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WO2025035073A1 - Systèmes, dispositifs, et procédés de détection d'analytes dans des échantillons - Google Patents

Systèmes, dispositifs, et procédés de détection d'analytes dans des échantillons Download PDF

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Publication number
WO2025035073A1
WO2025035073A1 PCT/US2024/041667 US2024041667W WO2025035073A1 WO 2025035073 A1 WO2025035073 A1 WO 2025035073A1 US 2024041667 W US2024041667 W US 2024041667W WO 2025035073 A1 WO2025035073 A1 WO 2025035073A1
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WIPO (PCT)
Prior art keywords
layer
pfas
microwells
microwell
polymer
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PCT/US2024/041667
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English (en)
Inventor
Rohit N. Karnik
Tioga Jasper Laird BENNER
Jongwan Lee
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Massachusetts Institute of Technology
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Massachusetts Institute of Technology
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Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/508—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above
    • B01L3/5085—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above for multiple samples, e.g. microtitration plates
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/02—Adapting objects or devices to another
    • B01L2200/025—Align devices or objects to ensure defined positions relative to each other
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/02—Adapting objects or devices to another
    • B01L2200/026—Fluid interfacing between devices or objects, e.g. connectors, inlet details
    • B01L2200/027—Fluid interfacing between devices or objects, e.g. connectors, inlet details for microfluidic devices
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0689—Sealing
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/04—Closures and closing means
    • B01L2300/041—Connecting closures to device or container
    • B01L2300/044—Connecting closures to device or container pierceable, e.g. films, membranes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B01L2300/0627—Sensor or part of a sensor is integrated
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0829—Multi-well plates; Microtitration plates

Definitions

  • PFAS per- and polyfluoroalkyl substances
  • PFAS per- and polyfluoroalkyl substances
  • PFAS per- and polyfluoroalkyl substances
  • 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).
  • PFAS per- and polyfluoroalkyl substances
  • 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 mm 3 ); and a layer domain above the structural material and the microwells, wherein: the layer domain seals the microwells.
  • 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 mm 3 ); and a layer domain above the structural material and the microwells, wherein: the layer domain seals the microwells.
  • a system for detecting analytes in an aqueous sample 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 mm 3 ); 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
  • a septum layer above the thin layer wherein the septum layer is configured to reseal after insertion of a needle
  • 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.
  • the analyte comprises a per- and polyfluoroalkyl substance (PF AS).
  • PF AS per- and polyfluoroalkyl substance
  • 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.
  • the microwells comprise a cutout section, wherein the conjugated polymer is localized in the cutout section.
  • the cutout section is configured to increase the diffusion time of PFAS to the conjugated polymer
  • 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.
  • the conjugated polymer comprises a molecule with a structure selected from the group consisting of the structures shown in FIG. 4.
  • the microwells are single use.
  • the microwells are hermetically sealed.
  • the microwells 30 pm diameter with 10 nm thickness In one embodiment of the disclosed system, the microwells 30 pm diameter with 10 nm thickness.
  • the system comprises 30-500 individual microwells fitting in a single sheet.
  • the sheet has a size 25 X 25 mm 2 .
  • the system is configured to deliver the water sample to a microwell by insertion of the needle through the septum layer.
  • the structural layer comprises polypropylene.
  • the thin layer comprises aluminum tape.
  • epoxy adhesive binds the thin layer to the structural layer.
  • epoxy adhesive binds the glass layer to the structural layer.
  • 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.
  • One aspect of the disclosure herein is a method of detecting an analyte in an aqueous sample using the disclosed system.
  • the analyte comprises a PFAS.
  • FIG. 1 shows a cartridge for detecting analytes, in accordance with some embodiments.
  • FIGS. 2A-2B show a microwell and a layer domain above the micro well, in accordance with some embodiments.
  • 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.
  • FIG. 5 shows a system for detecting analytes, in accordance with some embodiments.
  • FIG. 6 shows PFAS chemical classification, in accordance with some embodiments.
  • 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.
  • FIG. 9 shows a diagram of needle -based system, in accordance with some embodiments.
  • FIGS. 10A-10B show graphs of fluorescence of polymer after 10 pl drop of 100 ppb PFBA, in accordance with some embodiments.
  • FIGS. 11A-1 IB show graphs of fluorescence of polymer after 10 pl drop of 100 ppb PFOA, in accordance with some embodiments.
  • 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.
  • FIG. 13 shows graph of interaction between zonyl and pyridine polymer with and without PFOA, in accordance with some embodiments.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • PFOA perfluorooctanic acid
  • 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.
  • PFOA perfluorooctanic acid
  • Figure 22A color ratio difference of water subtracted out
  • 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.
  • 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.
  • PFOA perfluorooctanic acid
  • 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.
  • PFOA perfluorooctanic acid
  • FIG. 24B 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.
  • 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.
  • 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.
  • FIGS. 27A-27H show diagrams of FRAP diffusion, the expected outcome from these tests.
  • FIG. 28 shows a graph of pixel grayscale after PFOA Kapton tape drop testing over time, in accordance with some embodiments.
  • FIG. 29 shows a graph of pixel grayscale after PFBA Kapton tape drop testing over time, in accordance with some embodiments.
  • 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.
  • FIG. 32 shows a drawing of sensor dimensions (dimensions in mm), in accordance with some embodiments.
  • 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.
  • FIGS. 34A-34B show diagrams of material layers for two microwell designs, in accordance with some embodiments.
  • FIGS. 35A-35B show images of a fabrication setup for single section polypropylene microwells, in accordance with some embodiments.
  • FIGS. 36A-36B show images of current fabrication setup for delrin cartridges, in accordance with some embodiments.
  • FIG. 37 shows a fluid system diagram, in accordance with some embodiments.
  • 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.
  • FIG. 39 shows a solidworks model of system parts, with corresponding photo, in accordance with some embodiments.
  • FIG. 40 shows a spotted water droplet in 1 mm delrin cutout, arrow shows location of droplet, in accordance with some embodiments.
  • FIG. 41 shows a setup of spotting head with microscope underneath, in accordance with some embodiments.
  • FIG. 42 shows a diagram of microfab technologies ink-jet microdispenser, in accordance with some embodiments.
  • FIG. 43 shows parameters used in jetserver to spot droplets, in accordance with some embodiments.
  • FIG. 44 shows a fabrication protocol for a system including a microwell, in accordance with some embodiments.
  • FIG. 45 shows a septum layer and aluminum sheet being attached together, in accordance with some embodiments.
  • 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.
  • FIGS. 48A-48E show plots of wavelength shift ratios used to detect PFOA, in accordance with certain embodiments.
  • FIGS. 49A-49B are plots of fluorescent intensity as a function of time, in accordance with certain embodiments.
  • FIG. 49C is a plot of a ratio of fluorescent intensities as a function of time, in accordance with certain embodiments.
  • FIG. 49D is a plot of wavelength shift ratio as a function of PFOA concentration, in accordance with certain embodiments.
  • FIGS. 50A-50B are plots of fluorescent intensity as a function of time, in accordance with certain embodiments.
  • FIG. 50C is a plot of a ratio of fluorescent intensities as a function of time, in accordance with certain embodiments.
  • FIG. 50D is a plot of wavelength shift ratio as a function of PFOA concentration, in accordance with certain embodiments.
  • PF AS per- and polyfluoroalkyl substances
  • 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.
  • more than a hundred individual microwells fitting in a single 25 X 25 mm 2 sheet can be employed, allowing many tests to be done before requiring manual replacement of the cartridge.
  • the cartridges can be easily removed and replaced for ease of use in the field.
  • a cartridge is used that comprises a plurality of microwells.
  • the microwells can comprise, for example, a chamber and an optional cutout section.
  • 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.
  • 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.
  • the layer domain may seal the chamber of the microwell (e.g., such that liquid does not traverse the layer domain).
  • 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).
  • 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).
  • 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.
  • the cartridge comprises a multiplicity of microwells.
  • cartridge 100 has a multiplicity of microwells 102.
  • 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.
  • 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.
  • 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.
  • each micro well within the multiplicity of micro wells comprises a chamber.
  • 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.
  • a fluid e.g., a liquid
  • solid boundaries e.g., walls
  • chamber 201 includes walls 221, 222, 223, and 224 and the volume within those walls.
  • the chambers are adjacent to the structural material of the cartridge.
  • 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).
  • 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.
  • the structural material is in the form of a structural layer.
  • structural material 202 is shown in the form of a layer.
  • 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).
  • the structural layer has the thickness of the microwells.
  • 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.
  • 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).
  • the use of a structural material in the form of a structural layer helps form a multiplicity of micro wells with uniform thicknesses.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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)
  • the aspect ratio of the chamber 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.
  • 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.
  • 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.
  • FIG. 2B One example of such a microwell is shown schematically 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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).
  • 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.
  • the timescale for the analyte to diffuse to the sensing material is proportional to the square of the characteristic chamber length scale and inversely proportional to the diffusivity of the target analyte.
  • 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).
  • 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.
  • the microwell comprises a layer domain.
  • the layer domain can be above the structural material and the microwell.
  • microwell 102 comprises layer domain 203 above structural material 202 and chamber 201.
  • 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.
  • 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.
  • 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).
  • the layer domain seals the microwells.
  • the layer domain provides a seal that prevents PFAS from the environment traversing the layer domain while the seal remains intact.
  • 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.
  • 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
  • the seal 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.
  • the layer domain is configured to reseal after insertion of a needle.
  • reseal does not necessarily mean a hermetic seal is established after the resealing.
  • the resealing can, for example, prevent PFAS from traversing the layer domain.
  • resealing of the layer domain prevents PFAS from traversing the layer domain during the measurement.
  • resealing of the layer domain prevents liquids (e.g., water or other liquids) from traversing the layer domain.
  • resealing of the layer domain results in the formation of a hermetic seal.
  • 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.
  • 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.
  • the layer domain may comprise a single layer or a plurality of layers between the chamber of the microwell and the external environment.
  • layer domain 203 includes a single layer between chamber 201 of microwell 102 and external environment 211.
  • layer domain 203 includes a plurality of layers between chamber 201 of microwell 102 and external environment 211.
  • the layer domain includes a single layer (or a single set of multiple, stacked layers) that covers a plurality of microwells.
  • layer domain 203 may extend laterally over additional microwells 102 (not shown in these figures) in the cartridge.
  • Multiple layer domains may be present, each layer domain covering a subset of chambers (or a single chamber) within the cartridge.
  • 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.
  • 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.).
  • 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.
  • an adhesive agent e.g., epoxy adhesive
  • the layer domain comprises a first layer.
  • the first layer forms the top surface of the microwell.
  • layer domain 203 comprises first layer 305, which forms the top surface of microwell 102.
  • the first layer seals (e.g., hermetically seals) the microwell.
  • 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.
  • the layer domain is configured to block at least some electromagnetic radiation.
  • the layer domain e.g., aluminum tape
  • electromagnetic radiation e.g., stray visible light
  • the layer domain comprises metal foil and/or tape (e.g., aluminum foil and/or tape).
  • 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.
  • metal foil and/or tape e.g., aluminum tape
  • other metals e.g. stainless steel
  • polymer-based tapes e.g., Kapton tape, polyethylene tape, polypropylene tape, Delrin, Viton, Kalrez
  • ceramic based layers e.g., glass, silicon
  • the first layer of the layer domain is a thin layer.
  • 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).
  • the layer domain comprises a septum layer.
  • cartridge portion 103 comprises a multi-layer layer domain 203 comprising first layer 305 and septum layer 301.
  • 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).
  • the septum layer is above the first layer.
  • 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.
  • 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).
  • the septum layer may be made of a resealable material such that the microwell is resealed after removing the needle.
  • 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.
  • the septum layer are silicone sheets.
  • the septum layer is free of PFAS, which can reduce potential contamination of samples.
  • the septum layer is made of a non-polymeric material (e.g., carbon foam, etc.).
  • the layer domain has an outer layer.
  • the outer layer may be the top layer of the layer domain.
  • 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.
  • the outer layer is rigid and has holes.
  • the use of a rigid outer layer can, for example, provide mechanical robustness to the cartridge.
  • the holes may be positioned over the microwells.
  • the holes are configured to guide the needle or other injector (e.g., pipettes, capillary tubes, microchannels, etc.) into the microwells.
  • outer layer 301B can have hole 306, according to some embodiments.
  • the outer layer is above the septum layer.
  • outer layer 301B is above septum layer 301.
  • the outer layer is the top layer.
  • the outer layer may be a rigid outer layer that provides structural support to the septum layer and thereby to the cartridge.
  • 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).
  • 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.
  • 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.
  • each microwell within the cartridge includes the same sensing material.
  • two or more microwells contain different sensing materials (e.g., more than one type of sensing material).
  • 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).
  • there is a single type of sensing material in each microwell there is a first type of sensing material in a first microwell and a second type of sensing material in a second microwell.
  • layer 204 (e.g., a glass layer) is adjacent to chamber 201.
  • layer 204 (e.g., a glass layer) forms the bottom surface of the microwells.
  • 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.
  • 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.
  • microwell 102 contains sensing material 303.
  • sensing material 303 is within chamber 201.
  • 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.
  • 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.
  • the sensing material changes its infrared properties in response to an interaction with the target analyte.
  • the sensing material changes its ultraviolet properties in response to an interaction with the target analyte.
  • the sensing material changes its magnetic properties in response to an interaction with the target analyte.
  • the sensing material changes its Raman properties in response to an interaction with the target analyte.
  • 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).
  • the sensing material e.g., conjugated polymer
  • the sensing material is in some or all of the multiplicity of micro wells.
  • 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
  • the sensing material in some or all of the multiplicity of microwells contains more than one type of sensing material.
  • the sensing material can be present as one or more sections (e.g., spots of sensing material) within a microwell.
  • 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.
  • more than one type of sensing material may be present in a particular section.
  • the sensing material may have a first section of sensing material, a second section of sensing material, a third section of sensing material, etc.
  • the sections of sensing material are aggregates, crystals, glasses, segmented thin films, or the like.
  • 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.
  • 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).
  • the sensing material is configured to interact (e.g., react and/or bind) with PFAS to produce a detectible signal.
  • the sensing material contains an enzyme or catalyst that interacts with the target analyte to produce a signal (e.g., electromagnetic radiation).
  • 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.
  • 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.
  • 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).
  • the property and/or change in property is a change in fluorescence emission intensity at one or more wavelengths.
  • the property and/or change in property is a relative change in fluorescence emission intensities between two or more wavelengths.
  • the property and/or change in property is an absorption and/or a change in absorption.
  • the property and/or change in property is a refractive index and/or a change in refractive index.
  • the property and/or change in property is a scattering of light and/or a change in scattering of light.
  • the property and/or change in property is a an isotropy and/or a change in isotropy of the sensing material (e.g., birefringence).
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the sensing material is a conjugated polymer.
  • the conjugated polymer is a conjugated polymer falling within the scope of molecules shown in FIG. 4.
  • the conjugated polymer may have the following structure: Py* wherein
  • the conjugated polymer may have the following structure:
  • the sensing material e.g., conjugated polymer
  • the sensing material has a backbone moiety comprising poly(p-phenylene ethynylene) (PPE), polyfluorene (PF), and/or fluorinated poly(p-phenylene ethynylene) ( F PPE).
  • the sensing material e.g., conjugated polymer
  • the conjugated polymer has a moiety capable of binding to PFAS, where such moiety is pyridine and/or thiophene-functionalized pyridine.
  • PFAS 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.
  • 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”).
  • the analyte comprises a fluoroalkyl substance.
  • the analyte comprises a per- and polyfluoroalkyl substance (PFAS).
  • 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).
  • binding between the PFAS and conjugated polymer may produce a signal (e.g., fluorescent signal) that is indicative of such binding.
  • the sample is an aqueous sample.
  • 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).
  • the system and/or methods are configured to distinguish between different types of analytes.
  • the system and/or methods are configured to distinguish between long chain PFAS and short chain PFAS.
  • the length of the analyte e.g., the length of the PFAS
  • the length of the PFAS may impact the diffusion time of that analyte within the volume of the microwell.
  • a first type of analyte e.g., a first type of PFAS
  • a longer chain e.g., perfluorooctanoic acid
  • a second type of analyte e.g., a second type of PFAS
  • a shorter chain e.g., perfluorobutanoic acid
  • 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.
  • 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.
  • 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.
  • 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).
  • the senor 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).
  • the sensing material e.g., birefringence
  • 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).
  • PFAS analyte
  • the sensor may be used for a variety of purposes (e.g., detect the presence of PFAS, quantify the amount of PFAS present, etc.).
  • the senor 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).
  • the senor 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.
  • the cartridge and/or the microwells are single use.
  • the microwells may be used once and then may be disposed of.
  • the cartridge may have a multiplicity of microwells, where each microwell is single use.
  • a second glass layer separates the structural material from the layer domain.
  • a second layer forms the upper surface of the optional cutout section.
  • second layer 304 forms the upper surface 307 of cutout section 205.
  • the second layer is a glass layer.
  • the system has a multiplicity of microwells.
  • the system e.g., a cartridge for detecting analytes
  • the system may have 30-500 individual microwells.
  • 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.
  • 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).
  • the multiplicity of wells fit in a sheet.
  • the sheet is a single sheet.
  • the single sheet of microwells has a certain size.
  • the sheet has a size of 25 X 25 mm 2 (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.
  • the sheet could be of a different size, for example, greater than or equal to 1 mm 2 (e.g., greater than or equal to 1 X 1 mm 2 ), greater than or equal to 4 mm 2 (e.g., greater than or equal to 2 X 2 mm 2 ), greater than or equal to 25 mm 2 (e.g., greater than or equal to 5 X 5 mm 2 ), greater than or equal to 100 mm 2 (e.g., greater than or equal to 10 X 10 mm 2 ), greater than or equal to 625 mm 2 (e.g., greater than or equal to 25 X 25 mm 2 ), or greater than or equal to 2500 mm 2 (e.g., greater than or equal to 50 X 50 mm 2 ).
  • 1 mm 2 e.g., greater than or equal to 1 X 1 mm 2
  • 4 mm 2 e.g., greater than or equal to 2 X 2 mm 2
  • 25 mm 2 e.g., greater than or equal
  • the sheet can have a size of less than or equal to 10,000 mm 2 (e.g., less than or equal to 100 X 100 mm 2 ), less than or equal to 2500 mm 2 (e.g., less than or equal to 50 X 50 mm 2 ), less than or equal to 625 mm 2 (e.g., less than or equal to 25 X 25 mm 2 ), less than or equal to 100 mm 2 (e.g., less than or equal to 10 X 10 mm 2 ), less than or equal to 25 mm 2 (e.g., less than or equal to 5 X 5 mm 2 ), or less than or equal to 4 mm 2 (e.g., less than or equal to 2 X 2 mm 2 ). Combinations of these ranges are also possible.
  • 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.
  • movement of the belt positions microwells for readout by a sensor (e.g., an optical sensor and/or electromagnetic sensor).
  • the belt is transparent to light.
  • the belt is made of a plastic (e.g., polypropylene), woven or non-woven cloth, elastomer, metal, or mesh.
  • the belt forms a closed loop.
  • the belt is open-ended.
  • the belt is configured as a mobius strip.
  • the microwells are located on both sides of the belt.
  • 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.
  • the microwells are arranged in a pattern (e.g., a predetermined pattern such as a line of microwells).
  • the microwells are arranged in a rectangular array.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • at least a portion of the sample may be removed with the needle.
  • the structural material comprises polypropylene.
  • the structural material may also be any of a variety of other materials that can form microwells.
  • 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.
  • one or more layers within the cartridge are bound by an adhesive, such as an epoxy adhesive.
  • 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.
  • adhesive binds the bottom layer to the structural material.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • multiple microwells contain a sensing material.
  • samples are injected into each of the microwells containing the sensing material.
  • 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.
  • a target analyte e.g., a PFAS
  • 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).
  • the systems described herein can, in accordance with some embodiments, detect analytes present in the injected sample at very low concentrations.
  • 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.
  • 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.
  • PFAS perfluorooctanoic acid
  • PFOA perfluorooctanoic acid
  • 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).
  • 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).
  • 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).
  • 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.
  • 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).
  • PFOS 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).
  • LOAEL lowest observed adverse effect level
  • 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).
  • PFAS 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).
  • 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.
  • PFAS PFAS
  • 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.
  • 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.
  • SGS Swiss testing and inspection company
  • SGS 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.
  • PFOS includes a large variety of different molecules including both polymer and non-polymer materials with differing carbon chain lengths.
  • PFAS sensor 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).
  • 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.
  • 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.
  • exciton excited state
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the slope of the PFOA lOOppb test 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • microwells were 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.
  • 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.
  • 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.
  • FRAP Fluorescence recovery after photobleaching
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • monitoring the change in signal over time can help to distinguish between different classes of PFAS.
  • 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.
  • 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.
  • microwell dimensions 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.
  • FIGS. 33A-33D 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 40% of laser source power 40 W
  • 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.
  • a glass coverslip was first spin-coated with sensing polymer.
  • BONDiT B-482 was applied on the surface of the machined Delrin sheet by gently stamping the resin using a porous, clean wipe.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the 4-way valve 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Currently devices use 10 pl of fluid exposed to a polymer of around 12.5 mm 2 resulting 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 mm 2 with around the same thickness.
  • 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.
  • 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.
  • 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.
  • the spotting head occasionally clogged due to the presence of small particles. Using filtered water was found to be important.
  • 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.
  • PVC polyvinyl chloride
  • PVC polyvinyl chloride
  • 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.
  • THF tetrahydrofuran
  • 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.
  • FIG. 44 Additional testing was performed using a system schematically shown in FIG. 44 and further shown in FIGS. 45-46D.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • “at least one of A and 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.
  • PFAS update State-by- state groundwater regulations - May 2023.” Retrieved May 11, 2023, from JD Supra website: https://www.jdsupra.com/legalnews/pfas-update-state-by-state-groundwater-2004872/. Paisner, B., Kindschuh, J., Lee, T., & Voyen, E. (2022).
  • PFAS update State-by-state regulation of PFAS substances in drinking water. Retrieved from JD Supra website: https://www.jdsupra.com/legalnews/pfas-update-state-by-state-regulation-4639985/.
  • Method 533 Determination of per-and polyfluoroalkyl substances in drinking water by isotope dilution anion exchange solid phase ex- traction and liquid chromatography/tandem mass spectrometry.” Retrieved from Epa.gov website: https://www.epa.gov/sites/default/files/2019-12/documents/method-533- 815bl9020.pdf

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Abstract

L'invention concerne de manière générale des systèmes, des dispositifs, et des procédés de détection d'analytes tels que des substances per-et polyfluoroalkyle (PFAS) dans des échantillons. Certains aspects de la présente divulgation concernent des systèmes de détection d'analytes dans des échantillons. Dans certains modes de réalisation, une cartouche est utilisée qui comprend une pluralité de micropuits. Les micropuits peuvent comprendre, par exemple, une chambre et une section découpée facultative. Dans certains modes de réalisation, le micropuits présente un volume de 0,001 à 1 000 pl (par exemple, 0,1 à 100 pl), qui peut, par exemple, fournir un volume suffisant pour détecter la présence de PFAS tout en n'étant également pas suffisamment grand pour nécessiter une longue durée avant de produire un résultat. Dans certains modes de réalisation, la cartouche comprend un domaine de couche, qui peut sceller hermétiquement les micropuits. Le domaine de couche peut être, dans certains modes de réalisation, configuré pour se refermer après l'insertion d'une aiguille.
PCT/US2024/041667 2023-08-10 2024-08-09 Systèmes, dispositifs, et procédés de détection d'analytes dans des échantillons Pending WO2025035073A1 (fr)

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US20050233472A1 (en) * 2003-09-19 2005-10-20 Kao H P Spotting high density plate using a banded format
US20100261184A1 (en) * 2007-11-30 2010-10-14 Bioneer Corporation Micro-Chamber Plate, Manufacturing Method Thereof
US20130123139A1 (en) * 2010-07-23 2013-05-16 Namyong Kim Apparatus and method for multiple reactions in small volumes
US20190193076A1 (en) * 2016-07-14 2019-06-27 Hewlett-Packard Development Company, L.P. Microplate lid
WO2024119082A1 (fr) 2022-12-01 2024-06-06 Massachusetts Institute Of Technology Compositions pour la détection de fluorocarbones et articles, systèmes et procédés associés

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