Solid-state assay for detection of PFAS compounds

Abstract

Methods and kits are provided for an assay for detecting and quantifying a PFAS compound or a mixture of PFAS compounds in a sample. The method includes the steps of providing a solid cyclodextrin polymer adsorbed on or linked to a substrate, contacting an indicator molecule with the solid cyclodextrin polymer or the cyclodextrin adsorbed on or covalently linked to the substrate to form a complex, contacting the complex with the sample and measuring a parameter of the indicator molecule. The parameter is correlated with displacement of the indicator molecule from the solid polymerized cyclodextrin or the cyclodextrin adsorbed on or covalently linked to the substrate upon binding of the PFAS compound to the cyclodextrin, thereby indicating presence and quantity of the PFAS compound or the mixture of PFAS compounds in the sample.

Description

Solid-State Assay for Detection of PFAS CompoundsCROSS-REFERENCE TO RELATED APPLICATION

[0001] The present application claims the benefit of priority to US Patent Application No. 63 / 713,190, filed on October 29, 2024, which is incorporated herein by reference in its entirety.TECHNICAL FIELD

[0002] The invention relates to quantification of perfluorinated and polyfluorinated substances which are commonly known as PFAS compounds. Assays and kits for this purpose are described.BACKGROUND

[0003] Polyfluoroalkyl and perfluoroalkyl substances (PFAS) make up a large group of persistent anthropogenic chemicals used in industrial processes and commercial products over the past 60 years. Widespread use and extreme resistance to degradation have resulted in the ubiquitous presence of these compounds in the environment. The 2011— 2012 U.S. National Health and Nutrition Examination Survey reported detectable serum PFAS concentrations in virtually all individuals (97%). Human PFAS exposure has been linked to cancer, elevated cholesterol, obesity, immune suppression, and endocrine disruption. Health concerns have prompted manufacturers in Europe and North America to phase out production of some long-chain PFAS. Limited available data suggest that widespread exposure to replacement (short chain) PFASs may also adversely affect human health. Human PFAS exposure includes dietary sources, household dust, air, and drinking water. Exposure from drinking water is a serious concern because of the high aqueous solubility of many PFAS compounds. Relatively low PFAS concentrations can lead to elevated exposures in the general population. Elevated PFAS concentrations in U.S. drinking water have been reported in numerous regions, especially near industrial sites that produce or use them (Hu et al., Environ. Sei. Technol. Lett. 2016, 3, 10, 344- 350, incorporated herein by reference in its entirety).

[0004] Current regulatory methods and technologies for detecting PFAS compounds can reliably quantify about 50 specific PFAS, but these technologies are unable to detect or quantify the thousands of other PFAS known to exist. These technologies typically use lab-scale analytical chemistry techniques to separate, identify and quantify PFAS substances in different water matrices (landfill leachate, industrial waste, groundwater, drinking water, and surface water). Less common approaches do not separate components prior to detection. Due to the abundant nature of PFAS and its derivatives with their uniquely specific chemical structures and properties, different methods have been used based on the species of PFAS tested. All of the different EPA methods use mass spectrometry and differ by the way the molecules are separated: liquid chromatography (LC-MS / MS), gas chromatography (GC-MS), or High-Performance liquid chromatography (HPLC) coupled with mass spectrometry. The most notable method, used in EPA 1633 and EPA 537, is LC-MS / MS (liquid chromatography with tandem mass spectrometry). By applying standards, the PFAS molecules can later be quantified. Typical detection limits for the LC-MS / MS method, combined with sample preparation using SPE cartridges, range from parts-per-billion (ppb) to low parts-per-trillion (ppt), depending on the specific PFAS compound and instrument sensitivity.

[0005] In the last 5-10 years, different processes have been developed to bring PFAS testing into the field where it is most desirable to obtain rapid results in detection and quantification of PFAS compounds. The most notable example is Starfire's Centurion® system (Starfire Industries, LLC, Champaign, IL, USA), which despite its heavy weight (350 kg), can facilitate detection at parts-per-trillion levels. The advantage of this system resides in its speed, as it would provide results within minutes. Recently, MITRE (Bedford, MA, USA), and other academic researchers have investigated development of PFAS sensors based on molecularly imprinted polymers (MIPs) (Lu et al. Sensors and Actuators B, 2022, 352, 131055; Tasfaout et al., Taianta, 2023, 258, 124434).

[0006] Some of these Ml Ps-based sensors have shown a very low limit of detection for specific molecules such as GenX (Glasscot et al., Crit. Rev. Anal. Chem., 2022, 52, 1258, having a limit of detection of 0.083 ppt), but no application of these sensors in the field has been disclosed to date. Metal-organic frameworks (MOFs) are used to bind PFAS molecules such as PFOS which are proposed to be used to create MOF-based sensors, using microfluidics and electrochemical detection (Cheng et al., ACS Appl. Mater.Interfaces, 2020, 12, 10503). An overview of the different methods used to detect PFAS compounds is described in Menger et al., Chem Eng. J. 2021, 417, 128133.

[0007] There is a need for improved technologies for detection of PFAS compounds, particularly detector systems which can be operated outside of laboratories.SUMMARY

[0008] According to one embodiment, there is provided a solid-state assay method for detecting and quantifying a PFAS compound or a mixture of PFAS compounds in a sample, the method comprises the steps of: providing a solid cyclodextrin polymer or a cyclodextrin polymer adsorbed on or linked to a substrate; contacting an indicator molecule with the solid cyclodextrin polymer or the cyclodextrin polymer adsorbed on or covalently linked to the substrate to form a complex; and contacting the complex with the sample and measuring a parameter of the indicator molecule, wherein the parameter is correlated with an extent of displacement of the indicator molecule from the solid cyclodextrin polymer or the cyclodextrin polymer adsorbed on or covalently linked to the substrate upon binding of the PFAS compound to the solid cyclodextrin polymer or the cyclodextrin polymer adsorbed on or covalently linked to the substrate, thereby indicating presence and quantity of the PFAS compound or the mixture of PFAS compounds in the sample.

[0009] The solid cyclodextrin polymer or the cyclodextrin polymer adsorbed on or covalently linked to the substrate may be formed from p-cyclodextrin.

[0010] In some embodiments, the p-cyclodextrin comprises a non-hydrogen substituent. The non-hydrogen substituent may be selected from the group consisting of: hydroxypropyl, acetyl, azido, hydroxyamino, quaternary amine and amino.

[0011] In some embodiments, the p-cyclodextrin polymer comprises a linker. The linker may be a cyclic ether or a hydrophobic linker. The cyclic ether may be epichlorohydrin or a substituted or unsubstituted oxacyclopropane, oxacyclobutane, oxacyclopentane, oxacyclohexane or furan. The linker may be a hydrophilic linker. The hydrophilic linker may comprise a carbamate crosslinked by diisocyanates, a carbonate, an ester or an ether.

[0012] In some embodiments, the indicator molecule is a colorimetric dye molecule or a fluorescent dye molecule. The fluorescent dye molecule may be a cyanine dye, a xanthene dye, a pyrene dye, or a naphthalene dye. The naphthalene dye may be 8- anilinonaphthalene-1 -sulfonic acid (ANS) or bis-8-anilinonaphthalene-1 -sulfonic acid (bis- ANS). The colorimetric dye molecule may be Acid Blue 120 or Acid Black 24.

[0013] In some embodiments, the substrate is paper, cellulose, nitrocellulose, glass, quantum dot, or metal nanoparticles. The metal nanoparticles may be gold, silver or iron.

[0014] In some embodiments, the substrate is paper, cellulose or nitrocellulose and is provided as a disposable stick or is contained within a housing for a lateral flow assay.

[0015] In some embodiments, the method further comprises providing a scavenger molecule for sequestering one or more interferents present in the sample by treatment of the sample separately prior to contacting the indicator molecule with the solid cyclodextrin polymer. The interferents may be compounds having a hydrocarbon chain of a length greater than 6 carbons.

[0016] In some embodiments, the interferents may be hydrocarbons or surfactants.

[0017] In some embodiments, the scavenger molecule is solid polymeric a-cyclodextrin adsorbed on or linked to a second substrate or soluble monomeric a-cyclodextrin.

[0018] In some embodiments, the soluble monomeric a-cyclodextrin is adsorbed on or linked to a second substrate. The second substrate may be provided within a disposable syringe filter.

[0019] According to another embodiment, there is provided a kit for preparing an assay mixture for detecting and quantifying one or more PFAS compounds in a sample, the kit comprising a mixture of assay components, the mixture comprising: a solid cyclodextrin polymer or a cyclodextrin polymer adsorbed on or linked to a substrate and an indicator molecule bound to the solid cyclodextrin polymer or the cyclodextrin polymer adsorbed or covalently linked to a substrate which is displaced by a PFAS compound.

[0020] In some embodiments of the kit, the cyclodextrin polymer is p-cyclodextrin polymer. The p-cyclodextrin polymer may comprise a non-hydrogen substituent. The nonhydrogen substituent may be selected from the group consisting of: 2- hydroxy propyl, acetyl, amino and quaternary amine.

[0021] In some embodiments of the kit, the mixture further comprises a scavenger molecule for scavenging interferents in the sample. The scavenger molecule may be solid polymeric a-cyclodextrin adsorbed on or linked to a second substrate or may be soluble monomeric a-cyclodextrin adsorbed on or linked to a second substrate.

[0022] In some embodiments of the kit, the second substrate is provided within a filter.

[0023] According to another embodiment, there is provided an inline process sampling sensor system for detection of PFAS in a process flow, the sampling sensor system comprising: a branch conduit extending from a process flow conduit, the branch conduit configured to deliver a sample of the process flow to a sensor, the sensor comprising: a housing containing a solid cyclodextrin polymer or a cyclodextrin polymer adsorbed on or covalently linked to a substrate with an indicator molecule bound to the cyclodextrin polymer; and a detector for measuring a parameter of the indicator molecule, wherein the parameter is correlated with an extent of displacement of the indicator molecule from the solid cyclodextrin polymer or the cyclodextrin polymer adsorbed on or covalently linked to the substrate upon binding of the PFAS compound to the cyclodextrin, thereby indicating presence and quantity of the PFAS in the sample.

[0024] In some embodiments of the inline process sampling sensor system, the detector is a fluorescence detector for detecting a fluorescent indicator molecule or a spectrophotometer for detecting absorbance of a colorimetric indicator molecule.BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In the examples section below, one or more embodiments of the present technology are described in relation to the attached figures. These embodiments are intended to provide a better understanding of the invention, how the invention may be put into practice, and to demonstrate certain advantages of the invention.Figure 1A, Scheme A illustrates binding of an indicator molecule (IND) to polymeric p-cyclodextrin (p-p-CD) to provide a complex which provides elevated fluorescence relative to the indicator molecule in aqueous solution. Scheme B illustrates displacement of the indicator molecule (IND) from the complex by a PFAS molecule.Figure 1B, Scheme C illustrates competitive binding of an interferent (INT) with the indicator molecule (IND) for binding to p-p-CD, resulting in reduced fluorescence which affects the indicator displacement assay. Scheme D illustrates inclusion of monomeric a- cyclodextrin (m-a-CD) as a scavenger to sequester the interferent (INT) without affecting binding of the indicator molecule (IND) to p-p- CD), thereby rescuing the elevated fluorescence required for an accurate indicator displacement assay in Scheme D.Figure 2A is a plot of concentration of solid p-p-cyclodextrin (s-p-p-CD) beads vs. relative fluorescence (excitation: 405 nm and emission 510 nm) for a series of indicator molecules including bis-ANS, acid blue 120 (AB 120), and acid black 24 (AB 24) in an experiment performed to investigate binding of the indicator molecules to the s-p-p-CD beads with binding indicated by increased relative fluorescence.Figure 2B is a plot of concentration of s-p-p-CD beads vs. relative fluorescence (excitation: 405 nm and emission 510 nm) for bis-ANS alone in an experiment performed to investigate binding of bis-ANS to the s-p-p-CD beads. The data is normalized to absorbance at 800 nm to subtract background absorbance from the s-p-p-CD beads.Figure 3A is a plot of concentration of s-p-p-CD beads vs. absorbance at 675 nm for a series of indicator molecules including bis-ANS, acid blue 120 (AB 120), and acid black 24 (AB 24) in an experiment performed to investigate binding of the indicator molecules to the s-p-p-CD beads with binding indicated by absorbance.Figure 3B is a plot of concentration of s-p-p-CD beads vs. absorbance at 675 nm for a series of indicator molecules including bis-ANS, acid blue 120 (AB 120), and acid black 24 (AB 24) with absorbance at 800 nm subtracted in an experiment performed to investigate binding of the indicator molecules to the s-p-p-CD beads with binding indicated by absorbance. The data is normalized to absorbance at 800 nm to subtract background absorbance from the s-p-p-CD beads.Figure 4A is a plot of concentration of bis-ANS vs. relative fluorescence (excitation: 405 nm and emission 510 nm) in an experiment performed to investigate binding of bis-ANS to a constant concentration of 10 mg / mL of s-p-p-CD beads. The series indicating “dye Ctrl” is a control series measured in the absence of the s-p-p-CD beads.Figure 4B is a plot of concentration of bis-ANS vs. relative fluorescence (excitation: 405 nm and emission 510 nm) in an experiment performed to investigate binding of bis-ANS to a constant concentration of 10 mg / mL of s-p-p- CD beads with background fluorescence of the control series subtracted.Figure 5A is a plot of concentration of colorimetric indicator molecules (labelled as “dyes”) vs. the change in absorbance at 675 nm measured upon binding of the indicator molecules to a constant concentration of 10 mg / mL of s-p-p-CD beads. The data series labelled “Ctrl” are a control series measured in the absence of the s-p-p-CD beads.Figure 5B is a plot of concentration of colorimetric indicator molecules (labelled as “dyes”) vs. the change in absorbance at 675 nm measured upon binding of the indicator molecules to a constant concentration of 10 mg / mL of s-p-p-CD beads with background absorbance of the control series subtracted.Figure 6 is a plot of relative fluorescence (excitation: 405 nm and emission 510 nm) vs. PFOA concentration at two different concentrations of bis-ANS in an experiment performed to determine displacement of bis-ANS from 2.5 mg / mL s-p- P-CD beads by PFOA.DETAILED DESCRIPTIONIntroduction and Rationale

[0026] This technology relates to the detection of PFAS compounds within environmental samples. PFAS compounds, one of the classes of “forever chemicals,” are toxic and recalcitrant. These compounds are characterized by the presence of fluorine atoms along a carbon backbone. They have many commercial uses such as waterproofing, fire suppression, manufacturing, and inclusion in various products as stability additives. They enter the environment from multiple sources, including through the breakdown of PFAS- containing products over time (i.e., in landfills), through runoff from manufacturing processes, and through the cleaning after fire suppression events at airports, military bases, and ports.

[0027] The detection of PFAS compounds in the laboratory is cumbersome and labour intensive. Detection in the field at the location of contamination is currently non-existent. Conventional monitoring methods used in analytical labs rely on LC-MS / MS or combustion ion chromatography. These are expensive, time-consuming, require highly trained personnel, and rely on significant infrastructure investments. Current turnaround times for PFAS lab testing runs between 3 and 16 weeks depending on the exact test performed, and costs hundreds, sometimes thousands, of dollars per test. Rush testing is available with results available in one week, but this is very expensive. While testing is in progress, companies often attempt to remediate PFAS contamination blindly and are required to pay for onsite personnel and to store large volumes of water contaminated with PFAS that may or may not be at a concentration where it can be discharged.

[0028] The technology described herein uses advances in sample preparation and an assay developed using a cyclodextrin molecule and an indicator molecule in a field- deployable assay system in combination with a raw sample concentrating process which can detect PFAS molecules at low concentrations.

[0029] A recent review of sensors for detecting PFAS compounds has recognized that cyclodextrins have been used for adsorption and removal of PFAS compounds to remediate water sources but have not yet been developed and evaluated as a sensor for detecting PFAS compounds (Menger et al., Chem. Eng. J., 2021 , 417, 129133).

[0030] The present inventors have been engaged in development of field deployable electrochemical detectors for analyzing environmental samples for various analytes (see for example, US Patents 9,689,046 and 10,415,102, which are each incorporated herein by reference in entirety). During research in development other assays, modifications of P-cyclodextrin to increase affinity for PFAS molecules were considered and examined. It was surprisingly discovered that certain modifications to p-cyclodextrin produced higher affinity for a group of PFAS molecules relative to unmodified p-cyclodextrin, thereby providing advantageous limits of detection and high reproducibility. The inventors sought to explore if these differences were a result of the cyclodextrin-PFAS interaction or a result of some other interaction in the assay by utilizing an alternate assay that specifically addresses cyclodextrin guest-host interactions. This led to the surprising discovery that an indicator displacement assay based on displacement of a cyclodextrin-bound indicator molecule by a PFAS compound could serve as the basis for a sensitive PFAS sensorcapable of quantification of common PFAS molecules, or mixtures thereof. It was then recognized by the present inventors that such an assay could be developed and commercialized at relatively low cost and that it could be readily configured for use with a field deployable analysis system that could provide rapid PFAS detection and quantification results to guide and improve the efficiency of remediation efforts. The assay would not distinguish between different PFAS molecules but would provide a process for quantifying one PFAS compound, if only one was present and would also provide a process for quantifying a total amount of all PFAS compounds in a given sample if multiple PFAS molecules were present.

[0031] The present applicant has described embodiments of an indicator displacement assay in U.S. Provisional Patent Application No. 63 / 610,171 , filed on Dec. 14, 2023, U.S. Provisional Patent Application No. 63 / 679,782, filed on Aug. 6, 2024, and U.S. Provisional Patent Application No. 63 / 689,017, filed on August 30, 2024, each of which is incorporated herein by reference in its entirety. These patent applications describe experiments which establish proof of concept of a useful and efficient indicator displacement assay for detection of PFAS based on displacement of an indicator molecule by PFAS from a soluble P-cyclodextrin (P-CD) with the extent of displacement of the indicator molecule being indicated by the change in fluorescence of the indicator molecule as it is displaced from the p-CD. In some of the embodiments described in these patent applications, the assay is enhanced by using a soluble polymerized form of p-CD (p-p-CD).

[0032] While these indicator displacement assays are expected to be useful in many scenarios, the importance of rapid, repeatable and field deployable assays for detecting PFAS has inspired the inventors to consider additional assay formats, some of which will require re-configuration of at least some of the components of the initially developed indicator displacement assays. In considering such alternative assay formats, the present inventors recognized that the ability to conduct indicator displacement assays for PFAS on solid substrates would increase the scope of applications of the indicator displacement assays.

[0033] In one example of such an additional application, aqueous film-forming foams (AFFFs) containing PFAS have been used as fire retardants in firefighting applications and such use of AFFFs has resulted in significant environmental contamination, which has led to discontinuation of usage of these AFFFs. However, firefighting equipment such aspumps and hoses remain contaminated with PFAS compounds from the prior use of AFFFs and efforts to remove such contamination requires continuous analysis of rinses to determine if PFAS contamination is being reduced to acceptable low-risk levels. The current laborious methods using LC-MS / MS are too slow and expensive to be widely used for monitoring of AFFF decontamination efforts. When they are used, the firefighting equipment is generally deemed to be unusable during the waiting period for the results of the analyses.

[0034] The inventors conceived of an adaptation of the PFAS indicator displacement assays involving a solid substrate-based cyclodextrin such as a substrate-linked cyclodextrin or a bead formed from cyclodextrin which is not linked to a substrate but otherwise polymerized with or without cross-linking until it is in a solid form and suitable for adsorption or linkage to a solid substrate. If such a solid cyclodextrin remains functional in binding an indicator which is displaced by PFAS in such a solid form, it could be used to rapidly assay samples the presence and quantity of PFAS. In some embodiments, the substrate is provided on a stick which is dipped into a sample or provided within a housing which is configured to accept a drop or aliquot of sample for analysis. Other embodiments are configured to be placed into a flow of fluid to determine levels of PFAS. In each case, the measurement of the displaced indicator molecule may be conveniently performed by fluorescence or colorimetric analysis. Other assay embodiments include filter-based assays and on-line assays which may be configured for permanent installations where regular monitoring is required, such as monitoring of municipal or regional water resources, for example.

[0035] Cyclodextrins polymerized into a solid and provided in the form of beads for capture of PFAS molecules have been used for removal of PFAS from the environment in remediation efforts but to date the inventors remain unaware of any efforts to use solid cyclodextrins for detection and quantification of PFAS species in a field setting.

[0036] In experiments outlined hereinbelow, the inventors describe the use of p- cyclodextrins that are polymerized until they are solid, although in other embodiments p- cyclodextrin may also be polymerized onto solid beads formed of other polymers such as polystyrene, for example. In the solution-based indicator displacement assay described in U.S. Provisional Application Nos. 63 / 610,171 , 63 / 679,782, and 63 / 689,017 the applicant has clearly demonstrated that the incorporation of positively charged functional groups(most notably quaternary amines) results in improved binding (lower limits of detection) towards the anionic PFAS species tested thus far. It is therefore reasonably predicted that solid polymerized cyclodextrins further modified with positively charged functional groups and other functional groups will likewise contribute to improved assay dynamics and improve limits of detection of PFAS.

[0037] Another similarity between the soluble and solid phase indicator displacement assays using solid cyclodextrins, is in detection modalities. The inventors demonstrate herein that solid-phase cyclodextrins can bind to bis-8-anilino-1 -naphthalenesulfonate (bis-ANS), one of the preferred indicator molecules, in a concentration dependent manner, as would be expected for such a guest-host interaction. Binding to two different colorimetric dyes is also demonstrated herein.

[0038] It is reasonably predicted that optimization of the amounts of solid polymeric cyclodextrin will be a key factor in assay development and characterization of kinetics and diffusion will likely play a larger role with the solid cyclodextrins because binding is expected to first occur on the surface of the bead and then diffuse inwards, in contrast to the more homogenous nature of the soluble assay where the cyclodextrin is dissolved uniformly in the assay solution.PFAS Compounds

[0039] The United States Environmental Protection Agency (EPA) curates a master list of PFAS compounds on its internet site currently at comptox.epa.gov. Some of the more common PFAS compounds include perfluorononanoic acid (PFNA), perfluorooctanoic acid (PFOA), perfluorooctane sulfonate (PFOS), perfluorohexane sulfonate (PFHxS), perfluorobutane sulfonate (PFBS), and hexafluoropropylene oxide dimer acid (HFPO-DA, also commonly known as GenX). Notably, each of these compounds includes at least one ionizable functional group which will be protonated or deprotonated, depending on the pH of the solution and the pKa value of each individual ionizable group. For the sake of simplicity, the compound names listed above are intended herein to refer to both the protonated and deprotonated forms as well as salts thereof.Cyclodextrins and Derivatives Thereof

[0040] Cyclodextrins are a family of cyclic oligosaccharides, consisting of a macrocyclic ring of glucose subunits joined by a-1 ,4 glycosidic bonds. Cyclodextrins areproduced from starch by enzymatic conversion. They are used in food, pharmaceutical, drug delivery, and chemical industries, as well as agriculture and environmental engineering. Cyclodextrins are composed of 5 or more a-D- glucopyranoside units linked 1->4, as in amylose (a fragment of starch). Typical cyclodextrins contain a number of glucose monomers ranging from six to eight units in a ring, creating a cone shape. Common examples include a-(alpha)-cyclodextrin, which has 6 glucose subunits, p-(beta)-cyclodextrin, which has 7 glucose subunits, and y-(gamma)- cyclodextrin, which has 8 glucose subunits. Larger cyclodextrins with more than 8 glucose subunits exist and may be used in alternative embodiments. The cyclodextrins have toroidal shapes, with the larger and the smaller openings of the toroid exposing to the solvent secondary and primary hydroxyl groups respectively. Because of this arrangement, the interior of the toroids is not completely hydrophobic, but considerably less hydrophilic than the aqueous environment and thus able to host other hydrophobic molecules, thereby forming what is known as an inclusion complex or a host-guest complex. In contrast, the exterior is sufficiently hydrophilic to provide the cyclodextrins with solubility in water and other polar solvents.

[0041] Without being bound by any particular theory, the inventors postulated that a cyclodextrin with high affinity for PFAS compounds may be provided when the cyclodextrin is provided with one or more functional groups which can interact with the head group of a given PFAS compound (for example -COOH or -SO3 if PFOA or PFOS, respectively) or with the backbone (i.e., -CF2- units). In one embodiment, there is provided a cyclodextrin modified with a long -CF2- tail on one end of the toroid of the cyclodextrin to allow the cyclodextrin to interact with long chain PFAS (for example, greater than ten carbons in length, such as perfluorododecanoic acid, which is 12 carbons long). In another embodiment, strong positive charges provided by functional groups such as -NH2, -NH3+, or quaternary amines such as -NR3+, to promote interactions with the negatively charged acid or sulfonyl groups. In other embodiments, where the cyclodextrin is in polymeric form, the monomer units are connected by linkers which include functional groups that can increase affinity for PFAS compounds. Examples of such linker modifications are described in Wang et al., ACS Central Science, 2022, 8, 663, which is incorporated herein by reference in its entirety.

[0042] PFAS compounds may be classified into subsets according to their chemical properties. There is currently a strong need to detect compounds such as PFOA and PFOS, which are negatively charged at neutral pH. It is expected that detection of positively charged PFAS compounds will become important as well, as this subset of PFAS is newer and less well regulated. A cyclodextrin with anionic functional groups to provide a negatively charged cyclodextrin could be used to improve affinity for positively charged PFAS compounds. For detection of zwitterionic PFAS compounds, the inventors have conceived of solution by designing a cyclodextrin with suitable spacing of carboxylic acid and amino groups to increase the affinity of the modified cyclodextrin for a given zwitterionic PFAS compound, to lower the detection limit of an indicator displacement assay. In another embodiment, a pair of cyclodextrins could be used to create a sensor for cationic and anionic PFAS by having respective carboxylic and amino substituents on their outer surfaces such that the amino-modified cyclodextrin is positively charged at pH 7 while the carboxyl-modified cyclodextrin is negatively charged at pH 7. This combination of cyclodextrins will be useful for detecting zwitterionic PFAS compounds in the indicator displacement assay. While some of these modified cyclodextrins are known in the art, they are described in terms of absorption and remediation and have not been incorporated into an assay to use as a sensor for quantification of PFAS compounds. It is to be understood that such modifications may be applied to cyclodextrin monomers and polymers.

[0043] Alternate linkers provided to link at least some of the monomers within the solid cyclodextrin matrix are expected to improve the limits of detection of embodiments of the solid-state assay. In some embodiments, hydrophobic cyclodextrin linkers are used. Such linkers provide a hydrophobic environment for the tail region of a bound PFAS molecule extending from the cyclodextrin to provide stronger binding affinity for the PFAS molecule. In other embodiments, the linkers are hydrophilic linkers such as urethane (carbamate) linkers which are crosslinked by diisocyanates. These linkers have a rigid structure which is resistant to swelling in various chemical environments. Carbonate linkers may be used in other embodiments. These linkers are crosslinked by active carbonyl compounds. The crosslinks are short and have limited swelling capacity. These linkers are chemically resistant to acid environments. In other embodiments, linkages are provided via esters and ethers. Ester linkages between cyclodextrins are synthesized by addition of cyclodextrins to dianhydrides or dicarboxylic acids or polycarboxylic acids. The resultingpolymers absorb water to for hydrogels. Ether linkages between cyclodextrins are produced in reactions of cyclodextrins to produce epoxides such as epichlorohydrin linkages. Some examples of hydrophilic linkers of cyclodextrins are described in Ciesielska, et al., Appl. Sc / . 2020, 10, 8463, which is incorporated herein by reference in its entirety. An example of an ether-CD with branched epoxide, trimethylolpropane glycidyl ether (TMPGDE), is described in Demirci et al., ACS Appl. Polym. Mater. 2021 , 3, 12, 6238-6251 , incorporated herein by reference in its entirety.

[0044] All of the hydrophilic linkers described above, may vary in terms of polymer branch structure and may accommodate additional substituents and chemical modifications.

[0045] As disclosed by the present inventors in U.S. Provisional Application Nos. 63 / 610,171 , 63 / 679,782, and 63 / 689,017, it is expected that modifications to the functional groups of the cyclodextrin will increase affinity towards PFAS and create a more sensitive solid-state assay, with the most promising candidate being strongly positively charged groups such as quaternary amines.

[0046] Some embodiments of the solid-state assay described herein are configured for detection and quantification of cationic PFAS by incorporation of negatively charged functional groups on the solid-state cyclodextrins. Cationic PFAS species are not currently regulated but are an emerging area of active research and it is expected that detection of these compounds will become important in remediation efforts. Conversely to the positively charged cyclodextrin modifications for the detection of anionic PFAS species, a negatively charged cyclodextrin modification (e.g., carboxyl, succinyl, phosphatyl, sulfyl, or other anionic functional groups) creates a more sensitive assay towards cationic PFAS species. Furthermore, it is feasible to use both systems in parallel to quantify all PFAS present in a sample.

[0047] As disclosed in U.S. Provisional Application Nos. 63 / 610,171 , 63 / 679,782, and 63 / 689,017, a scavenger cyclodextrin is used in some assay embodiments to interact with common interferents found in water samples, such as alkanes, alkenes, and detergents, for example. The advantage to this arrangement is simplification of manufacture of assay components.

[0048] The size and morphology, as well as the density and porosity, of the solid cyclodextrin are predicted to play key roles in their functionality as sensors for a solid-stateindicator displacement assay. Similarly, selection of an optimized amount of solid cyclodextrin within the bead or particle is predicted to be an important parameter.

[0049] Initial investigations described in the examples below have employed a preparation of unmodified soluble p-CD polymer crosslinked with epichlorohydrin of molecular weight of -250 kDa which is subjected to polymerization conditions until it attains a solid form, followed by processing to form beads. Hereinafter, the term “beads” refers to solid particles of this solid polymerized p-cyclodextrin (also abbreviated herein as s-p- -CD). Other preparations of soluble cyclodextrins may be used to prepare solid polymeric forms suitable for use in embodiments of the solid-state indicator displacement assay where PFAS molecules present in an analyte solution displace indicator molecules from the solid polymeric cyclodextrin. Other examples of such soluble cyclodextrins include, but are not limited to, polymers based on p-cyclodextrin which include carboxymethyl, (2-hydroxy-3- N,N,N-trimethylamino)propyl and sulfobutylated derivatives. Such soluble polymeric derivatives may also be further polymerized into solid forms suitable for use of production of beads which are convenient for use in solid-state indicator displacement assay embodiments.Linkage and Adsorption of Cyclodextrins to Solid Substrates

[0050] Various detection platforms may be used in solid-state assay embodiments of the indicator displacement assay. The solid polymeric cyclodextrins used for detection and for scavenging may be adsorbed or linked to substrates including, but not limited to paper materials such as cellulose, nitrocellulose (which are compatible with lateral flow assays), as well as glass, semiconductors such as quantum dots (for use with Forster resonance energy transfer (FRET) analysis), and gold, silver or iron nanoparticles. The substrates themselves can be in the form of a bead or a planar substrate. Additional substrates may be incorporated into alternative embodiments. Such additional substrates include, but are not limited to, cellulose, polypropylene, polystyrene, and other plastic polymers, wood fibers, fabrics or any other solid substrate providing a compatible surface. In embodiments of the solid-state assay which include a-CD as an interferent scavenger, selection of the substrate depends on practicalities which do not require consideration of detection modalities because the scavenger is likely to be employed in a pre-treatment step. In one such example, the a-CD (which may be in monomeric or polymeric form) is linked or adsorbed onto filter paper provided in a disposable or regeneratable filter syringe systemwhich provides the ability to draw a raw sample through the filter with the syringe such that the interferents are captured by the scavenger on the filter paper and remain there while the analyte sample is drawn into the syringe body, and then the cleaned sample is applied to another PFAS detection substrate having the solid polymeric p-cyclodextrin adsorbed or linked thereto.

[0051] Certain embodiments may require special linkages. For example, gold and silver nanoparticles may require a thiol cap and iron nanoparticles may require a carboxyl cap. Other well-known biological conjugates such as biotin-streptavidin may be used to provide linkages between the substrate and the polymeric cyclodextrin provided that compatibility with the cyclodextrin polymer crosslinker (such as epichlorohydrin) is established through routine experimentation.Indicator Molecules

[0052] The present technology employs indicator molecules to provide a measurable parameter which indicates binding of a PFAS compound. In this context, “binding” refers to entry of another molecule into the cavity of a cyclodextrin in a host-guest interaction, which is presumed to be dominated by hydrophobic interactions and other non-covalent interactions. The parameter provided by the indicator molecule may be any measurable physical parameter. Examples of such parameters of indicator molecules include, but are not limited to, color change (colorimetric analysis), fluorescence, and change in oxidation state (redox chemistry). Indicator molecules for colorimetric analysis and fluorescence detection include dye molecules (also known as chromophores or fluorophores), which have conjugated multi-ring structures. Some examples of fluorescence indicator dyes include, but are not limited to, cyanines such as Cy3-COOH and Cy5-COOH, xanthenes such as fluorescein and rhodamine, pyrenes such as 8-hydroxypyrene-1 ,3,6-trisulfonic acid, trisodium salt (HPTS) and 6,8-dihydroxy-1 ,3-pyrenedisulfonic acid (DHPDS), napthalenes such as ANS, bis-ANS, Acid Blue and Acid Black, and other fluorescence indicators such as SYPRO™ Orange (Thermo Fisher (S6650), BODIPY-FL, 7-nitrobenz- 2-oxa-1 , 3-diazole-4-yl, naphthalimide (lucifer yellow), and acridine orange. Colorimetric indicator molecules are also used in the indicator displacement assays. Examples of such colorimetric molecules include dyes such as Acid Blue 120 and Acid Black 24. Indicator molecules for redox detection are also known as electroactive analytes. Examples of suchelectroactive analytes are described in US Patents 9,689,046 and 10,415,102. Additional examples include metallocenes containing metals such as iron, cobalt, chromium, nickel ruthenium and vanadium. Ferrocene and derivatives such as ferrocenemethanol, ferrocenedimethanol, ferrocenecarboxaldehyde, ferrocenecarboxylic acid, and aminoferrocene are expected to be useful as electroactive compounds in the indicator displacement assays described herein. Such compounds are capable of being reduced and / or oxidized when a voltage is applied to an electrical cell and provide a measurable electrical current in the electrical cell. Other examples of indicator molecules may be bifunctional by providing a chromophore while being electroactive. Some embodiments may include a mixture of indicators to provide a visual quality control metric to a user while a more quantifiable result is produced electrochemically using an electroactive indicator molecule.Indicator Displacement Assays

[0053] An indicator displacement assay is based on a supramolecular assembly of an indicator molecule that is reversibly bound to a host molecule. In the presence of an analyte, the indicator molecule is displaced from the host, resulting in a measurable change in a physical parameter. The target analyte must also have a higher affinity than the affinity of the indicator molecule at a particular concentration. The term “affinity” refers to the extent that the target analyte is bound relative to the indicator molecule, and thus the affinity is related to the association constant (sometimes referred to as the “formation constant”) and concentration of the host, guest, and indicator molecule, to achieve an effective displacement of the indicator and provide a measurable change of a parameter (Sedgewick et al. Chem. Soc. Rev., 2021 , 50, 9). The change of the parameter will indicate the amount(s) of the target analyte(s) in the sample being assayed.

[0054] While guest / host interactions with a fluorescent dye and two colorimetric dyes have been demonstrated, it is reasonably and soundly predicted that the solid-state assays can be extended to include electrochemical molecules which have been confirmed to be useful in prior versions of solution-based indicator displacement assays.

[0055] In terms of assay formats, the functionality of solution-based assays has been confirmed in 96-well microplates. It is reasonably predicted that conversion into other formats, such as adsorption onto filter paper to permit dispensing or flow of a sample,followed by a colorimetric analysis to permit a user to observe a color change with comparison of the observed color with a color chart that is correlated with PFAS concentration. In other embodiments, the solid-state assay is configured to simply provide a “yes or no” answer in context of a defined concentration threshold, using a lateral flow assay similar to common versions of rapid antigen tests, where the cyclodextrins are immobilized, either physically or chemically, on a substrate contained in a lateral flow assay cartridge, for example. An alternative embodiment has the PFAS immobilized on the test strip and the cyclodextrin immobilized on a gold nanoparticle, for example.

[0056] Some embodiments of the solid phase indicator displacement assay are configured to provide continuous on-line monitoring by a sensor provided within permanent or semi-permanent infrastructure such as a system of conduits where a sample is autonomously extracted from a conduit and applied to a sensor, followed by detector measurement such as colorimetric, fluorescence or electrochemical detection.

[0057] The advantage of the solid-state assays over the solution-based assays is that a flow-through system could be used with a basic inexpensive filter system to keep the assay particles / beads within the detection range of the sensor transducer (i.e., a photomultiplier tube or photodiode for a fluorescent system). Each sensor could be treated as disposable and replaced after use, or alternatively such a sensor could be regenerated by flowing a high concentration of the dye / indicator molecule to outcompete the PFAS molecule and displace the PFAS molecule from the cyclodextrin, resulting in a regenerated dye / cyclodextrin bead complex that is ready for another assay.Kits Including Containers for Indicator Displacement Assay Components

[0058] Another embodiment provides kits which include containers which are formed of materials such as plastics which are substantially free of PFAS compounds, in order to avoid assay interference. In one embodiment, separate containers separately containing cyclodextrin and an indicator molecule are provided. In a preferred embodiment, the cyclodextrin is provided in a mixture with an indicator molecule such that an extra step of mixing the cyclodextrin with the indicator is not required at the point of analysis. The mixture may be provided in a single container in either solution form or lyophilized form. A lyophilized form would provide a way to further increase the concentration of the PFAS sample being analyzed. The kits are configured to facilitate conducting indicatordisplacement assays in the field where environmental samples such as water samples requiring analysis for the presence of PFAS compounds.

[0059] The cyclodextrin of the kit may be any one of the cyclodextrins described herein, including but not limited to m- -CD, p- -CD, 2HP- -CD, TAc- -CD, 6-amino-p-CD, polymeric quaternary amine p-CD (pQ- -CD) and any derivative or polymer (including polymers with various linkers describe herein) thereof, which have suitable affinity for an indicator molecule. The polymer may be solid and may be in the form of beads or may be linked or adsorbed to a substrate. The indicator molecule of the kit may be a colorimetric indicator molecule, a fluorescence indicator molecule, or an electroactive molecule. The indicator molecule may be any one of the indicator molecules described herein, including but not limited to the fluorescence indicator Nile Red, ANS or bis-ANS, colorimetric indicators such as Acid Blue 120 or Acid Black 24, or the electroactive indicators PAP, ferrocene or ferrocenemethanol.

[0060] The kit may include an empty container which is formed of a material such as a plastic which is substantially free of PFAS compounds, which is provided for the purpose of collecting a water sample for analysis to detect and quantify the presence of one or more PFAS compounds.

[0061] The kit may further include other components to generate a concentrated version of a collected environmental sample. Such additional components may include a resin such as a weak anion exchange (WAX) resin, which may be provided in a pre-packaged column such as an Agilent Bond Elut PFAS WAX column, which is capable of binding PFAS compounds while eluting other common environmental contaminants. One or more syringes or reservoirs may be provided with the kit to facilitate transfer of the sample to the chromatography column. The kit may also include an evaporator to remove methanol from an eluted sample suspected of containing one or more PFAS compounds, to allow the PFAS compounds to be resuspended in another solvent at a reduced volume, thereby raising the concentration of the PFAS compounds for subsequent detection. The evaporator may be in the form of a heating block, a vacuum line, a line to provide a stream of dry gas. or any other device configured to evaporate a solvent such as water and / or other polar or nonpolar solvents from a sample.I nterf erents of PF AS-Contaminated Sites

[0062] Sites contaminated with significant concentrations of PFAS, such as sites where aqueous film-forming foams (AFFFs) have been used, are being tested to determine the extent of PFAS contamination and the related effectiveness of remediation efforts being conducted at these sites. Examples of such sites include firefighting training areas or sites of industrial fires where significant volumes of AFFFs have been released into the environment. As a result, the PFAS-contaminated sites also include significant amounts of various surfactants which may have been included in the AFFF formulations or used in previous remediation efforts or present in the environment prior to the release of the AFFFs. A common remediation strategy is a process known as foam fractionation which involves separation of organic compounds, colloidal particles and suspended solids from aqueous solution using a rising stream of bubbles and may include detergents as surfaceactive agents to increase the efficacy of foam fractionation processes. The inventors recognized that common surfactants based on alkyl groups with lengths of 6 or more carbons, would be likely to bind to cyclodextrins and interfere with embodiments of the indicator displacement assay described herein by reducing the range of fluorescence provided by the complex of the indicator molecule with the cyclodextrin used in the indicator displacement assay. As a result, the inventors recognized that it would be advantageous to modify the assay to include a scavenger molecule which selectively binds hydrocarbons and / or surfactants having alkyl chains with 6 or more carbons and which does not significantly bind the indicator molecule, and which does not significantly bind PFAS compounds. An example of such a surfactant expected to be present at significant concentrations at PFAS-contaminated sites is sodium dodecyl sulfate (SDS). Other structurally similar surfactants may include, but are not limited to, lauryldimethylamine N-oxide (LDAO); 3-laurylamido-N,N"-dimethylpropylaminoxide (LAPAO) dodecylphosphocholine (DPC, also known as Foscholine-12 (FC12)), p-octyl glucoside (P-OG), dodecyl maltoside (DDM) and members of the DOWFAX™ family of ionic surfactants manufactured by Dow Chemical / Dupont. Since these detergents have alkyl chains longer than about 6 carbons in length, they are more likely to interfere with cyclodextrin-based displacement assays than compounds with alkyl groups less than 6 carbons in length. Therefore, interferent compounds are co-contaminating compounds which may include, for example, hydrocarbons and surfactants having alkyl chains greater than 6 carbons in length. Such interferent compounds are currently considered among themost concerning of interferents with respect to assay interference. However, as used herein, the term “interferent” refers to any substance whose presence interferes with an analytical procedure via any mechanism. Although many other hydrocarbons such as benzene, toluene, xylene, polychlorinated biphenyls, dioxins and furans are typically present at PFAS-contaminated sites, such compounds are less likely to interfere with cyclodextrin-based displacement assays, due to the sizes of the ring structures of these compounds. However, the possibility remains that certain examples of such compounds may function as interferents by causing other issues with the cyclodextrin-based displacement assays, such as agglomeration and precipitation of assay components, for example through non-specific interactions or through specific chemical reactions which could occur between the interferents and assay components.

[0063] US Provisional Patent Application No. 63 / 689,017, incorporated herein by reference in its entirety, describes the result of experiments performed to develop an interferent scavenger to prevent interference with a PFAS detection assay based on displacement of an indicator molecule from a cyclodextrin. As used herein, the term “scavenger” refers to any chemical compound included in an assay for the purpose of sequestering an interferent which would otherwise interfere with the assay. It is to be understood that while the example scavengers described herein are based on a- cyclodextrins, which are investigated in terms of sequestering long chain hydrocarbons and surfactants, if a given PFAS-contaminated site includes another class of interferents which are found to interfere with the assay, an appropriate scavenger appropriate for that class of interferents can be identified and adapted for use in the assay without undue experimentation.

[0064] Figures 1A and 1B illustrate the binding principles involved in the indicator displacement assay with and without a scavenger exemplified by monomeric a- cyclodextrin (m-a-CD).

[0065] Scheme A of Figure 1A shows how contact of polymeric p-cyclodextrin (p-p-CD) with an indicator molecule (IND), results in the baseline fluorescence of the indicator molecule becoming elevated as a result of binding of the indicator to the polymeric p- cyclodextrin. Scheme B of Figure 1A indicates how a PFAS molecule will displace the indicator molecule from the polymeric p-cyclodextrin to result in the fluorescence of the indicator molecule returning to its baseline. Scheme C of Figure 1 B indicates aninterference situation where an interferent (I NT), which could be a long chain hydrocarbon or a surfactant having a hydrocarbon chain of at least 6 carbons, competes with the indicator molecule for binding to the polymeric p-cyclodextrin. The result is that the fluorescence provided by the complex of the indicator molecule with polymeric p- cyclodextrin is undesirably reduced because a fraction of the available polymeric p- cyclodextrin is occupied by the interferent or because this situation causes false positives. Scheme D of Figure 1 B indicates how the undesirable effect of the interferent is mitigated by inclusion of a scavenger molecule exemplified by monomeric a- cyclodextrin (m-a- cyclodextrin). The scavenger molecule sequesters the interferent (I NT) such that it cannot compete with the indicator molecule and the result is that the desirable elevated fluorescence resulting from binding of the indicator molecule to polymeric p-cyclodextrin is provided such that the presence of PFAS in a sample being measured using the indicator displacement assay will displace the indicator molecule and reduce fluorescence in a manner which accurately reflects the amount of PFAS in the sample.

[0066] While this embodiment of the scavenger system was developed in a solutionbased indicator displacement assay, it is now reasonably predicted that it will be reliably configured to operate with a solid-state embodiment of the indicator displacement assay where the scavenger such as a-cyclodextrin is provided in either a soluble monomeric form which is mixed with the analyte solution suspected of containing PFAS, or provided as a solid polymeric form (s-p-a-CD), where the latter is introduced prior to contacting the analyte sample with s-p-p-CD.Removal of Non-Specific Interferents by Pre-Treatment of Samples

[0067] An aspect of this technology is allowing the assay to be performed in the field, and to function adequately in environmental samples which will include many additional compounds in addition to the PFAS target molecules. As noted above, at least some cocontaminating compounds are expected to act as interferents.

[0068] It will be appreciated that the most controlled environment is a laboratory setting, where chemicals are stored as methanolic solutions in freezers or in flammable cabinets. Alternatively, there are analytical labs that can detect PFAS in environmental samples, but that requires off-site shipping.

[0069] Current systems in place for PFAS monitoring rely on analytical laboratory methodologies. EPA methods 537.1 and 1633 are LC-MS / MS methods that can quantitate PFAS compounds in environmental samples. These technologies first extract PFAS from a sample utilizing solid phase extraction columns, followed by elution, then evaporation, and then resuspension as a concentrated solution that is then injected into a LC-MS / MS system. This provides exceptional sensitivity but is slow and most analytical labs are experiencing significant backlogs for analysis of environmental samples being analyzed for the presence of PFAS compounds.

[0070] The inventors have identified that EPA methods 537.1 and 1633 may not always function adequately for some environmental samples. The inventors identified that at least some of the issues with the EPA methods related to the presence of interferents which negatively alter the response of the assay.

[0071] To isolate the target of interest, the present technology may include one or more of the following adaptations:The utilization of carbon adsorbents after methanol elution to remove organic contaminants that may interfere with the indicator displacement assay or the binding of PFAS molecules to the solid phase extraction column.The utilization of dilute methanolic rinses to the adsorbed PFAS on a solid phase extraction column to remove interferents that were co-adsorbed.The utilization of alternate solid phase extraction columns such as weak anion exchange (WAX), or hydrophobic columns using hydrophobic resins presenting hydrophobic moieties such as styrene divinyl benzene (SDVB), for example, to provide different mechanisms to capture PFAS molecules and co-adsorbents in different sample matrices.

[0072] For example, PFAS-containing samples may be treated by foam fractionation with the objective of creating a concentrated PFAS-containing “foamate” layer and a PFAS- free layer. It is advantageous to have the capacity to analyze such foamate layers using embodiments of the cyclodextrin-based indicator displacement assay described herein. It was found that weak anionic exchange (WAX) columns may provide the best functionality for embodiments of the indicator displacement assay, as evaluated by the quantification of PFAS from the assay compared to the gold-standard LC-MS / MS, in some commercialfoamates, but in groundwater samples it was found that SDVB (styrene divinyl benzene, a hydrophobic resin) was found to be a more effective column resin for removal of interferents in some environmental groundwater samples. These are particularly surprising results because the conventional laboratory LC-MS / MS procedures do not make such distinctions. Therefore, according to one embodiment, if the environmental sample is a foamate sample, the sample is subjected to a pre-treatment step comprising elution of the sample from a WAX column. According to another embodiment, if the environmental sample is a groundwater sample, the sample is subjected to a pretreatment step comprising elution of the sample from a hydrophobic column. Additionally, the composition of foamates is not homogenous between samples, and there may be some samples in which SDVB is a preferred column resin, with similar non-homogeneity noted for groundwater samples.

[0073] In some more varied sample matrices (e.g., landfill leachate), there were examples where sample pre-treatment steps using WAX columns provided more favorable results and other examples where the sample pre-treatment steps using the SDVB column provided more favorable results. As used herein, the term “leachate” refers to water that has percolated through a solid matrix (such as soil and landfill materials, for example) and leached out some of the constituent components present in the matrix. It is to be understood that if the most common interferents present in the leachate are known, this information may be used to guide the selection of an appropriate sample pre-treatment step.Storage and Transport of Components of the Indicator Displacement Assay

[0074] As noted above, the components of the cyclodextrin-based indicator displacement assay include a cyclodextrin, an indicator molecule and optionally an interferent scavenger compound. Control / calibration samples comprising known concentrations of PFAS compounds are also used. In one embodiment a premade mixture containing the main assay cyclodextrin, the indicator molecule and optionally the interferent scavenger compound is provided. It was established that this premade mixture can be stored in both a dry or a liquid format, as well as at room temperature or at 4 °C, with room temperature being preferable to avoid cold chain requirements during shipment.

[0075] In some embodiments, the premade mixtures include PFAS compounds for controls and calibration. Since the PFAS compounds are in methanolic solutions, the shipment of liquid formats presents a challenge due to the flammable nature of the methanol, which makes a dried format preferable in some situations. Unexpectedly, the optimal configuration for storage of a premade mixture in a dried format includes both the assay components and the PFAS standards together prior to drying in order to create optimal results. Without being bound by any particular theory, it is believed that the optimal results are enhanced by an unexpected beneficial rehydration artifact.

[0076] An advantage of having solid-state assay components is that drying and rehydration of the components is not necessary. This simplifies packaging and transport and also provides the components in a more stable form relative to soluble components.Field-Operable Apparatus for Performing the Indicator Displacement Assay

[0077] Embodiments of the technology described herein are adapted for implementation using a field-operable apparatus. Of note, SDVB and WAX columns or SPE cartridges require specific flow rates across the columns in order to ensure adequate contact time and proper binding of the PFAS compounds. The inventors have identified embodiments that enable evaporation of column eluents in a manner similar to the procedures of EPA 1633. Various embodiments of a field operable apparatus may include peristaltic pumps to control column elution flow rates, scaffolding to hold columns, resistive heating elements to maintain temperatures and evaporate eluents, as well as vacuum systems to apply negative pressure and increase eluent evaporation speed. These embodiments may be powered using alternating current or battery packs, the latter which is expected to provide a significant advantage for field analysis. Embodiments of the field-operable apparatus represent a dramatic improvement over standard laboratory practices that require bulky vacuum manifolds, ovens, and / or dedicated and automated solid phase extraction equipment.Examples

[0078] Example 1: Investigation of Binding of Indicator Molecules to Solid Polymerized 13- Cvclodextrin - This series of experiments was conducted using a preparation of soluble P-CD polymer crosslinked with epichlorohydrin of molecular weight of -250 kDa which is subjected to further polymerization conditions until it attains a solid form, followed byprocessing to form beads. Hereinafter within this example, the term “beads” refers to solid particles of this solid polymerized p- cyclodextrin (also abbreviated herein as s-p-p-CD). Preliminary experiments indicated that it was challenging to accurately quantify dispensed volumes of beads due to difficulties in evenly distributing them in the solutions and directly dispensing them into plates. The goals of the present series of experiments were to compare binding of the fluorescent indicator molecule bis-ANS and the colorimetric indicator molecules Acid Blue 120 and Acid Black 24 to the s-p-p-CD beads and to compare the detection of different amounts of s-p-p-CD beads and different amounts of indicator molecules. The conditions investigated in the first experiment are shown in Table 1 below.Table 1 : Conditions of First Experiment

[0079] In a first experiment, a dilution series of the s-p-^-CD beads were prepared. The beads are difficult to transfer from a 15 mL tube because they settle rapidly after vortexing and pipetting. For this reason, 1 mL was removed using a p1000 pipette after vigorously resuspending in the tube and this aliquot was used to prepare the dilution series. The beads are too large to be dispensed using a conventional p200 pipette tip. To dispense the beads into the wells of the plate, a cut was made on the tip of a p200 pipette to accommodate the larger bead size and this cut pipette tip was used to transfer 60 pL directly to the wells of a clear-bottomed 96-well plate for each dilution series. Mill iQ water was used as a control solution for a background measurement. Each concentration was dispensed in duplicate. Dilutions of indicator molecules were prepared according to Table 1 and added to each well in 60 pL volumes. The samples were then measured at the excitation and emission wavelengths indicated in the Table 1 for fluorescence and for absorbance. The results of the fluorescence measurements as a function of beadconcentration are shown in Figures 2A and 2B and the results of the absorbance measurements as a function of bead concentration are shown in Figures 3A and 3B.

[0080] The results of Figures 2A and 2B demonstrate that there is a useful working interaction between the s-p-^-CD beads and the bis-ANS indicator molecule with a maximum bead concentration visible in the signal response. This indicates that the s-p- ?- CD beads and the bis-ANS may be used in a solid-state indicator displacement assay based on fluorescence detection of bis-ANS and it is predicted that other similar fluorescence indicator molecules will also function in a similar manner in the solid-state indicator displacement assay.

[0081] The results of Figures 3A and 3B demonstrate that there is a useful working interaction between the s-p-^-CD beads and two different colorimetric indicator molecules acid blue 120 (AB 120) and acid black 24 (AB 24) with absorbance measured at 675 nm. This indicates that the s-p-^-CD beads and colorimetric indicator molecules may be used in a solid-state indicator displacement assay based on absorbance detection of the indicator molecules. It is predicted that other similar colorimetric indicator molecules will also function in a similar manner in the solid-state indicator displacement assay.

[0082] In a second experiment, a dilution series of the colorimetric indicator molecules was prepared by dilution of 1 in 2 over 5 dilutions and the same conditions outlined in Table 1 were employed using 10 mg / L of the s-p-p-CD beads.

[0083] The results of Figures 4A and 4B indicate that a concentration of 10 mg / mL of beads will produce a useful fluorescence response at a concentration approaching 0.01 mM bis-ANS, which is in a similar range of bis-ANS concentrations used in the solutionbased assay previously described in U.S. Provisional Patent Application Nos. 63 / 610,171 , 63 / 679,782, and 63 / 689,017.

[0084] The results of Figures 5A and 5B indicate that concentrations of the two tested colorimetric indicator molecules Acid Blue 120 (AB 120) and Acid Black 24 (AB 24) also provide useful absorbance responses at concentrations approaching 0.1 mM, with Acid Blue 120 providing a better response. Notably, a color change is visible in the wells of the 96-well plate, indicating the likelihood that an embodiment of a solid-state indicator displacement assay will be developed to provide a qualitative “yes / no” result which can be provided without the use of an absorbance or fluorescence detector, simply byvisualization of a color on a test strip in a dipstick type test (in a manner similar to conventional pH paper test) or in lateral flow assay in a format similar to conventional rapid antigen tests where a cyclodextrin-indicator complex is provided on the test substrate to provide a colored line on the test substrate and the colored line is observed to disappear upon binding of a PFAS compound to the cyclodextrin, which causes displacement of the indicator molecule, resulting in the loss of color.

[0085] The experiments described in this example indicate that indicator molecules such as fluorescent or colorimetric indicator molecules effectively bind to a solid polymeric form of ^-cyclodextrin, indicating proof of concept for development of various types of solid- state assays where the solid polymeric form of ^-cyclodextrin is adsorbed or otherwise linked to a substrate where the substrate is used in dipstick or lateral flow tests.

[0086] Example 2: Investigation of Displacement of an Indicator Molecule from Solid Polymeric / ?-Cyclodextrin by a PFAS molecule

[0087] Detection of PFOA with a solid-state assay using 2.5 mg / mL of s-p-^-CD beads was investigated using fluorescence detection of the indicator molecule bis-ANS at concentrations of 0.008 mM and 0.012 mM bis-ANS. A concentration series of PFOA ranging from 0.001 ppm to 1000 ppm was investigated. The results shown in Figure 6 indicate that the detection response begins near 10 ppm PFOA and that a sharper response is seen for the series using 0.012 mM bis-ANS.

[0088] This example indicates an excellent proof-of-concept first result of detection of a PFAS molecule based on solid polymeric forms of cyclodextrin in an indicator displacement assay. It is therefore reasonably predicted that the limit of detection of PFAS molecules using such solid-state assays will be lowered to provide more sensitive assays through optimization of parameters such as bead concentrations, chemical modifications of the solid cyclodextrin polymers and inclusion of a scavenger of interferents at appropriate concentration levels with the scavenger being provided in either a soluble form or a solid polymeric form.

[0089] Example 3: Solid-state Indicator Displacement Assay in Dipstick Format

[0090] This example describes a general embodiment of an indicator displacement assay configured for rapid detection of PFAS which is suitable for use in a remediation effort toremove PFAS from equipment by rinsing. In this example, an assay kit is provided which includes a pre-filter configured for connection to the inlet of a syringe. The pre-filter includes filter paper with monomeric or polymeric a- cyclodextrin adsorbed or chemically linked thereto. The kit further includes a test dipstick formed of polystyrene having the solid polymeric p-cyclodextrin (s-p-p-CD) described in Example 1 adsorbed or chemically linked thereto, with the colorimetric indicator molecule Acid Blue 120 bound to the s-p-p- CD to form a colored complex. The colored complex provides a blue strip on the test dipstick. A sample of rinse solution from a tank of a fire truck which was used previously to dispense a PFAS-containing aqueous film forming foam (AFFF) is obtained and first cleaned by drawing it through a pre-filter attached to the syringe, which causes detergents and other similar compounds to be scavenged by the a-cyclodextrin. The pre-filter is removed from the syringe and the cleaned sample residing in the syringe body is then dispensed across the test strip of the dipstick. Rapid visual disappearance of the blue strip indicates that the sample of rinse solution contains at least 10 ppm PFAS, which is an unacceptable level of PFAS. Visual appearance of an intermediate intensity blue strip indicates a concentration between about 1 ppm and about 10 ppm PFAS.

[0091] Rinsing of the tank of a fire truck continues and another sample is obtained after a given time interval. The cleaning and analysis steps outlined above are repeated using another syringe and test dipstick with the result indicating that the blue line does not change in intensity. This result indicates that the sample of rinse solution contains less than 1 ppm PFAS, indicating that the rinsing has been effective in lowering the concentration of PFAS of water being transferred through the fire hose to an acceptable risk level.

[0092] It is to be understood that the pre-filter and syringe cleaning step may be considered optional in situations where significant concentrations of interferents are not expected to be present. In such situations, the dipstick may simply be immersed in the solution or water sample to be tested where observation of disappearance of the blue strip indicates the presence of PFAS at a concentration level of at least 10 ppm and failure of the blue strip to disappear indicates less than 1 ppm PFAS. Visual appearance of an intermediate intensity blue strip indicates a concentration between about 1 ppm and about 10 ppm PFAS.

[0093] Example 4: Solid-state Indicator Displacement Assay in Lateral Flow Format

[0094] This example describes a general embodiment of an indicator displacement assay configured for rapid detection of PFAS which is suitable for various rapid applications in a lateral flow format. The pre-filter described in Example 3 may be optionally used in this example as well. The lateral flow assay format has a length of paper substrate contained in a plastic housing to facilitate handling. The paper substrate includes an appropriate amount of adsorbed or chemically-bound s-p-p-CD with the colorimetric indicator molecule Acid Blue 120 bound to the s-p-p-CD, thereby forming a blue strip on the paper substrate. The housing includes a sample port to accept a drop of test sample onto the paper substrate, in an arrangement similar to common rapid antigen tests. The test sample spreads and migrates along the paper substrate until it encounters the blue strip. If the test sample includes PFAS at a concentration level of about 10 ppm or higher, the Acid Blue 120 indicator molecule will be substantially displaced from the s-p-p-CD, thereby causing the blue strip to disappear. If the test sample includes PFAS at a concentration level at or below 1 ppm, there will be insufficient displacement of the Acid Blue 120 indicator molecule, and the blue strip will appear unchanged. Visual appearance of an intermediate intensity blue strip indicates a concentration between about 1 ppm and about 10 ppm PFAS.

[0095] In alternative embodiments, the lateral flow format of the indicator displacement assay is configured to include at least one additional separate paper substrate arranged in a similar manner to accept a control solution of PFAS at a specified concentration level at a control port, for confirmation that displacement of the indicator molecule from the s- p-P-CD occurs.

[0096] Alternative embodiments may include an additional colorimetric system where displacement of the indicator molecule from s-p-p-CD by a PFAS molecule results in binding of the indicator molecule to a different host molecule which provides a different color signature to indicate that PFAS is present in the test sample. Various additional embodiments may be calibrated to indicate a spectrum of color changes to correlate with a range of concentrations of PFAS in tested samples.

[0097] Example 5: Solid-state Indicator Displacement Assay in an In-Line Process FlowFormat

[0098] This example describes another solid-state assay format in the form of a sensor which may be of a more permanent construction and configured to be placed within a flow conduit drawn from a process flow such as treatment of drinking water for example. Such an assay sensor system may be placed in a switchable branch of a flow conduit to enable an operator to draw samples for assay measurements when desired. In one general embodiment, there is provided a valve in a flow conduit which draws part of the flow of water as an analyte sample into a side conduit, thereby enabling it to pass it over a sensor which includes a glass substrate with chemically-bound s-p-p-CD which itself has a fluorescent indicator molecule such as bis-ANS bound thereto. If the flow of water sample includes PFAS, the fluorescent indicator molecule will be displaced at a higher rate than would be observed in the absence of PFAS and the expected drop in the baseline fluorescence will be detected by a fluorescence detector which is part of the sensor system.

[0099] Since the arrangement is of at least a semi-permanent nature for routine in-line analysis of a process flow, incorporation of a fluorescence detector is a reasonable aspect of the system. Furthermore, the solid-state sensor permits regeneration of the complex with the fluorescent indicator molecule by providing a port to inject a solution of a high concentration of the fluorescent indicator molecule to outcompete and displace the PFAS molecule such that the sensor is primed for the next measurement. Alternatively, a plurality of sensor cartridges could be incorporated into the semi-permanent unit in a replaceable fashion such that a technician could replace used sensor cartridges on a routine (e.g., monthly) basis and dispose of the used sensor cartridges.

[0100] In some embodiments, the analyte sample drawn into the sensor may be flushed out into the original flow or flushed out into a disposal flow. In the embodiment of replaceable sensor cartridges, the waste could be retained and disposed of with the sensor cartridge upon replacement.Equivalents and Scope

[0100] Other than described herein, or unless otherwise expressly specified, all numerical ranges, amounts, values and percentages, such as those for amounts of materials, elemental contents, times and current rate, ratios of amounts, and others, in the following portion of the specification and attached claims may be read as if prefaced by the word“about” even though the term “about” may not expressly appear with the value, amount, or range. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0101] The terms “approximately,” "about," “substantially,” and similar terms are intended to have a broad meaning in harmony with the common and accepted usage by those of ordinary skill in the art to which the subject matter of this disclosure pertains. It should be understood by those of skill in the art that these terms are intended to allow a description of certain features described and claimed without restricting the scope of these features to the precise numeral ranges provided. Accordingly, these terms should be interpreted as indicating that insubstantial or inconsequential modifications or alterations of the subject matter described and are considered to be within the scope of the disclosure.

[0102] The articles “the”, “a” and “an” are not necessarily limited to mean only one, but rather are inclusive and open ended so as to include, optionally, multiple such elements.

[0103] “At least one,” in reference to a list of one or more entities should be understood to mean at least one entity selected from any one or more of the entities in the list of entities, but not necessarily including at least one of each and every entity specifically listed within the list of entities and not excluding any combinations of entities in the list of entities. This definition also allows that entities may optionally be present other than the entities specifically identified within the list of entities to which the phrase “at least one” refers, whether related or unrelated to those entities specifically identified. Thus, as a nonlimiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and / or B”) may refer, to at least one, optionally including more than one, A, with no B present (and optionally including entities other than B); to at least one, optionally including more than one, B, with no A present (and optionally including entities other than A); to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other entities). In other words, the phrases “at least one,” “one or more,” and “and / or” are open-ended expressions that are both conjunctive and disjunctive in operation. For example, each of the expressions “at least one of A, B and C,” “at least one of A, B, or C,” “one or more of A, B,and C,” “one or more of A, B, or C” and “A, B, and / or C” may mean A alone, B alone, C alone, A and B together, A and C together, B and C together, A, B and C together, and optionally any of the above in combination with at least one other entity.

[0104] Where two or more ranges are used, such as but not limited to 1 to 5 or 2 to 4, any number between or inclusive of these ranges is implied.

[0105] As used herein, the phrase, “for example," the phrase, "as an example," and / or simply the term "example," when used with reference to one or more components, features, details, structures, and / or methods according to the present disclosure, are intended to convey that the described component, feature, detail, structure, and / or method is an illustrative, non-exclusive example of components, features, details, structures, and / or methods according to the present disclosure. Thus, the described component, feature, detail, structure, and / or method is not intended to be limiting, required, or exclusive / exhaustive; and other components, features, details, structures, and / or methods, including structurally and / or functionally similar and / or equivalent components, features, details, structures, and / or methods, are also within the scope of the present disclosure.

[0106] The term “comprising” is intended to be open and permits but does not require the inclusion of additional elements or steps. When the term “comprising” is used herein, the term “consisting of” is thus also encompassed and disclosed. Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or subrange within the stated ranges in different embodiments, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. Where the term “about” is used, it is understood to reflect + / - 10% of the recited value. In addition, it is to be understood that any particular embodiment that falls within the prior art may be explicitly excluded from any one or more of the claims. Since such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein.

[0107] In addition, it is to be understood that any particular embodiment of the present disclosure that falls within the prior art may be explicitly excluded from any one or more ofthe claims. Since such embodiments are deemed to be known to those of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiments of compositions disclosed herein can be excluded from any one or more claims, for any reason, whether or not related to the existence of prior art.

[0108] All cited sources, for example, references, publications, databases, database entries, and art cited herein, are incorporated into this application by reference, even if not expressly stated in the citation. In case of conflicting statements of a cited source and the instant application, the statement in the instant application shall control.

Claims

CLAIMS1. A solid-state assay method for detecting and quantifying a PFAS compound or a mixture of PFAS compounds in a sample, the method comprising: providing a solid cyclodextrin polymer or a cyclodextrin polymer adsorbed on or linked to a substrate; contacting an indicator molecule with the solid cyclodextrin polymer or the cyclodextrin polymer adsorbed on or covalently linked to the substrate to form a complex; contacting the complex with the sample and measuring a parameter of the indicator molecule, wherein the parameter is correlated with an extent of displacement of the indicator molecule from the solid cyclodextrin polymer or the cyclodextrin polymer adsorbed on or covalently linked to the substrate upon binding of the PFAS compound to the solid cyclodextrin polymer or the cyclodextrin polymer adsorbed on or covalently linked to the substrate, thereby indicating presence and quantity of the PFAS compound or the mixture of PFAS compounds in the sample.

2. The method of claim 1 , wherein the solid cyclodextrin polymer or the cyclodextrin polymer adsorbed on or covalently linked to the substrate is formed from p- cyclodextrin.

3. The method of claim 2, wherein the - cyclodextrin comprises a non-hydrogen substituent.

4. The method of claim 3, wherein the non-hydrogen substituent is selected from the group consisting of: hydroxypropyl, acetyl, azido, hydroxyamino, quaternary amine and amino.

5. The method of any one of claims 1 to 4, wherein the p-cyclodextrin polymer comprises a linker.

6. The method of claim 5, wherein the linker is a cyclic ether or a hydrophobic linker.

7. The method of claim 6, wherein the cyclic ether is epichlorohydrin or a substituted or unsubstituted oxacyclopropane, oxacyclobutane, oxacyclopentane, oxacyclohexane or furan.

8. The method of claim 5, wherein the linker is a hydrophilic linker.

9. The method of claim 8, wherein the hydrophilic linker comprises a carbamate crosslinked by diisocyanates, a carbonate, an ester or an ether.

10. The method of any one of claims 1 to 9, wherein the indicator molecule is a colorimetric dye molecule or a fluorescent dye molecule.

11. The method of claim 10, wherein the fluorescent dye molecule is a cyanine dye, a xanthene dye, a pyrene dye, or a naphthalene dye.

12. The method of claim 11 , wherein the naphthalene dye is 8-anilinonaphthalene-1- sulfonic acid (ANS) or bis-8-anilinonaphthalene-1-sulfonic acid (bis-ANS).

13. The method of claim 10, wherein the colorimetric dye molecule is Acid Blue 120 or Acid Black 24.

14. The method of any one of claims 1 to 13, wherein the substrate is paper, cellulose, nitrocellulose, glass, quantum dot, or metal nanoparticles.

15. The method of claim 14, wherein the metal nanoparticles are gold, silver or iron.

16. The method of claim 14 or 15, wherein the substrate is paper, cellulose or nitrocellulose and is provided as a disposable stick or is contained within a housing for a lateral flow assay.

17. The method of any one of claims 1 to 16, further comprising providing a scavenger molecule for sequestering one or more interferents present in the sample by treatment of the sample separately prior to contacting the indicator molecule with the solid cyclodextrin polymer.

18. The method of claim 17, wherein the interferents are compounds having a hydrocarbon chain of a length greater than 6 carbons.

19. The method of claim 18, wherein the interferents are hydrocarbons or surfactants.

20. The method of any one of claims 17 to 19, wherein the scavenger molecule is solid polymeric a-cyclodextrin adsorbed on or linked to a second substrate or soluble monomeric a-cyclodextrin.21 . The method of claim 20, wherein the soluble monomeric a-cyclodextrin is adsorbed on or linked to a second substrate.

22. The method of claim 20, wherein the second substrate is provided within a filter.

23. A kit for preparing an assay mixture for detecting and quantifying one or more PFAS compounds in a sample, the kit comprising a mixture of assay components, the mixture comprising: a solid cyclodextrin polymer or a cyclodextrin polymer adsorbed on or linked to a substrate and an indicator molecule bound to the solid cyclodextrin polymer or the cyclodextrin polymer adsorbed or covalently linked to a substrate which is displaced by a PFAS compound.

24. The kit of claim 23, wherein the cyclodextrin polymer is p- cyclodextrin polymer.

25. The kit of claim 23 or 24, wherein the - cyclodextrin polymer comprises a nonhydrogen substituent.

26. The kit of claim 23, wherein the non-hydrogen substituent is selected from the group consisting of: 2-hydroxypropyl, acetyl, amino and quaternary amine.

27. The kit of any one of claims 23 to 26, wherein the mixture further comprises a scavenger molecule for scavenging interferents in the sample.

28. The kit of claim 27, wherein the scavenger molecule is solid polymeric a- cyclodextrin adsorbed on or linked to a second substrate or soluble monomeric a- cyclodextrin adsorbed on or linked to a second substrate.

29. The kit of claim 28, wherein the second substrate is provided within a filter.

30. An inline process sampling sensor system for detection of PFAS in a process flow, the sampling sensor system comprising:a branch conduit extending from a process flow conduit, the branch conduit configured to deliver a sample of the process flow to a sensor, the sensor comprising: a housing containing a solid cyclodextrin polymer or a cyclodextrin polymer adsorbed on or covalently linked to a substrate with an indicator molecule bound to the cyclodextrin polymer; and a detector for measuring a parameter of the indicator molecule, wherein the parameter is correlated with an extent of displacement of the indicator molecule from the solid cyclodextrin polymer or the cyclodextrin polymer adsorbed on or covalently linked to the substrate upon binding of the PFAS compound to the cyclodextrin, thereby indicating presence and quantity of the PFAS in the sample.

31. The inline process sampling sensor of claim 30, wherein the detector is a fluorescence detector for detecting a fluorescent indicator molecule or a spectrophotometer for detecting absorbance of a colorimetric indicator molecule.

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