Conductive and redox-active molecularly imprinted polymer for quantification of perfluoro alkyl substances, method of making and using

Abstract

The present disclosure provides a molecularly imprinted polymer (MIP) selective for a perfluoroalkyl and polyfluoroalkyl substance (PFAS), an article comprising such an MIP, methods of making the same, and methods of using the same. The MIP comprises moieties of a monomer having a first portion for an electrically conductive polymer chain and a second portion comprising a N-oxyl side group for redox activity. The MIP is obtained through polymerization of the monomer in the presence of the specific PFAS as a template. The MIP is configured to be selective to the PFAS for a measurement of a concentration of the PFAS.

Description

DOCKET NO.: E8144-00097 CONDUCTIVE AND REDOX-ACTIVE MOLECULARLY IMPRINTED POLYMER FOR QUANTIFICATION OF PERFLUOROALKYL SUBSTANCES, METHOD OF MAKING AND METHOD OF USING THE SAME PRIORITY CLAIM AND CROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 560,874, filed March 4, 2024, which application is expressly incorporated by reference herein in its entirety. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under 1R01ES032671-01 awarded by the U.S. National Institute of Health (NIH) and SERDP ER23-3593 awarded by the U.S. Department of Defense (DoD) though the Strategic Environmental Research and Development Program. The government has certain rights in the invention. FIELD OF THE INVENTION

[0003] The disclosure relates to detection and accurate measurement of a chemical generally. More particularly, the disclosed subject matter relates to a composition, a product comprising the same, and a method for directly detecting and measuring a per- or perfluoroalkyl substance (PFAS). BACKGROUND

[0004] Per- and polyfluoroalkyl substances (PFAS), also known as “forever chemicals,” have recently risen to the forefront of environmental issues. PFAS are a group of man-made chemicals which are potentially toxic and bioaccumulate in the bloodstream of most humans and animals throughout the world. PFAS have been shown to disrupt the normal endocrine activity, reduce immune function, accumulate in the liver, and have been shown to cause developmental DM2\20971811.1DOCKET NO.: E8144-00097 problems in rodent offspring exposed in the womb. PFAS may comprise a mixture of substances, most of which are anionic, but may also contain nonionic, zwitterionic, and cationic fluoroalkyl species. PFAS have unique chemical properties as being both hydrophobic and oleophobic and may form a separate phase in water or oil mixtures.

[0005] PFAS are a suite of ionizable synthetic organofluorine surfactants that have been widely used in various industrial and consumer applications over the past few decades (e.g., fire- fighting foam, fabrics, and paper goods). Some PFAS are highly resistant to environmental degradation (i.e., highly recalcitrant), bioaccumulative, and toxic. As a result, the U.S. Environmental Protection Agency has set maximum contaminant levels (MCLs) for six PFAS in drinking water, with the MCLs for perfluorooctanesulfonic acid (PFOS) and perfluorooctanoicacid (PFOA) at 4 ng L–1 each as of April 2024. Reports have indicated that PFAS are present inthe drinking water of 200 million Americans.

[0006] However, technologies that can rapidly and accurately detect ultralow PFAS concentrations in the environment are lacking. The current gold standard of PFAS detection relies on expensive instrumentation i.e., liquid chromatography with triple quadrupole tandem mass spectrometry (LC-MS / MS), which requires specialized operator training and labor- intensive sample processing, and is cost-prohibitive (e.g., hundreds of dollars per sample).

[0007] Consequently, optical and electrochemical PFAS sensors have been developed, but their selectivity remains problematic. This aspect is particularly important given that many interfering ions co-exist with PFAS in water. Concerns over the high cost of Surface Enhanced Raman Spectroscopy (SERS) substrates and the interference between PFAS signals and its fluorescence peaks remain. Furthermore, all existing platforms fail to address the urgent need for rapid and simultaneous quantification of multiple PFAS.

[0008] A method of directly and rapidly measuring the existence and the concentration of PFAS is needed. SUMMARY

[0009] The present disclosure provides a molecularly imprinted polymer (MIP) selective for one or more perfluoroalkyl and polyfluoroalkyl substances (PFAS), an article comprising such an MIP, methods of making the same, and methods of using the same. 2 DM2\20971811.1DOCKET NO.: E8144-00097

[0010] In accordance with some embodiments, such a molecularly imprinted polymer (MIP) comprises moieties of a monomer having a first portion for an electrically conductive polymer chain and a second portion comprising a N-oxyl side group for redox activity. The MIP is configured to be selective to the specific PFAS for a measurement of a concentration of the PFAS. The MIP is obtained by polymerizing the monomer in the presence of the PFAS as a template. The template will be removed post-polymerization, resulting in an MIP with cavities and / or some fingerprinting structures related to the specific PFAS as the template for the polymerization.

[0011] The monomer may be any suitable monomer as described herein. In some embodiments, the first portion of the monomer comprises 3,4-ethylenedioxythiophene or any other base units for a conductive polymer.

[0012] In some embodiments, the second portion comprises a monocyclic or a bicyclic structure as described herein. In some embodiments, the second portion comprises piperidinyloxy. For example, in some embodiments, the monomer is 3,4- ethylenedioxythiophene-2,2,6,6-tetramethylpiperidinyloxy (EDOT-TEMPO) monomer.

[0013] One or more PFAS used as a template and to be tested can be any PFAS. Examples of PFAS include, but are not limited to, any of 40 PFAS listed in the EPA draft method 1633 as shown in Table 1 or any other PFAS to be added.

[0014] In another aspect, the present disclosure also provides an article comprising the MIP as described herein. For example, the article is an electrode. The MIP is coated on a substrate of the article.

[0015] In another aspect, the present disclosure also provides a method of making the MIP as described herein and a method of making the article as described herein. Such a method comprises steps of providing the monomer and polymerizing the monomer in the presence of the PFAS as a template. In some embodiments, the monomer is provided through a condensation reaction between a first precursor for the first portion and a second precursor for the second portion. Electrochemical polymerization of the monomer may be used to make the MIP. After the polymerization, the PFAS can be removed, for example, by washing with water or a suitable solvent.

[0016] In another aspect, the present disclosure also provides a method of detecting and quantifying the concentration of the PFAS using the MIP as described herein. The PFAS to be DM2\20971811.1DOCKET NO.: E8144-00097 tested is the same as the PFAS used as a template in the polymerization of the monomer during the synthesis of the MIP. For example, such a method includes a step of measuring current or current density using an article coated with the MIP. The change in the current density such as that of anodic current peak compared to a control using the MIP without PFAS is calculated. Before a measurement, a standard curve (or calibration curve) is established between PFAS at different concentrations and changes in current response. The change in the current or current density versus different concentrations of one specific PFAS species is in a linear relationship.

[0017] This method does not need an expensive instrument such as a LC-MS / MS. A potentiostat can be used, and the testing can be performed on-site. The potentiostat can be portable. Such a method has high sensitivity and selectivity. The method can be tailored for any specific PFAS based on the choice of the PFAS as the template.

[0018] In another aspect, the present disclosure also provides a testing kit and a method of using the same for directly detecting the existence and measuring the concentration of a perfluoroalkyl substance (PFAS).

[0019] In another aspect, the present disclosure also provides a device comprising an array of MIP-coated electrodes. A different PFAS is used a template for each MIP. Each electrode is coated with a different MIP to quantify the respective PFAS. The device is configured to detect and quantify concentrations of multiple PFAS simultaneously by connected to a multi-channel potentiostat. The present disclosure also provides a scheme for making the device. The method comprises assembling the array of MIP-coated electrodes. The present disclosure also provides a method of using the device for detecting and measuring the concentrations of multiple PFAS. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The present disclosure is best understood from the following detailed description when read in conjunction with the accompanying drawings. It is emphasized that, according to common practice, the various features of the drawings are not necessarily to scale. On the contrary, the dimensions of the various features are arbitrarily expanded or reduced for clarity. Like reference numerals denote like features throughout specification and drawings.

[0021] FIG.1 is a schematic illustration of PFOA occupying MIP's cavity. DM2\20971811.1DOCKET NO.: E8144-00097

[0022] FIG.2 illustrates the proton-blocking mechanism by PFOA, which hinders the conversion of the redox-active moieties of MIP (i.e., TEMPOH / TEMPO+) and thus results in a decrease of MIP's current response.

[0023] FIG.3 shows1H nuclear magnetic resonance (NMR) of 3,4- ethylenedioxythiophene-2,2,6,6-tetramethylpiperidinyloxy (EDOT-TEMPO) monomer, which was prepared through the esterification reaction between 3,4- ethylenedioxythiophene hydroxymethyl (EDOT-MeOH) and 4-carboxy-2,2,6,6-tetramethyl piperidinyloxy (TEMPO- COOH).

[0024] FIG.4 shows Fourier transform infrared (FTIR) spectroscopy of the EDOT- TEMPO monomer compared to those of the reactants including EDOT-MeOH and TEMPO- COOH.

[0025] FIG.5 shows the CV scans of bare glassy carbon electrode, afterelectropolymerization, after template removal, and after exposure to 4.14×10-4 g L-1 PFOA forpoly (3,4-ethylenedioxythiophene-2,2,6,6-tetramethylpiperidinyloxy) (PEDOT-TEMPO) molecularly imprinted polymer (PEDOT-TEMPO-MIP).

[0026] FIG.6 shows the CV scans of bare glassy carbon electrode, afterelectropolymerization, after water-washing, and after exposure to 4.14×10-4 g L-1 PFOA forPEDOT-TEMPO non-molecularly imprinted polymers (PEDOT-TEMPO-NIP).

[0027] FIG.7 shows a calibration curve of the current density decreases at the anodicpeak of TEMPO (y-axis) vs. PFOA concentrations ranging from 0.4×10-9 to 4.14×10-4 g L-1 (x-axis).

[0028] FIG.8 shows the relative changes in the current density of TEMPO's anodic peak at 0.87 V over 5 cycles of washing (template removal) and rebinding (when exposed to 4.14×10-9g L-1 PFOA). Results from the washing and rebinding cycles are abbreviated as RX and WX,where X represents the number of cycles.

[0029] FIG.9 shows the percentage of decrease in current density at the anodic peak of TEMPO of PEDOT-TEMPO-MIP after rebinding with (i) a single PFOA, PFBA, PFOS or 6:2FTAB (each at 4.14×10-4 g L-1) in DI water, (ii) a mixture of PFOA / PFBA, PFOA / PFOS orPFOA / 6:2 FTAB (each at 4.14×10-4 g L-1) in DI water, and (iii) spiked a known concentration ofPFOA (4.14×10-4 g L-1) into the surface water sample for 240 minutes.5 DM2\20971811.1DOCKET NO.: E8144-00097

[0030] FIG.10 shows calibration curves of the current density decrease at the cathodic peak (y-axis) of EDOT-TEMPO MIPs made separately using three PFAS, including PFOA, PFBA, and PFOS, as template versus the corresponding PFAS concentrations (x-axis). Differential pulse voltammetry (DPV) was employed for PFAS quantification and to minimize the contribution of non-Faradaic current to the electrode response.

[0031] FIGS.11A-11C show the reusability and robustness of the MIP sensors including the EDOT-TEMPO MIPs made separately using three PFAS, including PFOA, PFBA, and PFOS, as a template, over multiple cycles of washing (template removal) and rebinding (whenexposed to (A) PFOA (4.14 × 10-10 g L ¹), (B) PFBA (2.14 × 10-10 g L ¹) and (C) PFOS (5 × 10-10 g L ¹) over 2 minutes). Results from the washing and rebinding cycles are abbreviated as RXand WX, where X represents the number of cycles.

[0032] FIG.12 shows the selectivity of the EDOT-TEMPO MIPs made separately using three PFAS, including PFOA, PFBA, and PFOS, as template and used for the corresponding PFAS.

[0033] FIG.13 illustrates an exemplary device comprising a MIP sensor array having a plurality of channels for multi-PFAS detections and measurements. DETAILED DESCRIPTION

[0034] For purposes of the description hereinafter, it is to be understood that the embodiments described below may assume alternative variations and embodiments. It is also to be understood that the specific articles, compositions, and / or processes described herein are exemplary and should not be considered as limiting.

[0035] In the present disclosure the singular forms “a,” “an,” and “the” include the plural reference, and reference to a particular numerical value includes at least that particular value, unless the context clearly indicates otherwise. Thus, for example, a reference to “a nano structure” is a reference to one or more of such structures and equivalents thereof known to those skilled in the art, and so forth. When values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment. As used herein, “about X” (where X is a numerical value) preferably refers to ±10% of the recited value, inclusive. For example, the phrase “about 8” preferably refers to a value of 7.2 to DM2\20971811.1DOCKET NO.: E8144-00097 8.8, inclusive; as another example, the phrase “about 8%” preferably (but not always) refers to a value of 7.2% to 8.8%, inclusive. Where present, all ranges are inclusive and combinable. For example, when a range of “1 to 5” is recited, the recited range should be construed as including ranges “1 to 4”, “1 to 3”, “1-2”, “1-2 & 4-5”, “1-3 & 5”, “2-5”, and the like. In addition, when a list of alternatives is positively provided, such listing can be interpreted to mean that any of the alternatives may be excluded, e.g., by a negative limitation in the claims. For example, when a range of “1 to 5” is recited, the recited range may be construed as including situations whereby any of 1, 2, 3, 4, or 5 are negatively excluded; thus, a recitation of “1 to 5” may be construed as “1 and 3-5, but not 2”, or simply “wherein 2 is not included.” It is intended that any component, element, attribute, or step that is positively recited herein may be explicitly excluded in the claims, whether such components, elements, attributes, or steps are listed as alternatives or whether they are recited in isolation.

[0036] Unless specifically indicated otherwise, the term “conductive polymer” used herein refers to a polymer that is electrically conductive. Such a conductive polymer may have conjugated structures in the backbone chain and is referred as an intrinsically conducting polymer. The conductive polymer may be doped for higher electrical conductivity. For example, the conductivity of a conductive polymer or a doped counterpart may have a suitable conductivity, for example, in a range of from 1x102S cm-1to 1x 105S cm-1.

[0037] N-oxyl radicals are a group of long-living free radicals. For example, TEMPO (2,2,6,6-tetramethylpiperidin-1-oxyl) is a stable free radical, which can be used as an oxidizing agent in organic synthesis. The term “N-oxyl side group” used herein refers to a group comprising a nitrogen-oxygen single bond and being in a form of an N-oxyl radical (-N-O.) or configured to provide an N-oxyl radical. The N-oxyl side group may be in a form of an N-oxyl radical or a chemical group such as -N-OH. The nitrogen atom is further connected with other structural moieties as described herein.

[0038] A method of directly and rapidly measuring the existence and the concentration of PFAS is needed. In addition, a consensus is that technologies for rapid detection of multiple PFAS at ultralow concentrations are critical but lacking.

[0039] The present disclosure provides a molecularly imprinted polymer (MIP) selective for a perfluoroalkyl and polyfluoroalkyl substance (PFAS), an article comprising such an MIP, methods of making the same, and methods of using the same. DM2\20971811.1DOCKET NO.: E8144-00097

[0040] Molecularly imprinted polymers (MIPs) offer high selectivity for PFAS due to the molecular “lock-and-key” binding mechanism, which is employed to concentrate PFAS from water samples. MIPs are cross-linked polymers synthesized in the presence of a template molecule (e.g., PFAS molecules), which is removed post-polymerization to create cavities with size, shape, and binding affinity tuned to the template used. This “lock-and-key” mechanism produces high selectivity for the template or compounds with similar physicochemical features.

[0041] MIPs are increasingly used in chemical sensing platforms to detect drug residues, organic dyes, pesticides, and pathogens. Combining MIPs with electrochemical detection has gained immense attention. However, several major obstacles exist for research translation and technology commercialization. First, existing MIPs coupled with electrochemical detection are not redox-active and thus require the addition and periodic replenishment of external redox probes (e.g., ferric / ferrocyanide, ferrocene carboxylate), adding complexity to the integration and downsizing of the sensor systems. Existing electrochemical platforms rely on external redox probes (e.g., ferric / ferrocyanide), which requires reagent replenishment, complicates their integration into the sensor platform, and limits the system’s portability. Moreover, existing platforms employ non-conductive MIPs (e.g., o-phenylenediamine), limiting electron transfer and signal transduction during subsequent measurements. Several conductive MIPs (e.g., polypyrrole and polyaniline) are not sufficiently stable in water, with the conductivity decreasing over time.

[0042] Furthermore, all existing platforms fail to address the urgent need for simultaneous quantification of multiple PFAS that often co-exist in environmental matrices.

[0043] One objective in the present disclosure is to develop a portable and highly adaptable sensor platform for rapid electrochemical detection of multiple PFAS at sub-ng L-1concentrations. To achieve the objective, the tasks include efforts to: (1) synthesize conductive and redox-active MIPs with N-oxyl moieties for individual PFAS, (2) select the best-performing MIP for individual PFAS with the highest selectivity and sensitivity, and (3) assemble MIP- microelectrode array for simultaneous quantification of multi-PFAS. This work leverages the novel PFAS sensing mechanism discovered by the inventor for direct electrochemical quantification, which enables the miniaturization of the developed sensor platform and opens the possibility of continuous monitoring. The project also highlights the seamless integration of DM2\20971811.1DOCKET NO.: E8144-00097 expertise from environmental engineering, electrochemistry, to electrical engineering that allows for innovation and convergent research.

[0044] This work in the present disclosure leverages the novel PFAS sensing mechanism for direct electrochemical quantification, which enables the miniaturization of the developed sensor platform and opens the possibility of continuous monitoring. The project also highlights the seamless integration of expertise from environmental engineering, electrochemistry, to electrical engineering that allows for innovation and convergent research. The developed sensor platform enables rapid multi-PFAS quantification, which is also highly adaptable to adding or removing PFAS analytes of interest to accommodate the fast-evolving PFAS regulatory landscape. A prototype portable sensor is described herein.

[0045] In accordance with some embodiments, a novel molecularly imprinted polymer (MIP) that is both conductive and redox-active has been developed for direct quantification of PFAS such as perfluorooctanoic acid (PFOA) via electrochemical detection. The redox property of MIP eliminates the need for the addition and replenishment of external redox probes.

[0046] The developed sensor platform also enables rapid multi-PFAS quantification, which is also highly adaptable to adding or removing PFAS analytes of interest to accommodate the fast-evolving PFAS regulatory landscape.

[0047] In accordance with some embodiments, such a molecularly imprinted polymer (MIP) comprises moieties of a monomer having a first portion for an electrically conductive polymer chain and a second portion comprising a N-oxyl side group for redox activity. The MIP has cavities imprinted by one or more specific PFAS. The cavities may have shape and / size matching with the one or more specific PFAS. The MIP is configured to be selective to the specific PFAS for a measurement of a concentration of the PFAS. The MIP is obtained by polymerizing the monomer in the presence of the one or more specific PFAS as template. The resulting MIP has cavities and / or some fingerprinting structures related to the specific PFAS as the template for the polymerization. The PFAS to be tested is the same as the PFAS used as a template in the polymerization of the monomer during the synthesis of the MIP.

[0048] The size and / or shape of each cavity depend on the type of the PFAS as the template, and the size may range from an Angstrom level to a nanometer level. In some embodiments, the size is less than 2 nanometers. DM2\20971811.1DOCKET NO.: E8144-00097

[0049] In some embodiments, the first portion of the monomer comprises 3,4- ethylenedioxythiophene or any other base units for a conductive polymer. The first portion of the monomer after polymerized provides the conductive backbone or matrix for the conductive polymer. The base unit may be optionally substituted, while the conductive polymer maintains good electrical conductivity.

[0050] In the first portion of the monomer, a precursor of the base unit for a conductive polymer, i.e., the first precursor, may be grafted with a chemical functional group for reacting with a precursor of the second portion of the monomer. In some embodiments, the functional groups may be -OH, -COOH, or other reactive group. For example, 3,4-ethylenedioxythiophene may be grated with hydroxylmethyl group. The precursor of the first portion of the monomer is 3,4- ethylenedioxythiophene hydroxymethyl abbreviated as EDOT-MeOH and having a formula (Ia):

[0051] In some embodiments, the second portion of the monomer comprises a monocyclic or a bicyclic structure. The precursor of the second portion of the monomer, i.e., the second precursor, may have a functional group, which can react with the functional group in the precursor for the first portion of the monomer. In some embodiments, the functional groups may be -COOH, -OH, or other reactive group. For example, when the precursor for the first portion has -OH, the precursor for the second portion may have -COOH so that both precursors can react with each other through an esterification reaction to provide the monomer.

[0052] Examples of a suitable second precursor for the second portion of the monomer containing an N-oxyl side group include, but are not limited to, a compound having formula (IIa), (IIb), (IIc), or any combination thereof:DM2\20971811.1DOCKET NO.: E8144-00097

[0053] In the formula (IIa), (IIb), and (IIc), Xn (n=1, 2, 3, or 4) may be selected from H, a C1-4 alkyl group such as -CH3, a substituted C1-4 alkyl group such as CF3, or any combination thereof.

[0054] In accordance with some embodiments, the monomer for the molecularly imprinted polymer (MIP) in the present disclosure is an esterification production of the compound having formula (Ia) with the compound having formula (IIa), (IIb), or (IIc). Thoiophene in the monomer will polymerize to form a backbone chain for electrical conductivity. The resulting polymer has a structure similar to poly(3,4-ethylenedioxythiophene) (PEDOT) in the backbone, except that the grafting structure from the second portion of the monomer such as that from the compound having formula (IIa), (IIb), or (IIc).

[0055] Various functional monomers containing an N-oxyl side group with variations in the numbers of -H, -CH3, -CF3 substitutes are reacted with EDOT to provide the MIP.

[0056] In some embodiments, the second portion comprises piperidinyloxy.

[0057] In some embodiments, the monomer is 3,4-ethylenedioxythiophene-2,2,6,6- tetramethylpiperidinyloxy (EDOT-TEMPO) monomer.

[0058] The exemplary reactions of synthesis of EDOT-TEMPO as an exemplary monomer and the resulting polymer are illustrated in Scheme 1: DM2\20971811.1DOCKET NO.: E8144-00097(Scheme 1)

[0059] In some embodiments, EDOT-MeOH and 4-carboxyl-TEMPO are used to make the monomer for EDOT-TEMPO polymer. One or more PFAS such as PFOA are used for the template to make the MIP. PFOA in Scheme 1 was used for illustration only. Any other PFAS can be used. The one or more PFAS are removed from the MIP, which are used to detect the presence of and measure the concentration of the specific PFAS used as the template.

[0060] Referring to Scheme 1, in some embodiments, 3,4-ethylenedioxythiophene- 2,2,6,6-tetramethylpiperidinyloxy (EDOT-TEMPO) monomer is first prepared by the esterification reaction between 3,4-ethylenedioxythiophene hydroxymethyl and 4-carboxy-2,2,6,6-tetramethyl piperidinyloxy in the presence of N-(3-dimethylaminopropyl)-N -ethylcarbodiimi dehydrochloride (EDC) and 4-(dimethylamino)pyridine under dry dichloromethane.

[0061] The PFAS can be any of the 40 PFAS listed in the EPA draft method 1633 or any other PFAS to be added to the list. More than 9,000 PFAS exist. Any of these PFAS can be tested using the MIP and the method described herein.

[0062] Examples of suitable a PFAS species include, but are not limited to, any of the following chemicals as shown in Table 1.

[0063] Table 1. DM2\20971811.1DOCKET NO.: E8144-00097DM2\20971811.1DOCKET NO.: E8144-00097

[0064] Another example of a suitable PFAS species is perfluorobutane sulfonamide (FBSA), which is neutral or in anion form. Its formula is CF3(CF2)3SO2NH2.

[0065] In some embodiments, examples of PFAS include, but are not limited to, perfluorooctanoic acid (PFOA), perfluorooctane sulfonic acid (PFOS), perfluorononanoic acid (PFNA), perfluorobutanoic acid (PFBA), perfluorohexane sulfonic acid (PFHxS), perfluoroheptanoic acid (PFHpA), perfluorodecanoic acid (PFDA), hexafluoropropylene oxide dimer acid (HFPO-DA), perfluorobutane sulfonic acid (PFBS), and any other PFAS chemicals as described herein.

[0066] A series of novel MIPs possessing redox-active N-oxyl moieties are prepared using individual PFAS templates, for example, the PFAS described herein and also shown in Table 1 and Table 2. The selectivity and sensitivity of each MIP are established. Results from the electrochemical detection are validated using the standard LC-MS / MS method. The best- performing MIP for each PFAS are coated on a microelectrode and assembled into a multi- channel microelectrode array compatible with a portable potentiostat, where the signal processing is optimized to further enhance the sensitivity. An MIP-enabled bench-scale prototype is built for rapid multi-PFAS quantification. The performance of the prototype sensor DM2\20971811.1DOCKET NO.: E8144-00097 is evaluated with PFAS-contaminated surface and groundwater samples, and assess the stability and recyclability of the MIPs.

[0067] PFAS analysis by LC-MS / MS. Seven PFAS compounds have been selected based on the EPA’s proposed National Primary Drinking Water Regulation, the occurrence of PFAS in the environment from existing literature, and the structure variations (charge, speciation, and chain length of PFAS). Integration of other PFAS will be considered pending the EPA’s regulatory outcome and the availability of PFAS in bulk quantity. All sample cleanup and analysis protocols are conducted in alignment with EPA draft Method 1633. Water samples will be passed through mixed-mode weak-anion exchange and solid-phase extraction (SPE) cartridges, followed by elution by acetonitrile with 5% ammonium hydroxide. Interfering substances in the extracts are removed by SPE and further cleaned up with powdered activated carbon. The extracts are evaporated under nitrogen gas and reconstituted for PFAS analysis by LC-MS / MS. Analytical sequences will include instrument blanks, instrument sensitivity checks, calibration verification standards, qualitative identification standards, method blanks, ongoing precision and recovery standards, and experimental samples. An Agilent 6495 LC-MS / MS is used for PFAS research and comparison.

[0068] Referring to Scheme 1, in some embodiments, 3,4-ethylenedioxythiophene- 2,2,6,6-tetramethylpiperidinyloxy (EDOT-TEMPO) monomer is first prepared by the esterification reaction between 3,4-ethylenedioxythiophene hydroxymethyl and 4-carboxy-2,2,6,6-tetramethyl piperidinyloxy in the presence of N-(3-dimethylaminopropyl)-N -ethylcarbodiimi dehydrochloride (EDC) and 4-(dimethylamino)pyridine under dry dichloromethane. The resulting monomer is purified and appear as orange color crystals.

[0069] Next, the electrochemical polymerization of the EDOT-TEMPO is performed in 0.1 mol L-1tetrabutylammonium hexafluorophosphate (TBAPF6) in DCM at a scan rate of 20 mV s-1for 10 sweep cycles on the surface of the glassy electrode under cyclic voltammetry (CV) with a potential range from 0-1.5 V. MIPs are prepared in the presence of individual PFAS, whereas NIP are synthesized under the same condition except in the absence of PFAS. After the completion of the electrochemical polymerization, the electrode is washed with a suitable solvent (e.g., methanol or deionized (DI) water) to remove the PFAS template. Electrochemical work employs a three-electrode setup following protocols (i.e., glassy carbon electrode as the working DM2\20971811.1DOCKET NO.: E8144-00097 electrode, Ag / AgCl reference electrode, and platinum wire as the counter electrode). The glassy carbon electrode coated with PEDOT-TEMPO-MIP has an apparent blue color.

[0070] In accordance with some embodiments, a novel redox-active molecularly imprinted polymer (MIP) has been developed for direct electrochemical quantification of PFAS such as perfluorooctanoic acid (PFOA), which can eliminate the need for external redox probes.

[0071] Referring to FIG.1, in some embodiments, a novel MIP was successfully prepared via the electropolymerization of 3,4-ethylenedioxythiophene-2,2,6,6- tetramethylpiperidinyloxy (EDOT-TEMPO) using PFOA as the template. The EDOT-TEMPO monomer was synthesized for electropolymerization on a glassy carbon electrode using PFOA as a template. The obtained MIP was abbreviated as PEDOT-TEMPO-MIP. A decrease in current density was observed for both the anodic and cathodic peaks of PEDOT-TEMPO-MIP when exposed to PFOA. Without bounded by any theory, it can be explained by the competition of proton between the anionic PFOA and redox-active moieties on the MIP (i.e., TEMPO: TEMPOH / TEMPO+), resulting in blockage of conversion between the oxidized and reduced forms of TEMPO. The extent of the decrease in current density showed excellent linearity withPFOA concentrations with a method detection limit of 0.28 ng L-1. The detection limit is alsocalled a limit of quantification (LoQ).

[0072] The obtained MIP also demonstrated high selectivity, stability, and recyclability. For example, PEDOT-TEMPO-MIP also exhibited high selectivity towards PFOA against its structural analog perfluorobutanoic acid (PFBA), perfluorooctanesulfonic acid (PFOS), and 6:2 fluorotelomer sulfonamide alkylbetaine (6:2 FTAB). The performance of PEDOT-TEMPO-MIP was highly reproducible, with a relative standard deviation of 6.5% after five consecutive measurements and regeneration cycles. Overall, this study in the present disclosure provides an innovative platform for rapid ex-situ PFAS quantification using redox-active MIP, which lays the groundwork for compact PFAS sensor development.

[0073] Without bounded by any theory, a novel PFAS sensing mechanism was proposed for the first time. The decrease in current intensity is attributed to the ability of PFOA to bind H+, preventing the conversion between the oxidized and reduced forms of the redox-active moieties of the MIP (i.e., TEMPOH to TEMPO+(FIG.2). The findings herein provide the intellectual foundation for direct PFAS quantification and thus eliminate the need for external DM2\20971811.1DOCKET NO.: E8144-00097 redox probes. This aspect helps simplify the component integration for the sensor platform and enables the development of portable devices.

[0074] The inventor leveraged the high conductivity and robustness of poly-3,4- ethylenedioxythiophene (PEDOT) in water and incorporated 2,2,6,6-tetramethylpiperidinyoxy (TEMPO), a redox-active N-oxyl derivative, into PEDOT to allow for direct quantification ofPFAS at sub-ng L-1 concentrations. The detection mechanism for PFOA relies on protonblocking, which hinders the conversion between TEMPOH to TEMPO+upon oxidation and the subsequent reduction (FIG.2). For example, the EDOT-TEMPO monomer was synthesized and purified. The monomer is electropolymerized to obtain the PEDOT-TEMPO-MIP using PFOA as a template. A calibration curve for the measurements was obtained. The selectivity, stability, and recyclability of PEDOT-TEMPO-MIP were assessed. The findings of this work provide an innovative platform for rapid ex-situ PFAS quantification by creating a redox-active MIP, eliminating the need for external redox probes and thus laying the groundwork for compact PFAS sensor development.

[0075] In one study, the inventor has prepared a novel molecularly imprinted polymer (MIP) that is both conductive and redox-active, which will be coupled with the electrochemical detector for direct quantification of POFA in water samples. Specially, the inventor synthesized novel MIPs deposited on the glassy carbon electrode (GCE) in the presence of PFOA as a template via. electropolymerization using voltammetry techniques. The MIP showed the oxidized and reduced peak at 0.30 and 0.18 V confirming the successful synthesis of MIP-GCE. After washing of MIP-GCE with DI water for 30 minutes, 14.1 % and 12.5 % increase in the oxidation and reduction peak was observed that confirmed the PFOA binds the active site of MIP-GCE and blocks the working electrode potential to transport the electron.

[0076] Additionally, MIP-GCE was further used to rebind the 1 mmol / L of PFOA for 30 minutes to determine the efficiency of electrodes towards PFOA rebinding, 54 % and 29 % decrease in the intensity of oxidation and reduction peak was observed which concludes that the rebinding of PFOA to the MIP-GCE film can increase the electrochemical impedance of the modified electrode. The PFOA was quantified by the reduction in the oxidation current intensity peak of MIPs polymer and decreased in current directly proportional to the concentration of POFA. Moreover, a linear dynamic range of 4.14 x 105- 0.41 ng / L with a limit of detection of 0.41 ng / L of perfluorooctanoic acid, was achieved. MIP-GCE sensor exhibited outstanding DM2\20971811.1DOCKET NO.: E8144-00097 selectivity toward its homologous structure perfluorobutanoic acid (PFBA) as well as natural organic interference such as humic acid and sodium chloride. MIP-GCE electrodes showed excellent reproducibility and reusability of an electrode with the repetition of five cycles having a residual standard deviation of 5.8 %. The MIPs sensor can be utilized for rapid and continuous screening of PFOA at different concentration levels in water and environmental samples for the field application due to its real-time, continuous, direct quantification and onsite feature.

[0077] Select the best-performing MIP for individual PFAS: To select the best- performing MIPs for individual PFAS compounds, efforts are made to assess (1) their sensitivity at a range of sub-ng L-1PFAS concentrations, (2) their selectivity toward the target PFAS versus its structural analogs, and (3) the reproducibility of these measurements. To quantify PFAS concentrations, square-wave voltammetry (SWV) and differential pulse voltammetry (DPV) methods are applied to minimize the contribution of non-Faradaic processes to the electrode response. With the same three-electrode setup, experiments are performed in 0.1 mol L-1NaClO4in water to ensure compatibility with water samples. The SWV and DPV parameters are optimized to achieve the highest sensitivity.

[0078] To determine the sensitivity of the MIP system, a solution containing PFAS (0-20 ng L-1) is added. The current density to decrease is observed in the presence of PFAS due to the proton-blocking mechanism. The ability of PFAS to compete for protons depends on the approximate pKa differences between PFAS and TEMPOH. The closer the pKa values, the stronger the formed H bond. The calibration curve of individual PFAS is constructed bymeasuring the relative changes in current density ( i) or direct charge transfer ( Q; moles of e-)in response to PFAS concentrations. The LoQ values are established for each PFAS. The selectivity of MIP is evaluated by monitoring the relative signal changes (e.g., i or Q) when they are exposed to (i) a single PFAS solution, (ii) a single PFAS solution that is not the template used for MIP synthesis, and (iii) a mixture of select PFAS solution (each at the same concentration as (i) and (ii)). To assess the reproducibility of the MIP system, PFAS bound to MIPs are removed by a suitable solvent (e.g., DI water or methanol). The same MIP is then exposed to a new PFAS solution and measured by electrochemical detection following the protocol discussed before. The same experiment is performed 3-5 times at the LoQ of each PFAS analyte to determine the relative standard deviation of the measurements. The differences DM2\20971811.1DOCKET NO.: E8144-00097 in signal changes among samples are compared. The results are validated by LC-MS / MS following the EPA draft method 1633.

[0079] In some embodiments, the medium containing a contaminant is soil or water. The products and the methods described herein are used to test PFAS in contaminated environments such as soil and water and then make them free of harmful contaminants.

[0080] In another aspect, the present disclosure also provides an article comprising the MIP as described herein. For example, the article is an electrode or a sensor. The MIP is coated on a substrate of the article.

[0081] In another aspect, the present disclosure also provides a method of making the MIP as described herein and a method of making the article as described herein. Such a method comprises steps of providing the monomer and polymerizing the monomer in the presence of the PFAS as a template. In some embodiments, the monomer is provided through a condensation reaction between a first precursor for the first portion and a second precursor for the second portion. Electrochemical polymerization of the monomer may be used to make the MIP. After the polymerization, the PFAS can be removed, for example, by water washing.

[0082] In another aspect, the present disclosure also provides a method of testing a concentration of the PFAS using the MIP as described herein. For example, such a method includes a step of measuring current density using an article coated with the MIP. The PFAS to be tested is the same as the PFAS used as a template in the polymerization of the monomer during the synthesis of the MIP. For example, such a method includes a step of measuring current density using an article coated with the MIP. The change in the current density such as that of anodic peak compared to a control using the MIP without PFAS is calculated. Before a measurement, a standard curve (or calibration curve) is established using the PFAS at different concentrations. The change in the current density versus different concentrations of one specific PFAS species is in a linear relationship.

[0083] This method does not need an expensive instrument such as LC-MS. A potentiostat can be used, and the testing can be performed on site. Such a method has high sensitivity and selectivity. The method can be tailored for any specific PFAS based on the choice of the PFAS as the template. DM2\20971811.1DOCKET NO.: E8144-00097

[0084] In another aspect, the present disclosure also provides a testing kit and a method of using the same for directly detecting the existence and measuring the concentration of a perfluoroalkyl substance (PFAS).

[0085] Assemble MIP-microelectrode array for simultaneous quantification of multiple PFAS. As described herein, the portable potentiostat or a device comprising an array of sensor are developed for rapid multi-PFAS quantification that is affordable and user-friendly, while still delivering the high-level performance of traditional benchtop instruments for multiplexed electrochemical measurements suited for field use. Specifically, the best-performing MIPs synthesized for individual PFAS are assembled into a microelectrode array, resulting in a multi- channel signal output with recorded current density over time. Each channel helps us differentiate PFAS species, whereas the current decrease enables the quantification of the specific PFAS concentration. A compact potentiostat will be designed. The potentiostat is capable of voltammetric sensing with a bandwidth of approximately 1 kHz. This device leverages an integrated electrochemical measurement system on a chip, specifically, the Analog Devices AD5941, which houses all the necessary components for precise, low-noise voltammetric measurements. This chip is mounted onto a tailor-made printed circuit board (PCB) that includes additional circuits for control and power management. The system is managed by a microcontroller through a serial-peripheral interface, and it features Bluetooth low-energy connectivity for wireless control and data retrieval via a smartphone application. Both 12-bit and 6-bit digital-to-analog converters are used to regulate the voltage bias applied to the reference and working electrodes. The design employs a three-electrode potentiostat configuration where signal of the sensor is amplified and then captured by a 16-bit analog-to- digital converter (ADC). To refine the signal, the inventor uses on-chip digital signal processing (DSP) for low-pass filtering and data decimation, yielding a clean, 150-Hz output that the microcontroller can easily handle. This on-chip DSP reduces the need for extensive post- processing and lessens the amount of data that needs to be transmitted, enhancing the system’s resource and power usage efficiency. One of the goals is to implement a system that can accurately generate and measure a broad spectrum of current signals, ranging from less than 1 nA to 3 mA, capable of up to multiple simultaneous measurements.

[0086] EXAMPLES

[0087] 1. Materials and Methods DM2\20971811.1DOCKET NO.: E8144-00097

[0088] 1.1. Chemicals, Material Characterization, and Chemical Analysis

[0089] 3,4-ethylenedioxythiophene hydroxymethyl (EDOT-MeOH, 95%), 4-carboxy- 2,2,6,6-tetra methylpiperidinyloxy (TEMPO-COOH, 97%), 4-(dimethylamino)pyridine(DMAP, 99%), N- (3-dimethylaminopropyl)-N -ethylcarbodiimide hydrochloride (EDC,97%) and anhydrous dichloromethane (DCM, 99%), were obtained from TCI (Tokyo, Japan) and used as received. Sodium chloride (NaCl, 99.5%) was purchased from Fisher Scientific (Pittsburgh, PA) and used as received. Perfluorooctanoic acid (PFOA, 97%), perfluorobutanoic acid (PFBA, 98%), Perfluorooctanesulfonic acid solution (PFOS, 97%), dry tetrabutylammonium hexafluorophosphate (TBAPF6, 99%), phenylhydrazine (97%), anhydrous magnesium sulfate (MgSO4, 99.7%), hexane (99.9%, HPLC Grade), dichloromethane (99.9%, HPLC Grade), and ethyl acetate (99.9%, HPLC Grade) were purchased from Sigma-Aldrich (Milwaukee, MI) and used as received.6:2 Fluorotelomer sulfonamide alkylbetaine (6:2 FTAB, 97%) was obtained from TRC (Connecticut, USA). Suwannee River natural organic matter (SRNOM) was purchased from the International Humic Substances Society (IHSS, MN, USA). Deionized (DI) water was purified by a Mili-Q-plus purificationsystem ( 18.2 M cm at 25 °C; Millipore Sigma, MO, USA). Surface water (SW) was collectedfrom a pond (latitude and longitude of 40.310329 and 74.644513°, respectively) in West Windsor Township, New Jersey.

[0090] Material Characterization: 1H nuclear magnetic resonance (NMR) spectra were recorded for EDOT-TEMPO monomer at 25 °C on a JEOL ECZ400S, 400 MHz spectrometer using deuterated chloroform as a solvent and tetramethyl silane as an internal standard. Fourier Transform Infrared Spectroscopy measurements (FTIR) were recorded on a Perkin Elmer 1600 Series FTIR Spectrophotometer. The spectrum was collected in 32 scans with a resolution of 4cm 1 in the 4000-400 cm-1 range.

[0091] Chemical Analysis: The non-purgeable organic carbon (NPOC) was analyzed with a TOC analyzer (TOC-L, Shimadzu, Japan). The cations (Na+, K+, Mg+, and Ca+) and anions (Cl- and SO42-were analyzed with an ion chromatography (IC) coupled with a conductivity detector using a Shodex YS-50 column (for cations) or Shodex SI-524E column (for anion) (Showa Denko, Tokyo, Japan), respectively. The turbidity and conductivity of the surface water sample were measured on a turbidity meter (2100Q, HACH) and a conductivity meter (HQ40d, HACH), respectively. DM2\20971811.1DOCKET NO.: E8144-00097

[0092] Standard PFAS analysis, other than the PFAS analysis technique developed in the present disclosure, was performed on an Agilent 6470A triple quadrupole LC / MS (LC / QQQ- MS) system C18 column (Agilent poroshell 120 EC, 50 × 3 mm, 1.8 m) with the mobile phases of solvent A (5 mM ammonium acetate in distilled water) and B (5 mM ammonium acetate in 100% methanol). The oven temperature was set at 40 °C. The injection volume of each samplewas 5 L with a flow rate of 0.5 ml min-1. Prior to analysis, all samples were filtered by 0.22- mPES filters with 1-mL syringes into polypropylene 1.5-mL autosampler vials (ThermoFisher) with polypropylene caps. An Agilent 6470A triple quadrupole LC- / MS / MS system was used to detect quantify the concentrations of EPA 537 standards, EPA 533 standards, and intermediates (C2 C7) based on USEPA Method 533 following mass calibration and initial calibration. Analytical sequences will include instrument blanks, instrument sensitivity checks, calibration verification standards, qualitative identification standards, method blanks, ongoing precision and recovery standards (OPRs), and experimental samples. A C18 column (Agilent poroshell 120 EC, 50 × 3 mm, 1.8 m) was used for separation at 40 °C using a mobile phase of solvent A (5 mM ammonium acetate in distilled water) and B (5 mM ammonium acetate in 100% methanol).The injection volume of each sample is 5 L with a flow rate of 0.5 ml min-1.

[0093] The EPA 533 standard (500 ppb) (Lot No.537PDSLR11021) purchased from Wellington laboratories were diluted with 100% methanol to, 100 ppt, 250 ppt, 500 ppt, 1 ppb, 2.5 ppb, 5 ppb, 10 ppb, 25 ppb and 50 ppb, respectively, to establish the calibration curve.

[0094] 1.2. Preparation of MIPs.

[0095] Synthesis and purification of 3,4-ethylenedioxythiophene-2,2,6,6- tetramethylpiperidinyloxy (EDOT-TEMPO) monomer:

[0096] Referring to Scheme 1, the EDOT-TEMPO monomer was prepared through the esterification reaction between 3,4- ethylenedioxythiophene hydroxymethyl (EDOT-MeOH) and 4-carboxy-2,2,6,6-tetramethyl piperidinyloxy (TEMPO-COOH) in the presence of N-(3-dimethylaminopropyl)-N -ethylcarbodiimidehydrochloride (EDC) and 4- (dimethylamino)pyridine under dry dichloromethane (DCM). TEMPO-COOH (1.4 mmol, 280 mg,) EDOT- MeOH (2.18 mmol, 380 mg), and DMAP (1.34 mmol, 17 mg) were dissolved in 10 mL of dry DCM in a 100 mL round bottom flask for 5 minutes under stirring at 0 ºC. After 5 min, EDC (1.52 mmol, 310 mg) was dissolved in 4 mL of dry DCM and added dropwise into the round bottom flask at 0ºC under stirring. The reactor was then warmed up to room temperature and DM2\20971811.1DOCKET NO.: E8144-00097 stirred for 72 h. After 72 h, the reaction solution was diluted with 20 mL DCM, and the organic phase was washed with NaCl (40 mL) and water (80 mL), then dried over anhydrous MgSO4 and filtered using 11 m Whatman paper. The filtrate was purified by column chromatography (SiO2, n-hexane / ethyl acetate 1:3 volume ratio) and followed by recrystallization in cold n- hexane. The obtained EDOT-TEMPO monomer appeared as orange color crystals. For 1H NMR analysis, phenylhydrazine was added to reduce the nitroxide free radical to its hydroxylamine derivative.

[0097] Preparation of poly-3,4-ethylenedioxythiophene-2,2,6,6- tetramethylpiperidinyloxy MIP (PEDOT-TEMPO-MIP) and non-molecularly imprinted polymers (PEDOT-TEMPO-NIP):

[0098] PEDOT-TEMPO-MIP and PEDOT-TEMPO-NIP were prepared by the electrochemical polymerization of the EDOT-TEMPO monomer. The reaction scheme with PFAS as a template for MIP was shown in Scheme 1. The glassy carbon electrode was first polished using 0.3 and 0.05 m alumina slurry and washed using deionized water for 5 min under a sonication bath. A standard three-electrode configuration was employed with a 3 mm glassy carbon working electrode (BASi, IN, USA), a coiled platinum wire counter electrode (BASi, IN, USA), and an Ag / AgCl reference electrode in 3 M KCl (BASi, IN, USA). Allpotentials were applied against Ag / AgCl reference electrode in 0.1 mol L-1 TBAPF6 DCMsolution.

[0099] A CH Instruments 630C potentiostat (Austin, TX) was used for electrochemical polymerization and PFOA quantification. Specifically, the electrochemical polymerization ofthe EDOT-TEMPO (1 mmol L-1) was performed in 0.1 mol L-1 TBAPF6 in DCM at a scan rateof 20 mV s-1 for 10 sweep cycles on the surface of the glassy electrode under cyclic voltammetry(CV) with a potential range from 0-1.5 V.

[0100] MIPs were prepared in the presence of 1 mmol L-1 of PFOA, whereas NIP wassynthesized under the same condition except in the absence of PFOA. After the completion of the electrochemical polymerization, the electrode was washed with deionized water to remove the PFOA template. In this experiment, DI water was chosen for PFOA template removal because organic solvents such as methanol, ethanol, and acetone can dissolve PEDOT-TEMPO- MIP. Moreover, the use of DI for template removal and MIP regeneration is also cost-effective compared to other technologies where organic solvents are often employed. DM2\20971811.1DOCKET NO.: E8144-00097

[0101] 1.3. Electrochemical Measurement

[0102] All potentials were reported relative to Ag / AgCl reference electrode. The cyclic voltammetry (CV) scans of glassy carbon electrode, PEDOT-TEMPO-MIP, and PEDOT-TEMPO-NIP were collected at potential range of 0.0-1.5 V with scan rate of 20 mV s-1 indichloromethane (DCM) solution containing 0.1 mol L-1 tetrabutylammoniumhexafluorophosphate (TBAPF6) as electrolyte. The rebinding of PFOA was conducted by exposing PEDOT-TEMPO-MIP to different PFOA concentrations (i.e., 0.41×10-9– 4.14×10-4g L-1) in DI water. Afterward, the PEDOT-TEMPO-MIP was transferred back to theelectrochemical cell for CV scans in DCM solution containing TBAPF6to investigate the impact of PFOA rebinding on the electrochemical signal.

[0103] Three cycles of CV scans were conducted to obtain a stable electrode response. The average value from the second and the third scans was used for each measurement. The baseline current density (i0) of PEDOT-TEMPO-MIP was taken at the anodic peak of TEMPO after the PFOA template removal whereas i was recorded after exposed to PFOA in DI water (also referred to as the “rebinding process”). A slight shift was noticed in the anodic peak (from 0.90 V to 0.81 V) and cathodic peak (0.71 V to 0.63 V) of PEDOT-TEMPO-MIP during the experiments; this is consistent with the literature for conductive polymers.

[0104] Due to the larger decrease in the current density of the anodic peak of TEMPO compared to its cathodic peak, the anodic peak was chosen for further analysis. The changes in current density (i.e., i = i – i0) were plotted against PFOA concentrations for the calibration curve. Each PFOA concentration was measured in triplicate.

[0105] 2. Results

[0106] 2.1. Characterization of EDOT-TEMPO monomer.

[0107] The successful synthesis of EDOT-TEMPO monomer was indicated by its orange color crystal and bynuclear magnetic resonance (NMR) and Fourier transform infrared (FTIR) spectroscopy (FIGS.3-4). As shown in FIG, 3, the1H NMR spectrum of EDOT- TEMPO shows two distinctive resonance peaks at 4.6 and 4.2 ppm corresponding to the methylene protons adjacent to the carbonyl carbon bonded to the ester oxygen (-CH2-COO-), suggesting the successful esterification of EDOT-MeOH and TEMPO-COOH.

[0108] The resonance at 1.2, 1.4, 1.8, and 2.6 ppm represent TEMPO-COOH backbone protons, whereas the resonances at 6.4 and 3.9 ppm correspond to EDOT-MeOH backbone DM2\20971811.1DOCKET NO.: E8144-00097 protons. The resonance peaks at 6.8 and 7.15 ppm (shown with a cross in FIG.3) represent the phenylhydrazine protons, which were used to reduce nitroxide free radical to its respective hydroxyl derivative.

[0109] The FTIR results further support these observations (FIG. 4). Specifically, thevibrational stretching peak shift from 1690 cm-1 to 1732 cm 1 corresponds to the conversion ofthe carboxylic carbonyl to the ester carbonyl. The disappearance of the hydroxyl peak at 3200cm 1 indicates the successful esterification of EDOT-MeOH and TEMPO-COOH.

[0110] 2.3. Preparation of PEDOT-TEMPO-MIP and PEDOT-TEMPO-NIP.

[0111] The CV scans were collected during the electropolymerization of PEDOT- TEMPO-MIP with PFOA. A slight shift was observed in the anodic peak of EDOT from 1.2 V to 1.4 V and from 0.86 V to 0.90 V for TEMPO over ten scans. The slight shift was attributed to the increased resistivity between the working electrode and counter electrode following the growth of PEDOT-TEMPO-MIP film. The increased current density following electropolymerization can be explained by the increased pseudocapacitance of the film by incorporating more redox-active moieties into the MIP. The glassy carbon electrode coated with the PEDOT-TEMPO-MIP shows an apparent blue color.

[0112] Two characteristic peaks were observed in the CV scans of PEDOT-TEMPO-MIP (FIG.5): the anodic and cathodic peaks appeared at 0.86 V and 0.69 V, respectively, corresponding to the redox-active TEMPO (i.e., TEMPO+, and TEMPOH). The peaks at 0.30 V and 0.18 V suggested the anodic and cathodic peaks of the PEDOT backbone. After the electropolymerization, PEDOT-TEMPO-MIP was washed with DI water to remove the PFOA template. It was observed an increase of 21.1% and 18.5% in current density at the anodic and cathodic peaks of PEDOT-TEMPO-MIP, respectively (FIG.5). When the PEDOT-TEMPO-MIP was exposed to 4.14×10-4 g L-1 PFOA in DI during the rebinding, a decrease of 40% and34% in current density was observed for the anodic and cathodic peaks of PEDOT-TEMPO- MIP, respectively (FIG.5). We postulate that the observed decrease in current density at TEMPO’s anodic and cathodic peaks in the presence of PFOA can be attributed to the interaction between PFOA and TEMPO moieties. Specifically, upon oxidation, the anionic PFOA (pKa=0.5- 3.8) may compete for protons with TEMPO+, interfering with the proton transfer to TEMPO+. As a result, the conversion from TEMPO+to TEMPOH is hindered, causing a decrease in current density for both anodic and cathodic peaks. DM2\20971811.1DOCKET NO.: E8144-00097

[0113] PEDOT-TEMPO-NIP was prepared under the same experimental conditions as PEDOT-TEMPO-MIP, but in the absence of PFOA. The CV scan for PEDOT-TEMPO-NIP showed CV characteristics similar to those of PEDOT-TEMPO-MIP (FIG.6). Instead of removing PFOA template using water, water-washing step was also used for comparaison. However, the current density after water-washing or rebinding with PFOA (FIG. 6) exhibited negligible changes at both the anodic and cathodic peaks of TEMPO, suggesting the lack of PFOA molecular imprinting cavities in PEDOT-TEMPO-NIP.

[0114] 2.2. Electrochemical measurements of PFOA and construction of calibration curve.

[0115] To determine the optimal rebinding time for PFOA quantification, time-dependent rebinding experiments were first performed by exposing PEDOT-TEMPO-MIP to two differentPFOA concentrations (i.e., 0.41×10-9 and 4.14×10-4 g L-1). Table 2 shows the decrease in currentdensity of the anodic peak of TEMPO during PFOA rebinding at different time intervals for different PFOA concentrations.

[0116] At the higher PFOA concentration (i.e., 4.14×10-4 g L-1), it was observed adecrease in current density by 37% to 85% from 30 min to 180 min, which plateaued off around 240 min as shown in Table 2. The current density decreased to a lesser extent at the lower PFOAconcentration (i.e., 0.41×10-9 g L-1). 240 min was chosen for all subsequent experiments forconsistency. It is worth noting that a shorter time could be used for PFOA detection if needed. For instance, we observed a current density decrease of 17.1±2.4% and 25.5±3.2% at 2 and 5min, respectively, at a 4.14×10-4 g L-1 PFOA concentration (Table 3).

[0117] Table 2.DM2\20971811.1DOCKET NO.: E8144-00097

[0118] The calibration curve of PFOA was constructed by obtaining i from the anodicpeak of TEMPO after rebinding with PFOA at a series of concentrations (i.e., 0.41×10-9 g L-1 to4.14×10-4 g L-1). FIG. 7 shows a calibration curve of the current density decreases at the anodicpeak of TEMPO (y-axis) vs. PFOA concentrations ranging from 0.4×10-9 to 4.14×10-4 g L-1 (x-axis). The decrease in current density (i ) of the anodic peak of TEMPO was observed after beingexposed to PFOA in a concentration range of 4.14×10-4 g L-1 to 4.14×10-9 g L-1 for 5 minutes.The time of 5 minutes can be changed to any other suitable time period such as 2 minutes. i0represents the current density at the anodic peak of TEMPO after template removal, i.e., the MIP without PFOA. The current density was recorded from the CV scans of PEDOT-TEMPO-MIP after being exposed to PFOA for 240 minutes. The regression analysis indicates a good linear relationship between the i and the corresponding PFOA concentrations. The error bar at each point was derived from triplicate measurements. The linear regression is y = 0.7528x – 0.1564 (R2= 0.9806).

[0119] A method detection limit (MDL) of 0.28×10-9 g L-1 was calculated for thePEDOT-TEMPO-MIP from seven replicates following an EPA standard method 1633. The MDL was calculated using the standard deviation (SD) of seven replicates at the lowest PFOAconcentration used for the rebinding experiments (i.e., 4.14×10-10 g L-1 and 4.14×10-9 g L-1)following the EPA standard method. Specifically, the standard deviation of the measured concentrations of seven replicates were determined using the calibration curve (i.e., Figure 2, R2=0.98), and the MDL was calculated following the equation: MDL = ( -1,1 =0.99) SD, where MDL represents the method detection limit, t(n-1,1- =0.99)represents Student’s t-value for a single DM2\20971811.1DOCKET NO.: E8144-00097 tailed 99thpercentile at the degrees of freedom of 6, and SD represents the standard deviation derived from 7 replicates.

[0120] The results suggest that as PFOA concentrations increase, more PFOA molecules were able to block proton transfer to TEMPO+moieties, hindering the conversion between TEMPO+ / TEMPOH, and thereby lowering the current density of the anodic peak of TEMPO.

[0121] 2.3. Reproducibility, stability, and selectivity of PEDOT-TEMPO-MIP.

[0122] The reproducibility of the PEDOT-TEMPO-MIP was assessed by evaluating the relative change in the current density of the anodic peak of TEMPO at 0.87 V during five successive measurements of the same PFOA sample (FIG.8). The relative standard deviation (RSD) of the five measurements was 6.5%. The current density ranged from -116 μA to -108 μA when PFOA was bound to the PEDOT-TEMPO-MIP. After removing PFOA, a consistent increase was observed in current density to the range of -125 μA to -117 μA. The results as shown in FIG.8 suggest that the PEDOT-TEMPO-MIP exhibited good stability for repeated use. For example, the MIP can be reused for at least 6-8 times.

[0123] Reproducibility was also evaluated and confirmed through measurement of PFOA (4.14×10-9g L-1) using three PEDOT-TEMPO-MIP electrodes.

[0124] The selectivity of PEDOT-TEMPO-MIP was evaluated by monitoring the current density decrease of the anodic peak of TEMPO at 0.87 V when exposed to (i) a single PFOAsolutiona single non-PFOA solution, namely, perfluorobutanoic acid(PFBA), PFOS, or 6:2 fluorotelomer sulfonamide alkylbetaine (6:2 FTAB) solution (4.14×10-4g L-1), and (iii) a mixture of PFOA / PFBA, PFOA / PFOS, or PFOA / 6:2FTAB solution (each at4.14×10-4 g L-1).

[0125] FIG.9 shows the percentage of decrease in current density at the anodic peak of TEMPO of PEDOT-TEMPO-MIP after rebinding with (i) a single PFOA, PFBA, PFOS or 6:2FTAB (each at 4.14×10-4 g L-1) in DI water, (ii) a mixture of PFOA / PFBA, PFOA / PFOS orPFOA / 6:2 FTAB (each at 4.14×10-4 g L-1) in DI water, and (iii) spiked a known concentration ofPFOA (4.14×10-4 g L-1) into the surface water sample for 240 minutes.

[0126] As shown in FIG.9, the current density decreased by 40.5±2% when PEDOT- TEMPO-MIP was exposed to a single PFOA solution. By contrast, the current density was only reduced by 3.0±0.6%, 5.5±0.9%, and 3.1±0.8% in the presence of a single PFBA, PFOS, or 6:2 FTAB solution, suggesting that neither compound was able to effectively block the proton DM2\20971811.1DOCKET NO.: E8144-00097 transfer and hinder the subsequent conversion between TEMPO+and TEMPOH. Without bound by any theory, this might be explained by the larger difference of pKa values between PFBA (pKa = -0.2-0.7), PFOS (pKa = -6.0-0.14), 6:2 FTAB (pKa = 9.2-11.1) and TEMPOH (pKa = 5.5- 6.2) compared to PFOA (pKa =0.5-3.8). The closer proximity of pKa values of two species, the stronger the formed H-bond. When PEDOT-TEMPO-MIP was exposed to a mixture of PFOA / PFBA, PFOA / PFOS, and PFOA / 6:2 FTAB, the current density decreased by 34.6±2.8%, 41.1±2.4% and 35.7±2.6%, respectively. The lesser extent of current density decrease compared to the single PFOA scenario (40.5±2%) can be attributed to that some of the non-template molecules (e.g., PFBA, PFOS, or 6:2 FTAB) may occupy the MIP cavity but may not be effective at blocking the proton transfer to TEMPO+compared to PFOA.

[0127] The performance of the PEDOT-TEMPO-MIP in a surface water sample was also evaluated. The chemical composition is provided in Table 3. The surface water contains a7.3±0.4 mg L-1 non-purgeable organic carbon (NPOC) and 7.8±0.1 mg L-1 chloride anion. Theabsence of PFOA in DI water and the collected surface water sample was confirmed by LC- MS / MS. To understand the impact of water matrices on the PFOA quantification using PEDOT-TEMPO-MIP, a known concentration of PFOA (4.14×10-4 g L-1) was spiked into the surfacewater sample. The relative current density change of the anionic peak of TEMPO after rebinding was monitored following the same protocol. It was observed a current density decrease (FIG. 9) by 22±2.8% in surface water sample, notably lower than the 40.5±2% reduction when MIP was exposed to same concentration of PFOA in DI water. This can be explained by interfering molecules (e.g., anions and cations) occupying MIP cavities but could not hinder H+transfer between TEMPO+and TEMPOH due to lack of ability to form H-bond.

[0128] Table 3DM2\20971811.1DOCKET NO.: E8144-00097

[0129] 2.4. Multiple-PFAS Detection and Related Sensor / Device

[0130] Referring to FIG.10, three molecularly imprinted polymers (MIPs) were obtained separately via electropolymerization of 3,4-ethylenedioxythiophene-2,2,6,6- tetramethylpiperidinyloxy (EDOT-TEMPO) in the presence of three different PFAS templates. These PFAS templates include perfluorooctanoic acid (PFOA), perfluorobutanoic acid (PFBA), and perfluorooctanesulfonic acid (PFOS). The same three-electrode setup as described wasapplied, except that sodium perchlorate (NaClO , 0.1 M) was used during PFAS measurements toensure the device’s compatibility with water samples. To increase the sensor’s sensitivity, differential pulse voltammetry (DPV) was employed to minimize the contribution of non- Faradaic current to the electrode response, with the following parameters: a potential range of 0.4–0.85 V, a potential increment of 0.005 V, a modulation amplitude of 0.025 V, and a pulse width of 0.05 seconds. For DPV measurements other than CV measurements, the cathodic (other than anodic) current change was monitored.

[0131] As shown in FIG.10, the calibration curves for three PFAS demonstrated good linearity between the relative changes in current and PFAS concentrations (ranged from 10-12to 10-8g L-1) with a 2-minute detection time. The calculated limits of quantification (LoQ) were three orders of magnitude lower than the proposed EPA MCLs for PFOA, PFOS, and PFBA, respectively (~10-12g L-1).

[0132] Referring to FIGS. 11A-11C, reusability and robustness of a sensor comprising one of the three MIP made using PFOA, PFBA, and PFOS as a template were evaluated. To determine the reusability and robustness of MIP sensors, PFAS bound to MIPs were removed by deionized water. The same MIP was then exposed to a new PFAS solution at the same DM2\20971811.1DOCKET NO.: E8144-00097 concentration (i.e., 41.4 ng L-1PFOA, 21.4 ng L-1PFBA, and 50 ng L-1PFOS) of and measured by electrochemical detection. The same experiment was repeated 6-10 times. The results suggested that the same MIP electrode can be repeatedly used for at least 6-8 times with good stability and reproducibility.

[0133] Referring to FIG.12, the selectivity of three MIPs was evaluated by monitoring the relative signal changes (i.e., i) when exposed to a single PFOA, including PFBA, PFOS,PFHxS, HFPO-DA, or 6:2 FTAB solution (41.4 ng L 1) with a 2-min detection time. All threeMIPs were highly responsive to their respective PFAS templates with a low level of cross- sensitivity to PFAS structural analogs (i.e., < 4%). The MIP imprinted with PFOA as a template and with PFOA removed is highly selective to PFOA, compared to other PFAS. The MIP imprinted with PFBA as a template and with PFBA removed is highly selective to PFBA. The MIP imprinted with PFOS as a template and with PFOS removed is highly selective to PFOS.

[0134] Using the sensors comprising the three different MIPs, the concentrations of PFAS (i.e., PFOA, PFBA, and PFOS) in a surface water sample impacted by PFAS were measured. The results were compared with the values obtained using LC-MS / MS following the EPA method 1633. Table 4 shows the measured PFAS concentrations by the sensors including the MIPs as described and LC-MS / MS in the surface water sample. The results show reasonable agreements.

[0135] Table 4.

[0136] Referring to FIG.13, an exemplary device or system 100 comprises an array of MIP sensors having a plurality of channels for multi-PFAS detections and measurements. The exemplary device or system 100 comprises a plurality of sensors 10, each of which comprises a microelectrode and a MIP as described herein and imprinted by a specific PFAS as a template during the polymerization. The template is removed after the polymerization and before the MIP or the sensor is used for detecting the corresponding PFAS. Each MIP are coated on a microelectrode. As illustrated in FIG.13, the plurality of sensors 10 may including eight sensors DM2\20971811.1DOCKET NO.: E8144-00097 from 10a to 10h, which include MIP imprinted by and used for different corresponding PFAS. The eight PFAS may include PFBA, PFOA, PFBS, HFPO-DA, FBSA, PFOS, 6:2 FTAB, and 6:2 FtSAam. The number of sensors and PFAS in FIG.13 is for illustration only. The number can be any suitable integer in a range of from 1 to 40.

[0137] The exemplary device or system 100 further comprises a support 20, which is configured to the plurality of sensors 10. Each sensor may be reversibly removable from the support 20.

[0138] The exemplary device or system 100 further comprises a potentipstat 30, which may be portable and includes a display window 32 (or screen) and control buttons 34. The control buttons 34 are configured to provide instructions when pressed by an operator. The display window 32 is configured to show the testing data for each PFAS. The MIPs for each PFAS are coated on a corresponding microelectrode and assembled into a multi-channel microelectrode array compatible with the portable potentiostat. The potentipstat 30 is configured to collect and process the signals from each electrode. In some embodiments, the data from the potentipstat 30 may be downloaded or sent to a computer for further analysis.

[0139] The exemplary device or system 100 can be used to detect the presence and measure the concentrations of different PFAS. For example, depending on the number of the sensors, multiple PFAS (e.g., 1-40 PFAS) can be measured simultaneously.

[0140] 2.5. Environmental Significance

[0141] This work demonstrates the feasibility of synthesizing PEDOT-TEMPO-MIP thatcan directly quantify sub-ng L-1 PFAS concentrations electrochemically without using externalredox probes. The obtained MDL for PEDOT-TEMPO-MIP as described herein is significantly lower than reported MDL values from potentiometric, fluorescence, and calorimetric sensors forPFAS (10-5 to 10-3 g L-1) and slightly lower than reported MDLs for photoelectrochemical,electrochemical impedance spectroscopy, and Raman spectroscopy sensors (10-9 to 10-8 g L-1).The calculated MDL of PEDOT-TEMPO-MIP has been further improved by applying other electrochemical techniques, for example, differential pulse voltammetry or square wave voltammetry, to reduce background noise and enhance sensitivity (FIG.10).

[0142] Moreover, a novel PFAS sensing mechanism was elucidated for the first time. Specifically, the inventor utilized the redox-active properties of PEDOT-TEMPO-MIP, namely the conversion between TEMPOH and TEMPO+upon oxidation and subsequent reduction, to DM2\20971811.1DOCKET NO.: E8144-00097 directly quantify PFAS. The direct quantifying PFAS is significance for environmental monitoring; existing electrochemical detection of PFAS relies on external redox probes (e.g., ferric / ferrocyanide), require periodic addition and replenishment. The ability to directly quantify PFAS helps simplify sensor integration and enables development of portable devices. The reversible redox activity of TEMPO eliminates reagent replenishment and lays the groundwork for continuous monitoring, potentially revolutionizing environmental monitoring.

[0143] In addition, the ability to rapidly quantify PFAS is highly desirable in the field. For instance, the current gold-standard LC-MS / MS requires complex sample preparation and analysis protocols, which take hours to days to obtain results. The Surface-Enhanced Raman Spectroscopy typically requires hours for sample preparation and testing, which relies on a diffusion-controlled process to accumulate sufficient PFAS for optimal signal enhancement between nanoparticles. In the proposed platform of the present disclosure, PFAS detection takes a few minutes (e.g., <2 min), enabling rapid and potentially near-real-time environmental monitoring, addressing an urgent need in the field.

[0144] Although the subject matter has been described in terms of exemplary embodiments, it is not limited thereto. Rather, the appended claims should be construed broadly, to include other variants and embodiments, which may be made by those skilled in the art. DM2\20971811.1

Claims

DOCKET NO.: E8144-00097 What is claimed is:

1. A molecularly imprinted polymer (MIP), comprising moieties of a monomer having a first portion for an electrically conductive polymer chain and a second portion comprising a N- oxyl side group for redox activity, wherein the MIP has cavities imprinted by one or more perfluoroalkyl and polyfluoroalkyl substance (PFAS), and is configured to be selective to the one or more PFAS for a measurement of a concentration of the one or more PFAS.

2. The MIP of claim 1, wherein the MIP is obtained by polymerizing the monomer in the presence of the one or more PFAS as a template, and the cavities are defined by the monomer as the template.

3. The MIP of claim 2, wherein the cavities have a size and / or shape matching with the one or more PFAS.

4. The MIP of claim 1, wherein the first portion of the monomer comprises 3,4- ethylenedioxythiophene.

5. The MIP of claim 1, wherein the second portion comprises a monocyclic or a bicyclic structure.

6. The MIP of claim 1, wherein the second portion comprises piperidinyloxy.

7. The MIP of claim 1, wherein the monomer is 3,4-ethylenedioxythiophene-2,2,6,6- tetramethylpiperidinyloxy (EDOT-TEMPO) monomer.

8. The MIP of claim 1, wherein the PFAS is selected from perfluorooctanoic acid (PFOA), perfluorooctane sulfonic acid (PFOS), perfluorononanoic acid (PFNA), perfluorobutanoic acid (PFBA), perfluorohexane sulfonic acid (PFHxS), perfluoroheptanoic acid (PFHpA), perfluorodecanoic acid (PFDA), hexafluoropropylene oxide dimer acid (HFPO-DA), perfluorobutane sulfonic acid (PFBS), or any other PFAS chemicals in Table 1. DM2\20971811.1DOCKET NO.: E8144-00097 9. An article comprising the MIP of any of claims 1-8.

10. The article of claim 9, wherein the article is an electrode or a sensor.

11. The article of claim 9, wherein the MIP is coated on a conductive substrate.

12. A method of making the MIP of any of claims 1-8, comprising providing the monomer, polymerizing the monomer in the presence of the one or more PFAS as a template, and removing the one or more PFAS.

13. The method of claim 12, wherein the monomer is provided through a condensation reaction between a first precursor for the first portion and a second precursor for the second portion.

14. The method of claim 12, wherein the monomer is polymerization through electrochemical polymerization.

15. A method of detecting presence of and / or testing a concentration of the one or more PFAS, comprising measuring current density using an article coated with the MIP of any of claims 1-8.

16. The method of claim 15, further comprising calculating a change in the current density compared to a control using the MIP without any PFAS.

17. The method of claim 16, wherein the current density is that of an anodic peak.

18. The method of claim 15, further comprising establishing a standard curve of the change in the current density versus different known concentrations of the one or more PFAS, wherein the standard curve is linear.

19. A device comprising an array of MIP-coated electrodes and a potentiostat electrically connected with the array of MIP-coated electrodes, wherein each electrode comprises a different DM2\20971811.1DOCKET NO.: E8144-00097 MIP according to any of claims 1-8, and each electrode is configured to selectively measure a specific PFAS, and the device is configured to detect and measure concentrations of multiple PFAS.

20. A method for making the device of claim 19, comprising assembling the array of MIP- coated electrodes.

21. A method of using the device of claim 19, comprising detecting and measuring the concentrations of multiple PFAS. DM2\20971811.1

Images