Systems, devices, and methods for the detection of analytes in samples

Abstract

Systems, devices, and methods for the detection of analytes such as per- and polyfluoroalkyl substances (PFAS) in samples are generally described. Certain aspects of the present disclosure are related to systems for detecting analytes in samples. In some embodiments, a cartridge is used that comprises a plurality of microwells. The microwells can comprise, for example, a chamber and an optional cutout section. In some embodiments, the microwell has a volume of 0.001-1,000 pl (e.g., 0.1-100 pl), which can, for example, provide sufficient volume to detect the present of PFAS while also not being so large as to require a long amount of time before producing a result. In certain embodiments, the cartridge comprises a layer domain, which can hermetically seal the microwells. The layer domain can be, in some embodiments, configured to reseal after insertion of a needle.

Description

[0001] SYSTEMS, DEVICES, AND METHODS FOR THE DETECTION OF ANALYTES IN SAMPLES

[0002] RELATED APPLICATIONS

[0003] This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 63 / 518,653, filed August 10, 2023, and entitled “Device to Detect Per- and Polyfluoroalkyl Substances (PFAS) in Water,” which is incorporated herein by reference in its entirety for all purposes.

[0004] TECHNICAL FIELD

[0005] Systems, devices, and methods for the detection of analytes, such as per- and polyfluoroalkyl substances (PFAS), in samples are generally described.

[0006] BACKGROUND

[0007] PFAS, or per- and polyfluoroalkyl substances, are a large group of man-made chemicals that have been used since the 1940s due to their stain and water-resistant properties. They have been used in a wide variety of consumer products without much initial thought given to the potential environmental and health impacts of the chemicals. While they are now beginning to be regulated and monitored, their properties still make them a chemical of concern (Blake & Fenton, 2020). Unfortunately, the negative health impacts of PFAS were not researched until well into their production and use, as PFAS were originally considered to be inert chemicals, leading to significant use before any form of regulation was implemented. This lack of regulation coupled with their widespread use has led to measurable PFAS levels in groundwater systems and the environment throughout the world (Brusseau et al, 2020). PFAS are ingested by animals, plants, and humans directly and spread through the food chain leading to significant health problems at high enough concentrations. Even low concentrations of PFAS are problematic due to bio-accumulation coupled with a long half-life that allows concentrations to continue to increase over time in the body eventually causing health problems (OECD, Organization for Economic Co-operation and Development).

[0008] PFAS were and are used in products ranging from non-stick pans and grease resistant fast-food paper to firefighting foams and stain resistant carpets and clothes. While government regulation and company actions have limited the use of PFAS recently, there are still many applications without an alternative forcing continued PFAS use (Gluge et al, 2020). Further issues come from the fact that PFAS does not easily degrade in the environment and build up in organic systems meaning that progress on removing PFAS from use and production has a slow impact on PFAS presence in people and the environment. All of this has led to studies finding that PFAS are present at detectable levels in 98% of the adult population of the United States (Calafat et al, 2007). At low concentrations this is not much of a concern but as PFAS levels continue to build up without significant degradation the health issues associated with them are likely to get worse. As a result, the EPA (Environmental Protection Agency) has started to put more significant work into a regulatory framework for PFAS products and waste while also looking into better monitoring and management of PFAS already within the environment (EPA, 2021).

[0009] SUMMARY

[0010] This Summary introduces a selection of concepts in simplified form that are described further below in the Detailed Description. This Summary neither identifies key or essential features, nor limits the scope, of the claimed subject matter.

[0011] Systems, devices, and methods for the detection of analytes, such as per- and polyfluoroalkyl substances (PFAS), in samples are generally described. The subject matter of the present disclosure involves, in some cases, interrelated products, alternative solutions to a particular problem, and / or a plurality of different uses of one or more systems and / or articles.

[0012] Certain aspects are related to systems for detecting analytes in a sample. In some embodiments, the system comprising a cartridge, wherein the cartridge comprises: a multiplicity of microwells within a structural material, wherein each microwell comprises: a chamber; an optional cutout section; and a volume of 0.001-1,000 pl (0.001-1,000 mm3); and a layer domain above the structural material and the microwells, wherein: the layer domain seals the microwells.

[0013] Some aspects are directed to methods of detecting an analyte in a sample. In some embodiments, the method comprises injecting the sample into a microwell of a system comprising a multiplicity of microwells, and based on the presence or absence of a change in a condition related to the binding and / or reaction of the analyte to a sensing material, determining whether the analyte is present in the sample, wherein the system comprises: the multiplicity of micro wells within a structural material, wherein each micro well comprises: a chamber; an optional cutout section; and a volume of 0.001-1,000 pl (0.001-1,000 mm3); and a layer domain above the structural material and the microwells, wherein: the layer domain seals the microwells.

[0014] One aspect of the disclosure herein is a system for detecting analytes in an aqueous sample, the system comprising a cartridge, wherein the cartridge comprises: a. a multiplicity of microwells, wherein each microwell comprises: i. a cylindrical chamber having a diameter and a thickness; ii. an optional cutout section; iii. a volume of 0.1-100 pl (0.1-100 mm3); b. a glass layer, wherein the glass layer forms the bottom surface of the microwells; c. a structural layer in contact with the glass layer, wherein the structural layer has the thickness of the microwells; d. a thin layer above the structural layer and over the microwells, wherein the thin layer forms the top surface of the microwells and hermetically seals the micro wells; e. a septum layer above the thin layer, wherein the septum layer is configured to reseal after insertion of a needle; f. an outer layer above the septum layer, wherein the outer layer is rigid and comprises holes, wherein the holes are configured to guide the needle to the micro wells.

[0015] In one embodiment of the disclosed system, the analyte comprises a per- and polyfluoroalkyl substance (PF AS).

[0016] In one embodiment, the disclosed system further comprises a conjugated polymer in some or all of the multiplicity of microwells, wherein the conjugated polymer is configured to bind to PFAS.

[0017] In one embodiment of the disclosed system, the microwells comprise a cutout section, wherein the conjugated polymer is localized in the cutout section.

[0018] In one embodiment of the disclosed system, the cutout section is configured to increase the diffusion time of PFAS to the conjugated polymer,

[0019] In one embodiment, the disclosed system further comprises an optical sensor, wherein the optical sensor is configured to detect fluorescence of the polymer conjugate through the glass layer.

[0020] In one embodiment of the disclosed system, the conjugated polymer comprises a molecule with a structure selected from the group consisting of the structures shown in FIG. 4.

[0021] In one embodiment of the disclosed system, the microwells are single use.

[0022] In one embodiment of the disclosed system, the microwells are hermetically sealed.

[0023] In one embodiment of the disclosed system, the microwells 30 pm diameter with 10 nm thickness.

[0024] In one embodiment of the disclosed system, the system comprises 30-500 individual microwells fitting in a single sheet. In one embodiment, the sheet has a size 25 X 25 mm2.

[0025] In one embodiment of the disclosed system, the system is configured to deliver the water sample to a microwell by insertion of the needle through the septum layer. In one embodiment of the disclosed system, the structural layer comprises polypropylene.

[0026] In one embodiment of the disclosed system, the thin layer comprises aluminum tape.

[0027] In one embodiment of the disclosed system, epoxy adhesive binds the thin layer to the structural layer.

[0028] In one embodiment of the disclosed system, epoxy adhesive binds the glass layer to the structural layer.

[0029] In one embodiment of the disclosed system, the microwells comprise a cutout section, wherein a second glass layer separates the structural layer from the thin layer, and wherein the second glass layer forms the upper surface of the cutout section.

[0030] One aspect of the disclosure herein is a method of detecting an analyte in an aqueous sample using the disclosed system.

[0031] In one embodiment of the disclosed method, the analyte comprises a PFAS.

[0032] The following Detailed Description references the accompanying drawings which form a part of this application, and which show, by way of illustration, specific example implementations. Other implementations may be made without departing from the scope of the disclosure. Moreover, other advantages and novel features of the present disclosure will become apparent from the following detailed description of various non-limiting embodiments of the disclosure when considered in conjunction with the accompanying figures. In cases where the present specification and a document incorporated by reference include conflicting and / or inconsistent disclosure, the present specification shall control.

[0033] BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Non-limiting embodiments of the present disclosure will be described by way of example with reference to the accompanying figures, which are schematic and are not intended to be drawn to scale unless otherwise indicated. In the figures, each identical or nearly identical component illustrated is typically represented by a single numeral. For purposes of clarity, not every component is labeled in every figure, nor is every component of each embodiment of the disclosure shown where illustration is not necessary to allow those of ordinary skill in the art to understand the disclosure.

[0035] FIG. 1 shows a cartridge for detecting analytes, in accordance with some embodiments.

[0036] FIGS. 2A-2B show a microwell and a layer domain above the micro well, in accordance with some embodiments.

[0037] FIGS. 3A-3D show microwell and a layer domain comprising multiple layers, in accordance with some embodiments. FIG. 4 shows conjugated polymer structures, in accordance with some embodiments.

[0038] FIG. 5 shows a system for detecting analytes, in accordance with some embodiments.

[0039] FIG. 6 shows PFAS chemical classification, in accordance with some embodiments.

[0040] FIG. 7 shows chemical structures of the fluorous conjugated polymers (x= 0.80, y= 0.20) and a conceptual scheme of the mechanism for the detection of PFAS in water: PFOA diffusion from water to the polymer induces protonation of the pyridine moieties and triggers changes in the emission of the polymers, in accordance with some embodiments.

[0041] FIGS. 8A-8B show fluorescence spectra of thin films upon exposure to aqueous solutions of PFOA, and the fluorescence photographs of the corresponding thin films: (FIG. 8 A) PPE-Py, (FIG. 8B) PPE-Py*, in accordance with some embodiments.

[0042] FIG. 9 shows a diagram of needle -based system, in accordance with some embodiments.

[0043] FIGS. 10A-10B show graphs of fluorescence of polymer after 10 pl drop of 100 ppb PFBA, in accordance with some embodiments.

[0044] FIGS. 11A-1 IB show graphs of fluorescence of polymer after 10 pl drop of 100 ppb PFOA, in accordance with some embodiments.

[0045] FIGS. 12A-12B show graphs of fluorescence of polymer after 10 pl drop of pure water (FIG. 12A) and 1 ppb PFOA (FIG. 12B), in accordance with some embodiments.

[0046] FIG. 13 shows graph of interaction between zonyl and pyridine polymer with and without PFOA, in accordance with some embodiments.

[0047] FIG. 14 shows a graph of polymer fluorescence before and after 10 pl drop of fluid at roughly 50 seconds (first test), in accordance with some embodiments.

[0048] FIG. 15 shows a graph of polymer fluorescence before and after 10 pl drop of fluid at roughly 50 seconds (second test), in accordance with some embodiments.

[0049] FIG. 16 shows a graph of polymer fluorescence before and after 10 pl drop of fluid at roughly 50 seconds (third test), in accordance with some embodiments.

[0050] FIG. 17 shows a graph of polymer fluorescence before and after 10 pl drop of fluid at roughly 50 seconds normalized by initial fluorescence (first test), in accordance with some embodiments.

[0051] FIG. 18 shows a graph of polymer fluorescence before and after 10 pl drop of fluid at roughly 50 seconds normalized by initial fluorescence (second test), in accordance with some embodiments.

[0052] FIG. 19 shows a graph of polymer fluorescence before and after 10 pl drop of fluid at roughly 50 seconds normalized by initial fluorescence (third test), in accordance with some embodiments. FIGS. 20A-20B shows (FIG. 20A) DAPI (left peak) and FITC (right peak) filter emission spectra and (FIG. 20B) the polymer emission spectra, in accordance with some embodiments.

[0053] FIGS. 21A-21B show color ratio difference for water and PFOA (perfluorooctanic acid) samples at different concentrations (FIG. 21A), in accordance with some embodiments. Color ratio difference for the PFOA samples on a log scale (FIG. 21B). Error bars denote standard deviation, with each droplet test treated as separate measurements.

[0054] FIG. 22A-22B show color ratio difference for PFOA (perfluorooctanic acid) samples at different concentrations, with the color ratio difference of water subtracted out (Figure 22A), in accordance with some embodiments. The same plot on a log scale is shown in FIG. 22B. Error bars denote standard deviation, with each droplet test treated as separate measurements.

[0055] FIGS. 23A-23B show color ratio difference for water and PFOA (perfluorooctanic acid) samples at different concentrations (FIG. 23A), in accordance with some embodiments. Color ratio difference for the PFOA samples on a log scale is shown in FIG. 23B. Error bars denote standard deviation, with each droplet test treated as separate measurements.

[0056] FIGS. 24A-24B show color ratio difference for PFOA (perfluorooctanic acid) samples at different concentrations, with the color ratio difference of water subtracted out (FIG. 24A), in accordance with some embodiments. The same plot on a log scale is shown in FIG. 24B. Error bars denote standard deviation, with each droplet test treated as separate measurements.

[0057] FIGS. 25A-25D show images of polymer after PFOA Kapton tape drop testing taken immediately (FIG. 25A), after 1 hour (FIG. 25B), after 3 hours (FIG. 25C) and 3 days (FIG. 25D), in accordance with some embodiments.

[0058] FIGS. 26A-26D show images of polymer after PFBA Kapton tape drop testing taken immediately (FIG. 26A), after 1 hour (FIG. 26B), after 3 hours (FIG. 26C) and 3 days (FIG. 26D), in accordance with some embodiments.

[0059] FIGS. 27A-27H show diagrams of FRAP diffusion, the expected outcome from these tests.

[0060] FIG. 28 shows a graph of pixel grayscale after PFOA Kapton tape drop testing over time, in accordance with some embodiments.

[0061] FIG. 29 shows a graph of pixel grayscale after PFBA Kapton tape drop testing over time, in accordance with some embodiments.

[0062] FIGS. 30A-30C show a magnified image of comer in PFBA tests taken immediately (FIG. 30A), after 23 hours (FIG. 30B) and after 71 hours (FIG. 30C), in accordance with some embodiments. FIGS. 31A-31B show a model of sensor geometry, with a simple cylinder shown in FIG. 31A and a cylinder with cutout to reduce impact of convection shown in FIG. 3 IB, in accordance with some embodiments.

[0063] FIG. 32 shows a drawing of sensor dimensions (dimensions in mm), in accordance with some embodiments.

[0064] FIGS. 33A-33D show estimated PF AS absorption into the polymer sensor over time for the microwell design in FIG. 3 IB for some PFAS molecules, in accordance with some embodiments.

[0065] FIGS. 34A-34B show diagrams of material layers for two microwell designs, in accordance with some embodiments.

[0066] FIGS. 35A-35B show images of a fabrication setup for single section polypropylene microwells, in accordance with some embodiments.

[0067] FIGS. 36A-36B show images of current fabrication setup for delrin cartridges, in accordance with some embodiments.

[0068] FIG. 37 shows a fluid system diagram, in accordance with some embodiments.

[0069] FIGS. 38A-38D show fluid flow diagrams with FIG. 38A showing test sample intake, FIG. 38B showing test sample injection, FIG. 38C showing ethanol intake, and FIG. 38D showing ethanol rinse, in accordance with some embodiments.

[0070] FIG. 39 shows a solidworks model of system parts, with corresponding photo, in accordance with some embodiments.

[0071] FIG. 40 shows a spotted water droplet in 1 mm delrin cutout, arrow shows location of droplet, in accordance with some embodiments.

[0072] FIG. 41 shows a setup of spotting head with microscope underneath, in accordance with some embodiments.

[0073] FIG. 42 shows a diagram of microfab technologies ink-jet microdispenser, in accordance with some embodiments.

[0074] FIG. 43 shows parameters used in jetserver to spot droplets, in accordance with some embodiments.

[0075] FIG. 44 shows a fabrication protocol for a system including a microwell, in accordance with some embodiments.

[0076] FIG. 45 shows a septum layer and aluminum sheet being attached together, in accordance with some embodiments.

[0077] FIGS. 46A-46D show an assembly of a micro well with a UV-curable resin, in accordance with some embodiments. FIG. 47 shows a fibrous substrate for applying an adhesive, in accordance with some embodiments.

[0078] FIGS. 48A-48E show plots of wavelength shift ratios used to detect PFOA, in accordance with certain embodiments.

[0079] FIGS. 49A-49B are plots of fluorescent intensity as a function of time, in accordance with certain embodiments.

[0080] FIG. 49C is a plot of a ratio of fluorescent intensities as a function of time, in accordance with certain embodiments.

[0081] FIG. 49D is a plot of wavelength shift ratio as a function of PFOA concentration, in accordance with certain embodiments.

[0082] FIGS. 50A-50B are plots of fluorescent intensity as a function of time, in accordance with certain embodiments.

[0083] FIG. 50C is a plot of a ratio of fluorescent intensities as a function of time, in accordance with certain embodiments.

[0084] FIG. 50D is a plot of wavelength shift ratio as a function of PFOA concentration, in accordance with certain embodiments.

[0085] DETAILED DESCRIPTION

[0086] Systems, devices, and methods for the detection of analytes such as per- and polyfluoroalkyl substances (PF AS) in samples are generally described.

[0087] Described herein, in accordance with some embodiments, is a PFAS measurement system that uses a needle-based delivery system for inserting microliter scale sample fluids into a cartridge of many single use micro wells. The microwells are, in some embodiments, part of multilayered devices with inner surfaces designed to not interact with or adsorb PFAS that can be integrated together into a cartridge. In some embodiments, more than a hundred individual microwells fitting in a single 25 X 25 mm2sheet can be employed, allowing many tests to be done before requiring manual replacement of the cartridge. In some embodiments, the cartridges can be easily removed and replaced for ease of use in the field.

[0088] Also disclosed herein are details of the engineering research efforts in creating an initial design and prototype for a portable PFAS testing device to be used in the field for long term PFAS measurement of both drinking and groundwater.

[0089] References cited herein are listed in the attached Citations which form a part of this Specification, and the literature is incorporated by reference herein. Certain aspects of the present disclosure are related to systems for detecting analytes in samples. In some embodiments, a cartridge is used that comprises a plurality of microwells. The microwells can comprise, for example, a chamber and an optional cutout section. In some embodiments, the microwell has a volume of 0.001-1,000 pl (or, in some embodiments, 0.1-100 pl), which can, for example, provide sufficient volume to detect the presence of PF AS while also not being so large as to require a long amount of time before producing a result. In certain embodiments, the cartridge comprises a layer domain, which can seal (e.g., hermetically seal) the microwells. The layer domain can be, in some embodiments, configured to reseal after insertion of a needle.

[0090] In some embodiments in which the layer domain is included, the layer domain may seal the chamber of the microwell (e.g., such that liquid does not traverse the layer domain). Generally, a microwell is considered to be sealed if the contents of the microwell are isolated from the external environment. Each of the plurality of the microwells may contain a reagent that can be used to detect whether a particular analyte (e.g., PFAS) is present within a sample that is introduced into the microwell (e.g., via injection). In some embodiments, the system and / or methods disclosed include a conjugated polymer that can bind to analytes (e.g., PFAS) and produce a signal (e.g., fluorescent signal). The system and / or methods disclosed herein may allow the detection of analytes in different types of samples (e.g., aqueous samples).

[0091] In certain embodiments, a detector can be used to determine whether a sample that is injected into the chamber includes an analyte of interest, such as PFAS. Examples of such detectors are described in more detail below.

[0092] Some aspects of this disclosure relate to a cartridge for detecting analytes in samples. One example of such a cartridge is shown in the perspective view schematic drawing in FIG. 1. In some embodiments, the cartridge comprises a multiplicity of microwells. As schematically shown in FIG. 1, cartridge 100 has a multiplicity of microwells 102. As shown in FIG. 1, cartridge 100 comprises a 7 x 21 array of 147 microwells 102, but in other embodiments, the cartridge could contain fewer or more micro wells.

[0093] In accordance with certain embodiments, the multiplicity of microwells can be within a structural material. Examples of microwells within structural materials are shown in the cross- sectional schematic illustrations of FIGS. 2A-2B and 3A-3D. In FIGS. 2A-2B and 3A-3D, for example, microwells 102 are formed in structural material 202. Any of a variety of materials can be used as the structural material (e.g., a polymer, such as polypropylene) as will be discussed in more detail below.

[0094] In some embodiments, each micro well within the multiplicity of micro wells comprises a chamber. As shown in FIGS. 2A-2B and 3A-3D, microwell 102 comprises chamber 201. The chamber generally includes a volume that can be occupied by a fluid (e.g., a liquid) and solid boundaries (e.g., walls) around the perimeter of the volume. In FIG. 2A, for example, chamber 201 includes walls 221, 222, 223, and 224 and the volume within those walls.

[0095] In some embodiments, the chambers are adjacent to the structural material of the cartridge. For example, in some embodiments, the structural material may form one or more walls of the chambers of each microwell within the multiplicity of microwells, where the structural material can determine certain physical parameters of the chamber (e.g., shape, size). In some embodiments, the chamber within each microwell may contain a volume, where, for example, a sample can be introduced and occupy such at least a portion of such volume.

[0096] According to some embodiments, the structural material is in the form of a structural layer. For example, in FIGS. 2A-2B and FIGS. 3 A and 3C, structural material 202 is shown in the form of a layer. In some such embodiments, the chambers can be formed by molding structural material such that the structural material includes the plurality of chambers and / or by cutting material away from the structural material (e.g., using a die) to form the chambers within the structural material).

[0097] In some embodiments, the structural layer has the thickness of the microwells. For example, referring to FIGS. 2A-2B, structural material 202 and chamber 201 are both shown as having thickness 206. According to some embodiments, the microwells may have a thickness (e.g., at least 0.2 mm) that is the same or similar (e.g., within 0.5 mm) as the thickness of the structural layer. The use of a structural material in the form of a structural layer may be advantageous for any of a variety of reasons. For example, using a layer of structural material can allow for relatively easy fabrication of chambers (e.g., by punch cutting chambers into the structural material), relatively easy handling of the structural material, and / or relatively easy fabrication of the cartridge (e.g., by building up the cartridge layer by layer). In some cases, the use of a structural material in the form of a structural layer helps form a multiplicity of micro wells with uniform thicknesses.

[0098] In some embodiments, each microwell has a thickness. As seen in FIG. 2A-2B, microwell 102 can have thickness 206, which according to some embodiments, can be the thickness of structural material 202 and chamber 201. In certain embodiments, the thickness of the microwell is at least 10 pm, at least 50 pm, at least 100 pm, at least 200 pm, at least 500 pm, at least 1 mm, or at least 3 mm. In certain embodiments, the thickness of the microwell is less than or equal to 3 mm, less than or equal to 1 mm, less than or equal to 500 pm, less than or equal to 200 pm, less than or equal to 100 pm, less than or equal to 50 pm, or less than or equal to 10 pm. Combination of these ranges are possible, e.g., at least 100 pm and less than or equal to 1 mm. In some embodiments, the chamber is cylindrical. It should be understood that a cylindrical chamber may have features that are not necessarily cylindrical, which may benefit the system and / or methods disclosed. For example, a chamber may have an optional cutout section in one of its cylindrical walls but may still be a cylindrical chamber. It should also be understood that the chamber may have other shapes and / or geometries besides cylindrical, e.g., polygonal (e.g., rectangular, triangular, etc.), elliptical, etc. In some embodiments, the cartridge comprises multiple chambers that are identical in geometry. In certain embodiments, the cartridge comprises sets of multiple identical chambers of a two or more different geometries.

[0099] In some embodiments, the chamber within each microwell has a diameter. The chamber may have a particular diameter depending on many factors (e.g., volume of the sample to be contained within the microwell). According to some embodiments, the diameter of the chamber within the microwell is at least 50 pm, at least 75 pm, at least 100 pm, at least 200 pm, at least 500 pm, at least 1 mm, or at least 3 mm. According to some embodiments, the diameter of the microwell is less than or equal to 3 mm, less than or equal to 1 mm, less than or equal to 500 pm, less than or equal to 200 pm, less than or equal to 100 pm, less than or equal to 75 pm, or less than or equal to 50 pm. Combinations of these ranges are also possible.

[0100] In some embodiments, the aspect ratio of the chamber (i.e., the ratio of the largest cross- sectional dimension of the chamber (e.g., the diameter of a cylindrical chamber) to the smallest cross-sectional dimension of the chamber (e.g., the chamber thickness)) is greater than or equal to 1:1, greater than or equal to 1.1:1, greater than or equal to 1.2:1, greater than or equal to 1.3:1, greater than or equal to 1.5:1, greater than or equal to 2:1, greater than or equal to 3:1, greater than or equal to 4:1, greater than or equal to 5:1, greater than or equal to 8:1, or greater. In some embodiments, the aspect ratio of the chamber is less than or equal to 10: 1, less than or equal to 9:1, less than or equal to 8:1, less than or equal to 7:1, or less. Combinations of these ranges are also possible (e.g., greater than or equal to 1:1 and less than or equal to 10:1.

[0101] In some embodiments, the microwells comprise an optional cutout section. The phrase “cutout section” is generally used herein to refer to a region of the microwell that can hold fluid and that extends into the structural material relative to the chamber of the microwell. One example of such a microwell is shown schematically in FIG. 2B. In FIG. 2B, cutout section 205 is adjacent to structural material 202 and in fluidic communication with chamber 201. The cutout section may, in some embodiments, contain a molecule that reacts and / or binds with an analyte in the sample that is to be analyzed. Generally, the cutout section, when present, will form a contiguous volume with the chamber of the microwell, such that molecules injected into the chamber of the microwell can diffuse or otherwise be transported to the cutout section. The cutout section can have at least one dimension that is smaller than the corresponding dimension of the chamber with which it is associated.

[0102] Generally, each microwell within the cartridge will have a volume. The volume of the microwell includes both the volume of the chamber and the volume of the cutout section (if present). For example, if a cutout section is present, and if the cutout section has a volume of 0.2 microliters (pl) and the chamber has a volume of 1 pl, then the volume of the microwell would be 1.2 pl (i.e., the sum of the volumes of the chamber and the cutout section).

[0103] In some embodiments, the microwell (and / or each microwell in the cartridge) has a volume of greater than or equal to 0.001 pl, greater than or equal to 0.01 pl, greater than or equal to 0.1 pl, greater than or equal to 0.5 pl, greater than or equal to 1 pl, greater than or equal to 5 pl, greater than or equal to 10 pl, or greater than or equal to 50 pl, greater than or equal to 100 pl and / or less than or equal to 1,000 pl, less than or equal to 100 pl, less than or equal to 50 pl, less than or equal to 10 pl, less than or equal to 5 pl, less than or equal to 1 pl, less than or equal to 0.1 pl or less than or equal to 0.01 pl. Combinations of these ranges are possible. For example, in some embodiments, the microwell (and / or each of the plurality of microwells, and / or each microwell in the cartridge) can have a volume of 0.001-1,000 pl, 0.001-100 pl, 0.001-10 pl, 0.001-1 pl, 0.01-1,000 pl, 0.1-1,000 pl, 0.1-100 pl, 1-100 pl, 10-100 pl, 0.1-10 pl, 0.1-100 pl, or 0.1-1 pl. In some embodiments, it can be particularly advantageous to use microwells having volumes of at least 0.1 pl (e.g., to provide a volume large enough to inject a sample into) and less than or equal to 100 pl (e.g., to provide a diffusion profile that allows the target analyte to reach the molecule used to sense the target analyte in a reasonably short amount of time).

[0104] Without wishing to be bound by any particular theory, it is now understood that the amount of target analyte in a sample available to interact with the sensing material in a chamber increases linearly with the volume of the sample. The sample volume in turn is limited by the chamber volume. So, a larger chamber can provide a larger amount of target analyte to interact with the sensing material. On the other hand, the timescale for the analyte to diffuse to the sensing material (in the absence of convection) is proportional to the square of the characteristic chamber length scale and inversely proportional to the diffusivity of the target analyte. In addition, the signal from the sensing material can depend on factors such as the amount of the sensing material, the properties of the sensing material, and the amount or concentration of the target analyte. Therefore, it is desirable to select a chamber size that allows for diffusion of the analyte to the sensing material on a desirable timescale for the sensing measurement (e.g., 10-30 min).

[0105] In the case of conjugated polymers as the sensing material for sensing PFAS, the wavelength shift in fluorescence emission can depend on the amount of the analyte bound to the conjugated polymer per unit mass of the conjugated polymer. Therefore, it is desirable, in accordance with certain embodiments, to minimize the amount of the sensing material while ensuring that its fluorescence signal can still be read out easily. For example, it was found that a 10 nm thick conjugated polymer with lateral dimensions visible in a microscope is sufficient to detect fluorescence signal. For a given amount of sensing material, a small chamber would provide faster readout but with a smaller amount of analyte and hence a smaller shift in the fluorescence emissions at different wavelengths, but a larger chamber would require more time to read out.

[0106] In some embodiments, the microwell comprises a layer domain. The layer domain can be above the structural material and the microwell. As shown in FIGS. 2A-2B, for example, microwell 102 comprises layer domain 203 above structural material 202 and chamber 201.

[0107] It should be understood that the word “above,” when used herein to describe a first feature’ s position relative to a second feature, means that there is at least one orientation in which the first feature is located above the second feature. For example, if feature B of an article is described as being “above” feature C of the article, and feature A of the article is described as being “above” feature B and feature C of the article, that means there is at least one orientation of the article in which feature B is above feature C and feature A is above both feature B and feature C. Similarly, the word “over,” when used herein to describe a first feature’s position relative to a second feature, means that there is at least one orientation in which the first feature is located over the second feature. Moreover, while the cartridge and cartridge portions are shown in the figures as having the layer domain over the microwells which are over a bottom layer, the cartridges can be in other orientations during use (e.g., lying flat / horizontal, lying on their side / vertical, or in any other orientation).

[0108] In some embodiments, the layer domain seals the microwells. For example, in some embodiments, the layer domain provides a seal that prevents PFAS from the environment traversing the layer domain while the seal remains intact. In some embodiments, the layer domain provides a seal that prevents liquids (e.g., water or other liquids) from traversing the layer domain while the seal remains intact. In some embodiments, the layer domain provides a hermetic seal. A hermetic seal is a seal that makes a given object airtight. The seal (e.g., hermetic or otherwise) can, in accordance with certain embodiments, isolate the chamber (and, if present, the optional cutout section) from the environment, contaminants (e.g., dirt), and / or other substances that may interfere with the use of the system and / or methods disclosed herein.

[0109] In certain embodiments, the layer domain is configured to reseal after insertion of a needle. It should be understood that “reseal,” in this context, does not necessarily mean a hermetic seal is established after the resealing. The resealing can, for example, prevent PFAS from traversing the layer domain. In some embodiments, resealing of the layer domain prevents PFAS from traversing the layer domain during the measurement. In some embodiments, resealing of the layer domain prevents liquids (e.g., water or other liquids) from traversing the layer domain. In certain embodiments, resealing of the layer domain results in the formation of a hermetic seal.

[0110] In some embodiments, the layer domain can include one or more self-sealing materials such that, when a needle is inserted through the layer domain and into the chamber of the microwell and subsequently removed from the microwell, the self-sealing material reseals. In some such embodiments, the resealing of the layer domain does not reestablish a hermetic seal, but it does inhibit the leaking of liquid out of the chamber of the microwell.

[0111] The layer domain may comprise a single layer or a plurality of layers between the chamber of the microwell and the external environment. For example, as shown in FIGS . 2A- 2B, layer domain 203 includes a single layer between chamber 201 of microwell 102 and external environment 211. In FIGS. 3A-3D, on the other hand, layer domain 203 includes a plurality of layers between chamber 201 of microwell 102 and external environment 211.

[0112] According to some embodiments, the layer domain includes a single layer (or a single set of multiple, stacked layers) that covers a plurality of microwells. For example, in some embodiments, referring to FIGS. 2A-2B and 3A-3D, layer domain 203 may extend laterally over additional microwells 102 (not shown in these figures) in the cartridge. In other embodiments, Multiple layer domains may be present, each layer domain covering a subset of chambers (or a single chamber) within the cartridge.

[0113] In some embodiments, the layer domain is a multi-layer domain. The layer domain may have one or more layers for a variety of reasons and / or purposes. As a non- limiting example, one layer of the layer domain may be used to establish a seal (e.g., a hermetic seal), and a second layer of the multilayer domain may be resealable. The use of multiple layers to provide these functionalities can provide flexibility in the choice of materials used in these layers. It should be understood, however, that multi-layer layer domains are not necessarily required, and in other embodiments, a single layer can be used in the layer domain while providing the desired functionalities (e.g., seal, ability to reseal, etc.).

[0114] In some embodiments, the layer domain is above the structural layer and the microwells. As shown in FIGS. 2A-2B, layer domain 203 is above structural layer 202 and microwell 102 (even though layer domain 203 does form the top boundary of microwell 102). The layer domain may be in direct contact with the structural layer or an intermediate material may be present between the layer domain and the structural layer. For example, in some embodiments, an adhesive agent (e.g., epoxy adhesive) may be between the layer domain and the structural layer and bind the layer and structural layer together.

[0115] In some embodiments, the layer domain comprises a first layer. In some embodiments, the first layer forms the top surface of the microwell. As schematically shown in FIGS. 3A-3D, layer domain 203 comprises first layer 305, which forms the top surface of microwell 102. According to some embodiments, the first layer seals (e.g., hermetically seals) the microwell. According to some embodiments, the first layer is above the structural layer. In some embodiments in which the first layer is above the structural layer, first layer 305 is adjacent to chamber 201 and structural material 202, as shown in FIGS. 3A-3D.

[0116] In some embodiments, the layer domain is configured to block at least some electromagnetic radiation. For example, the layer domain (e.g., aluminum tape) may block electromagnetic radiation (e.g., stray visible light) from entering the microwell.

[0117] In some embodiments, the layer domain comprises metal foil and / or tape (e.g., aluminum foil and / or tape). In some embodiments, the metal foil and / or tape is used to seal (e.g., hermetically seal) the microwells. The metal foil and / or tape may be used as the first layer within the layer domain, in some embodiments. It should be understood that metal foil and / or tape (e.g., aluminum tape) is not the only material that could be employed, and in other embodiments, other metals (e.g. stainless steel), polymer-based tapes (e.g., Kapton tape, polyethylene tape, polypropylene tape, Delrin, Viton, Kalrez), and / or ceramic based layers (e.g., glass, silicon) could be used.

[0118] In some embodiments, the first layer of the layer domain is a thin layer. For example, in some embodiments, the first layer has a thickness of less than or equal to 2 millimeters, less than or equal to 1 millimeter, less than or equal to 500 microns, less than or equal to 100 microns, less than or equal to 50 microns, less than or equal to 25 microns, less than or equal to 10 microns, less than or equal to 5 microns, less than or equal to 1 micron, or less (e.g., down to 0.1 microns, 0.01 microns, or less).

[0119] In some embodiments, the layer domain comprises a septum layer. As shown in FIGS. 3A-3D, cartridge portion 103 comprises a multi-layer layer domain 203 comprising first layer 305 and septum layer 301. In some embodiments, the septum layer is configured to reseal after insertion of a needle. In some cases, resealing may be desired (e.g., to keep samples isolated from the environment after removing a portion of the sample from the microwell, to keep samples airtight, to minimize contamination, and / or to avoid spilling liquid samples).

[0120] In some embodiments, the septum layer is above the first layer. Referring back to FIGS. 3A-3D, septum layer 301 is above first layer 305. It may be advantageous to have the septum layer above the first layer, for example, to isolate microwells. For example, the needle may puncture at least a portion of the first layer to introduce and / or remove a sample from the chamber, but the first layer might not be resealable (e.g., if it is in the form of a metal foil or other non-resealable material). In some such cases, the septum layer may be made of a resealable material such that the microwell is resealed after removing the needle.

[0121] In some embodiments, the septum layer is made of a polymer. The polymer may be a synthetic polymer, natural polymer, copolymer, cross-linked polymer, or any polymer known in the art that can help the system perform its intended functions. In some embodiments, the septum layer are silicone sheets. According to some embodiments, the septum layer is free of PFAS, which can reduce potential contamination of samples. In some embodiments, the septum layer is made of a non-polymeric material (e.g., carbon foam, etc.).

[0122] In some embodiments, the layer domain has an outer layer. The outer layer may be the top layer of the layer domain. In some cases, the outer layer has a surface that is exposed to the environment. As seen in FIGS. 3A-3B, outer layer 301 is exposed to environment 211. Similarly, in FIGS. 3C-3D, outer layer 301B is exposed to environment 211.

[0123] In some embodiments, the outer layer is rigid and has holes. The use of a rigid outer layer can, for example, provide mechanical robustness to the cartridge. In some embodiments in which the outer layer has holes, the holes may be positioned over the microwells. In some embodiments, the holes are configured to guide the needle or other injector (e.g., pipettes, capillary tubes, microchannels, etc.) into the microwells. As seen in FIGS. 3C-3D, outer layer 301B can have hole 306, according to some embodiments.

[0124] In some embodiments, the outer layer is above the septum layer. For example, in FIGS. 3C-3D, outer layer 301B is above septum layer 301. In some embodiments in which the outer layer is above the septum layer, the outer layer is the top layer. In some embodiments in which this arrangement is used, the outer layer may be a rigid outer layer that provides structural support to the septum layer and thereby to the cartridge.

[0125] In some embodiments, a material from which the cartridge is formed that is between the chamber and the external environment is transparent to at least one wavelength of electromagnetic radiation (e.g., a wavelength that the sensing material within the chamber is configured to emit upon interaction with the target analyte). For example, referring to FIGS. 2A-2B and 3A-3D, in some embodiments, the material from which region 240 is formed is transparent to at least one wavelength of electromagnetic radiation (e.g., a wavelength that the sensing material within chamber 201 is configured to emit upon interaction with the target analyte). Region 240 may be, for example, a region of layer 204. Layer 204 can be formed, for example, from the same material as structural material 202, in some embodiments. For example, in some embodiments, layer 204 and structural material 202 can be monolithic and formed from the same material (e.g., a transparent polymer, glass, etc.). In other embodiments, layer 204 can be formed of a different material than structural material 202. For example, in some embodiments, layer 204 can be a glass layer and structural material 202 can be a polymer material.

[0126] In some embodiments, each microwell within the cartridge includes the same sensing material. In other embodiments, two or more microwells contain different sensing materials (e.g., more than one type of sensing material). In certain embodiments, a single microwell may contain more than one spatially separated sensing material (e.g., more than one spatially separated sensing materials arranged in a specific pattern). In some embodiments, there is a single type of sensing material in each microwell. In some embodiments, there is a first type of sensing material in a first microwell and a second type of sensing material in a second microwell. In certain embodiments, within at least one microwell, there are multiple regions of sensing material arranged in a pattern.

[0127] As shown in FIGS. 2A-2B and 3A-3D, layer 204 (e.g., a glass layer) is adjacent to chamber 201. In some embodiments, layer 204 (e.g., a glass layer) forms the bottom surface of the microwells. As shown in FIGS. 2A-2B and 3A-3D, for example, glass layer 204 forms bottom surface 222 of microwell 102. Layer 204 may provide structural support to one or more of any components (e.g., structural material and / or conjugated polymer) of the cartridge.

[0128] In some embodiments, the systems and / or methods disclosed herein involve the use of a sensing material. The sensing material can, in accordance with certain embodiments, experience a change in optical and / or electromagnetic property in response to an interaction with the target analyte. The sensing material can, for example, emit electromagnetic radiation when it binds and / or reacts with the target analyte in the sample injected into the microwell. Referring back to FIGS. 3A-3D, microwell 102 contains sensing material 303. In FIGS. 3A and FIG. 3C, sensing material 303 is within chamber 201. In FIGS. 3B and 3D, sensing material 303 is within cutout section 205. The sensing material may be positioned on (e.g., deposited on) the bottom surface of the microwell. The sensing material may be injected after the bottom and side walls of the microwell have been formed, or it may be pre-deposited on layer 204 prior to adding the side walls and layer domain to the cartridge. For example, in some embodiments, the sensing material may be printed or otherwise deposited on layer 204 (e.g., a glass layer) and, subsequently, structural material 202 and layer domain 203 may be built up around the sensing material. In some embodiments, the sensing material changes its infrared properties in response to an interaction with the target analyte. In some embodiments, the sensing material changes its ultraviolet properties in response to an interaction with the target analyte. In some embodiments, the sensing material changes its magnetic properties in response to an interaction with the target analyte. In some embodiments, the sensing material changes its Raman properties in response to an interaction with the target analyte.

[0129] In some embodiments, the interaction of the sensing material with the target analyte is mediated by a different molecule or material (for example, the fluorescence of a sensing material may change when an enzyme or molecule with an interaction with the target analyte has an interaction with the sensing material or part of the sensing material).

[0130] In some embodiments, the sensing material (e.g., conjugated polymer) is in some or all of the multiplicity of micro wells. For example, according to some embodiments, at least a portion of the microwells (e.g., at least 70%, at least 80%, at least 90%, at least 99%, or all of the microwells) may contain sensing material.

[0131] In certain embodiments, the sensing material in some or all of the multiplicity of microwells contains more than one type of sensing material. In some embodiments, the sensing material can be present as one or more sections (e.g., spots of sensing material) within a microwell. For example, in some embodiments, the microwell can contain a first spot of sensing material and a second, discrete spot of sensing material. The sensing material can include the same type of sensing material or different types of sensing material in each section. In addition, more than one type of sensing material may be present in a particular section. In some embodiments, the sensing material may have a first section of sensing material, a second section of sensing material, a third section of sensing material, etc. In some embodiments, the sections of sensing material are aggregates, crystals, glasses, segmented thin films, or the like.

[0132] In some embodiments, the sensing material is configured to interact (e.g., react and / or bind) with a target analyte in a manner that produces a detectible signal. For example, in some embodiments, the sensing material is configured to interact (e.g., react and / or bind) with a target analyte in a manner that produces electromagnetic radiation (e.g., via fluorescence). In some embodiments, the sensing material is configured to interact (e.g., react and / or bind) with PFAS to produce a detectible signal. In some embodiments, the sensing material contains an enzyme or catalyst that interacts with the target analyte to produce a signal (e.g., electromagnetic radiation).

[0133] The sensing material can be configured to exhibit any of a variety of properties and / or changes in properties upon interaction with the target analyte. In some embodiments, the property and / or change in property of the sensing material that occurs upon interaction with the target analyte is an optical and / or electromagnetic property and / or a change in an optical and / or electromagnetic property. In some embodiments, the property and / or change in property is a fluorescence and / or a change in the fluorescence of the sensing material (e.g., fluorescence and / or change in fluorescence of a conjugated polymer through the glass layer). In some embodiments, the property and / or change in property is a change in fluorescence emission intensity at one or more wavelengths. In some embodiments, the property and / or change in property is a relative change in fluorescence emission intensities between two or more wavelengths. In some embodiments, the property and / or change in property is an absorption and / or a change in absorption. In some embodiments, the property and / or change in property is a refractive index and / or a change in refractive index. In some embodiments, the property and / or change in property is a scattering of light and / or a change in scattering of light. In some embodiments, the property and / or change in property is a an isotropy and / or a change in isotropy of the sensing material (e.g., birefringence).

[0134] In some embodiments, the sensing material is configured to emit and / or absorb at least one wavelength (or all wavelengths) of electromagnetic radiation having a wavelength of from 100 nanometers to 1 millimeter). In some embodiments, the sensing material is configured to emit and / or absorb at least one wavelength (or all wavelengths) of infrared light (i.e., electromagnetic radiation having a wavelength of from 750 nanometers to 1 millimeter). In some embodiments, the sensing material is configured to emit and / or absorb at least one wavelength (or all wavelengths) of visible light (i.e., electromagnetic radiation having a wavelength of from 400 nanometers to 750 nanometers). In some embodiments, the sensing material is configured to emit and / or absorb at least one wavelength (or all wavelengths) of ultraviolet light (i.e., electromagnetic radiation having a wavelength of from 100 nanometers to 400 nanometers).

[0135] In some embodiments, the sensing material (e.g., conjugated polymer) is configured to bind to PFAS. The sensing material may bind to PFAS and produce a signal (e.g., fluorescent signal) that may be indicative of such binding. The sensing material may have a moiety that can respond (e.g., quench) in the presence of PFAS. In some embodiments, the sensing material (e.g., conjugated polymer) is configured to react with PFAS. The sensing material may react with PFAS and produce a signal (e.g., fluorescent signal) that may be indicative of such reaction.

[0136] In some embodiments, the sensing material (e.g., conjugated polymer) is localized in the optional cutout section. As shown in FIGS. 3B and 3D, sensing material 303 is localized in optional cutout section 205. Locating the sensing material within the optional cutout section may provide various advantages. In some embodiments, locating the sensing material within the cutout section may help reduce the impact of fluid convection when a sample is introduced, which can provide a predictable interaction time between introduction of the sample and detection of a signal representative of an interaction between the sensing material and the target analyte within the sample.

[0137] In some embodiments, the sensing material has a thickness. In certain embodiments, the thickness of the sensing material is at least 10 nm, at least 15 nm, at least 20 nm, at least 30 nm, at least 50 nm, at least 75 nm, at least 100 nm, at least 200 nm, at least 300 nm, at least 500 nm, or at least 1 jam. In certain embodiments, the thickness of the sensing material is less than or equal to 5 pm, less than or equal to 1 pm, less than or equal to 500 nm, less than or equal to 300 nm, less than or equal to 200 nm, less than or equal to 100 nm, less than or equal to 75 nm, less than or equal to 50 nm, less than or equal to 30 nm, less than or equal to 20 nm, or less than or equal to 10 nm. Combinations of these ranges are also possible, e.g., at least 10 nm and less than or equal to 100 nm.

[0138] In some embodiments, the cutout section is configured to increase the diffusion time of the injected target analyte to the sensing material (e.g., the conjugated polymer). It may be advantageous to increase the diffusion time of the target analyte to the sensing material, for example, to detect analytes more efficiently and / or predictably. In some embodiments, the cutout benefits the system by providing a larger time frame to perform detection of analytes after introducing a sample, relative to microwells that do not include such cutout sections.

[0139] A variety of sensing materials can be used to detect analytes. In some embodiments, the sensing material is a conjugated polymer. In some embodiments, the conjugated polymer is a conjugated polymer falling within the scope of molecules shown in FIG. 4. In some embodiments, the conjugated polymer may have the following structure: Py* wherein

[0140] In some embodiments, the conjugated polymer may have the following structure:

[0141] wherein and wherein

[0142] In some embodiments, the sensing material (e.g., conjugated polymer) has a backbone moiety comprising poly(p-phenylene ethynylene) (PPE), polyfluorene (PF), and / or fluorinated poly(p-phenylene ethynylene) (FPPE). In some embodiments, the sensing material (e.g., conjugated polymer) comprises a PFAS selector, wherein the PFAS selector comprises pyridine

[0143] (Py) having the structure: wherein the PFAS selector comprises a thiophene-functionalized pyridine (Py*) having the structure:

[0144] In some embodiments, the conjugated polymer has a moiety capable of binding to PFAS, where such moiety is pyridine and / or thiophene-functionalized pyridine. Other examples of molecules that can be used as sensing materials are described in International Patent Application No. PCT / US2023 / 082090, filed on December 1, 2023, published as International Publication No. WO 2024 / 119082 on June 6, 2024, and entitled “Compositions for Detection of Fluorocarbons and Related Articles, Systems, and Methods,” which is incorporated herein by reference in its entirety for all purposes.

[0145] In some embodiments, the systems and / or methods disclosed herein can be configured and / or used to detect an analyte (also sometimes referred to herein as a “target analyte”). In some embodiments, the analyte comprises a fluoroalkyl substance. In some embodiments, the analyte comprises a per- and polyfluoroalkyl substance (PFAS). In some embodiments, the system may include a sensing material that can bind to and / or react with PFAS, thus helping the detection of PFAS in a sample (e.g., aqueous sample). In some embodiments, binding between the PFAS and conjugated polymer may produce a signal (e.g., fluorescent signal) that is indicative of such binding.

[0146] The systems and / or methods disclosed herein can be configured to analyze one or more different types of samples. According to some embodiments, the sample is an aqueous sample. According to some embodiments, the sample comprises an organic solvent, an emulsion, a suspension, water with solid particulates (e.g., sewage), and / or a mixture thereof (e.g., water and oil).

[0147] In some embodiments, the system and / or methods are configured to distinguish between different types of analytes. For example, in some embodiments, the system and / or methods are configured to distinguish between long chain PFAS and short chain PFAS. According to some embodiments, the length of the analyte (e.g., the length of the PFAS) may impact the diffusion time of that analyte within the volume of the microwell. For example, a first type of analyte (e.g., a first type of PFAS) with a longer chain (e.g., perfluorooctanoic acid) may have a shorter diffusion time than a second type of analyte (e.g., a second type of PFAS) with a shorter chain (e.g., perfluorobutanoic acid). In some embodiments, by measuring a property of the sensing material over time, one can detect the presence of multiple analytes based on the difference in diffusion time. In certain embodiments, the system may be configured in different ways (e.g., have an optional cutout section, increase the diameter of the chamber, etc.) to enhance the ability to detect different analytes based on differences in diffusion.

[0148] The system can comprise, in some embodiments, a sensor (e.g., an optical sensor and / or electromagnetic sensor). In some embodiments, it can be particularly advantageous to use an optical sensor, which can render the system easier to use relative to other sensor types. The sensor can be configured to detect any of a variety of properties and / or changes in properties of the sensing material. In some embodiments, the property and / or change in property of the sensing material that is detected is an optical and / or electromagnetic property and / or a change in an optical and / or electromagnetic property. In some embodiments, the system comprises a sensor configured to detect fluorescence and / or a change in fluorescence of the sensing material (e.g., a fluorescence and / or change in fluorescence of a conjugated polymer through the glass layer). In some embodiments, the sensor is configured to detect a change in fluorescence emission intensity at one or more wavelengths. In some embodiments, the sensor is configured to detect a relative change in fluorescence emission intensities between two or more wavelengths. In some embodiments, the sensor is configured to detect an absorption and / or a change in absorption. In some embodiments, the sensor is configured to detect a refractive index and / or a change in refractive index. In some embodiments, the sensor is configured to detect a scattering of light and / or a change in scattering of light. In some embodiments, the sensor is configured to detect an isotropy of the sensing material and / or a change in isotropy of the sensing material (e.g., birefringence).

[0149] As shown in FIG. 5, sensor 501 has field of view 502 configured to detect and / or receive a signal (e.g., fluorescent signal) of sensing material 303 (e.g., conjugated polymer). The sensor may provide data indicative of binding and / or reaction between an analyte (e.g., PFAS) and the sensing material (e.g., conjugated polymer). The sensor may be used for a variety of purposes (e.g., detect the presence of PFAS, quantify the amount of PFAS present, etc.).

[0150] In some embodiments, the sensor is configured to detect at least one wavelength (or all wavelengths) of electromagnetic radiation having a wavelength of from 100 nanometers to 1 millimeter). In some embodiments, the sensor is configured to detect at least one wavelength (or all wavelengths) of infrared light (i.e., electromagnetic radiation having a wavelength of from 750 nanometers to 1 millimeter). In some embodiments, the sensor is configured to detect at least one wavelength (or all wavelengths) of visible light (i.e., electromagnetic radiation having a wavelength of from 400 nanometers to 750 nanometers). In some embodiments, the sensor is configured to detect at least one wavelength (or all wavelengths) of ultraviolet light (i.e., electromagnetic radiation having a wavelength of from 100 nanometers to 400 nanometers).

[0151] Any of a variety of sensors can be used in the systems described herein. In some embodiments, the sensor is camera. Other types of sensors that could be used include photo optic sensors, photodiode sensors, EMF meters, magnetic sensors, microwave sensors, and the like. The sensor can comprise, in some embodiments, a substrate that includes a surface capable of converting electromagnetic radiation (e.g., visible light) into electronic signals.

[0152] In some embodiments, the cartridge and / or the microwells are single use. For example, in some embodiments, the microwells may be used once and then may be disposed of. In some embodiments, the cartridge may have a multiplicity of microwells, where each microwell is single use.

[0153] In some embodiments in which a cutout section is included, a second glass layer separates the structural material from the layer domain. In some embodiments, a second layer forms the upper surface of the optional cutout section. As shown in FIGS. 3B and 3D, second layer 304 forms the upper surface 307 of cutout section 205. In some embodiments, the second layer is a glass layer.

[0154] In some embodiments, the system has a multiplicity of microwells. For example, the system (e.g., a cartridge for detecting analytes) may have 30-500 individual microwells. According to some embodiments, the system may have at least 1 microwell; at least 2 microwells; at least 5 microwells; at least 10 microwells; at least 100 microwells; at least 500 micro wells; at least 1,000 micro wells; or at least 5,000 micro wells. According to some embodiments, the system may have less than or equal to 10,000 microwells; less than or equal to 5,000 microwells; less than or equal to 1,000 microwells; less than or equal to 500 microwells; less than or equal to 100 microwells; less than or equal to 10 microwells; or less than or equal to 5 microwells. Combinations of these ranges are also possible (e.g., at least 2 microwells and less than or equal to 1,000 microwells).

[0155] In some embodiments, the multiplicity of wells fit in a sheet. In some embodiments, the sheet is a single sheet. In some embodiments, the single sheet of microwells has a certain size. For example, according to some embodiments, the sheet has a size of 25 X 25 mm2(or greater). The size of the sheet may depend on the number of microwells in a cartridge, the diameter of the microwells, the spacing between the microwells, among other factors. According to some embodiments, the sheet could be of a different size, for example, greater than or equal to 1 mm2(e.g., greater than or equal to 1 X 1 mm2), greater than or equal to 4 mm2(e.g., greater than or equal to 2 X 2 mm2), greater than or equal to 25 mm2(e.g., greater than or equal to 5 X 5 mm2), greater than or equal to 100 mm2(e.g., greater than or equal to 10 X 10 mm2), greater than or equal to 625 mm2(e.g., greater than or equal to 25 X 25 mm2), or greater than or equal to 2500 mm2(e.g., greater than or equal to 50 X 50 mm2). According to some embodiments, the sheet can have a size of less than or equal to 10,000 mm2(e.g., less than or equal to 100 X 100 mm2), less than or equal to 2500 mm2(e.g., less than or equal to 50 X 50 mm2), less than or equal to 625 mm2(e.g., less than or equal to 25 X 25 mm2), less than or equal to 100 mm2(e.g., less than or equal to 10 X 10 mm2), less than or equal to 25 mm2(e.g., less than or equal to 5 X 5 mm2), or less than or equal to 4 mm2(e.g., less than or equal to 2 X 2 mm2). Combinations of these ranges are also possible.

[0156] In some embodiments, the system containing a multiplicity of microwells is flexible. In some embodiments, the cartridge is or is part of a flexible belt. In some embodiments, the system containing a multiplicity of microwells comprises rigid sections attached to a flexible belt. In some embodiments, the system containing a multiplicity of microwells comprises multiple sheets containing one microwell each attached to a flexible belt. In some embodiments, system containing a multiplicity of microwells comprises multiple sheets containing multiple micro wells each attached to a flexible belt. In some embodiments, system containing a multiplicity of microwells comprises multiple sheets attached to a flexible belt in a line. In some embodiments, movement of the belt positions microwells for readout by a sensor (e.g., an optical sensor and / or electromagnetic sensor). In some embodiments, the belt is transparent to light. In some embodiments, the belt is made of a plastic (e.g., polypropylene), woven or non-woven cloth, elastomer, metal, or mesh. In some embodiments, the belt forms a closed loop. In some embodiments, the belt is open-ended. In some embodiments, the belt is configured as a mobius strip. In some embodiments, the microwells are located on both sides of the belt. In some embodiments, the microwells occupy at least 1%, at least 3%, at least 10%, at least 30%, at least 50% or at least 80% of the area of the belt. In some embodiments, the microwells are arranged in a pattern (e.g., a predetermined pattern such as a line of microwells). In some embodiments, the microwells are arranged in a rectangular array. In some embodiments, the belt is sufficiently flexible that it can be folded back on itself such that an upstream portion of the belt can contact a downstream portion of the belt without plastically deforming, fracturing, and / or cracking the belt. In some embodiments, the belt is sufficiently flexible that it can be folded back on itself such that an upstream portion of the belt can contact a downstream portion of the belt without fracturing and / or cracking the belt. In some embodiments, the belt can be flexed such that it has a radius of curvature of less than or equal to 1 meter (or less than or equal to 50 cm, less than or equal to 25 cm, less than or equal to 10 cm, less than or equal to 5 cm, less than or equal to 1 cm, or less) without plastically deforming, fracturing, and / or cracking the belt. In some embodiments, the belt can be flexed such that it has a radius of curvature of less than or equal to 1 meter (or less than or equal to 50 cm, less than or equal to 25 cm, less than or equal to 10 cm, less than or equal to 5 cm, less than or equal to 1 cm, or less) without fracturing and / or cracking the belt.

[0157] In some embodiments, the system is configured to deliver the sample to a microwell by insertion of a needle through the layer domain (e.g., through a septum layer of the layer domain). The needle may temporarily penetrate the layer domain (e.g., the septum layer) and deliver the sample. In some embodiments, at least a portion of the sample may be removed with the needle.

[0158] Any of a variety of materials can be used as the structural material (e.g., structural material 202 in FIGS. 2A-2B and 3A-3D). In some embodiments, the structural material comprises polypropylene. The structural material may also be any of a variety of other materials that can form microwells. For example, the structural material may be a hydrogel (e.g., polyethylene glycol), a polymer (e.g., Delrin, Viton, Kalrez, silicone polymer (e.g., poly dimethylsiloxane), a crosslinked polymer, a natural polymer, and / or a synthetic polymer), glass, a ceramic, metal, or combinations of these.

[0159] In some embodiments, one or more layers within the cartridge are bound by an adhesive, such as an epoxy adhesive. In some embodiments, the adhesive binds the layer domain to the structural material. Referring back to FIG. 3A-3D for example, adhesive 302 can be used to bind layer domain 203 and structural material 202.

[0160] In some embodiments, adhesive binds the bottom layer to the structural material. As shown in FIGS. 3B and 3D, for example, adhesive 302 binds layer 204 and structural material 202. It is also possible that adhesive binds more than one layer to the structural material. For example, as shown in FIGS. 3B and 3D, adhesive 302 binds both layer 204 and layer 304 to structural material 202.

[0161] In some embodiments, the adhesive is an epoxy adhesive. The epoxy adhesive may be any glycidyl and non-glycidyl epoxy polymer that can act as an adhesive. Some examples of epoxy adhesives known in the art are: hydrogenated bisphenol- A, diglycidyl ether of bisphenol- F, etc. According to certain embodiments, other adhesives may also be advantageous. A nonlimiting example of adhesives that can be used include UV-curable resins (e.g., epoxy acrylates), according to some embodiments. FIGS. 44-46D show the fabrication of a device with UV- curable resins, according to some embodiments. In some embodiments, a fibrous substrate may help spreading the adhesive across a layer. FIG. 47 shows use of a fibrous substrates (e.g., cellulous sponge) for applying the adhesive on one or more layers.

[0162] In addition to systems, methods are also disclosed herein. In some embodiments, the method comprises detecting an analyte, for example, using any of the system described above or elsewhere herein. In some embodiments, the method is a method of detecting PFAS.

[0163] In some embodiments, the method comprises detecting an analyte by injecting a sample into a microwell. After injection, the method can comprise determining whether the analyte is present in the sample. In certain embodiments, the presence of the analyte is determined based on the presence or absence of a change in a condition related to the binding and / or reaction of the analyte to a molecule. For example, according to some embodiments, the presence of PFAS can be determined based on emission wavelength-dependent fluorescent quenching arising from the binding of PFAS with a molecule.

[0164] In certain embodiments, the method comprises providing a cartridge comprising a multiplicity of micro wells. The micro wells can be, for example, the same as any of the microwells described above or elsewhere herein. In some such embodiments, multiple microwells contain a sensing material. In some embodiments, samples are injected into each of the microwells containing the sensing material. In certain embodiments, the method comprises determining whether a target analyte (e.g., a PFAS) is present in each of the samples based on the presence or absence of a condition related to the binding and / or reaction of the target analyte to or with the sensing material. In some embodiments, the determining comprises detecting the presence of emission of electromagnetic radiation of a particular wavelength or wavelengths (indicating the presence of the target analyte) or the absence of emission of electromagnetic radiation of a particular wavelength or wavelengths (indicating the absence of the target analyte).

[0165] The systems described herein can, in accordance with some embodiments, detect analytes present in the injected sample at very low concentrations. In some embodiments, the systems and / or methods described herein are capable of detecting analytes present at a concentration of as little as 10 parts per billion (ppb), as little as 1 ppb, as little as 0.1 ppb, as little as 0.01 ppb, or less. In some embodiments, the systems and / or methods described herein are capable of detecting PFAS present at a concentration of as little as 10 parts per billion (ppb), as little as 1 ppb, as little as 0.1 ppb, as little as 0.01 ppb, or less.

[0166] U.S. Provisional Patent Application No. 63 / 518,653, filed August 10, 2023, and entitled “Device to Detect Per- and Polyfluoroalkyl Substances (PFAS) in Water,” is incorporated herein by reference in its entirety for all purposes.

[0167] The following description provides examples that are intended to illustrate certain embodiments of the present disclosure, but do not exemplify the full scope of the present disclosure.

[0168] Disclosed herein are experiments into polymer validation showing methods of differentiating different types of PFAS and with tests of polymer sensitivity using fluorescence images. These experiments found limits of detection close to 0.01 ppb or better. There are multiple promising ideas to improve that sensitivity.

[0169] Research on PFAS has generally been made difficult by the large number of different molecules within this category, many of which have only been created and put in use relatively recently. They are generally split into two categories, long and short chain. This distinction comes from the number of carbon atoms within the alkyl chain (Buck et al, 2011). Two of the most commonly used PFAS, both long chain, are PFOA and PFOS and do have more significant research around them. PFOA, or perfluorooctanoic acid, is a type of long chain perfluoroalkyl carboxylic acid that was used in stick-resistant pots and pans, and originally created as a byproduct of Teflon. PFOA was one of the earlier developed PFAS and its widespread uses have caused it to still be prevalent in watersheds even to this day. This prevalence along with some high-profile health problems caused by it are why PFOA is one of the two most researched PFAS today (Buck et al, 2011). The other commonly discussed and researched PFAS is PFOS, or perfluorooctane sulfonic acid, a long chain perfluoroalkane sulfonic acid, which was used in stain resistant fabrics and some food packaging and is still used in firefighting foams. PFOS’ use in firefighting foams, which causes PFOS to directly impact the environment and watersheds along with its past use in many consumer products means that it is still quite prevalent in the environment and more researched (Peshoria et al, 2020). When discussing PFAS long chain refers to the number of carbon atoms within the PFAS chain. Longer chains tend to be more stable and are often more hydrophobic causing them to be more likely to bond to organic systems when ingested (Zeng, 2022).

[0170] PFAS are generally absorbed orally through contaminated drinking water or food products and are then distributed through the body. PFAS normally concentrates in the liver or blood plasma but can also be regularly found in the kidney or lungs. In humans, PFOA generally has a half-life of around 2 to 4 years, but this number can change significantly between individuals. Other PFAS seem to have half-lives of around this same value as well, although longer carbon chains often result in longer half-lives (Kleinman, 2021).

[0171] Within the human body increased PFOA or other PFAS concentrations has been linked to increased cholesterol levels potentially leading to increased risk for obesity in adults and children (Kleinman, 2021 and Liu et al, 2018). PFOA and PFOS along with PFHxS and PFNA, perfluorohexane sulfonate and perfluorononanoic acid respectively, have also been linked to increased susceptibility to persistent infections, especially in adolescents (Bulka et al, 2021). Multiple studies have also shown significant correlation between increased PFOA levels and kidney and testicular cancer. Scott Bartell and Veronica Veira compiled the results of various experiments on the cancer risk of PFOA in order to attempt to find a causal link as well and found that an increase of only 10 ppb in PFOS concentration present in the body leads to around a 16% increase in risk of kidney cancer and around a 3% risk of testicular cancer (Bartell and Veira, 2021). There have also been some links between PFOA and various birth defects, but the evidence is not strong and other studies have shown essentially no connection.

[0172] PFAS also has impacts on the environment with similar health impacts on animals as in humans, with additional impact on the size and body weight of smaller animals (PFAS-TOX Database, 2021). PFAS also bioaccumulates up the food chain with humans often getting increased PFAS in their blood after eating contaminated food. Fish specifically have often been found to have more significant levels of PFAS within their bodies due to the aqueous nature of PFAS transport. In a similar manner PFAS have also been found to accumulate within plants that intake contaminated water, although the health impacts on plants, if there are any, are not well researched (McCarthy et al, 2017). Based on current research PFOS is the most impactful PFAS in terms of bioaccumulation and health impacts on species in the environment with a “lowest observed adverse effect level (LOAEL) for mammals [of] 0.4 mg / kg body weight / day” (McCarthy et al, 2017).

[0173] Exposure

[0174] With such widespread use coupled with a long half-life PF AS are found all over the world and seemingly in the majority of water systems and in most people. A test done on blood samples in the United States found that the vast majority of the population has detectable levels of PFAS in their blood. This experiment was done as part of the National Health and Nutrition Examination Survey and analyzed 2,094 serum samples across multiple demographics and found measurable PFAS in more than 98% of the tested samples. They found an average concentration of 20.7 ppb with some differences based on age, sex and race (Calafat et al, 2007). Even though regulation has significantly reduced the amount of PFOS and PFOA being produced those two molecules are still the most commonly found PFAS in water systems throughout the world (Brusseau et al, 2020). For humans the main intake is through diet from eating contaminated food, with bioaccumulation, even eating only a few contaminated animals or plants can lead to significant levels of PFAS in the human body. The second most common intake is through drinking contaminated drinking water, normally from groundwater contaminated by factory or landfill runoff, or in some occasions from firefighting foam runoff. The last and by far least common intake is through dust and air from indoor spaces contaminated with enough PFAS to become airborne in noticeable quantities. This intake path is only responsible for around 1-2% of PFAS intake however, at least for PFOA and PFOS, so it isn’t as much of a concern from a health standpoint (Domingo and Nadal, 2019).

[0175] Chemistry

[0176] PFAS is a term that corresponds to a large category of man-made chemicals characterized by carbon chains populated with fluorine atoms. While there are many different chemicals and subcategories within this classification, all PFAS are highly stable molecules that are both oleo and hydrophobic. This allows them to resist stains, water and grease when in film form but also pushes PFAS to absorb into surrounding surfaces when free floating in aqueous systems and to remain there without breaking down. Despite this PFAS are still soluble in water allowing them to be transported throughout the environment. However, their hydrophobic and fluorinated nature means that PFAS builds up within organic systems such as human bodies, leading to many of the health issues discussed earlier. From a chemistry standpoint PFAS are generally split into polymer PFAS and non-polymer PFAS and then further split into subcategories as shown in FIG. 6, taken from OECD (OECD & Buck et al, 2011).

[0177] The most commonly used and manufactured PFAS are generally non-polymers, and most are perfluoroalkyl acids. PFOA and PFOS are both non-polymer PFAS and PFOA is a PFCA (perfluoroalkyl carboxylic acid) and PFOS is a PFSA (perfluoroalkane sulfonic acid). Both of these polymers are known as long chain PFAS, which is based on the number of carbon atoms within the molecule, 8 for both, although the actual cut off for long chain is 7 carbons for PFCAs and 6 for PFSAs. This cutoff is based on the material properties of the chemicals with longer chains corresponding to more stable and hydrophobic molecules meaning they will absorb into organic systems more readily and strongly while also staying there longer before eventually breaking down. Only PFCAs and PFSAs with carbon chains longer than the cutoff or precursors that can form these compounds are referred to as long chain PFAS. Other molecules are defined as short chain PFAS regardless of chain length (Buck et al, 2011).

[0178] Current and Future Regulation

[0179] In September 2021, the EPA had an advisory limit of 70 ppt for PFOA and PFOS combined in drinking water. This advisory limit is a non-regulatory and non-enforceable limit but was used as a baseline for future enforceable regulation both for states and for potential future federal regulation (EPA, 2022). Multiple states had put in place their own enforceable laws on PFAS levels in drinking water near this level. Multiple states also have separate concentration regulations for other types of PFAS or have a single regulation that combines more than just PFOA and PFOS (Paisner et al, 2022).

[0180] However, in March of 2023 the EPA released a new drinking water regulation for PFAS that would be enforceable starting in 2026. This regulation is still only proposed and is open for public comment, perhaps changing over time, but as of writing this the limit is 4 ppt of both PFOA or PFOS. There is also a hazard index for the sum of four other PFAS: PFNA, PFHxS, PFBS and Genx, which corresponds roughly to 10 ppt for all but PFBS, which is less toxic and so requires a higher concentration to become a health issue (EPA, 2023). This increased EPA regulation also corresponds with increased statewide regulation and is likely the first step of a major national effort to fully monitor and begin to cleanup PFAS in the environment, which is mirrored in Europe providing a clear need for better on site PFAS testing that can be used on a nationwide level.

[0181] Existing PFAS Testing Devices

[0182] This example focuses on the development of a fluid system that can measure PFAS levels within already existing water systems down to a concentration of 70 ppt. The goal was to create a fluid sensing system that can be operated easily and function automatically without human oversight or intervention, taking regular measurements of PFAS concentration within groundwater over a period of weeks or months. Currently testing of PFAS in water is possible but requires samples to be collected from site and then tested in a laboratory. This means that tests take days in order to get results and require significant work by skilled technicians, which increases the cost of taking measurements. Beyond that, the presence of PFAS in a large number of commonly used products means that water technicians need to take significant extra steps in order to ensure that there isn’t additional contamination from external sources before the tests can be run.

[0183] Currently the EPA describes method 537.1 and 533 for measuring PFAS levels in drinking water. Both of these methods involve using solid phase extraction, liquid chromatography, and tandem mass spectrometry in order to get accurate results. This process is both time and equipment intensive requiring trained technicians, specialized software, and large and expensive machines. Extra difficulty occurs due to PFAS’ propensity to absorb into solid surfaces, forcing tests to be done quickly and in a very consistent manner while also requiring very regular and thorough cleaning of all equipment used for the tests, usually with a methanol or ethanol rinse. All of these steps make the process more time consuming and make it easier to make mistakes that lead to a set of tests being potentially inaccurate enough to be useless. There is an extra difficulty as well, in that these tests can only be done in full laboratory settings and there simply are not many labs set up to do these kind of tests on PFAS leading to long lead times on an already time-consuming process. The difficulty with PFAS absorption is also further exacerbated during the shipping process from site to labs with a significant chance of PFAS absorbing into even glass and other shear surfaces with enough time leading to potential issues with accuracy (Shoemaker and Tettenhorst, 2018 & Wendelkon, 2019).

[0184] These issues extend beyond testing and cause significant issues collecting PFAS samples. SGS (a Swiss testing and inspection company) has a full presentation on PFAS sensing detailing steps that need to be taken by water samplers in order to ensure accurate measurements of PFAs in drinking water. These steps range from the material used to collect and store the water to the clothes that the technician can wear, even noting that the technician shouldn’t use cosmetics, sunscreen, aluminum foil, prepackaged food, or water-resistant paper (Proffitt, 2018). All of this means that even with a new interest and focus on PFAS there still is not an effective way to get accurate measurements to the desired sensitivity on a large scale. In order to do that it is important to have some form of test that can be done on site and in a short amount of time without requiring expensive equipment or complicated training to allow for a large volume of tests being used by technicians across the country. Devices to this level do not exist in general production but there are some promising prototypes that could be very effective products in the future.

[0185] A group of researchers at Curtin University have developed a portable way to measure PFOS in water. With a preconcentration period of only 5 minutes the device can measure PFOS with a concentration of 67 ppb. They were able to measure this, or close to this concentration, in not only purified water but also tap and even sea water. The main issue, however is that the measurement is done using ion-transfer voltammetry and measurements are done using the corresponding ion-transfer behavior and properties of PFOS in order to measure concentration (Viada et al, 2020). The problem is that PFAS includes a large variety of different molecules including both polymer and non-polymer materials with differing carbon chain lengths. This would likely lead to different PFAS showing different results even at the same concentration. This type of issue could potentially be dealt with through further research to better understand the properties of each PFAS, but it is unclear whether it would be possible to accurately determine what differences are due to different PFAS versus simply differences in concentration.

[0186] Another interesting portable PFAS sensor is being developed by Pacific Northwest National Laboratory (Adkisson, 2020). This device is actually significantly more sensitive, able to measure PFOA down to a level of 2 ppt. This device also uses electrical signals to measure PFOA, however in this case they are using a proprietary sponge-like material that captures PFAS rather than through ion-transfer. The water flows through multiple channels blocked with this sponge-like material that is able to absorb the majority of the PFAS in the sample. Then, once the sample is through, an electrical signal is sent through the material, whose electrical properties change based on the amount of absorbed PFAS (Adkisson, 2020). This type of measurement could still have issues with different types of PFAS resulting in different signals, but it seems less likely than relying on the ionic properties of different PFAS being similar. The main issue with this device currently is simply a seeming lack of publicly available information leading to questions about the field utility of the device.

[0187] Goal

[0188] Both of these portable devices seem very promising and with a bit more research could go a long way towards providing a small and portable way to effectively measure PFAS at low concentrations. However, along with the issues described earlier the main issue with these sensors is that they still require significant setup for only one test and still have the issues associated with PFAS sampling, leading to extra time and training required for anyone operating these devices.

[0189] In order to test PFAS in drinking water on a large scale it is desired to develop an automated process that can be set up with limited training and can then take tests regularly over time without requiring constant oversight. A fluorescent polymer sensor could take hundreds of tests over months or years with limited required oversight or human interaction. This device could then act as an initial test site to determine areas with detectable levels of PFAS, which can be flagged for more detailed analysis in a laboratory. With a relatively cheap manufacturing process it should be possible to manufacture these devices at scale for use across the country to drinking water or water treatment facilities as well as eventually into the field giving an overall sense of PFAS levels throughout the environment.

[0190] Initial Design

[0191] The overall goal of this example was to develop a portable PFAS testing device that can be used to measure PFAS at 70 ppt in groundwater both in drinking water systems and in the field without requiring regular human interaction. The device was intended to be used as an initial test to determine whether the water in question should undergo further more detailed testing.

[0192] Sensing Polymer

[0193] The polymer used for this device was a conjugated polymer. The polymers are highly fluorous and will strongly attract the alkyl chains of PFAS. The polymer system uses ’’highly efficient excited state (exciton) transport” to allow easy and fast transport of excitons through the polymer and to neighboring polymers. When in contact with PFAS, the polymer acts as a quenching site, trapping excitons that come into contact with that polymer. One exciton can sometimes come into contact with thousands of polymers until it contacts the analyte, greatly increasing the sensitivity of testing and allowing detection at ppt scale concentrations. The polymer developed for this test use “highly fluorinated polymers with poly(p-phenylene ethynylene) (PPE) and polyfluorene (PF) backbones bearing pyridine or pyridine and thiophene based selectors” (Concellon and Castro Esteban, 2023) FIG. 7 shows the chemical structure and sensing process of the polymer. The testing here used the PPE backbone polymer with both the PY and later the PY* selectors.

[0194] When trapped the exciton acts as a fluorescence quencher decreasing the overall fluorescence of the polymer and shifting the emission wavelength. These polymers can be applied in thin films and allow visual measurement of PFAS by analyzing the change in wavelength using a fluorescence camera. FIGS. 8A-8B show the wavelength response of the polymers to different concentrations of PFAS measured with spin coated polymer on glass slides using a Horiba Quanta-phi fluorescence spectrophotometer per Concellon and Castro Esteban, 2023.

[0195] Design Space

[0196] The first step in designing this device was to create a list of requirements and targets to center our design around. Shown below is the list that was developed. The requirements and targets are split based on whether the device is designed for a drinking water system or for field operation.

[0197] Table 1: Initial Design Criteria

[0198] PFAS Detection Limits

[0199] These limits are based on the EPA’s previous health advisory limits of 70 ppt. Overall, this device may be used as an initial check to determine whether PFAS are present in the water at problematic levels. If more detailed information is desired, the water can be sent to labs for more in-depth information.

[0200] PFAS Differentiation

[0201] Different PFAS have different properties and health impacts. Long versus short chain

[0202] PFAS has historically had the largest differentiation from a regulatory standpoint, so it is important to differentiate between these two categories to fit regulatory frameworks. Currently proposed EU regulations are only based on total sum of PFAS, and proposed EPA regulations differentiate between individual PFAS molecules, so changes may be required for this metric in the future.

[0203] Contamination and Measurement Requirements

[0204] Currently there is significant difficulty in sampling for PFAS as the chemicals are so ubiquitous, and if special care is not taken samples can be easily contaminated by the sampler or sampling process. This device needs to simplify the sampling requirements to make it easier to use, enabling it to be used more broadly.

[0205] Time Between Measurements

[0206] PFAS levels do not change rapidly within water systems so results can be taken further apart. However, it is still important to quickly determine whether PFAS levels in drinking water systems have reached dangerous concentrations. Thus, a middle ground of 6 hours in between tests was selected for drinking water systems while the field use system can still only take tests every one or two days.

[0207] Time In Between Maintenance and Replacement

[0208] This device may regularly measure PFAS over a long period of time. This is somewhat difficult due to PFAS’ propensity to absorb into surfaces, but the proposed time frames should allow the device to operate on a large scale without requiring significant human interaction, while also limiting the chance of cross contamination.

[0209] Personnel Training

[0210] Initial installation is likely to be complicated and have a higher chance of contaminating the device, hence the desire for a fully trained technician. After the one-time installation though it is desired for the device to be used and maintained easily by the people already working on site or nearby.

[0211] These criteria led to the creation of a general system design, which is shown and discussed in detail below. With the goal of leaving these devices in the field for long periods of time, sensor size was a very important concern, and it was desired to make each sensor chamber very small. In order to increase the lifespan of the device, it was also desired to be able to easily replace the sensor chambers once all are used while keeping the overall device in place. The first four designs are flow through systems using consistent flow and timing to control the test fluid volume. The fifth design and the one pursued in depth instead uses a defined volume to directly control the test fluid volume.

[0212] The design that was pursued is a system using needle injection rather than flow through (FIG. 9). A needle would be installed into a fluid line and would inject the test fluid into a closed off microwell with the polymer sensor. This system allows for both a very simple and very inexpensive replacement process with a cartridge of sensors able to be removed in a batch but with every other component remaining with the device. This design is also beneficial in that it does not require any microvalves to function, simply a septum covering individual microwells. This design does allow for the possibility of cross contamination, however with the needle being inserted into every sensor without a clear way to rinse it out. The main challenge of this design is that it does not allow for flow through, meaning that the total amount of fluid that can be tested needs to be very limited in order to not have the individual sensors take up too much space making the device non-portable. Testing showed that the fluorescent change of the polymer can be measured even with spin coated thicknesses of only around 10 nm, and with microliter scale fluid volumes meaning the microwells can be small enough to keep the device portable.

[0213] Main Components of Selected Design

[0214] The initial diagram of the selected design is shown in FIG. 9 and work was done to further refine and redesign the initial concept. Some components are listed below and discussed in more detail throughout.

[0215] • Fluid sampling system connected to a small needle.

[0216] • Fluorescence camera.

[0217] • Sensor cartridge platform with many single use micro wells.

[0218] • Overall stage to hold sensor cartridge and move to correct microwell for each test.

[0219] The fluid sampling system connected the test fluid system to a needle that would inject it into the microwells. The fluid system reduced the chance of cross contamination between tests and limited the amount of standing water and other forms of external PFAS contamination as well as moved the test fluid through the system to actually perform tests. The fluorescence camera was the main measurement tool, using the visual change in the polymer in response to PFAS to provide an accurate concentration measurement. The fluorescence camera did not need to provide high resolution images, simply an overall brightness level. The sensor cartridge was a replaceable tray with many single use microwells, with each microwell containing polymer for a single test. The microwells were of a very small volume to allow portability and to be able to be pierced by the needle while avoiding external PFAS contamination. The stage held the sensor cartridge in place and moved it to the desired position so that the needle could pierce the correct microwell.

[0220] Initial Polymer Response Testing

[0221] Many different tests were run to better understand and characterize the polymer response to PFAS. Similar experiments were performed using a Horiba Quanta-phi fluorescence spectrophotometer, which allowed for full measurements of emission intensity through a band of wavelengths. This device provided a more accurate picture of the exact pattern of the polymer’s fluorescence as well as how it changes in the presence of different concentrations of PFAS. However, the device was both space and energy intensive making it non-ideal for portable use. For the eventual device the goal was to measure fluorescence visually using a fluorescence camera, thus multiple further tests were performed using a Nikon Eclipse TE2000-U Microscope. Images were taken at lOx magnification using an Andor Solis iXonEM+ EMCCD camera and used a DAPI emission and absorption filter.

[0222] Procedure

[0223] The goal of these initial tests was both to discern how effectively one could measure differences in PFAS concentration and to determine whether one could determine any difference between the polymer’s reaction to short and long chain PFAS. Tests were done using PFBA and PFOA both at 100 ppt, PFOA at 1 ppb, and pure water. The water used for all three types of test fluids was Mili-Q filtered water. All tests were done on glass slides with spin coated polymer with 10 pl placed droplets. The glass slides were large enough that multiple tests could be done on the same slide without direct interference and the order of tests was changed between tests to ensure that any cross contamination was minimal. Images were taken at the center of the droplet every second in order to get a time series of the fluorescent output. This did cause noticeable bleaching, which could impact results but was relatively consistent between different tests. A DAPI filter was used when taking measurements as its emission spectra matched the peak emission wavelength of the polymer. All measurements were taken while the microscope was covered to reduce the impact of changes in background illumination. All of the graphs are based on the grayscale of the image taken of the same area for each test, attempting to avoid any large and impactful features within the image. This grayscale value is a measure of the brightness of each pixel and the value used is the average grayscale value of all pixels in the selected region. The camera was set to the same settings for every test allowing it to be an objective measure of the overall brightness of the polymer during tests.

[0224] Pyridine Polymer Tests

[0225] Initial tests were done on a polymer with a poly(p-phenylene ethynylene) backbone and a pyridine selector shown in FIG. 7 (PPE-PY). Described below are a series of graphs of the grayscale over time of PFBA, PFOA and water droplets on the Pyrodine polymer. The first few data points of each graph have been removed as there was significant noise found while and immediately after the drop was placed.

[0226] FIGS. 10A-12B show a roughly linear decrease in fluorescence, but the actual slope of that decrease is very different between PFAS and water and even between PFOA and PFBA. PFBA at lOOppb showed an average decrease in fluorescence of around 0.52 per second over both tests and seemed consistent throughout all 200 seconds, although there were jumps in fluorescent value likely due to changes in background light. PFOA on the other hand has an average decrease of 1.18 per second over both tests, significantly higher than PFBA even at the same concentrations. The 200 second PFOA test also showed a sudden drop in fluorescence similar to the jumps in the PFBA test but also showed a definite tailing off of fluorescence drop towards the end of the 200 seconds. This would fit with the polymer absorbing the majority of the PFOA in sample with the remaining downward slope being due to bleaching. This did not occur in the PFBA test suggesting that the polymer did in fact absorb PFOA noticeably faster than PFBA. The tests were not continued long enough to see when PFBA reached this point, so the actual total amount of fluorescence drop between the two cases was not analyzed. Only one test was done with a 10 pl droplet of pure water (ACS Reagent grade ASTM type 2), which had an average drop of 0.18 grayscale per second, significantly less than either of the PFAS tests but still a noticeable drop likely due to polymer bleaching. Of note, the slope of the PFOA lOOppb test, even at the very end when the drop seemed to be flattening out, was around 0.25, still higher than the slope of pure water but much closer than any other test. This continued difference was likely due to small amounts of PFAS remaining in the sample with the system not quite reaching a true equilibrium. The 1 ppb PFOA test seemed to confirm that the drop in slope was due to the majority of PFAS being absorbed by the polymer as it flattened out significantly partway through the testing process. However, the slope of the initial 1 ppb test was actually 1.5 ppb, higher than either of the 100 ppb PFOA tests and it was unclear exactly why that was the case. This case also still shows a larger slope than pure water with a slope of around 0.26. The fluorescence drop of the 1 ppb test was around 105 before reaching the changing point, lower but not that much lower than the drop of 100 ppb to its changing point, which was around 158.

[0227] These results showed some promising ways to both measure overall fluorescence and differentiate between short and long chain PFAS. There were significant differences between different tests using the same procedure but there were more significant differences when actually using different fluid concentrations and types, which bodes very well. Further promise would be realized with improved precision. Different slides seemed to have different baseline fluorescence values, which was likely due to the lack of production quality spin coating at use in these prototypes leading to differences in thickness, which would be significantly improved in a final product. Even if this was not the case, taking a baseline measurement of the fluorescence could normalize the data between different tests. The fact that the reaction seemed to be mostly linear in all cases is also a large boon as it makes analysis and comparison much easier, requiring fewer calculations and reducing complexity. It was found that the pyridine selector did not have the required sensitivity to work effectively for the final device; thus, the focus of the project turned towards a pyridine selector with thiophene bridges for future testing. Similar sets of tests were done on the pyridine and thiophene polymer but with slightly different parameters to try and answer some of the questions that were still left open after these initial tests.

[0228] Pyridine and Thiophene Polymer Tests

[0229] The backbone of these polymers was identical as the previous case, but the selector included a thiophene bridge rather than being a simple pyridine selector. Again, the specific structure is shown in FIG. 7 (PPE-Py*). The move to a pyridine and thiophene polymer was impactful in terms of results but changed very little in terms of the actual procedure or desired information. However, as part of the change to the pyridine and thiophene polymer, zonyl surfactant was included in the solution at .01% weight to weight concentration. FIG. 13 shows results of tests done with pyridine-based polymers that show improving polymer sensitivity while not creating a signal without PFAS. This increased sensitivity is believed to be due to the fact that zonyl absorbs to surfaces in a very similar manner to PFAS. Tests had much higher concentrations of zonyl than PFOA or PFBA, so any absorbance onto surfaces such as scratches in the polymer layer or particles on the glass slides were significantly more likely to be zonyl than our analytes. This effectively increased the amount of PFAS absorbed into the polymer even at the same concentration, increasing sensitivity. Zonyl could be implemented into the final prototype and used in a final device.

[0230] The main goal of these tests was still to determine the reaction of the polymer to PFOA and PFBA, analyzing the full response of the polymer and determining whether there was a significant differentiation between short and long chain PFAS at the same concentration. In this case it was also desired to get a baseline of each slide before tests were run, so for each test initial images were taken of the dry polymer for around 50 seconds, before a 10 pl droplet was placed on the slide and tests were continued for another 150 seconds. Once again tests were done on PFBA and PFOA at 100 ppb as well as pure Mili-Q water. Also, with the increased brightness of the new polymers it was not important to have full fluorescent light on them, so a filter was used to reduce overall brightness while also reducing the rate of bleaching. FIGS. 14- 16 show the fluorescent result of one set of tests each. Each set of tests was performed on one glass slide with droplets placed in different sections. Six total droplets were placed on each slide, two each of PFBA, PFOA and pure water.

[0231] All of these tests showed an initial close to constant fluorescence before the droplet was placed, followed by a very sharp decrease as soon as the droplet was placed, and then another mostly constant (although slightly negative for most tests) fluorescence value for the remainder of the test while the droplet is on the slide. There was also a sharp but transient increase in fluorescence as the droplet was being placed, which was believed to have been due to the cover being removed and the droplet actually being placed, not a reaction within the polymer. While different tests do have different slopes, there did not seem to be any strong pattern between PFBA, PFOA, or water, and the differences were all small enough that the slope may be due to slightly different diffusion or bleaching rates. There was a significant amount of inaccuracy at the moment that the droplet was placed with the droplet being placed at slightly different times and with background light playing a much larger role making it harder to get solid information about this section to differentiate between any of the cases on the immediate impact of PFAS on the polymer. There was no pattern for the amount of fluorescence decrease before and after the droplet was placed. FIGS. 17-19 show results that were normalized linearly so that the average fluorescent value before the droplet was placed was equivalent through all tests.

[0232] None of these tests provided useful data or even much of a response from PFAS despite us knowing from the spectrophotometer tests that the polymer was functional. The issue was simply in the testing methodology used, specifically that overall brightness was being measured rather than wavelength change. The thiophene and pyridine polymer did generally decrease in fluorescence emission intensity with PFAS, but the main change was a shift in wavelength. This was true for the pyridine polymer as well, but in the thiophene and pyridine case the polymer also decreased in emission intensity (but did not shift wavelength in the presence of water, as seen in FIGS. 20A-20B). This was not a problem for detecting polymer fluorescence change in the spectrophotometer but did cause issues for these tests that were simply measuring overall fluorescence. The solution was to use two fluorescent filters in tandem as discussed below. Multiple Filter Polymer Testing

[0233] With the issues found during our previous tests using only a DAPI filter we altered our setup to take measurements with both a DAPI and FITC filter and compare the two sets of results. This new polymer had a slightly shifted peak wavelength from 450 nm to around 480 nm, slightly outside of the peak of the DAPI wavelength. The polymer also showed a small decrease in emission intensity when exposed to pure water, although without the change in wavelength associated when exposed to PFAS as shown in FIGS. 20A-20B. In order to use the polymer to visually measure PFAS, we needed to measure both the emission peak at 480 nm and the secondary peak from the wavelength changing, to get an approximate measurement of wavelength shift instead of overall fluorescent intensity. In order to accomplish this in our setup, we used a DAPI and FITC filter whose emission spectrums, taken from Chroma Technology, are shown in FIGS. 20A-20B. The DAPI fluorescent filter has an emission peak at 462 nm while the FITC fluorescent filter has an emission peak at 520 nm. By comparing the relative brightness of FITC and DAPI emissions before and after a sample droplet is placed on the polymer, the wavelength shift was detected without needing to measure the full emission spectrum. These filters were slightly off from the ideal fluorescence filter wavelengths but did function to get effective results.

[0234] Procedure

[0235] The procedure remained similar to the previous tests but with more time between images to give time to switch filters and with separate tests done on glass slides with no polymer to provide a baseline. In both the polymer and non-polymer cases images were taken of the slide using both FITC and DAPI filters before a droplet was placed. Images were taken 5 seconds apart to give enough time to switch from DAPI to FITC in an alternating pattern. Then after 10 images were taken or around 50 seconds, a 10 pl droplet of deionized water (Millipore MilliQ) was placed on the slide, with concentrations of PFOA in it ranging from 0, 0.1, 1 and 100 ppb. 20 seconds after the droplet was placed 15 images were taken, again alternating between DAPI and FITC at 5 second intervals. The same process was done 8 times on the same slide in different locations, i.e., 2 droplets each of 0, 0.1, 1 and 100 ppb. One test was also done for each concentration (i.e., one droplet per concentration) on a plain but identical glass slide as a control. The results of this control test were then directly subtracted from the test results in order to remove the impact of background light and any small changes in the fluorescent lamp. In all cases, the factor of interest was the ratio of FITC to DAPI before and after the droplet was placed, which was quantified by defining a Color Ratio Difference.

[0236] The difference in ratios gave an accurate sense of how much the emission spectra of the polymer changes from a peak at 450 nm to a peak at 500 nm. The FITC / DAPI color ratio for each drop was taken by dividing pairs of FITC and DAPI measurements, taken 5 seconds apart, and finding an average of all FITC / DAPI results before a droplet was placed and subtracting it by the average of all FITC / DAPI results after a 10 pl droplet was placed.

[0237] Spin coated Glass Slide Tests

[0238] The first set of tests were simply done on plain glass slides with polymer spin coated onto them similar to previous tests. FIGS. 21A-21B show the average results of three sets of tests, i.e., three glass slides, each with two droplets of a given concentration, with six droplets at each concentration. The graphs show the average of these results with the error bars representing one standard deviation. Both graphs show the exact same tests and information for the PFAS concentrations but the second is graphed on a log scale using the PFAS concentration. Shown in FIGS. 22A-2B are results using the same data but subtracting the mean Color Ratio Difference of the pair of water droplets on the same slide, in order to account for differences in light source or background for each day and set of tests. However, while there are differences between the results in FIGS. 22A-22B and FIGS. 21A-21B the difference is not significant. These results indicated that imaging the fluorescence of a thin (10 nm) layer of polymer by a simple color camera is promising for detection of PFAS down to sub-ppb concentrations. However, there were significant inconsistencies between tests, resulting in large standard deviations, which in a real sample could make measurement quite difficult. The error was especially present with the 0 and 100 ppb samples. However, despite these inconsistencies, the average values found for each concentration suggested a very strong correlation. Both of the graphs with log-based concentration axes show a very high logarithmic pattern with an R2 value above 0.995. This provided evidence that the tests and systems were accurate and that issues with inconsistencies and large errors were due to noise rather than any fundamental issues with the test. Multiple aspects of this test reduced sensitivity and increase inconsistency, such as the spin coating process and running tests on glass slides rather than cartridges, and further tests were performed to reduce those factors of external error to get more sensitive and useful PFAS testing.

[0239] Cylindrical Cutout Tests

[0240] The second set of tests were identical but performed on spin coated slides with 1 polypropylene sheets attached. The polypropylene sheets were 1 mm thick with 4 mm diameter holes drilled in them and attached using NOA88 UV curable adhesive. The goal of these tests was to perform tests more closely resembling the final microwell setup while still using spin coated polymer and allowing ease of fluid insertion. There was also a hope that having defined cutouts would normalize the amount of polymer in contact with the sample decreasing noise, but similar levels of error were seen. The results of these tests are shown in FIGS. 23A-23B (which shows the objective Color Ratio Results) and FIGS. 24A-24B (which shows the difference in color ratio between each PFAS concentration and water).

[0241] These results continued to show that measurements can be taken to 1 ppb or lower concentrations but still had a similar level of inconsistency leading to significant error, making actual use difficult. These results were very consistent with the spin coated glass slide tests when accounting for the color ratio difference of water, especially with the 100-ppb case with a difference of less than 7%. The objective Color Ratio Difference was quite different between tests, with the cylindrical cutout test having consistently lower values. It was unclear whether this was due to the tests themselves or some external difference, but it seemed to suggest that taking regular tests on samples with known quantities may be helpful, which was not ideal for the final design. It was hoped, however, that in a closed system with consistent testing parameters this would no longer be important as the value of water would remain constant even through multiple tests. These tests also showed a significant error in the result for 0.1 ppb perhaps demonstrating that the sensitivity of the device was not quite to that level but again the data followed a strong logarithmic pattern, which suggested that the large error was due to variations in the system rather than more fundamental issues with the testing itself.

[0242] Microwell Design

[0243] One of the most important steps of defining this design was to develop the single-use micro wells. Each of these microwells was identical and contained the sensing polymer inside it. The cartridge was designed to contain an array of dozens to up to 1000 microwells, and to be easily replaceable. Tests would be done using a fluorescence camera or sensor placed below the cartridge analyzing the change in emission wavelength of the polymer contained in the microwell. The cartridge was created using different layers for ease of fabrication, and each microwell was hermetically sealed to avoid contamination from the environment. Testing showed a strong fluorescence signal even with an estimated 10 nm thick polymer coating on slides, allowing for the use of small volumes of sensing polymer (and, consequently, small volume microwells) keeping the device portable even with a large numbers of micro wells. The setup of the cartridge is shown in FIG. 1.

[0244] Diffusion Tests

[0245] One of the important concerns when designing the microwells was how they could be used to differentiate between short and long chain PFAS. Initially the hope was that long and short chain PFAS would diffuse through the polymer at different rates but testing showed negligible diffusion for both short and long chain PFAS. Longer chain PFAS are characterized by longer fluoroalkyl chains and are thus generally more fluorous than short chain PFAS. It was hypothesized that long chain PFAS would interact more strongly with the polymer than short chain PFAS, enabling the short chain PFAS to diffuse more rapidly in the polymer. The goal was to use this faster diffusion to differentiate between the short and long chain PFAS.

[0246] In order to test this hypothesis multiple tests were done to measure the diffusion of a short and long chain PFAS within the polymer. Tests were done with both PFOA (Perfluorooctanoic acid), an eight-carbon long chain carboxylic acid and PFBA (perfluorobutanoic acid), a four-carbon short chain carboxylic acid. These tests were done with the first set of usable polymers, based on a pyridine connection, spin coated on glass slides and with the same procedure for both PFOA and PFBA. For one set of tests after the polymer was prepared a 10 pl droplet of lOOppb PFOA or PFBA solution using Mili-Q filtered water was added on top of the spin coated glass slide. The droplet was then left there for 10 seconds before the entire slide was washed with ACS reagent grade ASTM type 2 water. The second set of tests used a similar procedure but placed the droplet on the edge of a piece of Kapton tape placed on the glass slide to block half of the polymer. In both cases the slide was then dried and immediately placed on a Nikon Eclipse TE2000-U Microscope using a DAPI filter. Images were taken at lOx magnification using an Andor Solis iXonEM+ EMCCD. Multiple spots were picked and imaged immediately after the test and then images were taken of the same points for multiple hours and then days later. For both PFBA and PFOA tests were taken after 1, 3, and 72 hours after the droplet was placed.

[0247] For both PFOA and PFBA in both the Kapton tape and clear slide test, images showed essentially no measurable diffusion after the first image was taken. While individual features do form and are different between different tests those features do not change even after three days. Shown in FIGS. 25A-25D and FIGS. 26A-26D are two sets of images, one showing PFOA and one PFBA both from the Kapton tape test. Also included in FIGS. 27A-27H are diagrams of FRAP (Fluorescence recovery after photobleaching) diffusion taken from a paper by Niklas Loren et. al. to show the expected result if there was even minor diffusion.

[0248] Using image J the yellow lines were created in the image and the grayscale value was taken from each pixel in the line, which were then used to create the following graphs showing the fluorescence of each pixel along the line. FIG. 28 shows the fluorescence pattern of the line through FIGS. 25A-25D while FIG. 29 shows the fluorescence pattern of the line through FIGS. 26A-26D. The actual gray value shown is not accurate between different runs so it cannot be used to compare the fluorescence of each spot over time, but it does show that the qualitative similarity between visible features even after three days is backed quantitatively as all of the graphs show very similar features.

[0249] These tests show essentially no change in visible features even after three days. Looking specifically at one sharp comer does show some blurring of the edge but the time and length scale are minuscule making it unsuitable. The images in FIGS. 30A-30C are taken from the PFBA images at 40X rather than lOx magnification and further zoomed in to show one sharp comer of the polymer. These images were taken immediately, 23 hours and 72 hours after the droplet was placed. Even after 72 hours the sharp comer is still present showing very little diffusion, however, there does seem to be some small diffusion as the edge becomes more blurred over time. Part of this is likely due to the bleaching of the polymer from multiple measurements but the rest is likely from diffusion within the polymer. However, the time scale and amount of diffusion both made this impractical for use in the tested device.

[0250] These experiments showed that short and long chain PFAS have barely any diffusion within the polymer and measuring any diffusion would be time and resource intensive, not to mention the increased difficulty in then differentiating between short and long chain diffusion. Thus, another method was developed to differentiate short and long chain PFAS within the device. It is worth noting the difference shown between PFBA and PFOA in the images that were taken. Neither case showed diffusion after the initial images but the actual pattern of PFAS absorption for the two cases are different.

[0251] PFOA seemed to react as expected with a smooth transition from bright under the tape to dark where no tape was present. There are multiple individual features within the image that break this up such as the darker rectangular section seen in FIGS. 25A-25D, but these spots were likely due to differences or damage in the polymer rather than characteristics of PFAS. PFBA on the other hand reacted quite unexpectedly. There was still a definite difference in brightness between the polymer that was within the droplet compared to the polymer under the tape, but rather than a smooth transition, the edge of the droplet showed a region that was much darker than either of the surrounding regions. FIGS. 26A-26B also show two dark lines across the dark gap, but this was likely due to scratches rather than PFBA characteristics. PFAS in general are hydrophobic and so move towards the surface and edges when in a droplet of water. Thus, in a case like this it would be expected that the edge of the droplet would show a higher concentration of PFAS than the center. The amount of difference between the two is surprising in this case, especially since the pattern did not occur with PFOA but this may be because of their diffusion characteristics. The diffusivity of PFBA in water is more than twice that of PFOA causing it to move much faster towards areas of low concentration than PFOA. In this case, since the polymer does take time to absorb PFAS it is possible that PFBA was able to move to the edges of the droplet fast enough that the majority of the PFBA near the droplet edge concentrated into a very small band of polymer on the edge while PFOA still mostly just absorbed into the polymer directly below.

[0252] Microwell Geometry

[0253] The microwells were designed to be simple cylinders for ease of both manufacturing and measurement. Previous tests showed that diffusion through the polymer could not be used to differentiate between short and long chain PFAS but short and long chain PFAS also have differing diffusion rates through water. This metric, while effective, is dependent not only on the chain length of PFAS but also the head group potentially leading to more inconsistencies when compared to looking at diffusion in the polymer. However, even with differences in head group causing differing diffusion characteristics this method should still be enough to differentiate between the most common long and short chain PFAS. For this design the polymer would be spotted on one corner of the cylinder with the fluid inserted in the center to maintain a consistent diffusion length between tests. Alternatively, the polymer is placed at the end of a cutout section. The rate of change of signal and the time taken to reach steady state would depend on the diffusion rate of the PFAS. Thus, monitoring the change in signal over time can help to distinguish between different classes of PFAS. E.g., for a given steady state signal, short chain PFAS would lead to a shorter time to reach steady state signal, whereas long chain PFAS would take longer to reach a steady signal.

[0254] FIGS. 31A-31B show a proposed design that could be used if significant issues emerged differentiating between short and long chain PFAS. The concern was that having the micro well be a single cylinder could lead to the fluid injection and removal of the needle causing significant and inconsistent convection that would lead to different results making it impossible to differentiate PFAS based on diffusion through water. A solution is to have a cutout section that would have much more limited convection and use that geometry to differentiate between short and long chain PFAS. Other than this additional rectangular cutout the exact dimensions of the cylindrical section are identical, and drawings of the exact dimensions are shown in FIG. 32.

[0255] The exact dimensions shown in FIG. 32 were determined by practical needle size, positioning, polymer fluorescence detection, and desired testing time. Since the inherent fluorescence signal of the sensing polymer depends on the amount of PFAS bound to the polymer, a larger amount of polymer will require a larger amount of PFAS and hence a larger sample volume to produce the same inherent signal (i.e., shift in emission spectrum). We observed that fluorescence of even a 10 nm thick polymer film was bright and readily imaged. Therefore, based on typical resolution of inkjet-type spotters, a 30 pm diameter spot with 10 nm thickness was selected as the nominal polymer sensor element in each microwell. Assuming that a mass ratio of 1% PFAS to polymer would give a detectable signal (based on prior literature with these polymers), 100 fg of PFAS is required to produce a signal. At 100 ppt (= 100 pg / g) concentration of PFAS, it translates to a sample volume of 1 pl (i.e., 1mm3).

[0256] Another concern when determining microwell dimensions was the time for PFAS diffusion through the water to the sensing polymer. Calculations were done using diffusivities taken from “Measurement of Aqueous Diffusivities for Perfluoro alkyl Acids” by Charles E. Shafer et. al. Calculations were done using a design with a rectangular cutout. For these calculations it was assumed that the fluid was stagnant without convection and that the polymer has instantaneous uptake of PFAS. It was also assumed that the main cylindrical section is well- mixed and maintains an even concentration of PFAS. With these assumptions and with a PFAS concentration of 70 ppt a set of dimensions was found that would allow more than 75% of the PFAS to be absorbed by the polymer in an hour for even some of the slowest diffusing PFAS. Graphs showing the percentage of PFAS absorbed over time are shown in FIGS. 33A-33D. These graphs also effectively show how PFAS diffusion through water can be used to differentiate between short and long chain PFAS. Measuring the PFAS concentration in the polymer 3-4 times during the test should give both the overall concentration of PF AS within the microwell and the general diffusivity characteristics of the PFAS to differentiate between short and long chain.

[0257] Microwell Materials

[0258] Beyond the dimensions of the microwell, it was also important to clearly define the materials that would be used. Many of the common laboratory plastics either have PFAS or fluorinated material in their manufacturing or would allow for PFAS absorption into their surface. Glass is an obvious choice without either of those impacts, but it is difficult to machine. Polypropylene is compatible with PFAS measurement (Shoemaker, 2018), and thus for the initial prototypes, polypropylene and glass were used for the microwell geometry. There was some difficulty in machining polypropylene, so for some prototypes, delrin was used instead of polypropylene. Like polypropylene, delrin does not contain PFAS or other fluorous particles but its absorption properties in terms of PFAS are less well known. The microwell also used an aluminum and septum layer to provide a hermetic seal and a temporary seal after being pierced by the needle in order to inject the sample. The overall setup of these materials in a single microwell is shown in FIGS. 34A-34B.

[0259] The bottom glass layer had the polymer spotted (or spin coated) on it, and the fluorescent camera measured the response through it. Polypropylene was selected for the secondary layer as it resists PFAS absorption and is one of a set of plastics that does not use PTFE; it is the EPA’s suggested plastic when working with PFAS (Shoemaker, 2018). Plastic was used here instead of glass for easier manufacturing. Polypropylene and delrin are fluorescent in the polymer’s response wavelength, so for the rectangular cutout design, another layer of glass was used to provide the important micro well shape without interfering with the results. All of these layers were bonded using NOA88 Polyimide UV curable adhesive. This adhesive was selected as it does not contain fluorine or PFAS and can provide a near-hermetic seal. During our testing BONDiT B-482 epoxy resin was also used and tested, which has the benefit of not requiring UV light, although its ability to form near hermetic seals is a bit more in question. Both adhesives have been tested with polymer to ensure continued functionality of PFAS sensitivity with no issues in either case. Aluminum tape was selected as the top layer to create a hermetic seal before use but one that can be pierced with a needle to insert the fluid. The tape adhesive used does not contain fluorine or PFAS but its interaction properties with PFAS are unknown. However, in this case the uncertainty was deemed acceptable as the adhesive component of the tape will only be used to connect to the septum layer and will not contact the fluid except for tiny amounts of fluid that may flow out during injection. The top layer was a septum layer to form a temporary seal after injection by the needle, and to prevent evaporation during tests. The septum used Thermolite Plus (Restek part number 23864) for these tests, but PTFE and silicone sheets could also be used in final products for easier manufacturing. The top aluminum layer has conical cutouts in it to direct the needle as it is lowering to ensure that it enters the test fluid area correctly and does not damage the needle by hitting the top glass layer instead. This top layer is less important and could be made from a variety of different materials as long as they do not contain PFAS. It is simply used as a channel to ensure that issues do not arise from the needle being slightly off center of the injection site.

[0260] Fabrication

[0261] Initial manufacture used 1 mm thick polypropylene sheets cut to the size of the glass slide and 4 mm holes were drilled through the polypropylene using a power drill. The polypropylene was then attached to a glass slide with spin coated polymer using NOA88 and put under 365 nm UV light from a Spectroline EA-160 handheld UV lamp in order to cure it. Tape was placed to block UV light from impacting the polymer within the microwell that would actually be tested, although testing found that the UV light at this wavelength and intensity from the UV lamp had negligible bleaching impact on the polymer. Aluminum tape was also added on top of the polypropylene sheet and attached using NOA88 with the same bonding process. In both cases care was taken to limit the amount of adhesive and try to ensure that no adhesive leaked into the micro well or was present on the aluminum directly above the micro well. After exposure to UV light, a thermolite septum was attached to the top of the aluminum and the whole glass, polypropylene, septum cartridge was placed in the device shown in FIGS. 35A- 35B. This device used screws and two large glass slides to apply pressure to the cartridge system while using a set of four springs to normalize the pressure across the device. This device was used after initial curing with UV to help ensure strong and even bonding of the cartridge.

[0262] This fabrication system worked well for initial testing but the process of manually drilling holes led to inconsistencies and was also quite difficult to scale. Machining polypropylene was also quite difficult. The laser cutters could not cut polypropylene due to the potential for melting and out gassing hazardous fumes. Instead, the material of construction was switched to delrin, which is another plastic that does not include fluorous material or PFAS but is not as resistant to PFAS absorption as polypropylene. Switching to delrin allowed for the use of laser cutters, which made manufacturing much easier, and tests with delrin have shown no decreases in PFAS measurement sensitivity. Delrin is not as translucent as polypropylene which prevented the use of NOA88 to bond delrin to the aluminum sheet. Instead, BONDiT B-482 epoxy resin was used for all adhesive connections, which had some initial concerns about PFAS interactions, but testing showed no difference between NOA88 and BONDiT B-482 in terms of PFAS sensitivity. 3 / 64 inch (or roughly 1.2 mm) delrin sheets were used. The septa were also changed to a sheet-based septum layer that covered multiple wells, which made it easier to have multiple wells on the same glass sheet. 0.6 mm thick JTR-SA-0.5 Silicone Isolators bought from Grace Bio-Labs were used, which provide less sealing than most other septa but do not have PTFE or other fluorous material. FIGS. 36A-36B show the fabrication process which remained similar to the one described above despite the use of different materials, although this manufacturing process allowed for many 1 mm holes to be formed in the same delrin sheet.

[0263] A bench-top laser cutting system (Fusion Maker Laser Machine 12, © Epilog Laser, USA) was used to fabricate a Delrin sheet with holes and a septum layer with engraved circles to indicate the outline of the chamber. For cutting through, 40% of laser source power (40 W) was exposed with 5,000 Hz of frequency and 20% of scanning speed (1.5 m / s). Furthermore, 10% laser power, 80% scanning speed, and 5,000 Hz of frequency were set for engraving the surface of the silicone sheet to create icons showing the location of cutouts for ease of sample insertion. For assembling each layer, a glass coverslip was first spin-coated with sensing polymer. Then BONDiT B-482 was applied on the surface of the machined Delrin sheet by gently stamping the resin using a porous, clean wipe. The silicone sheet, attached with a singleadhesive aluminum foil, was then attached to the Delrin sheet following the same stamping method. The assembled layers were placed underneath a 1.5 kg steel plate to provide uniform pressure causing better and more uniform bonding between the surfaces.

[0264] Fluid System

[0265] Another major component of the device is the actual fluid system to bring the water sample to the needle and then to the test site. This was not a trivial process as PF AS has a tendency to absorb into surfaces (even EPA approved surfaces) and would lead to cross contamination between tests. The most pressing issue was out and back flow, which left water in the tubing allowing PFAS, which is hydrophobic, to absorb into the tube walls away from the water. The plan was to use pass through flow for the majority of the system with only a small section connecting the main flow line to the needle that would need to be out and back. This helped mitigate the issue but did not fully solve it. Thus, a rinsing system was included with an attached ethanol reservoir that could flush out the sections of the tubing that were more likely to have PFAS build up. This same ethanol reservoir can be used to rinse off the tip of the needle, which injects the fluid into the containers and is also likely to have PFAS contamination. All of these design decisions led to the following fluid system diagram of the planned approach shown in FIG. 37.

[0266] The system used a push-pull syringe pump and a multi-position valve to perform fluidic operations. The system was set up so that the syringe pump channel was always open but was only connected to one of the other three ports at a time. Fluid was drawn from the ethanol reservoir or the main flow channel into the syringe pump and held there while the valve changes to the needle and then the syringe pump pushes fluid through the needle either into the sensor or into a waste receptacle to store used ethanol. The main flow channel connected directly to the test water source, allowing flow through, and began running before the 4-way valve was open allowing the test water to replace any PFAS build up. The fluid can be run through the needle multiple times removing any excess ethanol or PFAS within the system before a new test is run. If PFAS cross contamination proves to be worse than expected, another line from the ethanol reservoir can be added directly to the syringe pump (the dashed line in FIG. 37), which will keep an area of ethanol above the sample water to ensure that there is not any contamination within the syringe. For the entire system there is a desire to make the tubes as short as possible to allow for the ethanol reservoir to be as small as possible while still being able to regularly rinse out the system. The distance between the main flow channel and the 4-way valve is especially important as that cannot be rinsed out with ethanol. All of this allows the ethanol reservoir to rinse out the tubing regularly and automatically, increasing the useful life of the device without requiring human oversight, but it will still need a more thorough deep cleaning to remove PFAS build up when installed in an area long term. The diagrams shown in FIGS. 38A-38D show the process of a test and an ethanol rinse in a more visual manner. Thicker lines are paths of fluid flow with white triangles representing open valves in the multiposition valve while black triangles are closed valves.

[0267] The system was set up so that the syringe pump channel was always open but was only connected to one of the other three ports at a time. Fluid was drawn from the ethanol reservoir or the main flow channel into the syringe pump and held there while the valve changes to the needle and then the syringe pump pushes fluid through the needle either into the sensor or into a waste receptacle to store used fluid. The main flow channel connects directly to the test water source, allows flow through and begins running before the 4- way valve is open allowing the sample water to replace any previous PFAS build up. In order to reduce the required size of the ethanol reservoir the tubing diameter and distance will be as small as reasonably possible to allow the ethanol to rinse the entire system regularly while still keeping the device portable. The distance between the main flow channel and the 4-way valve is especially important as that cannot be rinsed out with ethanol. All of this allows the ethanol reservoir to rinse out the tubing regularly and automatically, increasing the useful life of the device without requiring human oversight. However, the system will still need occasional more thorough deep cleaning to remove PFAS build up when installed in an area long term.

[0268] During testing the main flow channel will be turned on well before tests are desired, to have fluid flowing consistently through that larger channel and to flush out any remnants from previous tests. Then the 4-way valve will be opened between the syringe pump and the main flow channel, and the pump will begin drawing test fluid into it. Once the system has enough fluid the 4-way valve will switch to connect the syringe pump to the needle. Fluid can then be injected through the needle. For most tests the diagrams in FIGS. 38A and 38B will be repeated multiple times injecting into a waste reservoir rather than into the microwell in order to flush out any ethanol remaining in the system. Then after multiple rinses the needle can be injected into the microwell, and the sample can be tested. Before the next test the 4-way valve will be opened to the ethanol reservoir allowing ethanol intake into the system. And finally similar to operation in FIG. 38C, the 4-way valve will connect the syringe pump to the needle to allow flushing the ethanol through the system cleaning the fluid system out and preparing for the next test. This phase also opens the pinch valve to the ethanol reservoir allowing ethanol to rinse the outside of the needle as well.

[0269] The 4-way valve that was used for the prototype was a Valeo C-25 4 position selector, and the syringe pump was a WPI UMP3 infuse / withdraw Microinjection syringe pump attached to a 700 series Hamilton Syringe. The needle was a 28-gauge 0.75 in long Hamilton needle with an attachment to 1 / 16” OD tubing. All the tubing besides the main flow and ethanol to cleaning sheath use 1 / 16”OD IDEX Peek tubing, chosen to avoid fluorous plastics and reduce chance for PFAS absorption. The ethanol line to the syringe where PFAS absorption is not too important uses 1 / 16” ID Polyethylene tubing, which still does not have fluorous particles but is not resistant to PFAS absorption. However, this does allow easier connection to the ethanol line pump, a Welco WPX1 inline pump, and the connecting valve, an ASCO 2842-Way Solenoid Pinch Valve. The current design of this system with the actual component dimensions, not including the ethanol or main lines is shown in FIG. 39 and a list of the parts used along with their functions is in Table 3.

[0270] Table 3: Fluid System Components

[0271] Polymer Spotting

[0272] To apply polymer, polymer was spin coated to cover the glass slide in a layer around 10 nm thick. Droplets are placed onto that surface for testing, but testing only needs visual measurements from a very small area of that polymer film, meaning that a relatively large amount of polymer leaches PFAS from the sample without actually improving the measurements. By spotting the polymer on one specific section of the glass slide and taking measurements of only that spot, one can increase the amount of PFAS interacting with that polymer even at the same concentration thus increasing sensitivity using very similar testing methods.

[0273] The system used to create spotted polymer used a Microfab Technologies MJ-ATP-030 ink-jet microdispenser with an orifice diameter of 30 pm and a Microfab Technologies Jetdrive V electronic control system. The droplets formed, however, were around 40 pm and when spotted on glass had a diameter of around 100 pm, requiring 4 droplets in order to get the same thickness of polymer as the spin coated slide version. Currently devices use 10 pl of fluid exposed to a polymer of around 12.5 mm2resulting in a decreased detectable concentration of 0.1 ppb. Spotting tests also used 0.8 pl of fluid with a spotted area of around 0.008 mm2with around the same thickness. This resulted in an increase of PFAS-to-polymer ratios by a factor of around 80, which should help detection of significantly lower concentrations even if further improvements are not made. FIGS. 40-42 are images of the spotting setup, droplets spotted inside a 1 mm well, and a diagram of the ink-jet microdispenser. The spotting head functioned by using a slight negative pressure to keep the fluid within the spotting head but with the edge of the meniscus trapped on the outside edge of the spotting head. It was advantageous for there to be a slight negative pressure on the fluid at the spotting head, drawing the fluid into the tip but not enough to disconnect the meniscus from the outer edge of the spotting head tip. With the meniscus trapped and the fluid at the very end of the spotting head an electric signal was then sent to the spotting head, which changed the surface properties of the liquid while causing small droplets to form. The electronic signal could be controlled very precisely allowing control of the frequency of the droplets and allowing the device to work with different fluids with different characteristics. Using this spotter, water and polymer in solution were effectively spotted.

[0274] Microfab Technologies has a kit that can be bought, which includes holders for the spotting head and tubing, a high-speed camera to view the droplets as they form and a vacuum pressure control to make the process easier. However, this setup is expensive and space inefficient and so it was decided to use, instead a custom made holder and tubing while trying to create the pressure difference using the height of the fluid, which was successful. The end of the tubing was at around the same height as the spotting head in order to produces spots. The fluid would not fill the tubing to the very edge, providing the negative pressure desired. When using this simplified setup it was important to initially provide positive pressure, in this case done with a syringe filled with liquid, either water or the polymer solution, that could be attached to the end of the tubing using a luer lock connection. This positive pressure created a small stream of water through the spotting head and once this was seen the syringe was removed from the tubing. This ensured that water was trapped at the spotting head tip but in this case it was found that often droplets, while still trapped by the spotting head edge, would begin to form outside of the spotting head tip rather than inside the tip as needed for use. This excess water was removed using kimtech wipes to reduce the chance of fibers being caught in the spotting head and extra care had to be taken to absorb the droplet without touching the fluid inside the spotting head and disturbing the fragile connection between the meniscus and the spotting head tip. Operations were conducted in which fluid was present at the spotting head tip with the negative pressure causing it to slightly recede into the spotting head allowing the device to function. Many different square voltage signals were sent to the spotting head to get droplets to form and to have them be the proper size. The eventual parameters used are shown in FIG. 43. A frequency of 250 Hz was used when testing to ensure that the droplets could be seen more easily and once the parameters were set correctly then individual or bursts of drops could be dispensed as desired.

[0275] The spotting head occasionally clogged due to the presence of small particles. Using filtered water was found to be important. In order to clean the spotting head, a Bacoengineering 1 stage vacuum pump was connected to the spotting head through a catch basin to prevent liquid from getting into the pump or oil getting from the pump to the spotting head. The spotting head was first submerged in filtered water with alconox in order to remove the particle from the tip, then the spotting head was submerged into pure filtered water to remove any remnants of the alconox from the spotting head and tubing.

[0276] One final issue that arose involved the use of flexible PVC (polyvinyl chloride) tubing in the spotting head setup. PVC was chosen as one of the few highly flexible plastic tubing that does not contain fluorous materials or additives. Highly flexible tubing was desired to reduce the amount of fluid that would be required for spotting. The actual fluid used in each microwell is very small, so the main volume requirements are the fluid system surrounding the spotting head rather than the use itself. The polymer that was used was dissolved in THF (tetrahydrofuran), which is not compatible with PVC. A polypropylene tube of the same size was obtained, which does not have the same issue with THF as flexible PVC. This significantly increased the volume of the spotting head fluid system. Another potential option was to dissolve the polymer in benzotrifluoride, which does not have the same compatibility issues with PVC.

[0277] Spotting droplets was a somewhat long and arduous process but once the system was functional with water it was easy to transfer to the polymer solution being used, with small changes to the signal parameters. This then allowed for spotting polymer in controlled locations and with defined amounts, which should increase the sensitivity of the device and is likely to be the process used for the final product. Polymer droplets have been successfully spotted into 1 mm wells.

[0278] Additional Testing

[0279] Additional testing was performed using a system schematically shown in FIG. 44 and further shown in FIGS. 45-46D. Using these systems, replacing sample-handling materials (glass to polypropylene (PP)) and using fresh PFOA solution, PFAS detection was improved such that PFAS concentrations of less than 0.01 ppb (FIGS. 48D and 48E), compared to previous experiments (FIGS. 48A-48C) where the limit for quantitatively detecting PFAS was higher (0.1 ppb, as shown in FIG. 48B). Some experiments also showed that the detection of PFAS can be improved by increasing the length of time steps (10 seconds (FIGS. 49A-49D) to 30 seconds (FIGS. 50A-50D)) between readings of the optical sensor and / or the time for an analyte to be sensed by the sensing material, as shown at smaller time steps in FIGS. 49A-49D compared to larger time steps in FIGS. 50A-50D.

[0280] Conclusion The goal of the testing was to create a functional design and prototype for a PFAS testing device. It was desired that the device be portable and able to be automated for use in the field without regular human intervention. A fluidic system and overall device design have been created, compared with other potential designs, and tested. This fluidic system is fully functional for manual dispensing of liquid and includes components that could be bought and used as is or built in house. Multiple contingencies are in place should issues come up that were not fully tested, such as PFAS diffusion through water and potentially more strict rinsing requirements. Initial testing has also confirmed that PFAS can be detected with the testing polymer using only fluorescent measurements taken from visual data at relatively low magnification, which can be collected without significant space or energy requirements. The testing shows a functional method of testing PFAS in water that is designed to be packaged in a portable system that could be used in the field to effectively measure PFAS on a large scale.

[0281] Samples of Raw Data

[0282] Tables 4 and 5 show raw data for some of the dapi and fitc tests done on glass slides. These include the direct fluorescence emission data taken from empty glass slide used as a baseline and the data from polymer slides with different PFAS concentrations. Each set of tables was data taken from one glass slide.

[0283] Table 4. Glass Slide Data

[0284] Table 5. Cylindrical cutout data

[0285] It should be understood that the subject matter defined in the appended claims is not necessarily limited to the specific implementations described above. The specific implementations described above are disclosed as examples only.

[0286] While several embodiments of the present invention have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and / or structures for performing the functions and / or obtaining the results and / or one or more of the advantages described herein, and each of such variations and / or modifications is deemed to be within the scope of the present invention. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and / or configurations will depend upon the specific application or applications for which the teachings of the present invention is / are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, the invention may be practiced otherwise than as specifically described and claimed. The present invention is directed to each individual feature, system, article, material, and / or method described herein. In addition, any combination of two or more such features, systems, articles, materials, and / or methods, if such features, systems, articles, materials, and / or methods are not mutually inconsistent, is included within the scope of the present invention.

[0287] Measurements of concentrations in terms of “parts” (e.g., parts per million, parts per billion, parts per trillion, etc.) are calculated on a mass basis, unless clearly indicated to the contrary.

[0288] The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.” The phrase “and / or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Other elements may optionally be present other than the elements specifically identified by the “and / or” clause, whether related or unrelated to those elements specifically identified unless clearly indicated to the contrary. Thus, as a non-limiting example, a reference to “A and / or B,” when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A without B (optionally including elements other than B); in another embodiment, to B without A (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0289] As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating items in a list, “or” or “and / or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of’ or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e. “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0290] As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting 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”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc. In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of’ and “consisting essentially of’ shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.

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Claims

CLAIMSWhat is claimed is:

1. A system for detecting analytes in a sample, the system comprising a cartridge, wherein the cartridge comprises: a multiplicity of microwells within a structural material, wherein each microwell comprises: a chamber; an optional cutout section; and a volume of 0.001-1,000 pl (0.001-1,000 mm3); and a layer domain above the structural material and the microwells, wherein: the layer domain seals the micro wells.

2. The system of claim 1, wherein each microwell has a volume of 0.1-100 pl (0.1-100 mm3).

3. The system of any one of claims 1-2, wherein the layer domain hermetically seals the micro wells.

4. The system of any one of claims 1-3, wherein the layer domain is configured to reseal after insertion of a needle.

5. The system of any one of claims 1-4, wherein the system is configured to distinguish different analytes.

6. The system of claims 1-5, wherein the system is configured to distinguish between different analytes by monitoring the temporal change of a signal.

7. The system of claims 1-6, wherein the system is configured to distinguish between long chain PFAS and short chain PFAS.

8. The system of any one of claims 1-7, wherein the layer domain comprises a plurality of layers.

9. The system of any one of claims 1-7, wherein the layer domain is a single layer.

10. The system of any one of claims 1-9, wherein the chamber is cylindrical.

11. The system of any one of claims 1-10, further comprising a glass layer, wherein the glass layer forms the bottom surface of the micro wells.

12. The system of any one of claims 1-11, wherein the structural material is in the form of a structural layer, and the structural layer has the thickness of the microwells.

13. The system of any one of claims 1-12, wherein the layer domain comprises a first layer above the structural layer and the microwells, wherein the first layer forms the top surface of the micro wells.

14. The system of claim 13, wherein the first layer hermetically seals the microwells.

15. The system of any one of claims 1-14, wherein the layer domain comprises a septum layer, wherein the septum layer is configured to reseal after insertion of a needle.

16. The system of claim 15, wherein the septum layer is above the first layer.

17. The system of any one of claims 1-16, wherein the layer domain further comprises an outer layer, wherein the outer layer is rigid and comprises holes, wherein the holes are configured to guide a needle to the micro wells.

18. The system of claim 17, wherein the outer layer is above the septum layer.

19. The system of any one of claims 1-18, wherein the analyte comprises a per- and polyfluoroalkyl substance (PF AS).

20. The system of any one of claims 1-19, wherein the microwell contains a sensing material, and the sensing material is configured such that one or more optical and / or electromagnetic properties of the sensing material change upon interacting with the analyte.

21. The system of claim 20, wherein there is a single type of sensing material in each microwell.

22. The system of claim 20, wherein there is a first type of sensing material in a first microwell and a second type of sensing material in a second microwell.

23. The system of any one of claims 20-22, wherein, within at least one microwell, there are multiple regions of sensing material arranged in a pattern.

24. The system of any one of claims 1-23, wherein the cartridge is or is part of a flexible belt.

25. The system of any one of claims 1-24, further comprising a conjugated polymer in some or all of the multiplicity of microwells, wherein the conjugated polymer is configured to bind to PFAS.

26. The system of any one of claims 1-25, wherein the microwells comprise the cutout section, wherein the conjugated polymer is localized in the cutout section.

27. The system of any one of claims 1-26, wherein the microwells comprise the cutout section, wherein the cutout section is configured to increase the diffusion time of the analyte to the conjugated polymer.

28. The system of any one of claims 25-27, further comprising a sensor, wherein the sensor is configured to detect fluorescence of the conjugated polymer through the glass layer.

29. The system of any one of claims 25-28, wherein the conjugated polymer comprises a molecule with a structure selected from the group consisting of:Py wherein andand wherein30. The system of any one of claims 1-29, wherein the microwells are single use.

31. The system of any one of claims 1-30, wherein the microwells are at least 50 pm in diameter and at least 10 jam in thickness.

32. The system of any one of claims 1-31, wherein the system comprises 30-500 individual microwells fitting in a single sheet.

33. The system of claim 32, wherein the sheet has a size 25 X 25 mm2.

34. The system of any one of claims 1-33, wherein the system is configured to deliver the sample to a microwell by insertion of a needle through the septum layer.

35. The system of any one of claims 1-34, wherein the structural material comprises polypropylene.

36. The system of any one of claims 1-35, wherein the layer comprises aluminum tape.

37. The system of any one of claims 1-36, wherein epoxy adhesive binds the layer to the structural material.

38. The system of any one of claims 1-37, wherein epoxy adhesive binds the glass layer to the structural material.

39. The system of any one of claims 1-38, wherein the microwells comprise the cutout section, wherein a second glass layer separates the structural material from the layer, and wherein the second glass layer forms the upper surface of the cutout section.

40. The system of any one of claims 1-39, wherein the sample comprises an aqueous sample.

41. A method of detecting an analyte in a sample using the system of any of claims 1-40.

42. The method of claim 41, wherein the analyte comprises a PFAS.

43. A method of detecting an analyte in a sample, comprising: injecting the sample into a microwell of a system comprising a multiplicity of microwells, and based on the presence or absence of a change in a condition related to the binding and / or reaction of the analyte to a sensing material, determining whether the analyte is present in the sample, wherein the system comprises: the multiplicity of microwells within a structural material, wherein each micro well comprises: a chamber; an optional cutout section; and a volume of 0.001-1,000 pl (0.001-1,000 mm3); and a layer domain above the structural material and the microwells, wherein: the layer domain seals the microwells.

44. The method of claim 43, wherein each microwell has a volume of 0.1-100 (0.1-100 mm3).

45. The method of any one of claims 43-44, wherein the layer domain hermetically seals the micro wells.

46. The method of any one of claims 43-45, wherein the layer domain is configured to reseal after insertion of a needle.

47. The method of any one of claims 43-46, wherein the analyte comprises a per- and polyfluoroalkyl substance (PF AS).

48. The method of any one of claims 43-47, wherein the sensing material is a conjugated polymer.

49. The method of any one of claims 43-48, wherein the system is the system of any one of claims 1-40.

50. A system for detecting analytes in an aqueous sample, the system comprising a cartridge, wherein the cartridge comprises a. a multiplicity of microwells, wherein each microwell comprises i. a cylindrical chamber having a diameter and a thickness; ii. an optional cutout section; iii. a volume of 0.1-100 pl (0.1-100 mm3); b. a glass layer, wherein the glass layer forms the bottom surface of the microwells; c. a structural layer in contact with the glass layer, wherein the structural layer has the thickness of the microwells; d. a thin layer above the structural layer and the microwells, wherein the thin layer forms the top surface of the microwells and hermetically seals the micro wells; e. a septum layer above the thin layer, wherein the septum layer is configured to reseal after insertion of a needle; and f. an outer layer above the septum layer, wherein the outer layer is rigid and comprises holes, wherein the holes are configured to guide the needle to the microwells.

51. The system of claim 50, wherein the analyte comprises a per- and polyfluoroalkyl substance (PFAS).

52. The system of claim 51, further comprising a conjugated polymer in some or all of the multiplicity of microwells, wherein the conjugated polymer is configured to bind to PFAS.

53. The system of claim 52, wherein the microwells comprise a cutout section, wherein the conjugated polymer is localized in the cutout section.

54. The system of claim 53, wherein the cutout section is configured to increase the diffusion time of PFAS to the conjugated polymer.

55. The system of claim 54, further comprising a sensor, wherein the sensor is configured to detect fluorescence of the polymer conjugate through the glass layer.

56. The system of claim 55, wherein the conjugated polymer comprises a molecule with a structure selected from the group consisting of57. The system of any one of claims 50-56, wherein the microwells are single use.

58. The system of any one of claims 50-57, wherein the microwells are hermetically sealed.

59. The system of any one of claims 50-58, wherein the system comprises 30-500 individual microwells fitting in a single sheet.

60. The system of claim 59, wherein the sheet has a size 25 X 25 mm2.

61. The system of any one of claims 50-60, wherein the system is configured to deliver the water sample to a microwell by insertion of the needle through the septum layer.

62. The system of any one of claims 50-61, wherein the structural layer comprises polypropylene.

63. The system of any one of claims 50-62, wherein the thin layer comprises aluminum tape.

64. The system of any one of claims 50-63, wherein epoxy adhesive binds the thin layer to the structural layer.

65. The system of any one of claims 50-64, wherein epoxy adhesive binds the glass layer to the structural layer.

66. The system of any one of claims 50-65, wherein the microwells comprise a cutout section, wherein a second glass layer separates the structural layer from the thin layer, and wherein the second glass layer forms the upper surface of the cutout section.

67. A method of detecting an analyte in an aqueous sample using the system of any of claims 50-66.

68. The method of claim 67, wherein the analyte comprises a PFAS.

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