Patent Application
20220011316
The invention relates to water or fluid treatment, sampling, and testing devices. The invention samples and tests water or other fluids for analytes of interest.
Quality testing of liquids, such as water, juice, milk, beer, wine, and others are commonly done by sending out samples to a lab or purchasing test kits and performing the testing in house. However, each of these methods has their shortcomings. Sending samples to a lab takes a lot of time, and test kits are not always as accurate and cannot always differentiate among substances of interest in the water. For example, some test kits may determine bacteria count in general but cannot separate normal bacteria from harmful bacteria such as Escherichia coli (E. coli)) or Legionella pneumophila. Further, some of these harmful substances may affect many water-based uses, including HVAC humidity regulation, agriculture, industrial, and other home uses, in addition to drinking water.
Other water testing devices and methods prepare samples for use by greatly concentrating analytes in the water. For example, the devices use a large water sample to collect and concentrate analytes and elute the concentrated sample from the filter for analysis. Some example devices use filters to trap retentate (e.g., larger particles such as dirt and analytes of interest such as bacteria) and allow the permeate (e.g., water) to pass through the filter. The trapped analytes in the filter are eluted to be collected for analysis of analyte levels in the original sample. These concentration and collection methods generate many inefficiencies. Some of the inefficiencies include longer testing times, lower sensitivity, and a lower percentage of recovery of the trapped analyte from the filter. The concentration and collection methods require longer testing times requiring larger volumes of permeate carrying analytes to concentrate analytes through filtration. The concentration and collection methods have lower sensitivity which requires higher concentrations of analytes in order be detected. These inefficiencies of current designs result in increased testing membrane costs, longer testing times, and increased intervention by those who need the testing results.
The methods and devices of the invention include inline water testing devices that are connected to a water source and benchtop testing devices for separate analyzing of water sources. The testing devices comprise an assay and a testing cartridge for analysis of water samples from water sources. The invention includes disposable testing cartridges that filter the water sample to be analyzed for analytes of interest (i.e., target analyte) in the water and are used for direct membrane interrogation. The invention provides improved testing systems for faster, more efficient (e.g., near real-time and higher sensitivity) water testing and need smaller sample sizes at potentially lower analyte levels. The invention further simplifies testing methods of analyte levels by removing the need to elute analytes from filters. The invention completely removes losses from the elution process.
The invention provides assays, which use optically detectable labels in the testing cartridges to determine target analyte levels in the samples of water. The invention provides automated testing of water in systems. The invention provides fluidic paths to filter the water samples, react analyte-specific labelled binding reagents to target analytes captured on the filter, as well as wash the filters of non-specifically absorbed analyte-specific binding reagent for direct membrane interrogation. The invention also provides a conjugate of analyte-specific binding reagent with the optically detectable labels for analysis of analytes captured on the filter. These fluidic paths may each include pumps, valves, and waste containers. The pumps and valves are in communication with a processor and/or controller to force fluids (e.g., water or solutions) through the testing cartridge. In one embodiment of the invention, the flow rate of fluids through the testing cartridge is capped at 30 mL/min. However, the fluid flow rate can be adjusted to allow effective collection of the targeted analyte by the filter, binding of the analyte-specific labelled binding reagent, as well as washing of analyte-specific labelled binding reagent without removal of the collected target analyte capped at 30 mL/min. In one embodiment, the system prevents pressure in the fluidic paths from exceeding a predetermined pressure threshold to prevent damage to the testing cartridge and to prevent false readings.
The invention provides testing cartridges which include a filter membrane. The labels may include colorimetric or phosphor labels including fluorescent molecules and/or particles, such as up-converting nanoparticles. The labels are conjugated to analyte-specific binding reagents, such as antibodies or nucleic acids, that bind specifically to target analytes in the sample. The labels and analyte-specific binding reagents form a conjugate which is introduced to the sample post-filtering after capturing of the target analyte and allowing the permeate to pass through the filter membrane (i.e., hereinafter the “conjugate”). The labels are selected to emit light after absorption of energy. In one example embodiment of the invention, the filter membrane of the testing cartridge has 0.22 μm-sized pores that are larger than the conjugate, but smaller than the target analyte to be captured by the filter membrane. The analyte-specific binding reagents, including antibodies, bind specifically to the target analyte, such as bacteria (e.g., Legionella pneumophila and E. coli). The analyte-specific binding reagents can also bind specifically to target analytes such as viruses, polyfluoroalkyl substances (PFAS), polymers, aggregates, proteins, nucleic acids, toxins, chemical contaminants, and other target analytes. Thus, the water samples are filtered through the membranes and target analytes. For example, bacteria are captured by the filter membrane. The labelled antibody conjugate specifically binds with the captured bacteria on the membrane. Excess, non-specifically bound conjugate is washed from the filter membrane, and the testing site may be dried prior to optical analysis. The assay uses a laser to excite the labelled conjugate remaining on the filter membrane such as conjugated antibodies bound to the bacteria. The labels, once excited by the laser, emit a fluorescence, which is optically detectable. Thus, the presence of fluorescence indicates the presence of the target analyte (such as bacteria, for example) and the amount (intensity) of fluorescence determines the bacteria levels in the water. In some embodiments, the filter membranes include a seal. In some embodiments, conjugate packets in the testing cartridge include a seal.
In some example embodiments of the invention, the testing cartridges are configured to seal in-line with the fluidic paths. The testing cartridge is also configured to provide sufficient filter membrane space. The membrane space is in terms of a surface area, to accurately capture the analytes in the water sample for determination of the analyte levels. Further, the testing cartridges include a sealed barrier to prevent adulteration of the cartridge prior to testing. Additionally, the filter membranes of the testing cartridges may be made of a polyvinylidene fluoride (PVDF), hydrophilic polyester (PETE), nitrocellulose, cellulose acetate, and other materials that meet the system requirements. The filter membrane has larger pores than the conjugate, but smaller pores than the target analyte of interest. In other words, the filter membrane collects and concentrates the target analyte while allowing unbound conjugate to pass through.
For example, when the target analyte is bacteria (e.g., E. coli, Legionella pneumophila, etc.) the pore size of the filter membrane may be 0.22 μm as described above. However, for smaller target analytes, such as viruses, the pore size of the filter membrane must be much smaller, e.g., 15 nm to prevent passing of the viruses through the filter membrane pores. Similarly, the conjugate must also be smaller than the pore size of 15 nm so that unbound conjugate passes through the filter membrane without being captured.
In one embodiment of the invention, a testing device stores many testing cartridges for single use testing of a sample of water. The device dispenses and positions each testing cartridge as testing is needed. The device also positions, as needed, the testing cartridges for drying, excitation using a laser, and optical analysis. In one example embodiment of the invention, the device dispenses a testing cartridge onto a rotating base for filtering the water sample, drying the filter, and measuring analyte levels. In one example embodiment, analyte levels are measured using laser excitation and optical analysis. In one embodiment, the testing cartridge is in a reel including many testing sites separated by hydrophobic regions. The device positions the reel by feeding the testing cartridge into a position for fluidic pathing of the water sample filtering, and further positioning for the excitation process and optical reader.
In some example embodiments of the invention, the testing cartridges accept 3 mL of antibody solution. In some embodiments, the testing cartridges may tolerate 15 psi or less pressure from the fluidic paths. In some embodiments, the testing cartridges are configured to include sufficient cross-sectional flow area to accept a fluidic path flow rate of 10 L/hr.
As used herein, the term “water” is generally used in reference to any subject fluid of interest that may contain target analytes. In other embodiments, the fluids may include human-ingestible fluids such as milk, beer, wine, or other solutions that may need analyte levels determined, such as laboratory buffers or test samples.
As used herein, the term “labels” is generally used in reference to any substance that is used to make an analyte-specific binding reagent optically detectable as a conjugate. More specifically, as used herein, the term “labels” refers to an optically detectable molecule or particle acting as a label that may include colorimetric elements, phosphor particles, fluorescent molecules, and/or other particles, including up-converting nanoparticles that exhibit photon up conversion for better optical detection. The label may be conjugated with analyte-specific binding reagents directed at binding with a target analyte in the water. The analyte-specific binding reagents may also be any other molecule which elicits a response to a targeted analyte in a subject fluid. The label may be an upconverting nanoparticle labelling an antibody in a conjugate.
As used herein, the term “analyte-specific binding reagents” is generally used in reference to any substance that is capable of specifically reacting or binding with a targeted analyte. More specifically, as used herein, the term “analyte-specific binding reagents” refers to an antibodies or nucleic acids for reacting to a particular bacterium. However, the “analyte-specific binding reagents” could refer to any number of reactants that bind specifically to targeted analytes that include bacteria, viruses, particles, chemicals, and other target analytes.
The methods of use of the assays of the invention include loading of a test cartridge to pass a sample of fluid from a fluid source through a filter membrane of the test cartridge. Once the sample is passed, a conjugate of labels and analyte-specific binding reagents is passed through the filter membrane to bind with a target analyte that was captured on the filter membrane. The assay interrogates the sample-passed filter membrane for the labels bound to the target analyte to determine a level of the target analyte in the sample. In some embodiments, the sample-passed filter membranes are prepped for interrogation by washing the sample-passed filter membrane with a solution to remove excess conjugate. In some embodiments, the sample-passed filter membranes are dried prior to interrogation. In some embodiments, the labels of the conjugate bound to the target analytes are excited and optically detected, by emitting light, to determine the level of the target analyte. In some embodiments, the excitable and optically detectable labels are up-converting nanoparticles. Further, in some embodiments, the target analytes are a bacteria or virus. In some embodiments, the labels are selected from a group consisting of colorimetric elements, phosphor molecules, and up-converting nanoparticles.
The assay includes a fluid flow path to provide a sample of fluid from a fluid source to detect analyte levels. The fluid flow path also provides a conjugate of analyte-specific binding reagents with labels to the test cartridge for collection on a filter membrane of the test cartridge. The conjugate collects on the filter membrane by specifically binding to a target analyte previously captured on the filter membrane from passing the sample of fluid. The fluid flow path may include a pump and valve to provide the sample and conjugate to the test cartridge. The assay includes a translational base to position the test cartridge for analysis by the assay. The assay includes an excitation mechanism to excite the labels in the conjugate for optical analysis by an optical detector of the assay. The optical detector detects optical frequencies of the excited labels to determine a level of the target analyte. Further, the assay may include a drying mechanism to dry sample-passed test cartridges.
The assays of the invention provide a way to test liquids for target analytes.
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In one embodiment, the testing device 101 includes containers which provide the water sample, such as from water source 109. Container(s) 111, wash container 113, and waste container 115 with a drain 117 are also included in the device 101. Each container includes different solutions for introduction to the filter membrane. These solutions may include analyte-specific binding reagents (i.e., antibody, nucleic acids, aptamers, nanobodies, streptavidin, avidin, proteins, lipoproteins, lectins, carbohydrates, polypeptide ligands of cellular receptors, polynucleotide probes, drugs, antigens, toxins, and the like) and/or other wash solutions, e.g., a solution or buffer with salt or detergent to prevent the conjugate from sticking to the filter membrane. Each container is connected to a valve which may open or close as each fluid from each container is needed. For example, when a sample is needed from water source 109, valve 107a opens, and the sample of water is collected for testing using pump 105a. Similarly, valve 107b is opened for conjugate in the reagent container 111, and pump 105b may be used to pump a wash from wash container 113 throughout the system 100. Further, valve 107c may be opened to provide fluids to the testing cartridge 103, and valve 107d may be opened to provide direct access to the waste container 115. Waste container 115 may include a drain 117 to easily dump waste from the system.
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In one embodiment, the labelled conjugate may be bound with the sample-passed filter membrane 307 by mixing of the conjugate with the sample-passed filter membrane. The mixing may be through directing flow of the conjugate forward and reverse through the sample-passed filter membrane multiple times to ensure more binding of the conjugate to the target analyte. For example, E. coli or Legionella pneumophila bacteria in a water sample filtered through the filter membrane 307 may bind to labelled antibodies by repeated mixing with a sufficient amount of the labelled antibodies. The testing cartridges 103 may then be washed with water, wash solution, or other reagent to remove excess conjugate in the filter membrane 307 and to remove other chemicals or biologics which may adulterate the test. The testing cartridges 103 may then be purged of fluids with air.
In other embodiments, the water samples are provided to the filter membranes 307 through flow paths 301, to filter and collect targeted analytes. The conjugates may be provided by either the packets 305 or the flow paths 301. The target analytes on the filter membranes 307 react to the conjugate, e.g., labelled antibodies bind to the E. coli or Legionella pneumophila bacteria and that are captured by the filter membranes. The filter membranes 307 are then washed to remove any excess conjugate and set to dry for optical analysis.
In some embodiments, the testing cartridges 103 may then be placed in a dry position to allow drying of the concentrated analytes, e.g., when using up-converting nanoparticles. Drying may aid in optical analysis of the up-converting nanoparticles. In one embodiment, the drying position may include drying mechanisms, such as heaters and fans which do not affect and/or denature the labelled analytes. Once dry, the testing cartridges 103 may be positioned for analysis and disposal. A laser 311 is used to excite the captured labels bound to the analytes and optical analysis through optical detection 313 of the excited nanoparticles provides a concentration level of the target analyte.
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A method of using the testing device 101 with a testing cartridge 103 is shown in blocks 801-807 of
In one embodiment, the testing device 101 measures the level of Legionella pneumophila cells in tap water by using up-converting nanoparticles conjugated with anti-L. pneumophila antibodies. The water and conjugate are passed through a 25 mm thick PVDF filter membrane with 0.22 μm pores at 30 mL/min. Once the Legionella pneumophila and conjugate are captured on the filter membrane, the filter membrane is washed and may be dried prior to optical analysis.
The invention addresses design and ease of use difficulties of many previously available water testing systems. The invention provides an economical and easy to use platform when performing tests of water samples for analyte levels.
This application claims the benefit of priority of U.S. Provisional Application No. 63/046,238 filed on Jun. 30, 2020.
| Number | Date | Country | |
|---|---|---|---|
| 63046238 | Jun 2020 | US |
