited fluorophore, preventing fluorescence.

[0194] Quenchers may be used in detection systems to modulate or monitor fluorescence signals, including systems designed to detect per- or poly-fluoroalkyl substances (PFAS) or other analytes.

[0195] As used herein, “Forster Resonance Energy Transfer (FRET)” refers to a radiationless energy transfer process in which energy from an excited donor fluorophore (D) is transferred to a nearby acceptor molecule (A) without photon emission. FRET requires spectral overlap between the donor’s emission spectrum and the acceptor’s excitation spectrum, and close proximity between the donor and acceptor — typically within 1-10 nanometers. The efficiency of energy transfer is highly sensitive to distance and is influenced by factors including the donor’s quantum yield, the acceptor’s extinction coefficient, spectral overlap, the refractive index of the medium, and the relative orientation of the donor and acceptor dipoles.

[0196] 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. 026389-0053-W001

[0197] 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.

[0198] 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 certain wavelengths to pass while blocking out undesirable wavelengths. A bandpass excitation filter transmits a narrow range of wavelengths and may be used for selective excitation.

[0199] 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 the 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 allows 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.

[0200] 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 which selectively blocks out the excitation light to reduce background noise may be used to maximize the signal collected. If multiple fluorophores are used in the sample, a band pass emission filter can be used to isolate the emission from each dye.

[0201] Generally, the methods of the present disclosure comprise the use of fluorophores to selectively detect and / or quantify per- or poly-fluoroalkyl substances (PFAS) in a sample. These methods include contacting a sample with fluorophores disclosed herein. If a PFAS is present in 026389-0053-W001 the sample, then the PFAS interacts with the fluorophores in a manner that induces a change in the ability of the fluorophore to fluoresce at particular emission wavelength(s). Changes in fluorescence intensity of the fluorophores when they come into contact with a sample thus can be used to detect and / or quantify the amount of PFAS in the sample. The fluorophores disclosed herein are therefore being used as detection reagents.

[0202] In the following, various embodiments of the methods are disclosed, showing the various embodiments of fluorophores that may be used for the methods, where the first embodiment is denoted E1 , another embodiment is denoted E2, and so forth.

[0203] E1. A method for detecting one or more per- or poly-fluoroalkyl substances (PFAS) in a sample, the method comprising: contacting the sample with a detection reagent comprising a compound of formula (I), or a salt thereof: wherein

[0204] R1is hydrogen, halogen, cyano, Ci-ealkyl, Ci-ehaloalkyl, -OR1a, -SR1a, -CC>2R1a, -C(O)R1a, -SO2R1a, -N(R1a)2, -CO2N(R1a)2, or -NO2, where:

[0205] R1a, at each occurrence, is independently, hydrogen, C^alkyl, or Ci.2haloalkyl;

[0206] R2, at each occurrence, is independently hydrogen, halogen Ci-ealkyl, Ci-ehaloalkyl, -OR1b, -SR1b, -CO2R1b, -C(O)R1b, -SO2R1b, -N(R1b)2, -CO2N(R1 b)2, or -NO2, where:

[0207] R1b, at each occurrence, is independently, hydrogen, Ci-ealkyl, or Ci-2haloalkyl;

[0208] G1, at each occurrence, is independently a 6- to 12-membered aryl or a 5- to 6-membered heteroaryl, wherein G1is optionally substituted with 1-5 substituents independently selected from the group consisting of Ci.4alkyl, Ci-2haloalkyl, halogen, cyano, -OCi-4alkyl, and -OCi.2haloalkyl;

[0209] G2, at each occurrence, is independently a 5- to 12-membered heteroarene or -N(GX)-, where:

[0210] Gx, at each occurrence, is a 6- to 12-membered aryl optionally substituted with 1-5 substituents independently selected from the group consisting of Ci-4alkyl, Ci. 2haloalkyl, halogen, cyano, -OCi-4alkyl, and -OCi.2haloalkyl; 026389-0053-W001

[0211] G3is a 5- to 6-membered heteroaryl or a 6- to 12-membered aryl, wherein G3is optionally substituted with 1-5 substituents independently selected from the group consisting of Ci-4alkyl, Ci-2haloalkyl, halogen, cyano, -OCi-4alkyl, and -OCi.2haloalkyl; m is 0-1 ; and n is 0-2; and quantifying a change in fluorescence intensity of the detection reagent being contacted with the sample, relative to a baseline fluorescence intensity of the detection reagent prior to contact with the sample.

[0212] E2. The method of E1 , wherein G3is the optionally substituted 5- to 6-membered heteroaryl.

[0213] E3. The method of E2, wherein the 5- to 6-membered heteroaryl is pyridinyl.

[0214] E4. The method of any one of E1-E3, wherein R1and R2are each hydrogen.

[0215] E5. The method of any one of E1-E4, wherein the compound of formula (I) is a compound of formula (la):

[0216] E6. The method of any one of E1-E5, wherein m is 0 and n is 0.

[0217] E7. The method of any one of E1-E5, wherein m is 1 and n is 1.

[0218] E8. The method of any one of E1-E5 or E7, wherein G2is the optionally substituted 5- to 12- membered heteroarene.

[0219] E9. The method of E8, wherein the 5- to 12-membered heteroarene

[0220] E10. The method of any one of E1-E5 or E7-E9, wherein G2is -N(GX)-.

[0221] E11. The method of E10, wherein Gxis the optionally substituted 6- to 12-membered aryl.

[0222] E12. The method of any one of E1-E5 or E7-E11 , wherein G1is the optionally substituted 6- to

[0223] 12-membered aryl.

[0224] E13. The method of E12, wherein the 6- to 12-membered aryl is phenyl.

[0225] E14. The method of any one of E1-E5 or E7-E11 , wherein G1is the optionally substituted 5- to 6-membered heteroaryl.

[0226] E15. The method of E14, wherein the 5- to 6-membered heteroaryl is pyridinyl. 026389-0053-W001

[0227] E16. The method of any one of E1-E15, wherein the compound of formula (I) is selected from the group consisting of:

[0228] E17. A method for detecting one or more per- or poly-fluoroalkyl substances (PFAS) in a sample, the method comprising: contacting the sample with a detection reagent comprising a compound of formula (II), or a salt thereof: wherein

[0229] X is O and n is 0, or X is N and n is 1 ;

[0230] R10is hydrogen, halogen, cyano, Ci-6alkyl, Ci-ehaloalkyl, or -OR10a, -SR10a, -CC>2R10a, -C(O)R10a, -SO2R10a, -N(R10a)2, -CO2N(R10a)2, or -NO2, where:

[0231] R10a, at each occurrence, is independently, hydrogen, Ci-salkyl, or Ci-2haloalkyl;

[0232] R20is hydrogen, halogen Ci-ealkyl, Ci-ehaloalkyl, or -OR10b, -SR10b, -CO2R10b, -C(O)R10b, -SO2R10b, -N(R10b)2, -CO2N(R10b)2, or -NO2, wherein:

[0233] R10b, at each occurrence, is independently, hydrogen, Ci-ealkyl, or Ci.2haloalkyl;

[0234] R30, at each occurrence is hydrogen or Ci.8alkyl;

[0235] G10is a 4- to 12-membered heterocyclylene, a C3-iocarbocyclylene, a 6- to 12-membered arene, or a 5- to 12-membered heteroarene, wherein G10is optionally substituted with 1-6 substituents, each independently selected from the group consisting of Ci-4alkyl, Ci-2haloalkyl, halogen, cyano, -OCi-4alkyl, and -OCi-2haloalkyl;

[0236] G20, at each occurrence, is independently a 5- to 12-membered heteroarene, wherein G20is optionally substituted with 1-5 substituents, each independently selected from the group consisting of Ci-4alkyl, Ci-2haloalkyl, halogen, cyano, -OCi-4alkyl, and -OCi-2haloalkyl; 026389-0053-W001

[0237] G30, at each occurrence, is independently a 5- to 12-membered heteroaryl or a 6- to 12- membered aryl, wherein G30is optionally substituted with 1-5 substituents, each independently selected from the group consisting of Ci.4alkyl, Ci-2haloalkyl, halogen, cyano, - OCi.4alkyl , and -OCi.2haloalkyl; w is 0-1 ; x is 1-2; n is 0-1 ; and quantifying a change in fluorescence intensity of the detection reagent being contacted with the sample, relative to a baseline fluorescence intensity of the detection reagent prior to being contacted with the sample.

[0238] E18. The method of E17, wherein G10is the 4- to 12-membered heterocyclylene or the 6- to 12- membered arene.

[0239] E19. The method of E18, wherein the 4- to 12-membered heterocyclylene is or

[0240] E20. The method of E18, wherein the 6- to 12-membered arene is phenylene.

[0241] E21. The method of any one of E17-E20, wherein R10and R20are each hydrogen.

[0242] E22. The method of any one of E17-E21 , wherein G30, at each occurrence, is the 5- to 12- membered heteroaryl.

[0243] E23. The method of E22, wherein the 5- to 12-membered heteroaryl is pyridinyl or pyrimidinyl.

[0244] E24. The method of any one of E17-E21 , wherein G30, at each occurrence, is the 6- to 12- membered aryl.

[0245] E25. The method of E24, wherein the 6- to 12-membered aryl is phenyl.

[0246] E26. The method of any one of E17-E25, wherein w is 0.

[0247] E27. The method of any one of E17-E25, wherein w is 1.

[0248] E28. The method of any one of E17-E25 or E27, wherein G20is the 5- to 12-membered heteroarene. 026389-0053-W001

[0249] E29. The method of E28, wherein, the 5- to 12-membered heteroarene is

[0250] E30. The method of any one of E17-E29, wherein X is O and n is 0.

[0251] E31. The method of any one of E17-E29, wherein X is N and n is 1. E32. The method of E31 , wherein R30is Ci.8alkyl.

[0252] E33. The method of any one of E17-E32, wherein the compound of formula (II), or a salt thereof, is selected from the group consisting of:

[0253] 026389-0053-W001

[0254] E34. A method for detecting one or more per- or poly-fluoroalkyl substances (PFAS) in a sample, the method comprising: contacting the sample with a detection reagent comprising a compound of formula (IV), or a salt thereof: wherein

[0255] X100is S, N(R110), or O;

[0256] L100is Co-3alkylene-C=C-Co-3alkylene or Ci-ealkylene;

[0257] R100is -N(GY)2, -N(R100a)2, -O-(GY)2, -O-(R100a), or -O-(GY) where:

[0258] GY, at each occurrence, is independently a 6- to 12-membered aryl or a 5- to 12- membered heteroaryl, wherein GYis optionally substituted with 1-5 substituents independently selected from the group consisting of hydrogen, halogen, Ci-4alkyl, Ci.2haloalkyl, cyano, -OCi.4alkyl, or -OCi.2haloalkyl; and

[0259] R100a, at each occurrence, is independently hydrogen or Ci.6alkyl;

[0260] R110is hydrogen, Ci.6alkyl, or Ci.2haloalkyl;

[0261] R120is hydrogen, halogen, Ci-4alkyl, Ci-2haloalkyl, cyano, -OCi^alkyl, or-OCi-2haloalkyl; and

[0262] R130is hydrogen, halogen, Ci.4alkyl, Ci.2haloalkyl, cyano, -OCi^alkyl, or-OCi.2haloalkyl; and

[0263] X- is a counterion; and 026389-0053-W001 quantifying a change in fluorescence intensity of the detection reagent being contacted with the sample relative to a baseline fluorescence intensity of the detection reagent prior to being contacted with the sample.

[0264] E35. The method of E34, wherein X100is S.

[0265] E36. The method of E34 or E35, wherein R120and R130, at each occurrence, are each independently hydrogen.

[0266] E37. The method of any one of E34-E36, wherein the compound of formula (IV) is a compound of formula (I a):

[0267] E38. The method of any one of E34-E37, wherein L100is Co-3alkylene-C=C-Co-3alkylene.

[0268] E39. The method of any one of E34-E38, wherein L100is Coalkylene-C=C-Coalkylene.

[0269] E40. The method of any one of E34-E39, wherein R110is Ci.6alkyl.

[0270] E41. The method of any one of E34-E40, wherein R100is -N(GY)2 or -N(R100a)2.

[0271] E42. The method of E41 , wherein GY, at each occurrence, is the 6- to 12-membered aryl.

[0272] E43. The method of E42, wherein the 6- to 12-membered aryl is phenyl.

[0273] E44. The method of E41 , wherein R100a, at each occurrence, is Ci.6alkyl.

[0274] E45. The compound of any one of E34-E44, wherein the compound of formula (IV) is selected from the group consisting of:

[0275] E46. A method for detecting one or more per- or poly-fluoroalkyl substances (PFAS) in a sample, the method comprising: contacting the sample with a detection reagent comprising a compound of formula (V), or a salt thereof: wherein 026389-0053-W001

[0276] R200is halogen, hydrogen, Ci^alkyl, Ci.2haloalkyl, or -OCi.2haloalkyl;

[0277] G200is a 4- to 12-membered heterocyclylene or a Ca- carbocyclylene, wherein G200is optionally substituted with 1-4 substituents, each independently selected from the group consisting of Ci.4alkyl, Ci.2haloalkyl, halogen, cyano, -OCi.4alkyl, and -OCi. 2haloalkyl;

[0278] G210is a 5- to 12-membered heteroaryl or a 6- to 12-membered aryl, wherein G210is optionally substituted with 1-4 substituents, each independently selected from the group consisting of Ci-4alkyl, Ci.2haloalkyl, halogen, cyano, -OCi.4alkyl, and -OCi. 2haloalkyl; and quantifying a change in fluorescence intensity of the detection reagent while being contacted with the sample relative to a baseline fluorescence intensity of the detection reagent prior to being contacted with the sample.

[0279] E47. The method of E46, wherein R200, at each occurrence, is hydrogen or halogen.

[0280] E48. The method of E46, wherein G200is a 4- to 12-membered heterocyclylene.

[0281] E49. The method of E48, wherein the 4- to 12-membered heterocyclylene is

[0282] E50. The method of any one of E46-E49, wherein G210is a 5- to 12-membered heteroaryl.

[0283] E51. The method of E50, wherein the 5- to 12-membered heteroaryl is

[0284] E52. The compound of any one of E46-E51 , wherein the compound of formula (V) is:

[0285] The fluorophores of the present disclosure may be covalently attached or non-covalently adhered to the surface of a solid support. In some embodiments, the solid support may comprise a nanoparticle, nanofiber, microparticle or microfiber having average sizes ranging from about 1 nm to about 500 pm in diameter. In some embodiments, the average particle size may be from 10 pm to about 15 pm. The nanoparticles, nanofibers, microparticles or microfibers may be soluble or soluble in a particular solvent, may form colloidal suspensions and / or or may adsorb or absorb liquids. In some embodiments, the solid support may be a solid phase extraction membrane or “SPE membrane.” SPE membranes may include porous structures having pore sizes ranging from about 0.1 pm to about 10 pm. In some embodiments, the SPE membrane may be housed in a filter disk and / or a filter column having diameters ranging from about 5 mm 026389-0053-W001 to about 100 mm. In some embodiments, the solid support may be used to form a column for use in chromatographic separations.

[0286] The solid support may be formed of any suitable material, including but not limited to a cellulose, a metal oxide, a polymer, a resin, a silica, and combinations thereof. The selection of a particular substrate generally depends on various criteria, including, but not limited to, chemical stability, photostability, the ability (or lack thereof) of the substrate to absorb excitation or emission light, ease of surface modification, cost, and environmental toxicity (or lack thereof). Suitable celluloses may include, but are not limited to, a nitrocellulose and a cellulose acetate. Suitable metal oxides may include, but are not limited to, titania (TiC>2), zinc oxide (ZnO) and alumina (AI2O3). Suitable polymers may include, but are not limited to a polyacrylate, a polyacrylonitrile, a polycarbonate, a polyimide, a polymethyl methacrylate, a polypropylene, a polytetrafluoroethylene, and a polyvinylidene difluoride. For example, the polymer may include a polystyrenedivinylbenzene and a sulfonated polystyrenedivinylbenzene. Resins may include any known cation or anion exchange resin. Silicas may include but are not limited to fumed silica, precipitated silica, non-porous silicas, and silicas produced through aerosol assisted selfassembly. Exemplary silicas may include, but are not limited to, Cs and Cis bonded silica.

[0287] As noted above, the fluorophores of the present disclosure may be non-covalently adhered to the surface of a solid support. For example, the fluorophores may be bound to the surface by a ligand that binds to the fluorophore via intermolecular interactions including, but not limited to, electrostatic interactions, ir-effects interactions, hydrogen bonding, van der Waals forces, and / or hydrophobic interactions, among others.

[0288] The fluorophores also may be covalently attached to the surface of the solid support through reactions with reactive functional groups that chemically react with the fluorophore to form a covalent bond. Upon reaction with a functional group, the fluorophore may then be coupled, tethered, or linked to the solid support with a linker. Many different chemistries have been developed to couple compounds to solid supports, and any such chemistry currently known or hereinafter devised may be used, provided the chemistry affords the use of an appropriate functional group that may react with a reactive moiety on the fluorophore.

[0289] In some embodiments, X is O and n is 0 in the fluorophores of Formula (II) and have a formula p(llla): 026389-0053-W001

[0290] In some embodiments, a fluorophore of Formula (pl I la) may be coupled to a solid support by functionalizing the surface of the solid support with a primary amine functional group, and then reacting the primary amine with the fluorophore of formula (pl I la) to form a fluorophore of Formula (HI): where Z is a solid support, L10is a linker, and L20is a linker.

[0291] Solid supports that have been functionalized with primary amine functional groups are referred to herein as “amine-functionalized solid supports.” The literature is replete with various methods for functionalizing solid supports with amine functional groups, including methods for amine-functionalizing cellulose-based materials, such as paper, cotton, and pure cellulose (see, e.g., Shaken et al., Adv. Mat. Interfaces 6(19): 1900940 (2019); Jiang et al., AIP Advances 11(2): 025127 (2021); de Oliveira et al., Cellulose 20(1): 217-226 (2013); Makowski, Cellulose 27(1): 1- 9 (2020); Khanjanzadeh et al., Int. J. Biol. Macromolecules 106: 1288-1296 (2018); and Voicu and Thakur, Cur. Opin. Green Sustainable Chem. 30: 100480 (2021); metal oxides such as titania, zinc oxide and alumina (see, e.g., Sypabekova et al., Biosensors 13(1): 36 (2023); Shaken et al., Adv. Mat. Interfaces 6(19): 1900940 (2019); Pujari et al., Angew. Chem. Int. Ed. 53(25): 6322- 6356 (2014); Ahangaran and Navarchian, Adv Colloid Interface Sci. 286: 102298 (2020); Guo et al, J. Environ. Chem. Eng. 9(6): 106800 (2021); Zou et al., J. Alloys Compounds 825: 153904 (2020); Nayak et al., Sep. Purif. Tech. 229: 115674 (2019)); polymers such as polymethyl methacrylate (see, e.g., Vlachopoulou et al., J. Micromech. Microeng. 19(1): 015007 (2009); Mai et al., J. Clin. Med. 8(11): 1831 (2019)); resins, such as epoxy resin (see, e.g., Li et al. Prog. Org. Coatings 174: 107228 (2023); Rajan et al. Polymer Bull. 75(1): 167-195 (2018)); and silica (see, e.g., Sypabekova et al., Biosensors 13(1): 36 (2023); Shaken et al., Adv. Mat. Interfaces 6(19): 1900940 (2019); Pujari et al., Angew. Chem. Int. Ed. 53(25): 6322-6356 (2014), each of which is 026389-0053-W001 incorporated by reference herein for such teachings. In various instances, solid supports functionalized with carbonyl may be purchased commercially.

[0292] In some embodiments, amine-functionalized solid supports may comprise -C- oalkylene- NH2chemically coupled to the solid support, such that, upon reaction of the amine with the compound of Formula (Ic), the linker L10comprises the Ci- alkylene. For example, silica may be amine-functionalized using 3-aminopropyl(trimethoxysilane) (APTMS) or 3- aminopropyl(trimethoxysilane) (APTES) according to reactions well-known in the art, and as shown in Scheme 2 of the examples, below. Reacting the compound of formula (pl I la) with the APTES-functionalized or APTMS-functionalized silica thus forms the compound of formula (III) where L10comprises Csalkylene.

[0293] Compounds of formula (III) may be used in various methods for detecting PFOA, according to the following numbered embodiments.

[0294] In some embodiments, X is N, n is 1, and R30is hydrogen in the fluorophores of Formula (II) and have a formula p(lllb): (plllb).

[0295] In some embodiments, a fluorophore of Formula (plllb) may be couple to a solid support by functionalizing the surface of the solid support with a carbonyl functional group, and then reacting the carbonyl with the fluorophore of formula (plllb) to form a fluorophore of Formula (III): where Z is a solid support, L10is a linker, and L20is a linker.

[0296] Solid supports that have been functionalized with primary amine functional groups are referred to herein as “carbonyl-functionalized solid supports.” The literature is replete with various methods for functionalizing solid supports with carbonyl functional groups, including methods for carbonyl-functionalizing resins (see, e.g., including methods for carbonyl-functionalizing resins (see, e.g., Metz et al., Bioorg. Med. Chem. Lett. 8(17): 2399-2402 (1998)); silica (see, e.g., 026389-0053-W001

[0297] Barczak, J. Porous Mater. 26: 291-300 (2019); Yang et al., Chem. Mater. 17: 5999 (2005); Han et al., J. Mater. Chem. 21 : 11033 (2011); Tsai et al., J. Hazard. Mater. 309: 236 (2016)); cellulose (see, e.g., Elias et al., J, Colloid Interface Sci. 533: 678-691 (2019)); cotton (see, e.g., Guo et al., ACS Appl. Nano Mater. 6(1): 261-269 (2022)); and beads (see, e.g., Saber-Samandari et al., Chem. Eng. J. 308: 1133-1144 (2017)), each of which is incorporated by reference herein for such teachings. In various instances, solid supports functionalized with carbonyl may be purchased commercially.

[0298] In some embodiments, carbonyl-functionalized solid supports may comprise -Ci- walkylene-COOH; -Ci- alkylene-C(O)H; -0-Ci-ioalkylene-C(0)H; or -O-Ci- alkylene-COOH chemically coupled to the solid support. The carbonyl group on the solid support may react with fluorophore of Formula (plllb) to form a linker L10comprising the Ci- alkylene; -Ci- alkylene- C(O)-; or -O-Ci-ioalkylene-C(O)-.

[0299] E53. A method for detecting one or more per- or poly-fluoroalkyl substances (PFAS) in a sample, the method comprising: contacting the sample with a detection reagent comprising a compound of formula (III), or a salt thereof, wherein

[0300] Z is a solid support;

[0301] L10is C ioalkylene; Ci-ioalkylene-C(O)-, or -O-Ci-i0alkylene-C(O)-; , where: m is 20-60;

[0302] R10is hydrogen, halogen, cyano, Ci-ealkyl, Ci-ehaloalkyl, -OR10a, -SR10a, -CO210a, -C(O)R10a, -SO2R10a, -N(R10a)2, -CO2N(R10a)2, or -NO2, where:

[0303] R10a, at each occurrence, is independently, hydrogen, Ci-ealkyl, or Ci.2haloalkyl;

[0304] R20is hydrogen, halogen Ci-ealkyl, Ci-ehaloalkyl, -OR10b, -SR10b, -CO2R10b, -C(O)R10b, -SO2R10b, -N(R10b)2, -CO2N(R10b)2, or -NO2, where:

[0305] R10b, at each occurrence, is independently, hydrogen, Ci-ealkyl, or Ci-2haloalkyl; 026389-0053-W001

[0306] G10is a 4- to 12-membered heterocyclylene, a Cs-iocarbocyclylene, a 6- to 12-membered, aryl, or a 5- to 12-membered heteroaryl, wherein G10is optionally substituted with 1-6 substituents independently selected from the group consisting of C^alkyl, Ci- 2haloalkyl, halogen, cyano, -OCi.4alkyl, and -OCi.2haloalkyl;

[0307] G20, at each occurrence, is independently a 5- to 12-membered heteroaryl, wherein G20is optionally substituted with 1-5 substituents independently selected from the group consisting of Ci.4alkyl, Ci.2haloalkyl, halogen, cyano, -OCi.4alkyl, and -OCi. 2haloalkyl;

[0308] G30, at each occurrence, is independently a 5- to 12-membered heteroaryl or a 6- to 12- membered aryl, wherein G30is optionally substituted with 1-5 substituents independently selected from the group consisting of Ci-4alkyl, Ci.2haloalkyl, halogen, cyano, — OCi-4alkyl , and -OCi-2haloalkyl; n is 0-1 ; w is 0-1 ; and x is 1-2; and quantifying a change in fluorescence intensity of the detection reagent while being contacted with the sample, relative to a baseline fluorescence intensity of the detection reagent prior to being contacted with the sample.

[0309] E54. The method of E53, wherein G10is the 4- to 12-membered heterocyclylene or the 6- to 12- membered arene.

[0310] E55. The method of E54, wherein the 4- to 12-membered heterocyclylene is E56. The method of E54, wherein the 6- to 12-membered aryl is phenyl.

[0311] E57. The method of any one of E53-E56, wherein R10and R20are each hydrogen.

[0312] E58. The method of any one of E53-E57, wherein G30, at each occurrence, is the 5- to 12- membered heteroaryl.

[0313] E59. The method of any one of E53-E58, wherein G30, at each occurrence, is independently pyridinyl or pyrimidinyl.

[0314] E60. The method of any one of E53-E59, wherein w is 0.

[0315] E61. The method of any one of E53-E59, wherein w is 1.

[0316] E62. The method of any one of E53-E59 or E61 , wherein G20is the 5- to 12-membered heteroarene. 026389-0053-W001

[0317] E63. The method of any one of E53-E59 or E61-E62, wherein the 5- to 12-membered heteroarene

[0318] E64. The method of any one of E53-E63, wherein the solid support comprises a silica, a polymer, a resin, or a combination thereof.

[0319] E65. The method of any one of E53-E64, wherein L10is C2-4alkylene.

[0320] E66. The method of any one of E53-E65, wherein n is 1 and m is 30-50.

[0321] E67. The method of any one of E53-E66, wherein the compound of formula (III), or a salt thereof, is the reaction product of an amine-functionalized solid support and a compound of formula (pl I la):

[0322] E68. The method of any one of E53-E67, wherein the amine-functionalized solid support comprises Ci-ioalkylene-NH2chemically coupled to the solid support, and wherein L10comprises Ci-walkylene.

[0323] The fluorophores used in the methods 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.

[0324] In some embodiments, the fluorophores disclosed herein may interact with one or more PFAS including, but not limited to: N-Ethyl-N-(2-hydroxyethyl)perfluorooctanesulfonamide; 8:2 Fluorotelomer Alcohol; Perfluorodecanoic Acid; Perfluorononanoic Acid; Lithium Perfluorooctanesulfonate; Potassium Perfluorooctanesulfonate; Perfluorooctanesulfonate; Potassium Perfluorohexanesulfonate; Hexafluoropropylene Oxide Dimer Acid (GenX); Fluorotelomer Alcohol; Perfluorohexanoic Acid; Perfluorooctanoic Acid; Perfluorohexanol; Perfluoroheptanoic Acid; Perfluorooctanesulfonamide; 6:2 Fluorotelomer Sulfonamide Betaine; 6:2 Fluorotelomer Alcohol; 6:2 Fluorotelomer Methacrylate; 7:1 Fluorotelomer Alcohol; Perfluorotetradecanoic Acid; Perfluoroundecanoic Acid; Sodium Perfluorohexanoate; 4:2 Fluorotelomer Alcohol; Perfluoropentanesulfonic Acid; Perfluorooctanesulfonamido Amine; 6:2 026389-0053-W001

[0325] Fluorotelomer Sulfonic Acid; N-Methylperfluorooctanesulfonamide; Perfluoroheptanesulfonamido Amine; Perfluoropentanoic Acid; Potassium Perfluoropentanesulfonate; Ammonium Perfluorononanesulfonate; Ammonium Perfluoroheptanesulfonate; N-Methyl-N-(2- hydroxyethyl)perfluorooctanesulfonamide; 10:2 Fluorotelomer Alcohol; Perfluorododecanoic Acid; Perfluorooctanesulfonic Acid; N-Ethylperfluorooctanesulfonamide; Potassium Perfluorobutanesulfonate; 10:2 Fluorotelomer Acrylate; Perfluorobutanesulfonic Acid; Ammonium Perfluorooctanoate; 6:2 Fluorotelomer Acrylate; Perfluorohexanesulfonic Acid; Perfluorodecanesulfonic Acid; Trifluoroacetic Acid; Perfluoro(4-methyl-3,6-dioxaoct-7- ene)sulfonyl Fluoride; Perfluoro-3-(1 H-perfluoroethoxy)propane; Perfluorobutanoic Acid; Perfluoropropanoic Acid; Perfluoroheptanesulfonic Acid; 8:2 Fluorotelomer Methacrylate; 10:2 Fluorotelomer Methacrylate; 2-(N-Ethylperfluorooctanesulfonamido)acetic Acid; Perfluorooctadecanoic Acid; 8:2 Fluorotelomer Acrylate; Perfluorohexane Sulfonamido Amine; Perfluorohexadecanoic Acid; Ammonium Perfluorooctanesulfonate; Potassium Perfluoroheptanesulfonate; Ammonium Perfluorodecanesulfonate; Ammonium Perfluorohexanesulfonate; Ammonium Perfluorobutanesulfonate; Perfluoropentane Sulfonamido Amine; Perfluoro-3-Methoxypropanoic Acid; p-Toluenesulfonic Acid; Perfluoro-4- (perfluoroethyl)cyclohexylsulfonic Acid; Ammonium Perfluoropentanesulfonate; Perfluorobutane Sulfonamido Amine; Ammonium Perfluoro-2-methyl-3-oxahexanoate; 9:1 Fluorotelomer Alcohol; 10:1 Fluorotelomer Alcohol; Perfluoro-3,6-Dioxaheptanoic Acid; Difluoro(perfluoromethoxy)acetic Acid; Perfluoro(4-methoxybutanoic) Acid; 6:2 Fluorotelomer Phosphate Monoester; Perfluoro- 3,6,9-TrioxatridecanoicAcid; Perfluoro-3,6-DioxadecanoicAcid; 3-PerfluoroheptylpropanoicAcid; Sodium Perfluorooctanesulfonate; Perfluoro-3,5,7,9,11-Pentaoxadodecanoic Acid; Perfluorooctanesulfonamido Ammonium; Perfluoroethanesulfonic Acid; Perfluorohexanesulfonate; 6:2 / 8:2 Fluorotelomer Phosphate Diester; Ammonium 4,8-Dioxa-3H- perfluorononanoate; Perfluoro-3,5,7,9-Butaoxadecanoic Acid; Difluoro(perfluoropropoxy)acetic Acid; Perfluorotridecanoic Acid; Ammonium Perfluorodecanoate; Sodium Perfluorooctanoate; Sodium Perfluorodecanoate; Sodium Perfluoropentanoate; Sodium Perfluorobutanoate; Silver Perfluorobutanoate; Ammonium Perfluoroheptanoate; Ammonium Perfluorohexanoate; Lithium Perfluorohexanesulfonate; 4,8-Dioxa-3H-perfluorononanoic Acid; Perfluorooctanesulfonamido Betaine; 2-Perfluorodecyl Ethanoic Acid; Perfluorooctanesulfonamido Ethanol; Ammonium Perfluorononanoate; Sodium Perfluoroheptanoate; 10:2 Fluorotelomer Sulfonamide Betaine; Potassium 9-Chlorohexadecafluoro-3-Oxanonane-1 -Sulfonate; 8:2 Fluorotelomer Thioether Amido Sulfonic Acid; Perfluoro-3,5,7-Trioxaoctanoic Acid; Perfluoro-3,6-Dioxa-4-Methyl-7- Octene-1-Sulfonic Acid; Perfluoro-2-(perfluorobutoxy)-2-(perfluoromethyl)propanoic Acid; 026389-0053-W001

[0326] Perfluoro-2,5-Dimethyl-3,6-Dioxanonanoic Acid; Potassium 11-Chloroeicosafluoro-3- Oxaundecane-1-Sulfonate; 5:3 Fluorotelomer Betaine; Perfluoro-2-{[Perfluoro-3- (perfluoroethoxy)-2-propanyl]oxy}ethanesulfonic Acid; Perfluoro-2-

[0327] [(Perfluoropentyl)oxy]propanoic Acid; Sodium Perfluorohexanesulfonate; Perfluorobutanoate; Perfluoroheptanoate; Perfluorooctanoate Ion (1-); Perfluorotridecanoate; Perfluoroheptanesulfonate; Perfluorononanesulfonic Acid; Lithium Perfluoroheptanesulfonate; Trifluoroacetate; 8:2 Fluorotelomer Sulfonic Acid; Perfluoro-(2,5,8-Trimethyl-3,6,9- Trioxadodecanoic) Acid; 6:1 Fluorotelomer Alcohol; 8:2 Fluorotelomer Phosphate Diester; 8:1 Fluorotelomer Alcohol; 11 :1 Fluorotelomer Alcohol; Perfluoro-3,6,9-Trioxadecanoic Acid; 5:1 Fluorotelomer Alcohol; 2-Perfluorooctyl Ethanoic Acid; 7:2 Fluorotelomer Alcohol; 6:2 Fluorotelomer Phosphate Diester; 2-Perfluorohexyl Ethanoic Acid; Perfluoro-2- (perfluoromethoxy)propanoic Acid; 2-(N-Methylperfluorooctanesulfonamido)acetic Acid; Perfluoro-4-lsopropoxybutanoic Acid; Perfluorooctanesulfonamido Amine Oxide; 2H-Perfluoro-2- Decenoic Acid; 8-Fluorosulfonylperfluoro(2,5-Dimethyl-3,6-Dioxaoctanoyl) Fluoride; Perfluoropropanesulfonic Acid; Perfluorodecanesulfonate; 8:2 Fluorotelomer Sulfonate; 8:2 Fluorotelomer Phosphate Monoester; Perfluorononanesulfonate; Perfluorobutanesulfonate; 6:2 Fluorotelomer Sulfonate; 2H,2H,3H,3H-Perfluorooctanoic Acid; Potassium Perfluorooctanoate; Silver Perfluorooctanoate; Ammonium 2-(N-Ethylperfluorooctanesulfonamido)acetate; Ammonium Perfluoro-9-(Methyl)decanoate; Sodium 2-(N-

[0328] Ethylperfluorooctanesulfonamido)acetate; Perfluoro-2-Methyl-3-Oxahexanoic Acid; 8:2 Fluorotelomer Sulfonamide Betaine; Ammonium Perfluoropentanoate; Perfluorosulfonic Acid, PTFE Copolymer; 4:2 Fluorotelomer Sulfonic Acid; 2H-Perfluoro-2-Octenoic Acid; 6:2 Fluorotelomer Thioether Amido Sulfonic Acid; 4:2 Fluorotelomer Thioether Amido Betaine; 4:2 Fluorotelomer Sulfonate; Perfluoro-4-(Perfluoroethyl)cyclohexylsulfonate; 6:2 Fluorotelomer Thioether Amido Sulfonate; 8:2 Fluorotelomer Thioether Amido Sulfonate; Perfluoro-3,5- Dioxahexanoic Acid; Perfluoro-2-(Perfluoropropoxy)-2-(Perfluoromethyl)propanoic Acid; Perfluoro-3-Ethoxypropanoic Acid; Sodium Perfluorodecanesulfonate; Sodium 4,8-Dioxa-3H- Perfluorononanoate; Perfluoro(2,5,8,10-Tetramethyl-3,6,9-Trioxaundecanoic) Acid; Perfluoro(2,5,8, 11 ,14-Pentamethyl-3,6,9, 12, 15-Pentaoxaoctadecanoic) Acid;

[0329] Perfluoroundecanoate; Perfluoropropanoate; Perfluorodecanoate; Perfluorohexanoate; Perfluoropentanoate; 2-(N-Methylperfluorooctanesulfonamido)acetate; 5:1:2 Fluorotelomer Betaine; Perfluoropentanesulfonate; Perfluorododecanoate; Perfluorononanoate; 7:3 Fluorotelomer Betaine; 8:2 Fluorotelomer Sulfonamide N,N-Dimethyl Amine Ion; 4:2 Fluorotelomer Thioether Amido Sulfonate; Perfluorobutane Sulfonamide Amino Carboxylates; 026389-0053-W001

[0330] Perfluorooctane Sulfonamide Amino Carboxylates; Perfluorooctanesulfonamido Ammonium; 12:2 Fluorotelomer Sulfonamide Betaine; 6:2 Fluorotelomer Thioether Hydroxyammonium; 9:3 Fluorotelomer Betaine; Perfluorotetradecanoate; 2-(N-Ethylperfluorooctanesulfonamido)acetate; 7:1 :2 Fluorotelomer Betaine; 9:1:2 Fluorotelomer Betaine; 6:2 Fluorotelomer Sulfonamido N,N- Dimethyl Amine; 4:2 Fluorotelomer Thioether Amido Sulfonic Acid; Perfluoroheptane Sulfonamide Amino Carboxylates; Perfluoropentane Sulfonamide Amino Carboxylates; Perfluorooctanesulfonamido Amine Oxide; ions thereof; and salts thereof.

[0331] The sample may be a liquid sample of any material. The liquid sample may be prepared by any means known to a person of ordinary skill. In some embodiments, the sample may be prepared experimentally or obtained from the natural environment. In some embodiments, the sample may be obtained from a river, lake, well, pond, stream, ocean, inlet, canal, loch, bay, fountain, sea, or a combination thereof. In other embodiments, the sample may be obtained from tap water, municipal water, municipal wastewater, industrial wastewater, run-off water, ballast water, water treatment water, agricultural water, or a combination of.

[0332] In some embodiments, the sample may be prepared by treating a solid sample with a liquid to dissolve an analyte. In some embodiments, the solid may be soil, rock, sand, or a combination thereof.

[0333] In some embodiments, the sample may be a biological sample obtained from an animal. In some embodiments, the biological sample may be serum, plasma, urine, or other bodily fluids. In some embodiments, the biological sample may be a sample obtained from tissue.

[0334] In some embodiments, the sample may be a sample from a device that contains material that may for an aqueous film forming foam (AFFF) containing PFAS. In some embodiments, the sample may be a sample obtained from an environment in which after an AFFF has been deployed. In some embodiments, the AFFF is used for firefighting.

[0335] In some embodiments, the sample may be a sample from tank rinsates containing rinsate from spill cleanups, sumps, leaks, or residual chemicals. In some embodiments, the rinsates may include, but are not limited to, surfactants (e.g., dodecyl sulfate and oleic acid), flocculants and coagulants (e.g., clays, polyaluiminum chloride (PACI), zirconiumoxychloride) and cleaning products (e.g., cationic and anionic polyacrylamides).

[0336] In some embodiments, the sample may be a sample collected from materials used to remove PFAS from a system or environment. In some embodiment, the materials used to remove PFAS may be carbon sorbents, ion exchange materials, or filters.

[0337] In some embodiments, the sample may be a sample prepared from a food, fish, carpet, apparel, upholstery, cookware, food wrappers, water bottles, medical supplies, lab supplies, 026389-0053-W001

[0338] PFAS-free lab supplies (e.g., sample vials and caps and solid-phase extraction resins and membranes), or any material that may contain or have been in contact with a PFAS.

[0339] The fluorophores described herein each have slightly different absorption spectra and slightly different emission spectra when excited at particular excitation wavelengths. Peak absorption wavelengths for the fluorophores range from about 350 nm to about 520 nm. When excited at peak absorption wavelengths, the fluorophores disclosed in the present application have peak emission wavelengths between about 425 nm and about 700 nm, such as between about 540 nm and about 600 nm.

[0340] In some embodiments, the fluorophores of the present disclosure may non-selectively interact with a PFAS in a manner that significantly changes their emission intensities at a particular emission wavelength. In some embodiments, the fluorophore may exhibit no fluorescence prior to exposure to the PFAS. In some embodiments, fluorophore interaction(s) with PFAS may increase the measured fluorescence intensity of the fluorophore.

[0341] In some embodiments, the fluorophore may exhibit fluorescence prior to exposure to PFAS. In some embodiments, fluorophore interaction(s) with PFAS may decrease the measured fluorescence intensity of the fluorophore.

[0342] In some embodiments, the interaction(s) between the fluorophores of the present disclosure and one or more PFAS may 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 fluorophores disclosed herein). These quenching of the fluorophore allow for use of the fluorophore to detect and / or quantify one or more PFAS in a sample with the limits of detection between about 1 and about 50 parts per billion (ppb). To detect and / or quantify one or more PFAS in a sample at these limits of detection, the detection reagent may be excited at an excitation wavelength of between about 350-430 nm (both in the presence and in the absence of the sample), and the fluorescence intensity may then be quantified at an emission wavelength of between about 425-700 nm.

[0343] In some embodiments, the interaction(s) between the fluorophores of the present disclosure and one or more PFAS may causes an increase in the fluorescence of the fluorophore at one or more emission wavelengths (i.e., PFAS functions as a fluorescence activator to the fluorophores disclosed herein). The activation of fluorescence of the fluorophore allows for use of the fluorophore to detect and / or quantify one or more PFAS in a sample with the limits of detection between about 1 and about 50 parts per billion (ppb). To detect and / or quantify one or more PFAS in a sample at these limits of detection, the detection reagent may be excited at an excitation wavelength of between about 350 and about 520 nm (both in the presence and in the 026389-0053-W001 absence of the sample), and the fluorescence intensity may then be quantified at an emission wavelength of between about 425 and about 580 nm.

[0344] In some embodiments, the fluorophores of the present disclosure may selectively interact with a PFAS in a highly sensitive manner that significantly changes their emission intensities at a particular emission wavelength. According to the present disclosure, the fluorophores do not interact with other PFAS in the same manner, thereby enabling these fluorophores to selectively detect a PFAS in a sample with, or without, other PFAS.

[0345] In some embodiments, the interaction(s) between the fluorophores of the present disclosure and PFOA may causes a decrease in the fluorescence of the fluorophore at one or more emission wavelengths (i.e., PFOA functions as a fluorescence quencher to the fluorophores disclosed herein). These quenching of the fluorophore allow for use of the fluorophore to detect and / or quantify PFOA in a sample with the limits of detection between about 1 and about 50 parts per billion (ppb). To detect and / or quantify PFOA in a sample at these limits of detection, the detection reagent may be excited at an excitation wavelength of between about 350-430 nm (both in the presence and in the absence of the sample), and the fluorescence intensity may then be quantified at an emission wavelength of between about 425-700 nm.

[0346] In some embodiments, the interaction(s) between the fluorophores of the present disclosure and PFOA may causes an increase in the fluorescence of the fluorophore at one or more emission wavelengths (i.e., PFOA functions as a fluorescence activator to the fluorophores disclosed herein). These activation of fluorescence of the fluorophore allow for use of the fluorophore to detect and / or quantify PFOA in a sample with the limits of detection between about 1 and about 50 parts per billion (ppb). To detect and / or quantify PFOA in a sample at these limits of detection, the detection reagent may be excited at an excitation wavelength of between about 350 and about 520 nm (both in the presence and in the absence of the sample), and the fluorescence intensity may then be quantified at an emission wavelength of between about 425 and about 580 nm.

[0347] In some embodiments, the fluorophores of the present disclosure selectively interact with PFOS in a highly sensitive manner that significantly changes their emission intensities at a particular emission wavelength. According to the present disclosure, the fluorophores do not interact with other PFAS in the same manner, thereby enabling these fluorophores to selectively detect PFOS in samples containing PFOS with, or without, other PFAS.

[0348] In some embodiment, the interaction(s) between the fluorophores of the present disclosure and PFOS causes a decrease in the fluorescence of the fluorophore at one or more emission wavelengths (i.e., PFOS functions as a fluorescence quencher to the PDI-based 026389-0053-W001 fluorophores disclosed herein. These changes in fluorescence of the fluorophore allow for use of the fluorophore to detect and / or quantify PFOS in a sample with the limits of detection between about 1 and about 50 parts per billion (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 between about 350 and about 490 nm (both in the presence and in the absence of the sample), and the fluorescence intensity may then be quantified at an emission wavelength of between about 575 nm and about 625 nm.

[0349] It will be apparent to those of ordinary skill in the relevant art that suitable modifications and adaptations to the methods, processes, and applications described herein can be made without departing from the scope of any embodiments or aspects thereof. The methods provided are exemplary and are not intended to limit the scope of any of the specified embodiments. All of the various embodiments, aspects, and options disclosed herein can be combined in any variations or iterations. The scope of the methods and processes described herein include all actual or potential combinations of embodiments, aspects, options, examples, and preferences herein described. The exemplary methods described herein may substitute any component disclosed herein, or include any component disclosed elsewhere herein. Should the meaning of any terms in any of the patents or publications incorporated by reference conflict with the meaning of the terms used in this disclosure, the meanings of the terms or phrases in this disclosure are controlling. Furthermore, the foregoing discussion discloses and describes merely exemplary embodiments. All patents and publications cited herein are incorporated by reference herein for the specific teachings thereof.

[0350] Various embodiments and aspects of the inventions described herein are summarized by the following clauses:

[0351] Clause 1 . A method for detecting one or more per- or poly-fluoroalkyl substances (PFAS) in a sample, the method comprising: contacting the sample with a detection reagent comprising a compound of formula (I), or a salt thereof: wherein 026389-0053-W001

[0352] R1is hydrogen, halogen, cyano, C^alkyl, Ci-ehaloalkyl, -OR1a, -SR1a, -CC>21a, -C(O)R1a, -SO2R1a, -N(R1a)2, -CO2N(R1a)2, or -NO2, where:

[0353] R1a, at each occurrence, is independently, hydrogen, Ci-salkyl, or Ci.2haloalkyl;

[0354] R2, at each occurrence, is independently hydrogen, halogen Ci.6alkyl, Ci.6haloalkyl, -OR1b, -SR1b, -CO2R1b, -C(O)R1b, -SO2R1b, -N(R1b)2, -CO2N(R1 b)2, or -NO2, where:

[0355] R1b, at each occurrence, is independently, hydrogen, Ci-salkyl, or Ci.2haloalkyl;

[0356] G1, at each occurrence, is independently a 6- to 12-membered aryl or a 5- to 6-membered heteroaryl, wherein G1is optionally substituted with 1-5 substituents independently selected from the group consisting of Ci-4alkyl, Ci-2haloalkyl, halogen, cyano, -OCi-4alkyl, and -OCi.2haloalkyl;

[0357] G2, at each occurrence, is independently a 5- to 12-membered heteroarene or -N(GX)-, where:

[0358] Gx, at each occurrence, is a 6- to 12-membered aryl optionally substituted with 1-5 substituents independently selected from the group consisting of Ci-4alkyl, Ci- 2haloalkyl, halogen, cyano, -OCi-4alkyl, and -OCi.2haloalkyl;

[0359] G3is a 5- to 6-membered heteroaryl or a 6- to 12-membered aryl, wherein G3is optionally substituted with 1-5 substituents independently selected from the group consisting of Ci-4alkyl, Ci.2haloalkyl, halogen, cyano, -OCi-4alkyl, and -OCi.2haloalkyl; m is 0-1 ; and n is 0-2; and quantifying a change in fluorescence intensity of the detection reagent being contacted with the sample, relative to a baseline fluorescence intensity of the detection reagent prior to contact with the sample.

[0360] Clause 2. The method of clause 1 , wherein G3is the optionally substituted 5- to 6-membered heteroaryl.

[0361] Clause 3. The method of clause 2, wherein the 5- to 6-membered heteroaryl is pyridinyl.

[0362] Clause 4. The method of clause 1 or 2, wherein R1and R2are each hydrogen.

[0363] Clause 5. The method of any one of clauses 1-4, wherein the compound of formula (I) is a compound of formula (la): 026389-0053-W001

[0364] Clause 6. The method of any one of clauses 1-5, wherein m is 0 and n is 0.

[0365] Clause 7. The method of any one of clauses 1-5, wherein m is 1 and n is 1.

[0366] Clause 8. The method of any one of clauses 1-5 or 7, wherein G2is the optionally substituted

[0367] 5- to 12-membered heteroarene.

[0368] Clause 9. The method of clause 8, wherein the 5- to 12-membered heteroarene is

[0369] Clause 10. The method of any one of clauses 1-5 or 7-9, wherein G2is -N(GX)-.

[0370] Clause H . The method of clause 10, wherein Gxis the optionally substituted 6- to 12- membered aryl.

[0371] Clause 12. The method of any one of clauses 1-5 or 7-11, wherein G1is the optionally substituted 6- to 12-membered aryl.

[0372] Clause 13. The method of clause 12, wherein the 6- to 12-membered aryl is phenyl.

[0373] Clause 14. The method of any one of clauses 1-5 or 7-11, wherein G1is the optionally substituted 5- to 6-membered heteroaryl.

[0374] Clause 15. The method of clause 14, wherein the 5- to 6-membered heteroaryl is pyridinyl.

[0375] Clause 16. The method of any one of clauses 1-15, wherein the compound of formula (I) is selected from the group consisting of:

[0376] Clause 17. The method of any one of clauses 1-16, wherein the compound is noncovalently adhered to a surface.

[0377] Clause 18. The method of clause 17, wherein the surface comprises a cellulose, a silica, a polymer, a resin, or a combination thereof. 026389-0053-W001

[0378] Clause 19. The method of any one of clauses 1-18, wherein the compound of formula (I), or a salt thereof, is dissolved in a solvent.

[0379] Clause 20. The method of any one of clauses 1-19, wherein the change in fluorescence intensity is quantified at an emission wavelength of 425-500 nm.

[0380] Clause 21. The method of any one of clauses 1-20, wherein the detection reagent is excited at an excitation wavelength of 275-400 nm.

[0381] Clause 22. The method of any one of clauses 1-21 , wherein the detection reagent has a limit of detection of about 1-50 ppb.

[0382] Clause 23. A method for detecting one or more per- or poly-fluoroalkyl substances (PFAS) in a sample, the method comprising: contacting the sample with a detection reagent comprising a compound of formula (II), or a salt thereof: wherein

[0383] X is O and n is 0, or X is N and n is 1 ;

[0384] R10is hydrogen, halogen, cyano, Ci-salkyl, Ci-ehaloalkyl, or -OR10a, -SR10a, -CO2R10a, -C(O)R10a, -SO2R10a, -N(R10a)2, -CO2N(R10a)2, or -NO2, where:

[0385] R10a, at each occurrence, is independently, hydrogen, Ci-ealkyl, or Ci.2haloalkyl;

[0386] R20is hydrogen, halogen C^alkyl, Ci-ehaloalkyl, or -OR10b, -SR10b, -CO2R10b, -C(O)R10b, -SO2R10b, -N(R10b)2, -CO2N(R10b)2, or -NO2, wherein:

[0387] R10b, at each occurrence, is independently, hydrogen, Ci.6alkyl, or Ci.2haloalkyl;

[0388] R30, at each occurrence is hydrogen or Ci.8alkyl;

[0389] G10is a 4- to 12-membered heterocyclylene, a Cs-wcarbocyclylene, a 6- to 12-membered arene, or a 5- to 12-membered heteroarene, wherein G10is optionally substituted with 1-6 substituents, each independently selected from the group consisting of Ci^alkyl , Ci.2haloalkyl, halogen, cyano, -OC- alkyl, and -OCi-2haloalkyl;

[0390] G20, at each occurrence, is independently a 5- to 12-membered heteroarene, wherein G20is optionally substituted with 1-5 substituents, each independently selected from 026389-0053-W001 the group consisting of Ci-4alkyl, Ci-shaloalkyl, halogen, cyano, -OCi-4alkyl, and -OCi-2haloalkyl;

[0391] G30, at each occurrence, is independently a 5- to 12-membered heteroaryl or a 6- to 12- membered aryl, wherein G30is optionally substituted with 1-5 substituents, each independently selected from the group consisting of Ci-4alkyl, Ci-2haloalkyl, halogen, cyano, -OCi-4alkyl, and -OCi-2haloalkyl; w is 0-1 ; x is 1-2; n is 0-1 ; and quantifying a change in fluorescence intensity of the detection reagent being contacted with the sample, relative to a baseline fluorescence intensity of the detection reagent prior to being contacted with the sample.

[0392] Clause 24. The method of clause 23, wherein G10is the 4- to 12-membered heterocyclylene or the 6- to 12-membered arene.

[0393] Clause 25. The method of clause 24, wherein the 4- to 12-membered heterocyclylene is

[0394] Clause 26. The method of clause 24, wherein the 6- to 12-membered arene is phenylene.

[0395] Clause 27. The method of any one of clauses 23-26, wherein R10and R20are each hydrogen.

[0396] Clause 28. The method of any one of clauses 23-27, wherein G30, at each occurrence, is the

[0397] 5- to 12-membered heteroaryl.

[0398] Clause 29. The method of clause 28, wherein the 5- to 12-membered heteroaryl is pyridinyl or pyrimidinyl.

[0399] Clause 30. The method of any one of clauses 23-27, wherein G30, at each occurrence, is the

[0400] 6- to 12-membered aryl.

[0401] Clause 31. The method of clause 30, wherein the 6- to 12-membered aryl is phenyl.

[0402] Clause 32. The method of any one of clauses 23-31 , wherein w is 0.

[0403] Clause 33. The method of any one of clauses 23-31 , wherein w is 1.

[0404] Clause 34. The method of clause 33, wherein G20is the 5- to 12-membered heteroarene.

[0405] Clause 35. The method of clause 34, wherein, the 5- to 12-membered heteroarene is 026389-0053-W001

[0406] Clause 36. The method of any one of clauses 23-35, wherein X is O and n is 0.

[0407] Clause 37. The method of any one of clauses 23-35, wherein X is N and n is 1 .

[0408] Clause 38. The method of any one of clauses 23-37, wherein R30is Ci-salkyl.

[0409] Clause 39. The method of any one of clauses 23-38, wherein the compound of formula (II), or a salt thereof, is selected from the group consisting of:

[0410] Clause 40. The method of any one of clauses 23-39, wherein the compound of formula (II) is noncovalently adhered or covalently attached to a surface.

[0411] Clause 41. The method of clause 40, wherein the surface comprises a cellulose, a silica, a polymer, a resin, or a combination thereof.

[0412] Clause 42. The method of any one of clauses 23-41 , wherein the compound of formula (II), or a salt thereof, is dissolved in a solvent. 026389-0053-W001

[0413] Clause 43. The method of any one of clauses 23-42, wherein the detection reagent is excited at an excitation wavelength of 400 nm.

[0414] Clause 44. The method of any one of clauses 23-43, wherein the change in fluorescence intensity is quantified at an emission wavelength of 400-475 nm.

[0415] Clause 45. The method of any one of clauses 23-44, wherein the detection reagent has a limit of detection of about 1 to about 50 ppb.

[0416] Clause 46. A method for detecting one or more per- or poly-fluoroalkyl substances (PFAS) in a sample, the method comprising: contacting the sample with a detection reagent comprising a compound of formula (III), or a salt thereof, wherein

[0417] Z is a solid support;

[0418] L10is Ci-ioalkylene; Ci-ioalkylene-C(O)-, or -O-Ci-ioalkylene-C(O)-;

[0419] R10is hydrogen, halogen, cyano, Ci-ealkyl, Ci-ehaloalkyl, -OR10a, -SR10a, -CO2R10a, -C(O)R10a, -SO2R10a, -N(R10a)2, -CO2N(R10a)2, or -NO2, where:

[0420] R10a, at each occurrence, is independently, hydrogen, Ci-ealkyl, or Ci.2haloalkyl;

[0421] R20is hydrogen, halogen Ci-ealkyl, Ci-ehaloalkyl, -OR10b, -SR10b, -CO2R10b, -C(O)R10b, -SO2R10b, -N(R10b)2, -CO2N(R10b)2, or -NO2, where:

[0422] R10b, at each occurrence, is independently, hydrogen, Ci.6alkyl, or Ci.2haloalkyl;

[0423] G10is a 4- to 12-membered heterocyclylene, a Cs- carbocyclylene, a 6- to 12-membered, aryl, or a 5- to 12-membered heteroaryl, wherein G10is optionally substituted with 1-6 substituents independently selected from the group consisting of Ci-4alkyl, Ci- 2haloalkyl, halogen, cyano, -OCi^alkyl, and -OCi.2haloalkyl;

[0424] G20, at each occurrence, is independently a 5- to 12-membered heteroaryl, wherein G20is optionally substituted with 1-5 substituents independently selected from the group 026389-0053-W001 consisting of Ci-4alkyl, Ci.2haloalkyl, halogen, cyano, -OCi^alkyl, and -OC1. 2haloalkyl;

[0425] G30, at each occurrence, is independently a 5- to 12-membered heteroaryl or a 6- to 12- membered aryl, wherein G30is optionally substituted with 1-5 substituents independently selected from the group consisting of Ci-4alkyl, Ci-2haloalkyl, halogen, cyano, -OCi-4alkyl, and -OCi.2haloalkyl; n is 0-1 ; w is 0-1 ; and x is 1-2; and quantifying a change in fluorescence intensity of the detection reagent while being contacted with the sample, relative to a baseline fluorescence intensity of the detection reagent prior to being contacted with the sample.

[0426] Clause 47. The method of clause 46, wherein G10is the 4- to 12-membered heterocyclylene or the 6- to 12-membered arene.

[0427] Clause 48. The method of clause 47, wherein the 4- to 12-membered heterocyclylene is

[0428] Clause 49. The method of clause 47, wherein the 6- to 12-membered aryl is phenyl.

[0429] Clause 50. The method of any one of clauses 46-49, wherein R10and R20are each hydrogen.

[0430] Clause 51. The method of any one of clauses 46-50, wherein G30, at each occurrence, is the

[0431] 5- to 12-membered heteroaryl.

[0432] Clause 52. The method of clause 51 , wherein G30, at each occurrence, is independently pyridinyl or pyrimidinyl.

[0433] Clause 53. The method of any one of clauses 46-52, wherein w is 0.

[0434] Clause 54. The method of any one of clauses 46-52, wherein w is 1.

[0435] Clause 55. The method of any one of clauses 46-52 or 54, wherein G20is the 5- to 12- membered heteroarene.

[0436] Clause 56. The method of clause 55, wherein the 5- to 12-membered heteroarene is

[0437] Clause 57. The method of any one of clauses 46-56, wherein the solid support comprises a silica, a polymer, a resin, or a combination thereof. 026389-0053-W001

[0438] Clause 58. The method of any one of clauses 46-57, wherein L10is C2-4alkylene.

[0439] Clause 59. The method of any one of clauses 46-58, wherein n is 1 and m is 30-50.

[0440] Clause 60. The method of any one of clauses 46-59, wherein the compound of formula (III), or a salt thereof, is the reaction product of an amine-functionalized solid support and a compound of formula (pllla): (pllla).

[0441] Clause 61. The method of clause 60, wherein the amine-functionalized solid support comprises Ci-walkylene-NH2chemically coupled to the solid support, and wherein L10comprises Ci-ioalkylene.

[0442] Clause 62. The method of clause 60 or 61 , wherein the amine-functionalized solid support is amine-functionalized silica.

[0443] Clause 63. The method of any one of clauses 46-62, wherein the compound of formula (III), or a salt thereof, is dissolved in a solvent.

[0444] Clause 64. The method of any one of clauses 46-63, wherein the detection reagent is excited at an excitation wavelength of 400 nm.

[0445] Clause 65. The method of any one of clauses 46-64, wherein the change in fluorescence intensity is quantified at an emission wavelength of 400-475 nm.

[0446] Clause 66. The method of any one of clauses 46-65, wherein the detection reagent has a limit of detection of about 1 to about 50 ppb.

[0447] Clause 67. A method for detecting one or more per- or poly-fluoroalkyl substances (PFAS) in a sample, the method comprising: contacting the sample with a detection reagent comprising a compound of formula (IV), or a salt thereof: wherein

[0448] X100is S, N(R110), or O;

[0449] L100is Co-3alkylene-C=C-Co-3alkylene or Ci-6alkylene;

[0450] R100is -N(GY)2, -N(R100a)2, -O-(GY)2, -O-(R100a), or -O-(GY) where: 026389-0053-W001

[0451] GY, at each occurrence, is independently a 6- to 12-membered aryl or a 5- to 12- membered heteroaryl, wherein GYis optionally substituted with 1-5 substituents independently selected from the group consisting of hydrogen, halogen, C^alkyl, Ci.2haloalkyl, cyano, -OCi.4alkyl, or -OCi.2haloalkyl; and

[0452] Ri°°a,at each occurrence, is independently hydrogen or Ci-ealkyl;

[0453] R110is hydrogen, Ci-ealkyl, or Ci-2haloalkyl;

[0454] R120is hydrogen, halogen, Ci-4alkyl, Ci-2haloalkyl, cyano, -OCi-4alkyl, or-OCi-2haloalkyl; and

[0455] R130is hydrogen, halogen, Ci-4alkyl, Ci-2haloalkyl, cyano, -OCi-4alkyl, or-OCi-2haloalkyl; and

[0456] X- is a counterion; and quantifying a change in fluorescence intensity of the detection reagent being contacted with the sample relative to a baseline fluorescence intensity of the detection reagent prior to being contacted with the sample.

[0457] Clause 68. The method of clause 67, wherein X100is S.

[0458] Clause 69. The method of clause 67 or 68, wherein R120and R130, at each occurrence, are each independently hydrogen.

[0459] Clause 70. The method of any one of clauses 67-69, wherein the compound of formula (IV) is a compound of formula (IVa):

[0460] Clause 71. The method of any one of clauses 67-70, wherein L100is Co-3alkylene-C=C-Co- 3alkylene.

[0461] Clause 72. The method of any one of clauses 67-71 , wherein L100is Coalkylene-C=C- Coalkylene.

[0462] Clause 73. The method of any one of clauses 67-72, wherein R110is Ci-ealkyl.

[0463] Clause 74. The method of any one of clauses 67-73, wherein R100is -N(GY)2 or -N(R100a)2.

[0464] Clause 75. The method of any one of clauses 67-74, wherein GY, at each occurrence, is the

[0465] 6- to 12-membered aryl.

[0466] Clause 76. The method of clause 75, wherein the 6- to 12-membered aryl is phenyl.

[0467] Clause 77. The method of any one of clauses 67-76, wherein R100a, at each occurrence, is Ci.

[0468] 6alkyl. 026389-0053-W001

[0469] Clause 78. The compound of any one of clauses 67-77, wherein the compound of formula (IV) is selected from the group consisting of:

[0470] Clause 79. The method of clause 78, wherein the compound of formula (IV), or a salt thereof, is noncovalently adhered or covalently attached to a surface.

[0471] Clause 80. The method of clause 79, wherein the surface comprises a cellulose, a silica, a polymer, a resin, or a combination thereof.

[0472] Clause 81. The method of any one of clauses 67-80, wherein the compound of formula (IV), or a salt thereof, is dissolved in a solvent.

[0473] Clause 82. The method of any one of clauses 67-81 , wherein the counterion is iodide, chloride, or bromide.

[0474] Clause 83. The method of any one of clauses 67-82, wherein the detection reagent is excited at an excitation wavelength of 520 nm.

[0475] Clause 84. The method of any one of clauses 67-83, wherein the change in fluorescence intensity is quantified at an emission wavelength of 577-700 nm.

[0476] Clause 85. The method of any one of clauses 67-84, wherein the detection reagent has a limit of detection of about 1 to about 50 ppb.

[0477] Clause 86. A method for detecting one or more per- or poly-fluoroalkyl substances (PFAS) in a sample, the method comprising: contacting the sample with a detection reagent comprising a compound of formula (V), or a salt thereof: wherein

[0478] R200is halogen, hydrogen, Ci-ealkyl, Ci-2haloalkyl, or -OCi-2haloalkyl;

[0479] G200is a 4- to 12-membered heterocyclylene or a Cs- carbocyclylene, wherein G200is optionally substituted with 1-4 substituents, each independently selected from the 026389-0053-W001 group consisting of Ci-4alkyl , Ci -shaloal kyl, halogen, cyano, -OCi-4alkyl, and -OCi. 2haloalkyl;

[0480] G210is a 5- to 12-membered heteroaryl or a 6- to 12-membered aryl, wherein G210is optionally substituted with 1-4 substituents, each independently selected from the group consisting of Ci-4alkyl, Ci-2haloalkyl, halogen, cyano, -OCi-4alkyl, and -OCi. 2haloalkyl; and quantifying a change in fluorescence intensity of the detection reagent while being contacted with the sample relative to a baseline fluorescence intensity of the detection reagent prior to being contacted with the sample.

[0481] Clause 87. The method of clause 86, wherein R200, at each occurrence, is hydrogen or halogen.

[0482] Clause 88. The method of clause 86 or 87, wherein G200is a 4- to 12-membered heterocyclylene.

[0483] Clause 89. The method of clause 88, wherein the 4- to 12-membered heterocyclylene is

[0484] Clause 90. The method of any one of clauses 86-89, wherein G210is a 5- to 12-membered heteroaryl.

[0485] Clause 91. The method of clause 90, wherein the 5- to 12-membered heteroaryl is .

[0486] Clause 92. The compound of any one of clauses 86-91 , wherein the compound of formula (V) is: .

[0487] Clause 93. The method of any one of clauses 86-92, wherein the compound of formula (V) is noncovalently adhered or covalently attached to a surface.

[0488] Clause 94. The method of any one of clauses 86-93, wherein the surface comprises a cellulose, a silica, a polymer, a resin, or a combination thereof.

[0489] Clause 95. The method of any one of clauses 86-94, wherein the compound of formula (V), or a salt thereof, is dissolved in a solvent.

[0490] Clause 96. The method of any one of clauses 86-95, wherein the detection reagent is excited at an excitation wavelength of 325-425 nm.

[0491] Clause 97. The method of any one of clauses 86-96, wherein the change in fluorescence intensity is quantified at an emission wavelength of 575-650 nm. 026389-0053-W001

[0492] Clause 98. The method of any one of clauses 86-97, wherein the detection reagent has a limit of detection of about 1 to about 50 ppb.

[0493] EXAMPLES

[0494] Example 1

[0495] Detection of PerfluoroctanoicAcid (PFOA) with Imidazole Pyridine (IPY)

[0496] Synthesis

[0497] Abbreviations

[0498] DCM is dichloromethane;

[0499] ACN is acetonitrile;

[0500] CDCh is deuterated chloroform;

[0501] NMR is nuclear magnetic resonance;

[0502] TLC is thin-layer chromatography; eq. or equiv. is equivalents; min or min. is minute(s); h or hr. is hour(s); rt, RT, or r.t. is room temperature; and sat’d or sat. is saturated.

[0503] Example Synthesis of Imidazole Pyridine (IPY)

[0504] 3-Phenyl-1-(pyridin-2-yl)imidazo[1 ,5-a]pyridine (IPY): Flasks were filled with 15 mL of glacial acetic acid and 0.32 g of 2,2'-dipyridyl ketone (1.7 mmol). All the solids were dissolved by stirring at room temperature under nitrogen. Afterwards, benzaldehyde (4.26 mmol) was added, followed by ammonium acetate (17 mmol). The reaction was monitored with thin-layer chromatography (TLC). The reaction mixture was stirred at 80 °C for 6 h and subsequently cooled to room temperature. After adding the mixture to ice-water, 200 mL of DCM solution was used to extract the reaction product from the mixture. The organic phase was washed with water and dried over sodium sulfate. The residue was purified using chromatography on a silica column followed by 026389-0053-W001 further recrystallization.1H NMR (400 MHz, CDCh) ppm 5: 8.72-8.70 (d, J = 8.0 Hz 1 H, ArCH), 8.64-8.62 (d, J = 8.0 Hz 1H, ArCH), 8.26-8.24 (d, J = 8 Hz 2H, ArCH), 7.85-7.84 (d, J = 4 Hz 2H, ArCH), 7.73 (m, 1 H, ArCH), 7.57-7.55 (t, J = 8.0 Hz 2H, ArCH), 7.48-7.46 (t, J = 4 Hz 1 H, ArCH), 7.11 (m, 1 H, ArCH), 6.94-6.93 (q, J = 4.0 Hz 1 H, ArCH), 6.66-6.65 (t, J = 4 Hz 1 H, ArCH);13C NMR (150 MHz, CDCI3) 6: 13.89, 119.91 ,120.43, 121.82, 128.37,128.91 ,129.04, 136.22, 148.96.

[0505] Spectroscopic Studies Evaluation of IPY

[0506] The photo-physical behavior of IPY towards PFOA was determined by measuring the absorption and fluorescence spectra in H2O:ACN (8:2 mL). Fluorescence emission from IPY solution was strongest at 460 nm, whereas the maximum absorption band was at 275 and 340 nm. When PFOA was added to IPY solution, a new absorption peak developed at 408 nm. A significant decrease was also noted in the absorption peaks at 275 nm, along with isosbestic points at 226 nm and 372 nm, indicating formation of new products. A strong fluorescence emission peak was observed at 460 nm under 350 nm excitation, with a Stokes shift of 185 nm (FIG. 1).

[0507] To determine the sensitivity of the probe, fluorescence spectra were recorded after adding various concentrations of PFOA to IPY. A linear relationship was observed between fluorescence intensity and PFOA concentration with a correlation coefficient of 0.988, as displayed in FIG. 2A.

[0508] After the addition of PFOA (0-150 pM) into IPY, the fluorescence intensity gradually decreased (87%) at the emission wavelength of 460 nm and reached the saturation point at the concentration of 150 pM. The detection limit of IPY toward PFOA was estimated to be 40.4 nM (16.7 ppb) using the formula of LOD = 3o / K, where o is the standard deviation and K is the slope of the line. FIG. 2B shows a Stern- Volmer plot of Z0 / / of IPY at an emission wavelength of 460 nm as a function of PFOA concentration. IPY exhibited a quenching constant (Ksv) of 1.9 x 105M-1for detecting PFOA in H2O:ACN (8:2 mL).

[0509] Absorption spectra of 10 pM IPY in the present of various concentrations of PFOA exhibited isosbestic points at 226 nm and 372 nm.

[0510] The IPY+PFOA complex binding constant was calculated using IPY fluorescence titration data with different PFOA concentrations. The binding constant (a) of PFOA to IPY was determined using the Benesi-Hildebrand plot slope, which was 2.94 x 104M’1(FIG 4).

[0511] Selectivity and Interference Studies

[0512] As shown by FIG. 5, the emission spectrum of IPY (2 pM in acetonitrile) showed significant changes in the presence of strong acids including HCI, H2SO4, perfluorohexanoic acid (PFHxA), 026389-0053-W001 perfluorooctanoic (PFOA), perfluorononanoic acid (PFNA), perfluorodecanoic acid (PFDA), perfluoroundecanoic acid (PFUDA), trifluoroacetic acid (TFA), and p-toluenesulfonic acid (p-TSA). In addition, IPY for PFOA demonstrated sensing ability in the presence of other interfering per- or poly-fluoro alkyl substances (PFAS) and metal ions. The fluorescent emission was quenched by other metal ions in the presence of PFOA. There was no interference from other metal ions in the emission intensity value (FIG. 6). As a result, IPY exhibited sensing selectivity for PFOA. Additionally, these results show that different interferent including other PFAS do not interfere with the sensing selectivity of IPY for PFOA. A summary of the structures of the different interferents are shown in FIG. 7.

[0513] Effect of Response Time of IPY

[0514] The kinetics of the sensing platform was determined by measuring the fluorescence profile of IPY solution over time after the presence of 150 pM PFOA. As shown in FIG. 8, IPY showed a rapid decrease in fluorescence intensity after the addition of PFOA. The lowest emission intensity was reached within 1 minute. These results exhibit that I PY and PFOA formed a complex immediately.

[0515] Effect of Ionic Strength of IPY

[0516] The effect of salt was examined by using different concentrations of sodium chloride (0- 30 mM) while keeping IPY and IPY+PFOA complex at fixed concentrations (FIG. 9). IPY did not show any significant changes in fluorescence intensity when 30 mM was present (6%). Both IPY and IPY + PFOA complexes were stable under conditions maintained by this complex; therefore, no adjustments in ionic strength were necessary.

[0517] Effect of Temperature on IPY to PFOA

[0518] Fluorescence experiments were performed to evaluate the thermal stability of 2 pM IPY towards PFOA (150 pM). The fluorescence intensity of IPY remained steady within a specified temperature range (25 to 70 °C); however, PFOA gradually dissociated from IPY fluorophore as the temperature increased (FIG. 10).

[0519] Binding Stoichiometry of IPY for PFOA

[0520] To determine the binding stoichiometry between PFOA and IPY, a Job’s plot was plotted between fluorescence data at 460 nm and the mole fraction (varying from 0.1 to 1.0) of PFOA 026389-0053-W001 added. The concentration of the resulting solution was maintained at the same level. Job’s plot showed the binding stoichiometry between IPY and PFOA as a 1 :1 ratio (FIG. 11).

[0521] Example 2

[0522] Detection of PerfluoroctanoicAcid (PFOA) with NA-3N.

[0523] Synthesis

[0524] Abbreviations

[0525] DCM is dichloromethane;

[0526] CDCh is deuterated chloroform;

[0527] NMR is nuclear magnetic resonance;

[0528] TLC is thin-layer chromatography; eq. or equiv. is equivalents; min or min. is minute(s); h or hr. is hour(s); and rt, RT, or r.t. is room temperature.

[0529] Example Synthesis of Imidazole Pyridine (NA-3N)

[0530] 6-(4-(Pyridin-2-yl)piperazin-1-yl)-1 H,3H-benzo[de]isochromene-1 , 3-dione 9 (NA-3N): Under a nitrogen (N2) atmosphere, 2.77 g (10 mmol) of 4-bromo-1,8-naphthalic anhydride (commercially available from TCI) was dispersed in 30 ml_ of 2-methoxyethanol. To the mixture, 1 -(2-pyridyl) piperazine (1.6 mL, 10 mmol) was added dropwise. The reaction was refluxed at 110 °C for 24 hours. After cooling, the resulting yellow solid was filtered and washed with 4 x 50 mL of deionized water. Further purification was performed by extracting the reaction product with 1 :1 DCM:H2O. The organic layer from the extraction was collected and dried under anhydrous sodium sulfate for 2 hours. The pure reaction product was obtained by evaporating the DCM solvent with a rotary 026389-0053-W001 evaporator and appeared as a yellow solid.1H NMR (CDCh): 8.63-8.61 (d, 1 H), 8.56-8.54 (dd, 2H), 8.27-8.25 (d, 1 H), 7.80-7.76 (t, 1 H), 7.59-7.54 (t, 1 H), 7.30-7.28 (d, 1 H), 6.78-6.71 (m, 2H), 3.89-3.86 (t, 4H), 3.46-3.44 (t, 4H) ppm.

[0531] Fluorescence Spectroscopic Studies for NA-3N

[0532] The fluorescence activation of NA-3N was monitored by detecting the fluorescence at an emission wavelength of 525 nm (when excited at 400 nm) of 3.3 pM NA-3N solution in acetonitrile (3.3 pM) in the absence and presence of PFOA at increasing concentrations (0 pM to 4 pM) (FIG. 12). No significant emission at an emission wavelength of 525 nm (when excited at 400 nm) was measured for the solution containing NA-3N alone. Upon addition of PFOA, fluorescence began to emerge, and the intensity increased as the concentration of PFOA increased.

[0533] Assuming 1 :1 complexation between NA-3N and PFOA, the increase in fluorescence intensity correlated with the concentration of PFOA following the linear Benesi-Hildebrand equation, which was employed as a calibration curve for quantitative analysis (FIG. 13). From the Benesi-Hildebrand equation, a limit of detection (LOD) of 15.7 nM (6.5 ppb) was obtained for PFOA by defining the minimal detectable signal as three times the standard deviation of measurement. The linear fitting provided a binding constant (Ka) of 1.5 x 106M-1, which implies strong binding between NA-3N and PFOA. The binding constant is consistent with the efficient fluorescence activation of NA-3N.

[0534] Without being bound by theory, the sensing mechanism of fluorescence activation of NA- 3N due to complexation with PFOA as schematically illustrated in FIG. 14. The NA-3N is a typical electron donor-acceptor (D-A) molecule with the piperazine as the donor and naphthalic anhydride as acceptor. NA-3N is non-fluorescent in its pristine state due to the intramolecular D- A electron transfer that quenches the emission intrinsic to the TT-TT* transition of naphthalene moiety. The charge transfer (CT) state is non-fluorescent as the free D-A rotation favored the non-radiative decay pathway. Once NA-3N is complexed with PFOA, the D-A rotation is restricted to some extent, thus activating the so-called twisted intramolecular charge transfer (TICT) emission, in which the decay of CT state becomes more favorable for the radiative pathway. Complexation between NA-3N and PFOA may be attributed to hydrogen bonding between the carboxylic group of PFOA and the two tertiary amine groups on piperazine and pyridine moiety of NA-3N as illustrated in FIG. 14 and confirmed by various spectrometric study.

[0535] Absorption Spectroscopic Studies for NA-3N 026389-0053-W001

[0536] The complexation between NA-3N and PFOA was also characterized by UV-vis absorption spectrometry, the results of which are shown in FIG. 15. Two isosbestic points were observed, indicating the quantitative conversion of the free molecular form of NA-3N to a complexed state. Assuming 1 :1 complexation between NA-3N and PFOA, the change in UV-vis spectra can be correlated with the concentration of PFOA with the linear Benesi-Hildebrand equation (FIG. 16). The linear fitting shown by FIG. 16 is consistent with the fluorescence spectral measurements.

[0537] Selectivity and Interference Studies for NA-3N

[0538] The NA-3N sensor exhibited a high level of specificity for PFIA as shown in FIG. 17. Even when the interferents were present at concentrations 10 times higher than that of PFOA, the activation of fluorescence was only slightly above the noise level of the instrument. Thus, the high specificity obtained may be attributable to the specific complexation between NA-3N and PFOA.

[0539] Binding Stoichiometry of NA-3N to PFOA

[0540] A 1 :1 molar ratio of complex was confirmed by Job plot as shown FIG. 18.

[0541] Example 3

[0542] Detection of Perfluoroctanoic Acid (PFOA) with DIPY

[0543] Synthesis

[0544] Abbreviations

[0545] DCM is dichloromethane;

[0546] DMSO is dimethyl sulfoxide;

[0547] NMR is nuclear magnetic resonance; eq. or equiv. is equivalents; min or min. is minute(s); h or hr. is hour(s); rt, RT, or r.t. is room temperature; and sat’d or sat. is saturated.

[0548] Example Synthesis of DIPY 026389-0053-W001

[0549] 1-Phenyl-3-(4-(1-(pyridin-2-yl)imidazo[1 ,5-a]pyridin-3-yl)phenyl)imidazo[1 ,5-a]pyridine (DIPY): A round-bottom flask was filled with 2.2'-dipyridyl ketone (0.5 g, 2.717 mmol). Glacial acetic acid (15 ml_) was added at room temperature (RT). Afterwards, terephthalaldehyde (0.87 g, 6.51 mmol) was added along with ammonium acetate (2.1 g, 27 mmol). The mixture was stirred at 110 °C for 12 hours. Following the reaction time, ice water was added to the reaction mixture. The reaction product was extracted with 200 ml_ of DOM solution. The DCM organic phase was dried over sodium sulfate. The reaction produced was purified with a silica column chromatography and recrystallization.1H NMR (400 MHz, DMSO-cfe) ppm 6: 8.76-8.75 (d, J = 4.0 Hz 2H, ArCH), 8.68-8.66 (d, J = 8.0 Hz 2H, ArCH), 8.58-8.56 (d, J = 8 Hz 2H, ArCH), 8.51- 8.49 (t, J = 8 Hz 2H, ArCH), 8.44-8.42 (d, J = 8 Hz 2H, ArCH), 8.12-8.10 (q, J = 8 Hz 5H, ArCH), 7.72-7.71 (t, J = 4.0 Hz 2H, ArCH), 7.50-7.48 (q, J = 8 Hz 2H, ArCH), 7.15-7.13 (t, J = 8 Hz 2H, ArCH).

[0550] Fluorescence Spectroscopic Studies for DIPY

[0551] DIPY showed a strong fluorescence emission at 480 nm when excited at 370 nm. Fluorescence intensity at 480 nm gradually decreased with increasing PFOA concentrations from 0 to 5 pM (FIG. 19). The emission intensity of DIPY decreased by approximately 92%, suggesting that the pyridine moiety may accept a proton from PFOA. Using the spectroscopic data, the limit of detection (LOD) was calculated from the linear plot using 3 / slope, where k represents the standard deviation from 10 blank measurements and the slope was obtained from the linear plot of the spectroscopic data. The fluorescence titration curve obtained from an Agilent Fluorescence Spectrophotometer revealed that the limit of detection of PFOA by DIPY was 28.5 nM (11.8 ppb) (FIG. 20)

[0552] Absorption Spectroscopic Studies for DIPY

[0553] A UV-visible absorption spectrum of DIPY was recorded in acetonitrile solvent (FIG. 21). There were intense spectral bands at 330 nm (36000 mol-1 cm-1) and 370 nm (52000 mol-1 cm-1) of the TT-TT* and n— TT* transitions, respectively. DIPY developed a significant hypochromic shift at 370 nm when exposed to PFOA, with a new peak appearing at 410 nm. An isosbestic 026389-0053-W001 point appeared at 393 nm, indicating the formation of the PFOA and DIPY complex. The PFOA binding constant (Ka) for DIPY was determined using Benesi-Hildebrand (B-H plot) linear graph plotted between Amax-Ao / Ax-Ao and 1 / [PFOA], which is represented in FIG. 22. The PFOA binding constant was calculated as 1.80 x 104M-1.

[0554] Selectivity and Interference Studies for DIPY

[0555] As shown by FIG. 23, the emission spectrum of DIPY (1 pM in acetonitrile) exhibited a significant change in the presence of all strong acids including HCI, H2SO4, PFHxA, PFOA, PFNA, PFUA, PFUDA, TFA, Oxalic acid, and p-TSA. A competition experiment was performed to determine the selectivity of DIPY in the presence and absence of PFOA. The fluorescence intensity of 1 pM DIPY in acetonitrile was measured in the presence of 15 pM PFOA and 15 pM of various competitive analytes (FIG. 24). As displayed in FIG. 24, none of the tested analytes showed any significant change in fluorescence emission intensity in the presence of PFOA recognition. These results show that DIPY may be selective for PFOA.

[0556] Effect of Ionic Strength on DIPY Binding to PFOA

[0557] For DIPY, the fluorescence intensity changed significantly at low ionic strength (0-1.4 mM NaCI). The fluorescence intensity of the DIPY + PFOA complex did not significantly change. Therefore, at lower concentrations of NaCI, DIPY + PFOA complex may not be affected by NaCI (FIG. 25).

[0558] Thermal Stability of DIPY and its PFOA Complex

[0559] As shown by FIG. 26 the fluorescence intensity of DIPY remained stable with increasing temperature, demonstrating that DIPY may withstand high temperatures. When the temperature of a solution containing the DIPY + PFOA complex increases above 30 °C, the fluorescence intensity increased as well. These results indicate that DIPY + PFOA complexes may be unstable at high temperatures. In view of these results, the final temperature used for the experiments described herein was 25 °C.

[0560] Binding Stoichiometry of DIPY to PFOA

[0561] DIPY+PFOA binding stoichiometry was determined using Job’s plot, as shown in FIG. 27. There were two potential binding sites for PFOA on DIPY. Both PFOA and DIPY appear to form a 1 : 1 complex due to the lengthy chain length of PFOA that attaches to a single site (FIG. 27). 026389-0053-W001

[0562] Example 4

[0563] Detection of PerfluoroctanoicAcid (PFOA) with Fluorescent Probe TPIPY

[0564] Synthesis

[0565] Abbreviations

[0566] DCM is dichloromethane;

[0567] NMR is nuclear magnetic resonance; eq. or equiv. is equivalents; min or min. is minute(s); h or hr. is hour(s); rt, RT, or r.t. is room temperature; and sat’d or sat. is saturated.

[0568] Example Synthesis of TPIPY / ,A / -Diphenyl-4-(1-(pyridin-2-yl)imidazo[1 ,5-a]pyridin-3-yl)aniline (TPIPY): 2'-Dipyridyl ketone (0.3 g, 1.63 mmol) and glacial acetic acid (15 mL) were added at room temperature to a roundbottom flask. To the round-bottom flask, 1.1 g (4.1 mmol) of diphenylaminobenzaldehyde and 1.25 g (16.3 mmol) of ammonium acetate were added. The mixture was heated to 110 °C for 12 hours. Following the reaction time, ice-water was added to the reaction mixture. The reaction product was extracted with 150 mL of DCM. The organic layer from the extraction was dried over sodium sulfate. The reaction product was purified with silica column chromatography and recrystallization.1H NMR (400 MHz, CDCI3) ppm 5: 8.60-8.58 (d, J = 8.0 Hz 2H, ArCH), 8.48- 8.46 (d, J = 8.0 Hz 1 H, ArCH), 8.11-8.09 (d, J = 8 Hz 1 H, ArCH), 7.82-7.80 (q, J = 8 Hz 3H, ArCH), 7.37-7.35 (t, J = 8 Hz 4H, ArCH), 7.19-7.02 (m, 10H, ArCH), 6.83-6.82 (t, J = 4.0 Hz 1 H, ArCH).

[0569] Fluorescence Spectroscopic Studies for TPIPY

[0570] A strong fluorescence emission was observed in response to TPIPY (2 pM) when excited at 350 nm. At increasing concentrations of PFOA (0-10 pM), there was a decrease in 026389-0053-W001 fluorescence intensity at 480 nm (FIG. 28). Approximately 89% of TPIPY fluorescence was quenched by PFOA. Using the spectroscopic data, the limit of detection (LOD) was calculated from the linear plot using 3 / r / slope, where k represents the standard deviation from 10 blank measurements and the slope was obtained from the linear plot of the spectroscopic data. The fluorescence titration curve obtained from an Agilent Fluorescence Spectrophotometer revealed that the limit of detection of PFOA by TPIPY was 20.5 nM (8.50 ppb) (FIG. 29). Additionally, the quenching constant (sv) of PFOA on TPIPY in acetonitrile was 3.54 x 105M“1(FIG. 30).

[0571] Absorption Spectroscopic Studies for TPIPY

[0572] In acetonitrile solvent, the UV-visible absorption spectrum of TPIPY was measured (FIG. 31). Strong bands at 310 nm (25000 mol-1cm-1) and 345 nm (31000 mol-1cm-1) were observed for the TT— TT* and n-n* transitions, respectively. In response to PFOA, TPIPY displayed a significant hypochromic shift at 345 nm, with a new peak appearing at 435 nm. In this study, an isosbestic point was spotted at 388 nm, indicating the formation of a PFOA and TPIPY complex. PFOA-binding constant (Ka) to TPIPY was determined using Benesi-Hildebrand plot (FIG 32). The binding constant of PFOA to TPI PY was 2.15 x 105M’1.

[0573] Selectivity and Interference Studies for TPIPY

[0574] As shown by FIG. 33, the emission spectrum of TPIPY (2 pM in acetonitrile) showed significant changes with all strong acids including HCI, H2SO4, PFHxA, PFOA, PFNA, PFUA, PFUDA, TFA, and p-TSA. To determine the selectivity of TPIPY in the presence and absence of PFOA, a competitive experiment was conducted. The fluorescence measurements were performed using acetonitrile as a solvent. Competition studies were performed with 2 pM TPIPY against 10 pM of PFOA and various other competitive analytes. FIG. 35 shows no significant changes in fluorescence emission intensity in response to any of the various other competitive analytes.

[0575] Effect of Ionic Strength on TPIPY Binding to PFOA

[0576] The fluorescence intensity of TPIPY was significantly altered at low ionic strength (0-30 mM NaCI). However, there was no significant change in the fluorescence intensity of the PFOA complex. Thus, when NaCI concentrations are low, the TPIPY + PFOA complex may not be affected (FIG. 35).

[0577] Thermal Stability of TPIPY and its PFOA Complex 026389-0053-W001

[0578] The fluorescence intensity of TPIPY was stable at increasing temperatures as shown in FIG. 36, demonstrating that TPIPY is highly stable. The fluorescence intensity of the TPIPY + PFOA increased as the temperature increased. At high temperatures, the TPIPY+PFOA complexes may be unstable, suggesting it may be beneficial to use TPIPY at 25 °C.

[0579] Binding Stoichiometry of TPIPY to PFOA

[0580] FIG. 37 shows the Job’s plot for TPIPY + PFOA binding stoichiometry. Job’s plot shows that PFOA and TPIPY form a 1 :1 complex with each other (FIG. 37).

[0581] Example 5

[0582] Detection of Perfluoroctanoic Acid (PFOA) with Fluorescent Probe BTD-3N

[0583] Synthesis

[0584] Abbreviations

[0585] DCM is dichloromethane;

[0586] CDCh deuterated chloroform;

[0587] NMR is nuclear magnetic resonance; eq. or equiv. is equivalents; min or min. is minute(s); h or hr. is hour(s); rt, RT, or r.t. is room temperature; and sat’d or sat. is saturated.

[0588] Example Synthesis of BTA-3N

[0589] 4-Bromo-7-(4-(pyridin-2-yl)piperazin-1-yl)benzo[c][1 ,2,5]thiadiazole (BTD-3N): In 10 mL of DMSO, 1-phenylpiperazine (0.5 mL, 3.4 mmol) and 3 drops of triethylamine were added to a solution of 4,7-dibromobenzo[d][1 ,2,3]thiadiazole (100 mg, 0.34 mmol). After degassing with nitrogen for 10 minutes at 110°C, the mixture was reacted for 18 hours. The reaction product was extracted with DCM (50 mL) and washed several times in water. The organic layer was dried over MgSCU, filtered, and concentrated. The reaction product was purified by column chromatography to obtain a white solid.1H NMR (400 MHz, CDCI3) ppm 6: 8.76-8.75 (d, J = 5.0 026389-0053-W001

[0590] Hz 2H, ArCH), 7.98-7.96 (d, J = 8.0 Hz 1 H, ArCH), 7.88-7.86 (d, J = 8 Hz 2H, ArCH), 7.68-7.66 (d, J = 8 Hz 1 H, ArCH). Exact mass calcd for CisHuBrNsS: 375.01. Found: 376.02 (MH+ ion peak).

[0591] Fluorescence Spectroscopic Studies for BTD-3N

[0592] BTD-3N exhibited a very weak fluorescent intensity due to strong intramolecular charge transfer. When PFOA was added to a solution of BTD-3N, the fluorescent intensity at 620 nm increased 7-fold (FIG. 38). Correspondingly, the fluorescence of the solution changed from colorless to red. This color change was visible under a 365 nm UV lamp. Thus, BTD-3N exhibited a large Stokes shift to induce a fluorescence emission response to PFOA. Due pyridine’s ability to accept a proton from PFOA, the fluorescence enhancement may result from interaction the PFOA. Using the spectroscopic data, the limit of detection (LOD) was calculated from the linear plot using 3 / slope, where k represents the standard deviation from 10 blank measurements and the slope was obtained from the linear plot of the spectroscopic data. The fluorescence titration curve obtained from an Agilent Fluorescence Spectrophotometer revealed that the limit of detection of PFOA by BTD-3N was 12.7 nM (5.26 ppb) (FIG. 39).

[0593] Absorption Spectroscopic Studies for BTD-3N

[0594] The UV-vis absorption spectrum (FIG. 40) of BTD-3N showed four bands centered at 260 nm, 300 nm, 310 nm, and 425 nm. A slight increase in absorption intensity was observed when PFOA was added to the probe solution.

[0595] Selectivity and Interference Studies for BTD-3N

[0596] The selectivity of BTD-3N towards metal salts and PFAS in acetonitrile solutions was investigated. As shown by FIG. 41, there were no significant changes after the addition of 20 pM of HCI, H2SO4, PFHxA, PFOA, PFNA, PFUA, PFUDA, TFA, and p-TSA (FIG. 41). Competition experiments were performed with 2 pM BTD-3N in the presence of with 10 pM PFOA and 20 pM of various PFAS and metal cations (FIG. 42). The BTD-3N+PFOA complex fluorescence was almost unaffected by other PFAS or other metal ions.

[0597] Effect of Ionic Strength on BTD-3N Binding to PFOA

[0598] Fluorescence intensity of BTD-3N was not significantly altered at low ionic strength (0-30 mM NaCI); however, the fluorescence intensity of the PFOA complex was significantly altered in 026389-0053-W001 the presence of NaCI (FIG. 43). Therefore, a low NaCI concentration may affect BTD-3N+PFOA complexes.

[0599] Thermal Stability of BTD-3N and its PFOA Complex

[0600] As shown by FIG. 44, the fluorescence intensity of BTD-3N was stable at increased temperatures. The fluorescence intensity of the BTD-3N + PFOA complex decreased when temperature was increased indicating that the BTD-3N + PFOA complex may be unstable at high temperatures. Thus, a temperature of 25 °C was used during experiments.

[0601] Example 6

[0602] Detection of Perfluorosulfonic Acid (PFOS) with Fluorescent Probe Benz-1+

[0603] Synthesis

[0604] Abbreviations

[0605] DMF is dimethylformamide;

[0606] DMSO is dimethylsulfoxide;

[0607] NMR is nuclear magnetic resonance;

[0608] TLC is thin-layer chromatography; eq. or equiv. is equivalents; min or min. is minute(s); mmol is millimole; h or hr. is hour(s); and rt, RT, or r.t. is room temperature.

[0609] Example Synthesis of Benz 1+ and TP-1+ Intermediate

[0610] 3-Ethyl-2-methylbenzo[d]thiazol-3-ium iodide: 3-Ethyl-2-methyl-1,3-benzothiazol-3-ium iodide was prepared by mixing 2-methylbenzothiazole (1.43 mL, 11 mmol) with iodoethane (3.22 mL, 40 mmol) in DMF (5 mL). The reaction mixture was heated at a reflux temperature of 90 °C for 5 hours. The reaction mixture was cooled, and diethyl ether and ethanol were added to extract the reaction product. The organic layer was washed several with water containing a desired salt. The reaction produced was subjected to vacuum drying under reduced pressure three times to 026389-0053-W001 obtain white crystal solids.1H NMR (400 MHz, DMSO-cfe) ppm 6: 8.45-8.43 (t, J = 8 Hz, 1H, ArCH), 8.34-8.32 (d, J = 8 Hz, 1 H, ArCH), 7.90-7.88 (t, J = 8 Hz, 1 H, ArCH), 7.81-7.79 (t, J = 8 Hz, 1 H, ArCH), 4.78-4.76 (q, J = 8.0 Hz, 2H, CH2), 3.20 (s, 3H, CH3), 1.45-1.44 (t, J = 4.0 Hz, 3H, CH3).

[0611] Example Synthesis of Benz 1+ and TP-1+ Fluorophores

[0612] (E)-2-(4-(Dimethylamino)styryl)-3-ethylbenzo[d]thiazol-3-ium iodide (Benz-1 +): 3-Ethyl-2-methyl- 1 ,3-benzothiazol-3-ium iodide (0.3 g, 1 mmol) and 4-dimethylaminobenzaldehyde (1.2 mmol) were added to a reaction vessel. In an oil bath at 120-150°C, the mixture was stirred for 5-7 hours. Progress of the reaction was monitored with thin-layer chromatography (TLC). Once the reaction product was formed, the reaction mixture was cooled slowly and produced a thick semisolid mass. The purification of Benz-1+ was then performed through recrystallization with methanol.1H NMR (400 MHz, DMSO-cfe) ppm 5: 8.30-8.28 (d, J = 8 Hz, 1H, ArCH), 8.13-8.11 (q, J = 8 Hz, 2H, ArCH), 7.93-7.91 (d, J = 8 Hz, 2H, ArCH), 7.78-7.76 (t, J = 8 Hz, 1 H, ArCH), 7.68-7.59 (m, 2H, ArCH), 6.83-6.81 (d, J = 8 Hz, 2H, ArCH), 4.84-4.83 (q, J = 4.0 Hz, 2H, CH2), 3.09 (s, 6H, 2xCH3), 1.42-1.41 (t, J = 4.0 Hz, 3H, CH3).

[0613] (E)-2-(4-(Diphenylamino)styryl)-3-ethylbenzo[d]thiazol-3-ium iodide (TP-1+): 3-Ethyl-2-methyl- 1 ,3-benzothiazol-3-ium iodide (0.15 g, 0.5 mmol) and 4-(diphenylamino)benzaldehyde (0.156 g, 0.52 mmol) added into 10 mL of anhydrous ethanol solvent. The mixture was refluxed at 80°C for overnight. In the meantime, the reaction progress was monitored by TLC. After cool down, the residue was poured into ethyl acetate and then stirred at r.t for 30 min. The brown precipitate slowly settled down and then filtered followed dried at hot-air oven.1H NMR (400 MHz, DMSO- d6) ppm 6: 8.38-8.36 (d, J = 8 Hz, 1 H, ArCH), 8.23-8.21 (d, J = 8 Hz, 1 H, ArCH), 8.15-8.13 (d, J = 026389-0053-W001

[0614] 8 Hz, 1 H, ArCH), 7.93-7.91 (d, J = 8 Hz, 2H, ArCH), 7.84-7.71 (m, 3H, ArCH), 7.42-7.40 (t, J = 8 Hz, 4H, ArCH), 7.19 (m, 6H, ArCH), 6.90-6.88 (d, J = 8 Hz, 2H, ArCH), 4.9I.4.90 (q, J = 4.0 Hz, 2H, CH2), 1.44-1.42 (t, J = 8.0 Hz, 3H, CH3).

[0615] Fluorescence Spectroscopic Studies for Benz 1+

[0616] Benz-1+ exhibited fluorescence at an emission wavelength of 590 nm when excited at 520 nm. When PFOS was added, the emission intensity of Benz 1+ gradually decreased. Due to the electrostatic interaction between Benz 1+ and PFOS, Benz 1+ became saturated at 10 pM PFOS in aqueous media (FIG. 45). The change in fluorescence intensity of Benz 1+ in the presence of PFOS at various concentrations from 0 to 2 pM are show by FIG. 46. Using the spectroscopic data, the limit of detection (LOD) was calculated from the linear plot using 3 / slope, where k represents the standard deviation from 10 blank measurements and the slope was obtained from the linear plot of the spectroscopic data. The fluorescence titration curve obtained from an Agilent Fluorescence Spectrophotometer revealed that the limit of detection of PFOS by Benz-1+ was 48 nM (25.8 ppb) (FIG. 46).

[0617] Absorption Studies for Benz 1 +

[0618] As depicted in FIG. 47, Benz-1 + exhibited UV-vis absorption peaks at 515 nm (FIG. 47). With the addition of PFOS to Benz-1 + solution, the absorption peak gradually decreased. Moreover, the color of the [Benz-1 + PFOS] solution became almost colorless, indicating the formation of PFOS adducts with Benz-1+.

[0619] Selectivity and Interference Studies for Benz 1 +

[0620] The emission intensity at 590 nm of the mixture containing 2 pM Benz 1+ decreased gradually with the addition 10 pM PFOS and PFOS K+. In the presence of various other analytes (10 pM), there was no appreciable change in color or fluorescence intensity (FIG. 48). Competition studies were performed to investigate the changes in the emission spectrum of Benz- 1+ (5 pM) in the presence of 10 pM PFOS and 20 pM of various PFAS and salts. FIG. 49 shows that the emission the Benz-1 + and PFOS complex was nearly the same in the presence of 20 pM of various other analytes (FIG. 49).

[0621] Effect of Ionic Strength on Benz 1+ Binding to PFOS

[0622] The effect of salt (0-70 mM) was examined with Benz-1 + alone and a complex formed by Benz-1+ and PFOS at fixed concentration levels (FIG. 50). There was no significant change in 026389-0053-W001 fluorescence intensity for Benz-1+ and its PFOS complex at 70 mM NaCI. Therefore, both Benz- 14- and Benz-1-i-PFOS complexes may be stable under the various ionic conditions and may not require ionic strength adjustment of the solution.

[0623] Fluorescence Spectroscopic Studies for TP-1-n

[0624] TP-1 - exhibits a very weak fluorescent intensity due to strong intramolecular charge transfer in the absence of PFOS (FIG. 51). The fluorescent intensity at an emission wavelength of 670 nm (when excited at 520 nm) of 5 pM TP-1 - showed an increased significantly as PFOS was added (0 pM to 10 pM) (FIG. 51). In response to the addition of PFOS, the fluorescence of TP-1 -r changed from colorless to red. Under a 365 nm UV lamp, this color change was visible. TP-1 -r exhibited a large Stokes shift and fluorescence activation in response to PFOS. A limit of detection of 7.2 nM (3.9 ppb) was calculated for TP-14- detection of PFOS (FIG. 52).

[0625] Absorption Studies for TP-14-

[0626] FIG. 53 shows the UV-visible absorption spectra of 5 pM TP-14- dissolved in water / ACN (9:1, v / v) in the presence of different concentrations of PFOS (0 pM to 10 pM). In response to PFOS, TP-1 -r displayed a significant hypochromic shift at 490 nm, with a new peak appearing at 612 nm. The absorption spectra of TP-14- in the presence of PFOS exhibits two isosbestic points at 407 nm, and 554 nm, respectively. This formation of the isosbestic points indicate the formation of the PFOS 4- TP- 14- complex.

[0627] Selectivity and Interference Studies for TP-14-

[0628] FIG. 54 shows a significant increase in fluorescence intensity of TP-14- (5 pM) at an emission wavelength of 670 nm (when excited with 520 nm) in the presence of PFOS, PFOS-K-i-, PFHxS, PFDA, PFUDA, and SDS. No appreciable change in fluorescence intensity of TP-14- was observed for with other analytes.

[0629] The selectivity of 5 pM TP-14- for 10 pM PFOS in water / ACN (9:1 , v / v) was determined in the presence of 10 pM of other analytes. No significant change in fluorescence emission intensity were detected in any of the tested analytes in response to PFOS recognition (FIG. 55).

[0630] Effect of Ionic Strength on TP-14- Binding to PFOS

[0631] The effect of salt (0-40 mM) was examined at an emission wavelength of 670 nm (when excited at 520 nm) for 5 pM TP-14- and a complex formed with 5 pM TP-14- and 5 pM PFOS (FIG. 56). No significant change in fluorescence intensity was observed for TP-14- alone or in complex 026389-0053-W001 with PFOS at 40 mM NaCI. This indicates that both TP-1+ alone and the PFOS + TP-1+ complex may not require any adjustment of ionic strength.

[0632] Effect of pH on TP-1+ Binding to PFOS

[0633] The pH-dependence of 5 pM TP-1+ alone and the complex formed by 5 pM TP-1+ and 10 pM PFOS at an emission wavelength of 670 nm (when excited at 520 nm) was examined over a pH range of 1-11 (FIG. 57). TP-1+ alone exhibited weak emission intensity across the pH range of 1 to 11. A higher emission intensity was observed in the pH range 4 to 9 for the PFOS + TP- 1+ complex; however, the fluorescence intensity decreased significantly from 9 to 11.

[0634] The Effect of Viscosity on the Fluorescence of TP-1 +

[0635] The effect of viscosity on the fluorescence of TP-1 + was examined. When the viscosity is low (0-30%), the styryl bond between the benzene and benzothiazole is not coplanar and produces weak fluorescence at 670 nm (when excited at 520 nm) (FIG. 58). Glycerol has a relatively high viscosity (40-90%) resulting in inhibition of free rotation of the styryl bond (FIG. 59). This allows for a significant increase in fluorescence intensity in TP-1 +. This experiment indicates that molecular twisting of fluorescent TP-1+ is restricted, thus offering insights into the twisted intramolecular charge transfer (TICT) mechanism.

[0636] Example 7: Detection of Perfluoroctanoic Acid (PFOA) with Fluorescent Probe NI-TPY

[0637] Synthesis

[0638] Abbreviations

[0639] Pd(PPh3)4is palladium triphenylphosphine;

[0640] DCM is dichloromethane;

[0641] THF is tetrahydrofuran;

[0642] CDCh is deuterated chloroform;

[0643] NMR is nuclear magnetic resonance;

[0644] TLC is thin-layer chromatography; eq. or equiv. is equivalents; min or min. is minute(s); h or hr. is hour(s); and rt, RT, or r.t. is room temperature. 026389-0053-W001

[0645] Example Synthesis of NI-TPY Intermediates

[0646] 6-Bromo-2-butyl-1 H-benzo[de]isoquinoline-1 ,3(2H)-dione: 4-Bromo-1 ,8-naphthalic anhydride (554.2 mg, 2 mM) was dissolved in 20 mL of ethanol. To the solution, n-butyl amine (197 pL, 2mM) was added at room temperature. The mixture was heated to 85 °C and stirred overnight. The reaction mixture was cooled to room temperature and the precipitate was filtered and washed with cold ethanol. The reaction product was dried under the vacuum at 60 °C.1H NMR (400 MHz, CDCh) ppm b: 8.66-8.64 (d, 1 H, ArCH), 8.57-8.55 (d, 1 H, ArCH), 8.42-8.40 (d, 1 H, ArCH), 8.04-8.02 (d, 1 H, ArCH), 7.86-7.82 (t, 1 H, ArCH), 4.19^1.15 (t, 2H, CH2), 1.75-1.68 (m, 2H, CH2), 1.49-1.40 (m, 2H, CH2), 1.0-0.96 (t, 3H, CH3).

[0647] 4-(2-Butyl-1 ,3-dioxo-2,3-dihydro-1 H-benzo[de]isoquinolin-6-yl)benzaldehyde: 6-Bromo-2-butyl- 1 H-benzo[de]isoquinoline-1 ,3(2H)-dione (0.1g, 0.3 mmol), (4-formylphenyl)boronic acid (67 mg, 0.45 mmol), Pd(PPh3)4 (34 mg, 0.03 mmol), K2CO3 (1 ml_ of 2 M solution), and THF (3 ml_) were heated to 100 °C under N2 atmosphere for 24 h. The reaction mixture was cooled, and 100 mL water was added. The reaction product was extracted with DCM (3*100 mL), and the organic extract was dried with MgSO4. The extracted reaction product was filtered and concentrated under vacuum. The resulting product (56%) was purified by column chromatography.1H NMR (400 MHz, CDCh) ppm 5: 10.15 (s, 1 H, CHO), 8.67-8.66 (t, J = 4 Hz, 2H, ArCH), 8.16-8.14 (d, J = 8 Hz, 1 H, ArCH), 8.08-8.06 (d, J = 8 Hz, 2H, ArCH), 7.75-7.73 (q, J = 8 Hz, 4H, ArCH), 4.23- 026389-0053-W001

[0648] 4.21 (t, J = 8.0 Hz, 2H, CH2), 1.76-1.72 (t, J = 8.0 Hz, 2H, CH2), 1.49-1.44 (q, J = 8.0 Hz, 2H, CH2), 1.01-0.99 (t, J = 8.0 Hz, 3H, CH3);13C NMR (150 MHz, CDCh) 5:13.83, 20.37, 30.20, 40.33, 76.67, 76.98, 77.30,127.25, 127.80, 129.92, 130.59, 131.32, 136.01 , 136.28, 145.03, 145.10, 162.30,164.01 , 196.17.

[0649] Example Synthesis of NI-TPY Fluorophores

[0650] 6-(4-([2,2':6',2"-Terpyridin]-4'-yl)phenyl)-2-butyl-1 H-benzo[de]isoquinoline-1 ,3(2H)-dione (Nl- TPY): 4-(2-Butyl-1 ,3-dioxo-2,3-dihydro-1 H-benzo[de]isoquinolin-6-yl)benzaldehyde (80 mg, 0.24 mmol) and 2-acetylpyridine (0.11 ml_, 0.9 mmol) were dissolved in methanol (20 mL). To the solution, 15 % aq. KOH (10 mL) and ammonium acetate (10 mmol) were added and stirred vigorously for three days. The reaction product was filtered and washed copiously with water until no base was found in the precipitate. The precipitate was dissolved methanol (CHCI3) and washed with NaHCOs. The organic layer was dried with MgSO4, and the methanol was evaporated. The reaction product was recrystallized from absolute ethanol.1H NMR (400 MHz, CDCh) ppm 5: 8.79 (s, 2H, ArCH), 8.71 (m, 6H, ArCH), 8.27-8.25 (d, J = 8 Hz, 1 H, ArCH), 8.05- 8.03 (d, J = 8 Hz, 2H, ArCH), 7.88-7.86 (t, J = 8 Hz, 2H, ArCH), 7.73-7.66 (q, J = 8 Hz, 2H, ArCH), 7.35-7.33 (t, J = 8.0 Hz, 2H, ArCH), 7.21-7.19 (d, J = 8, Hz 2H, ArCH), 4.20-4.18 (t, J = 8.0 Hz, 2H, CH2), 1.75-1.73 (q, J = 8.0 Hz, 2H, CH2), 1.48-1.31 (m, 2H, CH2), 0.97-0.95 (t, J = 8.0 Hz, 3H, CH3);13C NMR (150 MHz, CDCh) 6: 13.85, 20.39, 30.20, 40.27,118.80, 121.35, 121.92, 123.93, 126.93, 127.60, 127.80, 130.46, 131.16, 132.44, 136.96, 138.69, 139.44, 149.13, 149.39, 156.07, 164.00, 164.2, ArCH);13C NMR (150 MHz, CDCh) 6: 13.89, 119.91 , 120.43, 121.82, 128.37, 128.91 , 129.04, 136.22, 148.96. 026389-0053-W001

[0651] Fluorescence Spectroscopic Studies for NI-TPY

[0652] In the absence of PFOA, NI-TPY produced a strong fluorescence emission at 435 nm when excited at 350 nm. It is indicated that the naphthalic anhydride segments fluoresce at 435 nm. When PFOA was added to the NI-TPY solution, the fluorescence intensity at 435 nm gradually decreased. The fluorescence emission intensity decreased by approximately 88% when the PFOA concentration was increased from 0 to 100 pM. This suggests that the pyridine moiety may accept a proton from PFOA (FIG. 60 A-C). Using the spectroscopic data, the limit of detection for NI-TPY was calculated as 23.3 nM by applying o / K, where o is the standard deviation and K is the slope obtained from the linear plot of the spectroscopic data. FIG. 61 shows the Stern-Volmer plot for PFOS. The Ksv value of NI-TPY for PFOA was 6.20 * 105M-1.

[0653] Absorption Spectroscopic Studies for NI-TPY

[0654] In a 2:8 acetonitrile / water mixture, the UV-visible absorption spectrum of NI-TPY was recorded (FIG. 62A). The absorption spectrum showed intense spectral bands at 225 nm (64000 mol-1cm-1), 283 nm (35000 mol-1cm-1), and 350 nm (25000 mol-1cm-1) for the TT-TT* and n— TT* transitions, respectively. In the presence of PFOA, NI-TPY developed a significant hypochromic shift at 283 nm, with a new peak appearing at 350 nm. Absorption at 225 nm was reduced significantly. An isosbestic point was identified at 293 nm, indicating the formation of the PFOA and NI-TPY complex. The binding constant (a) of NI-TPY with PFOA was calculated as 7.1 104M-1using the Benesi-Hildebrand (B-H plot) linear graph drawn between Amax-Ao / Ax-Ao and 1 / [PFOA] shown in FIG. 62B.

[0655] Selectivity and Interference Studies for NI-TPY

[0656] As shown by Figure 63, the emission spectrum of 2 pM NI-TPY in acetonitrile showed significant changes in fluorescence in the presence of with all strong acids including HCI, H2SO4, PFHxA, PFOA, PFNA, PFUA, PFUDA, TFA, and p-TSA.

[0657] Competitive experiments were conducted to examine the selectivity of NI-TPY for PFOA. The fluorescence intensity of 2 pM NI-TPY in CH3CN in the presence of 100 pM PFOA and 200 pM of PFAS and metals (FIG. 64). As illustrated in FIG. 64, no significant changes in fluorescence emission intensity were observed for any of the PFAS or metals.

[0658] Effect of Response Time of NI-TPY

[0659] A fluorescence experiment in a CH3CN solvent system was performed to investigate the effect of response time of 2 pM NI-TPY response to 100 pM PFOA. As shown by FIG. 65, a 026389-0053-W001 significant decrease in fluorescent intensity was immediately observed. The fluorescence intensity stabilized within 1 minute and remained stable for 20 minutes. These results demonstrate NI-TPY fast fluorometric response to PFOA.

[0660] Effect of Ionic Strength on NI-TPY Binding to PFOA

[0661] A low ionic strength (0-30 mM NaCI) quenched approximately 12% of the fluorescence induced by NI-TPY (FIG. 66). There was no considerable change in fluorescence intensity for the NI-TPY+PFOA complex. These results indicate that NaCI may not influence the fluorescence behavior of NI-TPY and its PFOA complex.

[0662] Thermal Stability of NI-TPY + PFOA Complex

[0663] As shown by FIG. 67, the fluorescence intensity of NI-TPY was stable at increased temperatures, demonstrating that free sensor probes are stable at high temperatures. However, when the temperature of the NI-TPY+PFOA complex exceeds 30 °C, the fluorescence intensity increased with temperature. Based upon these results, NI-TPY+PFOA complexes may not stable at high temperatures. As a result, 25 °C was selected as the temperature for experiments.

[0664] Binding Stoichiometry of NI-IPY for PFOA

[0665] Job’s plot was used to determine the binding stoichiometry of NI-TPY+PFOA complex, as shown in FIG. 68. Job’s plot showed that NI-TPY and PFOA forms a 1 :1 complex.

[0666] Example 8

[0667] Detection of PFOA with NA-3N Immobilization on an Example Surface

[0668] Synthesis

[0669] Abbreviations

[0670] EPTES is 3-(2,3-epoxypropoxy) propyltriethoxysilane;

[0671] NMP is N-methylpyrrolidone;

[0672] DCM is dichloromethane; mmole is millimole;

[0673] M is molar; mL is milliliter; eq. or equiv. is equivalents; min or min. is minute(s); 026389-0053-W001 h or hr. is hour(s); and rt, RT, or r.t. is room temperature.

[0674] Example Synthesis of PEG-Functionalized Linker with Fluorophore

[0675] 5pSG-PEG44: Flash silica gel (Surface area: 480 m2 / g; Particle size: 60 pm) was coated with 3- (2,3-epoxypropoxy) propyltriethoxysilane (EPTES). The coating reaction was conducted at 100 °C in toluene for 24 hours. Afterwards, toluene and ethanol were used to wash the coated particles. Following washing, the dried silica was transferred to a carbonate buffer containing polyethylene glycol)diamine (PEG) (50 mL, 0.1 M, pH 9.5). The mixture was stirred overnight at 50°C. The resulting product was washed with the same carbonate buffer and stored at 4 °C dry.

[0676] 5pSG-PEG44-NI-3N: 5pSG-PEG44 (5 equiv., 20 equiv., or 50 equiv.) were dispersed in / - methylpyrrolidone (NMP) (3 mL) under N2atmosphere, and NA-3N (1 equivalent) was subsequently added. The mixture was stirred and refluxed for 3 hours at 100 °C. After cooling the reaction solution to room temperature, the modified particles were filtered and washed several times with DMF followed by ethanol. The wash product was dried overnight in a vacuum oven at 60 °C. Reactions produced materials with a 5pSG-PEG44 to NA-3N ratios of 50: 1 , 20: 1 , and 5: 1.

[0677] Confirmation of PEG Attachment to Fluorophore

[0678] Confirmation of PEG attachment was achieved by using Fluorescamine (FA) (0-130 pM), a fluorescence activating fluorophore. Fluorescamine was reacted with the primary amine groups of Si@EPTMS+PEG and switched the non-fluorescent fluorescamine into a strong blue, fluorescent product (FIG. 69-71).

[0679] Spectroscopic Properties of 5pSG-PEG44-NI-3N for the Detection of PFOA

[0680] Using 5pSG-PEG44-NA-3N (porous particle) fluorescence titrations were performed with different perfluorooctanoic acid (PFOA) concentrations. When PFOA (0-700 pM) was gradually added to 5pSG-PEG44-NI-3N in acetonitrile, the emission maxima at 524 nm increased 026389-0053-W001 significantly, indicating the formation of a complex between PFOA and 5pSG-PEG44-NI-3N. Consequently, PFOA emission color changed from blue to green under UV-lamp irradiation. These results indicate that the pyridine moiety of NA-3N accepts a proton from PFOA and forms pyridinium ions, which was responsible for fluorescent activation of 5pSG-PEG44-NA-3N (5:1 , 20:1, and 50:1) (FIG. 72, 75, and 78). A calibration plot was also drawn between fluorescence intensity and PFOA concentration (FIG. 73, 76, and 79). Accordingly, the limit of detection (LOD) of PFOA was 26 pM (10.7 ppm), 31 pM (13.0 ppm), and 34.6 pM (14.3 ppm) for 5:1 , 20:1 , and 50:1 of 5pSG-PEG44-NA-3N (FIG. 74, 77 and 80), respectively.

[0681] The emission response of non-porous 5pSG-PEG44-NA-3N (3 pm, non-porous) for detection of PFOA in acetonitrile was in agreement with porous 5pSG-PEG44-NA-3N (FIG. 81).

[0682] The limit of detection (LOD) of non-porous particle for PFOA was 35 pM (15 ppm) (FIG. 82).

Claims

026389-0053-W001CLAIMSWhat is claimed:

1. A method for detecting one or more per- or poly-fluoroalkyl substances (PFAS) in a sample, the method comprising: contacting the sample with a detection reagent comprising a compound of formula (I), or a salt thereof:whereinR1is hydrogen, halogen, cyano, Ci-ealkyl, Ci-ehaloalkyl, -OR1a, -SR1a, -CC>2R1a, -C(O)R1a, -SO2R1a, -N(R1a)2, -CO2N(R1a)2, or -NO2, where:R1a, at each occurrence, is independently, hydrogen, Ci-ealkyl, or Ci-2haloalkyl;R2, at each occurrence, is independently hydrogen, halogen Ci-ealkyl, Ci-ehaloalkyl, -OR1b, -SR1b, -CO2R1b, -C(O)R1b, -SO2R1b, -N(R1b)2, -CO2N(R1 b)2, or -NO2, where:R1b, at each occurrence, is independently, hydrogen, Ci-ealkyl, or Ci-2haloalkyl;G1, at each occurrence, is independently a 6- to 12-membered aryl or a 5- to 6-membered heteroaryl, wherein G1is optionally substituted with 1-5 substituents independently selected from the group consisting of Ci-4alkyl, Ci-2haloalkyl, halogen, cyano, -OCi-4alkyl, and -OCi-2haloalkyl;G2, at each occurrence, is independently a 5- to 12-membered heteroarene or -N(GX)-, where:Gx, at each occurrence, is a 6- to 12-membered aryl optionally substituted with 1-5 substituents independently selected from the group consisting of Ci-4alkyl, Ci- 2haloalkyl, halogen, cyano, -OCi-4alkyl, and -OCi-2haloalkyl;G3is a 5- to 6-membered heteroaryl or a 6- to 12-membered aryl, wherein G3is optionally substituted with 1-5 substituents independently selected from the group consisting of Ci-4alkyl, Ci-2haloalkyl, halogen, cyano, -OCi-4alkyl, and -OCi-2haloalkyl; m is 0-1 ; and n is 0-2; and026389-0053-W001 quantifying a change in fluorescence intensity of the detection reagent being contacted with the sample, relative to a baseline fluorescence intensity of the detection reagent prior to contact with the sample.The method of claim 1 , wherein G3is the optionally substituted 5- to 6-membered heteroaryl.

3. The method of claim 2, wherein the 5- to 6-membered heteroaryl is pyridinyl.

4. The method of claim 1 , wherein R1and R2are each hydrogen.

5. The method of claim 1 , wherein the compound of formula (I) is a compound of formula(la):

6. The method of claim 1 , wherein m is 0 and n is 0.

7. The method of claim 1 , wherein m is 1 and n is 1.

8. The method of claim 1 , wherein G2is the optionally substituted 5- to 12-membered heteroarene.

9. The method of claim 8, wherein the 5- to 12-membered heteroarene10. The method of claim 1 , wherein G2is -N(Gx)-.

11. The method of claim 10, wherein Gxis the optionally substituted 6- to 12-membered aryl.026389-0053-W00112. The method of claim 1, wherein G1is the optionally substituted 6- to 12-membered aryl.

13. The method of claim 12, wherein the 6- to 12-membered aryl is phenyl.

14. The method of claim 1 , wherein G1is the optionally substituted 5- to 6-membered heteroaryl.

15. The method of claim 14, wherein the 5- to 6-membered heteroaryl is pyridinyl.

16. The method of claim 1 , wherein the compound of formula (I) is selected from the group consisting of:

17. The method of claim 1 , wherein the compound of formula (I), or a salt thereof, is noncovalently adhered to a surface.

18. The method of claim 17, wherein the surface comprises a cellulose, a silica, a polymer, a resin, or a combination thereof.

19. The method of claim 1, wherein the compound of formula (I), or a salt thereof, is dissolved in a solvent.

20. The method of claim 1 , wherein the change in fluorescence intensity is quantified at an emission wavelength of 425-500 nm.

21. The method of claim 1 , wherein the detection reagent is excited at an excitation wavelength of 275-400 nm.

22. The method of claim 1 , wherein the detection reagent has a limit of detection of about 1- 50 ppb.026389-0053-W00123. A method for detecting one or more per- or poly-fluoroalkyl substances (PFAS) in a sample, the method comprising: contacting the sample with a detection reagent comprising a compound of formula (II), or a salt thereof:whereinX is O and n is 0, or X is N and n is 1 ;R10is hydrogen, halogen, cyano, Ci-ealkyl, O-ehaloalkyl, or -OR10a, -SR10a, -CC>2R10a, -C(O)R10a, -SO2R10a, -N(R10a)2, -CO2N(R10a)2, or -NO2, where:R10a, at each occurrence, is independently, hydrogen, Ci-ealkyl, or Ci-2haloalkyl;R20is hydrogen, halogen Ci-ealkyl, Ci-ehaloalkyl, or -OR10b, -SR10b, -CO2R10b, -C(O)R10b, -SO2R10b, -N(R10b)2, -CO2N(R10b)2, or -NO2, wherein:R10b, at each occurrence, is independently, hydrogen, Ci-ealkyl, or Ci.2haloalkyl;R30, at each occurrence is hydrogen or Ci-salkyl;G10is a 4- to 12-membered heterocyclylene, a Cs-iocarbocyclylene, a 6- to 12-membered arene, or a 5- to 12-membered heteroarene, wherein G10is optionally substituted with 1-6 substituents, each independently selected from the group consisting of Ci-4alkyl, Ci-2haloalkyl, halogen, cyano, -OCi-4alkyl, and -OCi-2haloalkyl;G20, at each occurrence, is independently a 5- to 12-membered heteroarene, wherein G20is optionally substituted with 1-5 substituents, each independently selected from the group consisting of Ci.4alkyl, Ci.2haloalkyl, halogen, cyano, -OCi.4alkyl, and -OCi.2haloalkyl;G30, at each occurrence, is independently a 5- to 12-membered heteroaryl or a 6- to 12- membered aryl, wherein G30is optionally substituted with 1-5 substituents, each independently selected from the group consisting of Ci-4alkyl, Ci.2haloalkyl, halogen, cyano, -OC- alkyl, and -OCi-2haloalkyl; w is 0-1 ; x is 1-2; n is 0-1 ; and026389-0053-W001 quantifying a change in fluorescence intensity of the detection reagent being contacted with the sample, relative to a baseline fluorescence intensity of the detection reagent prior to being contacted with the sample.

24. The method of claim 23, wherein G10is the 4- to 12-membered heterocyclylene or the 6- to 12-membered arene.

25. The method of claim 24, wherein the 4- to 12-membered heterocyclylene is26. The method of claim 24, wherein the 6- to 12-membered arene is phenylene.

27. The method of claim 23, wherein R10and R20are each hydrogen.

28. The method of claim 23, wherein G30, at each occurrence, is the 5- to 12-membered heteroaryl.

29. The method of claim 28, wherein the 5- to 12-membered heteroaryl is pyridinyl or pyrimidinyl.

30. The method of claim 23, wherein G30, at each occurrence, is the 6- to 12-membered aryl.

31. The method of claim 30, wherein the 6- to 12-membered aryl is phenyl.

32. The method of claim 23, wherein w is 0.

33. The method of claim 23, wherein w is 1.

34. The method of claim 33, wherein G20is the 5- to 12-membered heteroarene.026389-0053-W00135. The method of claim 34, wherein, the 5- to 12-membered heteroarene is36. The method of claim 23, wherein X is 0 and n is 0.

37. The method of claim 23, wherein X is N and n is 1.

38. The method of claim 37, wherein R30is Ci-sal kyl.

39. The method of claim 23, wherein the compound of formula (II), or a salt thereof, is selected from the group consisting of:026389-0053-W00140. The method of claim 23, wherein the compound of formula (II) is noncovalently adhered or covalently attached to a surface.

41. The method of claim 40, wherein the surface comprises a cellulose, a silica, a polymer, a resin, or a combination thereof.

42. The method of claim 23, wherein the compound of formula (II), or a salt thereof, is dissolved in a solvent.

43. The method of claim 23, wherein the detection reagent is excited at an excitation wavelength of 400 nm.

44. The method of claim 23, wherein the change in fluorescence intensity is quantified at an emission wavelength of 400-475 nm.

45. The method of claim 23, wherein the detection reagent has a limit of detection of about 1 to about 50 ppb.

46. A method for detecting one or more per- or poly-fluoroalkyl substances (PFAS) in a sample, the method comprising:026389-0053-W001 contacting the sample with a detection reagent comprising a compound of formula (III), or a salt thereof,whereinZ is a solid support;L10is Ci-walkylene; Ci-ioalkylene-C(O)-, or -O-Ci-ioalkylene-C(O)-;, where: m is 20-60;R10is hydrogen, halogen, cyano, Ci-ealkyl, C-i-ehaloalkyl, -OR10a, -SR10a, -CC>210a, -C(O)R10a, -SO2R10a, -N(R10a)2, -CO2N(R10a)2, or -NO2, where:R10a, at each occurrence, is independently, hydrogen, Ci-ealkyl, or Ci-2haloalkyl;R20is hydrogen, halogen Ci-ealkyl, Ci-ehaloalkyl, -OR10b, -SR10b, -CO2R10b, -C(O)R10b, -SO2R10b, -N(R10b)2, -CO2N(R10b)2, or -N02, where:R10b, at each occurrence, is independently, hydrogen, Ci-ealkyl, or Ci.2haloalkyl;G10is a 4- to 12-membered heterocyclylene, a Cs- carbocyclylene, a 6- to 12-membered, aryl, or a 5- to 12-membered heteroaryl, wherein G10is optionally substituted with 1-6 substituents independently selected from the group consisting of Ci^alkyl, Ci- 2haloalkyl, halogen, cyano, -OCi-4alkyl, and -OCi-2haloalkyl;G20, at each occurrence, is independently a 5- to 12-membered heteroaryl, wherein G20is optionally substituted with 1-5 substituents independently selected from the group consisting of Ci.4alkyl, Ci.2haloalkyl, halogen, cyano, -OCi.4alkyl, and -OCi. 2haloalkyl;G30, at each occurrence, is independently a 5- to 12-membered heteroaryl or a 6- to 12- membered aryl, wherein G30is optionally substituted with 1-5 substituents independently selected from the group consisting of Ci.4alkyl, Ci-2haloalkyl, halogen, cyano, -OCi.4alkyl, and -OCi.2haloalkyl; n is 0-1 ; w is 0-1 ; and x is 1-2; and026389-0053-W001 quantifying a change in fluorescence intensity of the detection reagent while being contacted with the sample, relative to a baseline fluorescence intensity of the detection reagent prior to being contacted with the sample.

47. The method of claim 46, wherein G10is the 4- to 12-membered heterocyclylene or the 6- to 12-membered arene.

48. The method of claim 47, wherein the 4- to 12-membered heterocyclylene49. The method of claim 47, wherein the 6- to 12-membered aryl is phenyl.

50. The method of claim 46, wherein R10and R20are each hydrogen.

51. The method of claim 46, wherein G30, at each occurrence, is the 5- to 12-membered heteroaryl.

52. The method of claim 51 , wherein G30, at each occurrence, is independently pyridinyl or pyrimidinyl.

53. The method of claim 46, wherein w is 0.

54. The method of claim 46, wherein w is 1.

55. The method of claim 46, wherein G20is the 5- to 12-membered heteroarene.

56. The method of claim 55, wherein the 5- to 12-membered heteroarene i026389-0053-W00157. The method of claim 46, wherein the solid support comprises a silica, a polymer, a resin, or a combination thereof.

58. The method of claim 46, wherein L10is C2.4alkylene.

59. The method of claim 46, wherein n is 1 and m is 30-50.

60. The method of claim 46, wherein the compound of formula (III), or a salt thereof, is the reaction product of an amine-functionalized solid support and a compound of formula (pllla):(pllla).

61. The method of claim 60, wherein the amine-functionalized solid support comprises Ci. i0alkylene-NH2chemically coupled to the solid support, and wherein L10comprises Ci. iOalkylene.

62. The method of claim 60, wherein the amine-functionalized solid support is amine- functionalized silica.

63. The method of claim 46, wherein the compound of formula (III), or a salt thereof, is dissolved in a solvent.

64. The method of claim 46, wherein the detection reagent is excited at an excitation wavelength of 400 nm.

65. The method of claim 46, wherein the change in fluorescence intensity is quantified at an emission wavelength of 400-475 nm.

66. The method of claim 46, wherein the detection reagent has a limit of detection of about 1 to about 50 ppb.026389-0053-W00167 A method for detecting one or more per- or poly-fluoroalkyl substances (PFAS) in a sample, the method comprising: contacting the sample with a detection reagent comprising a compound of formula (IV), or a salt thereof:whereinX100is S, N(R110), or O;L100is Co-3alkylene-C=C-Co-3alkylene or Ci-ealkylene;R100is -N(GY)2, -N(R100a)2, -O-(GY)2, -O-(R100a), or -O-(GY) where:GY, at each occurrence, is independently a 6- to 12-membered aryl or a 5- to 12- membered heteroaryl, wherein GYis optionally substituted with 1-5 substituents independently selected from the group consisting of hydrogen, halogen, Ci-4alkyl , Ci-2haloalkyl, cyano, -OCi-4alkyl, or -OCi-2haloalkyl; andR100a, at each occurrence, is independently hydrogen or Ci-ealkyl;R110is hydrogen, Ci-eal kyl, or Ci-2haloalkyl;R120is hydrogen, halogen, Ci-4alkyl, Ci.2haloalkyl, cyano, -OC^alkyl, or-OCi-2haloalkyl; andR130is hydrogen, halogen, Ci-4alkyl, Ci-2haloalkyl, cyano, -OCi^alkyl, or-OCi-2haloalkyl; andX" is a counterion; and quantifying a change in fluorescence intensity of the detection reagent being contacted with the sample relative to a baseline fluorescence intensity of the detection reagent prior to being contacted with the sample.

68. The method of claim 67, wherein X100is S.

69. The method of claim 67, wherein R120and R130, at each occurrence, are each independently hydrogen.

70. The method of claim 67, wherein the compound of formula (IV) is a compound of formula (IVa):026389-0053-W00171. The method of claim 67, wherein L100is Co-3alkylene-C=C-Co-3alkylene.

72. The method of claim 67, wherein L100is Coalkylene-C=C-Coalkylene.

73. The method of claim 67, wherein R110is Ci-ealkyl .

74. The method of claim 67, wherein R100is -N(GY)2 or -N(R100a)2.

75. The method of claim 74, wherein GY, at each occurrence, is the 6- to 12-membered aryl.

76. The method of claim 75, wherein the 6- to 12-membered aryl is phenyl.

77. The method of claim 67, wherein R100a, at each occurrence, is Ci^alkyl.

78. The compound of claim 67, wherein the compound of formula (IV) is selected from the group consisting of: / 9. The method of claim 78, wherein the compound of formula (IV), or a salt thereof, is noncovalently adhered or covalently attached to a surface.

80. The method of claim 79, wherein the surface comprises a cellulose, a silica, a polymer, a resin, or a combination thereof.

81. The method of claim 67, wherein the compound of formula (IV), or a salt thereof, is dissolved in a solvent.026389-0053-W00182 The method of claim 67, wherein the counterion is iodide, chloride, or bromide.

83. The method of claim 67, wherein the detection reagent is excited at an excitation wavelength of 520 nm.

84. The method of claim 67, wherein the change in fluorescence intensity is quantified at an emission wavelength of 577-700 nm.

85. The method of claim 67, wherein the detection reagent has a limit of detection of about 1 to about 50 ppb.

86. A method for detecting one or more per- or poly-fluoroalkyl substances (PFAS) in a sample, the method comprising: contacting the sample with a detection reagent comprising a compound of formula (V), or a salt thereof:whereinR200is halogen, hydrogen, Ci^alkyl, Ci-zhaloalkyl, or -OCi-2haloalkyl;G200is a 4- to 12-membered heterocyclylene or a Ca- carbocyclylene, wherein G200is optionally substituted with 1-4 substituents, each independently selected from the group consisting of Ci-4alkyl, Ci-2haloalkyl, halogen, cyano, -OCi.4alkyl, and -OCi. 2haloalkyl;G210is a 5- to 12-membered heteroaryl or a 6- to 12-membered aryl, wherein G210is optionally substituted with 1-4 substituents, each independently selected from the group consisting of Ci.4alkyl, Ci-2haloalkyl, halogen, cyano, -OCi.4alkyl, and -OCi. 2haloalkyl; and quantifying a change in fluorescence intensity of the detection reagent while being contacted with the sample relative to a baseline fluorescence intensity of the detection reagent prior to being contacted with the sample.026389-0053-W00187. The method of claim 86, wherein R200, at each occurrence, is hydrogen or halogen.

88. The method of claim 86, wherein G200is a 4- to 12-membered heterocyclylene.

89. The method of claim 88, wherein the 4- to 12-membered heterocyclylene is90. The method of claim 86, wherein G210is a 5- to 12-membered heteroaryl.

91. The method of claim 90, wherein the 5- to 12-membered heteroaryl is92. The compound of claim 86, wherein the compound of formula (V) is:

93. The method of claim 86, wherein the compound of formula (V) is noncovalently adhered or covalently attached to a surface.

94. The method of claim 86, wherein the surface comprises a cellulose, a silica, a polymer, a resin, or a combination thereof.

95. The method of claim 86, wherein the compound of formula (V), or a salt thereof, is dissolved in a solvent.

96. The method of claim 86, wherein the detection reagent is excited at an excitation wavelength of 325-425 nm.

97. The method of claim 86, wherein the change in fluorescence intensity is quantified at an emission wavelength of 575-650 nm.026389-0053-W00198. The method of claim 86, wherein the detection reagent has a limit of detection of about 1 to about 50 ppb.

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