WO2025059638A1 - Dispositif et procédés de détection d'un organisme - Google Patents

Dispositif et procédés de détection d'un organisme Download PDF

Info

Publication number
WO2025059638A1
WO2025059638A1 PCT/US2024/046881 US2024046881W WO2025059638A1 WO 2025059638 A1 WO2025059638 A1 WO 2025059638A1 US 2024046881 W US2024046881 W US 2024046881W WO 2025059638 A1 WO2025059638 A1 WO 2025059638A1
Authority
WO
WIPO (PCT)
Prior art keywords
detection
substrate
detection apparatus
agent
collection surface
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2024/046881
Other languages
English (en)
Inventor
Pravansu S. Mohanty
Shari RADFORD
Laura Bronsart
Mary RETZLAFF
Tejasvi CHUNDURI
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Ambient Biosciences Inc
Original Assignee
Ambient Biosciences Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Ambient Biosciences Inc filed Critical Ambient Biosciences Inc
Publication of WO2025059638A1 publication Critical patent/WO2025059638A1/fr
Anticipated expiration legal-status Critical
Pending legal-status Critical Current

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/02—Devices for withdrawing samples
    • 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
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5029—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures using swabs
    • 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
    • B01L2200/16—Reagents, handling or storing thereof
    • 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
    • B01L2200/18—Transport of container or devices
    • B01L2200/185—Long distance transport, e.g. mailing
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/06—Valves, specific forms thereof
    • B01L2400/0677—Valves, specific forms thereof phase change valves; Meltable, freezing, dissolvable plugs; Destructible barriers
    • B01L2400/0683—Valves, specific forms thereof phase change valves; Meltable, freezing, dissolvable plugs; Destructible barriers mechanically breaking a wall or membrane within a channel or chamber
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/02—Devices for withdrawing samples
    • G01N2001/028—Sampling from a surface, swabbing, vaporising

Definitions

  • the present disclosure generally concerns apparatuses and methods for detecting the presence of an agent such as a chemical or biological agent or organism.
  • Detection agents are commonly liable to degradation during storage and prior to use. Degradation of detection agents is prevented by the use of cold chain storage the increase the lifetime of the detection agent. Cold chain storage, however, is not always available, particularly in the field in areas where electrical service is irregular or absent or other considerations prevent the presence of infrastructure suitable to allow for such cold chain storage. Thus, when analyzing a surface or space for the presence or absence of an agent or organism, samples must be collected and shipped to a separate location for analysis. This increases the time to detection and introduces potential issues with the sample during transfer to an analysis location. [0005] Accordingly, a need exists to develop devices and methods to allow for remote detection of agents or organisms and without the need for expensive and often unreliable infrastructure.
  • Example embodiments disclosed herein are directed to devices and methods for rapidly analyzing a biological sample for the presence of an agent or organism.
  • Embodiments of the present disclosure overcome obstacles in microbial detection by providing devices and methods for rapid microbial detection using a detection apparatus with vitrified detection agents built directly into the device such that the detection agents are immediately available for detection of the agent or organism and without the need for cold chain storage of such detection agents.
  • detection apparatuses that include a sample collection device having a collection surface and an elongate shaft, coupled to a proximal end of the collection surface.
  • the detection apparatus also includes a substrate fluidly coupled to said collection surface, where said substrate includes at least one detection agent vitrified thereto.
  • a method includes collecting a sample to be tested for the organism on the collection surface, contacting the substrate with elution buffer, where the elution buffer rehydrates the vitrified detection agent, whereby the agent or organism, if present in the sample, reacts with or otherwise contacts the detection agent, and detecting the presence or absence of the agent or organism by the presence or absence of a detectable signal.
  • FIG. 1A illustrates an exemplary device according to one or more embodiments described herein;
  • FIG. IB illustrates the exemplary device of FIG. 1A, wherein the internal partition has been ruptured according to one or more embodiments described herein;
  • FIG. 1C illustrates an exemplary sample collection device according to one or more embodiments described herein;
  • FIG. 2A illustrates an exemplary configuration of a sample collection surface and substrate in a sample collection device according to one or more embodiments described herein;
  • FIG. 2B illustrates an exemplary configuration of a sample collection surface and substrate in a sample collection device according to one or more embodiments described herein;
  • FIG. 2C illustrates an exemplary configuration of a sample collection surface and substrate in a sample collection device according to one or more embodiments described herein;
  • FIG. 3 illustrates an exemplary device according to one or more embodiments described herein;
  • FIG. 4 graphically illustrates comparative results of organism detection according to one or more embodiments described herein.
  • the devices employ a collection surface whereby a substrate housing one or more detection agents therein is fluidly connected to the collection surface, optionally within the collection surface, adjacent thereto, or the collection surface itself.
  • the collection surface is optionally on an end of an elongate shaft such that the device may be contacted to a surface or space for analysis of the presence or absence of an agent or organism.
  • the presence of the detection agents in stable form that does not require cold chain storage to the collection surface allows for rapid and robust detection upon dissolving the one or more detection agents with a fluid. This allows for rapid, remote, and robust detection of the presence or absence of an agent or organism on a surface optionally without the need for separately housed detection agents.
  • 0 VitrificationO is a process of converting a material into an amorphous material.
  • the amorphous solid may be free of any crystalline structure.
  • 0 Vitrification mixtureO as used herein, means a heterogeneous mixture of biological material(s) and a vitrification medium containing vitrification agents and optionally other materials.
  • Vitrification agentO is a material that forms an amorphous structure, or that suppress the formation of crystals in other material(s), as the mixture of the vitrification agent and other material(s) cools or desiccates.
  • the vitrification agent(s) may also provide osmotic protection or otherwise enable cell survival during dehydration.
  • the vitrification agent(s) may be any water soluble solution that yields a suitable amorphous structure for storage of biological materials.
  • the vitrification agent may be imbibed within a cell, tissue, or organ.
  • Hydrophilic means attracting or associating preferentially with water molecules. Hydrophilic materials with a special affinity for water, maximize contact with water and have smaller contact angles with water relative to hydrophobic materials.
  • Hydrophobic means lacking affinity for water. Materials that are hydrophobic naturally repel water, causing droplets to form, and have large contact angles with water.
  • Bio sample refers to a sample that may contain an organism or portion of an organism of interest or any biologically derived agent.
  • the biological sample may be any sample capable of flowing through the matrices described herein and potentially harboring the agent or organism of interest.
  • the biological sample comprises saliva, blood, serum, plasma, urine, bronchoalveolar lavage fluid, sputum, nasal fluid, skin secretions, or combinations thereof.
  • the biological sample is collected from a surface.
  • the terms 0 surfaceO or 0 environmental surfaceO generally refer to any surface from which a sample can be collected.
  • Exemplary, non-limiting examples of surfaces include walls, doors, floors, ceilings, drains, appliances, furniture, refrigeration systems, ducts, vents, toilet seats, handles, doorknobs, handrails, bedrails, countertops, tabletops, eating surfaces, working surfaces, equipment surfaces, clothing, etc.
  • the devices and methods as provided herein may be used to detect the presence or absence of an agent or organism on a surface or within a space or the environment.
  • the organism is a microbial organism.
  • the microbial organism may be a bacteria or portion thereof.
  • the organism is a fungi or portion thereof.
  • the organism is a virus or a portion thereof.
  • the presence or absence of the microbial organism is determined by the presence or absence of a target analyte on a collection surface after collection.
  • a target analyte or agent may be any chemical or biological material.
  • a target analyte may be a biomolecule.
  • BiomoleculeO refers to proteins, polysaccharides, nucleic acids, and nucleotides such as ATP, GTP, NAD, and NADP, that indicate the presence of one or more organisms.
  • An exemplary bacteria used and exemplified throughout this disclosure for exemplary purposes alone is Escherichia coli (E. coll).
  • the devices and methods as provided herein are equally described with respect to any other bacteria, virus, fungus, or portion thereof. It is further appreciated that the devices and methods as provided herein are equally described as detecting one or more chemical agents, including but not limited to toxins, chemical warfare agents, industrial chemicals, or other. This disclosure provides devices and methods that can specifically detect the presence or absence of an agent or organism or other target analyte in a specific and robust system.
  • FIGS. 1A-1C depict an exemplary embodiment of a detection apparatus 10 in accordance with the present disclosure. It is noted that positional terms such as 0 upper, 0 0 lower, 0 0 proximal, 0 distal, 0 0 top,0 and 0 bottomO are used herein to assist in describing an embodiment of the present disclosure but are not limiting.
  • the detection apparatus 10 may be inverted or positioned differently than shown.
  • the detection apparatus 10 generally includes a sample collection device 100.
  • the sample collection device 100 generally includes an elongate shaft 102 and a collection surface 104. coupled to the elongate shaft 102.
  • the elongate shaft 102 may be constructed from a variety of materials, such as plastic, metal, wood, or combinations thereof. In some embodiments, the elongate shaft 102 is flexible such that it can reach sampling sites that would otherwise be inaccessible. In other embodiments, the elongate shaft 102 can be relatively rigid, so that force can be applied to the elongate shaft 102 to collect a sample.
  • the sample collection device 100 also includes the collection surface 104.
  • the collection surface 104 is adapted for receiving a biological sample.
  • the collection surface 104 is formed from one or more liquid permeable materials, such as cotton, fibers, sponges, foams, non-woven fabrics, bamboo, hydrophilic or hydrophobic polymers, and the like.
  • the collection surface 104 is sterile to prevent contamination of the sample or inaccurate results.
  • the collection surface 104 may be coupled to the elongate shaft 102.
  • a proximal end 105 of the collection surface 104 is coupled to the elongate shaft 102.
  • the collection surface 104 may be coupled to the elongate shaft 102 using any suitable mechanism, including, but not limited to fasteners, adhesives, heat sealing, thermal bonding, and the like.
  • the collection surface 104 is coupled to the elongate shaft 102 by entanglement of the liquid permeable material on and/or around the elongate shaft 102.
  • the collection surface 104 is coupled to the elongate shaft 102 by one or more threaded fasteners.
  • the sample collection device 100 is used to contact a sampling site.
  • the biological sample may be collected by contacting the collection surface 104 to the sampling site.
  • the collection surface 104 is gently rubbed or rotated over the sampling site, wherein the collection surface 104 will absorb or collect the biological sample.
  • Exemplary sampling sites include skin, nails, mucous membranes, wounds, environmental surfaces, and the like.
  • the sample collection device 100 may include a substrate 106 fluidly coupled to the collection surface 104.
  • the substrate 106 may be a membrane scaffold or any other structure operable to function within this disclosure.
  • the substrate is present in the form of a fibrous random or ordered mesh that defines channels or other continuous or discontinuous routes of access for a fluid or a target analyte.
  • a substrate is in the form of a nonwoven fibrous mesh with high porosity (e.g. greater than 50% by volume).
  • the substrate is a foam material.
  • suitable substrates can be found in International Patent Application Publication No. WO 2020/086812.
  • the substrate 106 may be formed from one or more matrix materials into or onto which one or more vitrified components may be associated at one or more locations.
  • the matrix material may be formed into an underlying patterned ridged support or of a porous material such as a membrane may be made of a material that is not toxic and not reactive to the biomaterials or biological samples and does not react chemically or physically with the vitrification medium.
  • the material can be of a suitable polymer, metal, ceramic, glass, or a combination thereof.
  • a matrix material is formed from a material of polydimethylsiloxane (PDMS), polycarbonate, polyurethane, polyethersulphone (PES), polyester (e.g. polyethylene terephthalate), among others.
  • matrix materials include polymers, optionally including but not limited to, collagen, elastin, hyaluronic acid and derivatives, sodium alginate and derivatives, chitosan and derivatives, gelatin, starch, cellulose polymers (for example, nitrocellulose (e.g.
  • Polymers that may be used in a substrate herein optionally can be formed into a porous mesh, such as in the form of a filter, symmetric mesh, or other such porous sheet-like material.
  • a polymer is formed into a fibrous network such as by methods that include electrospinning. In electrospinning, desired polymers are placed in a desired solvent (e.g.
  • the substrate includes 2 or more layers of polymer, optionally 3, 4, 5, 6, or more layers of polymer.
  • the substrate comprises a pore size, the pore size optionally suitable for controlling sample flow or regulating the size of molecules that may pass through the system.
  • the pore size within the matrix ranges from about 5 micrometers (pm) to about 500 pm, or any value or range therebetween.
  • the pores may be of an average opening of from about 20 pm to about 0.1 pm, including about 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, and 0.2 pm.
  • a capillary channel may have a length optionally defined by the thickness of a substrate that forms the channels or by one or a plurality of individual channels themselves.
  • a capillary channel length is optionally about one millimeter or less, but is not to be interpreted as limited to such dimensions.
  • a capillary channel length is of about 0.1 microns to about 1000 microns, or any value or range therebetween.
  • a capillary channel length is of about 5 to about 100 microns, optionally of about 1 to about 200 microns, and/or optionally of about 1 to about 100 microns.
  • a capillary channel length is optionally about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 microns.
  • the length of the capillary channels varies throughout a plurality of capillary channels, optionally in a non-uniform variation.
  • the pore size of the substrate is selected to permit the flow of a liquid and/or any reporters or target analytes of interest present in the biological sample.
  • different layers of the substrate component may comprise different pore sizes.
  • the cross-sectional area of the capillary channel(s) may be of about 2000 pm 2 or less.
  • a cross-sectional area is of about 0.01 pm 2 to about 2000 pm 2 , optionally of about 100 pm 2 to about 2000 pm 2 , or any value or range therebetween.
  • a cross- sectional area of the capillary channel(s) is of about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 pm 2 or less.
  • the substrate is of a hydrophilic material.
  • the substrate may be of a hydrophobic material and further treated to be hydrophilic or more hydrophilic in nature, such as through exposure to a plasma.
  • a substrate is or includes polycaprolactone (PCL), collagen, or combinations thereof.
  • PCL polycaprolactone
  • the primary characteristic of such water-stable polymers is that they are able to form networks or fibers such that one or more components may be vitrified thereon or therein.
  • the substrate must have sufficient stability in an aqueous environment so as to serve as a suitable surface for vitrification of an aqueous vitrification medium containing one or more desired molecules such as detection agents.
  • the substrate 106 is disposed on the sample collection device. As shown, the collection surface 104 is in fluidic contact with the substrate 106. Optionally, the substrate 106 is coupled to the elongate shaft 102. In some embodiments, the substrate 106 is disposed on a proximal surface of the collection surface 104. In some embodiments, the substrate 106 is disposed at least partially between the elongate shaft 102 and the collection surface 104. Optionally, the substrate 106 is a layer disposed underneath the collection surface 106. In some embodiments, the collection surface 104 partially or completely covers the substrate 106.
  • the substrate 106 and the collection surface 104 are coupled to the elongate shaft at discrete positions.
  • the substrate 106 and the collection surface 104 may be disposed on diametrically opposite points.
  • the substrate 106 and the collection surface 104 may be offset from one another by an angle, optionally 90 degrees.
  • the substrate 106 optionally contains or supports one or more vitrified components, such as one or more detection agents, buffering agents, or other desired material.
  • Components may be vitrified to the substrate using any suitable method known in the art. For example, and not as a limitation, methods may include those detailed in U.S. Patent No. 10,433,540, U.S. Patent No. 10,568,318, International Patent Application No. PCT/US2022/035892, and International Patent Application No PCT/US2021/060164, the contents of each of which are incorporated by reference in their entirety.
  • a vitrification medium including one or more materials to be vitrified into or onto a substrate are combined and placed into or onto the matrix.
  • a vitrification medium may include at least one vitrification agent.
  • a vitrification agent include, but are not limited to, dimethylsulfoxide, glycerol, sugars (e.g. trehalose, etc.), polyalcohols, methylamines, betines, antifreeze proteins, synthetic anti-nucleating agents, polyvinyl alcohol, cyclohexanetriols, cyclohexanediols, inorganic salts, organic salts, ionic liquids, or combinations thereof.
  • 1, 2, 3, 4, or more vitrification agents are included in the vitrification medium.
  • the vitrification agent is included in the vitrification medium at a concentration that is dependent on the identity of the vitrification agent.
  • concentration of the vitrification agent below that which would be toxic to the material(s) being vitrified.
  • 0 toxicO means that functional or biological viability is not achieved upon subsequent sample use, or the material is not suitable for subsequent use or analyses.
  • concentration of the vitrification agent is greater than or equal to 500 micromolar (p.M) and less than or equal to 6 molar (M), or any value or range therebetween.
  • trehalose is included in various aspects in a concentration of greater than or equal to 1 millimolar (mM) and less than or equal to 6 M, optionally greater than or equal to 150 mM and less than or equal to 6 M.
  • the total concentration of all vitrification agents when combined is greater than or equal to 1 mM and less than or equal to 6 M, optionally greater than or equal to 1 mM and less than or equal to 6 M.
  • the vitrification medium may further include other components, such as, by way of example and not limitation, water or other solvents, a buffering agent, one or more salts, RNase or DNAse inhibitors, or combinations thereof.
  • a buffering agent is any agent with a pKa of 6 to 8.5 at 25 °C.
  • Illustrative examples of buffering agents include choline, betaine, HEPES, TRIS, PIPES, MOPS, among others.
  • the buffering agent is a buffering agent that contains large organic ions (greater than 120 kDa), such as choline, betaine, or HEPES.
  • the buffering agent is provided at a concentration suitable to stabilize the pH of the vitrification medium to a desired level.
  • a vitrification medium may include one or more salts.
  • Salts can include, by way of example and not limitation, magnesium salts, sodium salts, potassium salts, chloride salts, or combinations thereof.
  • the salts can be provided at a concentration of from greater than or equal to 1 mM to less than or equal to 500 mM.
  • a detection agent, reporter, buffering agent or any other desired material may be vitrified into or onto a particular device, optionally according to the teachings of U.S. Patent No. 10,433,540.
  • the material(s) to be vitrified may be combined in a vitrification solution including a sugar (e.g. trehalose) and that further includes a combination of divalent metal ions and a chelator.
  • a sugar e.g. trehalose
  • divalent metal ions includes salts of Ca, Mg, Co, Fe, Zn, Mn among others.
  • Illustrative salts include chloride, sulfate, acetate, etc.
  • Chelators include polyols, ethylenediaminetetraacetic acid (EDTA), egtazic acid (EGTA), among others.
  • EDTA ethylenediaminetetraacetic acid
  • EGTA egtazic acid
  • the molar ratio of divalent metal salt to the chelator in the vitrification solution may be 10:90 to 90:10, optionally 50:50.
  • the detection of the analyte may be performed by detection of the reporter in the detection apparatus 10 by any suitable method.
  • detection includes chemiluminescence, a fluorescence reader matched to the detection agent, colorimetric assay, direct detection of label on the reporter molecule (e.g. gold or other), or other such assay systems.
  • Illustratively detection of an analyte is performed using an enzyme/substrate system (e.g. luciferase coupled with luciferin, horseradish peroxidase (HRP) or alkaline phosphatase (AP) coupled with a substrate such as 4-chloro-l -naphthol (4-CN), 3, 30 -diaminobenzidine (DAB), p-Nitrophenyl Phosphate (PNPP), CSPD and CDP-Star substrates, DynaLight Substrate with RapidGlow Enhancer, o-phenylenediamine dihydrochloride (OPD), 3, 3', 5,5'- tetramethylbenzidine (TMB) or its derivatives (e.g.
  • an enzyme/substrate system e.g. luciferase coupled with luciferin, horseradish peroxidase (HRP) or alkaline phosphatase (AP) coupled with a substrate such as 4-
  • the enzyme/substrate system is luciferase coupled with luciferin.
  • the substrate 106 and/or the collection surface 104 may include one or more vitrified assay reagents therein.
  • vitrified assay reagents may include buffers, detergents, salts, or any other needed reagent.
  • the collection surface may include vitrified therein a detergent and/or a buffer whereby rehydration of the collection surface allows the stored assay reagents to be functional to alter one or more characteristics of the sample applied (e.g. pH adjustment, lysing one or more components, etc.).
  • the microbial detection apparatus includes one or more lysing reagents.
  • the lysing reagents may be vitrified to the collection surface or scaffold.
  • the lysing reagents may be present as a liquid lysis buffer.
  • Cell lysis reagents can be added to the moistening liquid to help permeabilize biological cells and facilitate the detection of an analyte associated with the cells.
  • intracellular analytes e.g., nucleic acids, proteins, oligopeptides, and small molecules such as ATP
  • cell wall- associated or cell-membrane associated molecules e.g., polysaccharides and proteins
  • the cell lysis reagent does not substantially inhibit the activities (e.g., interfere with an enzyme activity or the ability of a protein to recognize and/or bind to another molecule) of proteins (e.g., enzymes, antibodies), if the protein is present in the sample or the test device.
  • Cell lysis reagents and their effective concentrations are known in the art. Examples of cell lysis agents include detergents (e.g., TRITON X-100), biocides (e.g., chlorhexidine gluconate, benzalkonium chloride), enzymes (e.g., phospholipases, lysozyme, lysostaphin), and cytolytic peptides (e.g., phylloxin).
  • detergents e.g., TRITON X-100
  • biocides e.g., chlorhexidine gluconate, benzalkonium chloride
  • enzymes e.g., phospholipases, lysozyme,
  • the lysing reagents include reducing agents that may assist in improving signal results in the assay system, maintain the reduced state of proteins, and prevent disruption of the lysis process.
  • Illustrative reducing agents include, but are not limited to dithiothreitol (DTT), P- mercaptoethanol, glutathione, ascorbic acid, and the like.
  • the sample collection device 100 may further include a plunger 108.
  • the elongate shaft 102 is sized and shaped to be inserted into or is formed into the shape of the plunger 108 so as to be integral therewith.
  • the plunger 108 serves as a grip or handle for collecting a sample with the sample collection device 100, optionally without contaminating the collection surface.
  • the elongate shaft 102 may be inserted into the plunger 108 after sample collection.
  • the plunger 108 forceably contacts a wall of the housing meaning that the plunger or a portion thereof or appendix thereof contacts a wall of a housing with a force that creates a pressure between the wall and the plunger or portion thereof, optionally forming a seal.
  • the plunger 108 forms a seal, optionally a seal that is sufficient to prevent fluid passing through or past the seal, with a housing 110 of the detection apparatus 10.
  • the plunger 108 includes one or more features (e.g., O. rings) to form the seal with the housing.
  • the plunger 108 is formed from a material that is relatively flexible and/or malleable such that the plunger 108 itself forms the seal with the housing 110, optionally when forceably contacting a wall of the housing.
  • the detection apparatus 10 may further include a housing 110 for analyzing the biological sample.
  • the housing 110 is configured to receive the sample collection device 100.
  • the housing 110 defines a first chamber 112 that is sized and shaped to receive the elongate shaft 102 of the sample collection device 100.
  • the housing 110 also defines a second chamber 114, fluidly coupled to the first chamber 112.
  • the first chamber 112 and second chamber 114 are divided by an internal partition 116.
  • the internal partition 116 is coupled to the housing 110.
  • the internal partition 116 is displaced for sample analysis.
  • the internal partition 116 may be formed from a rupturable material designed to break, tear, or rupture when force is applied to the internal partition 116.
  • the internal partition 116 is ruptured by the sample collection device 100 upon depressing the plunger.
  • the internal partition 116 is ruptured by an external force on the housing 110, such as pinching, twisting, bending, or snapping of a portion of the housing 110.
  • Exemplary rupturable materials include, but are not limited to foils, polymer films, gelatin, glass, gels, waxes, metal films, and/or any pressureO sensitive seal.
  • the detection apparatus 10 includes an elution liquid 118 disposed within the housing 110.
  • An elution liquid is optionally any liquid suitable for dissolving the vitrified material in the substrate.
  • an elution liquid is water or aqueous material.
  • an elution liquid is or includes one or more organics solvents.
  • elution liquid includes one or more buffering agents.
  • the elution buffer 118 may be present in the second chamber 114 as shown in FIG. 1A. Alternatively, and discussed in greater detail herein, the elution buffer 118 may be present in the first chamber 112, as shown in FIG. 3.
  • Illustrative examples of elution buffer agents include but are not limited to choline, betaine, HEPES, TRIS, PIPES, MOPS, among others.
  • the elution buffer can include, by way of example and not limitation, magnesium salts, sodium salts, potassium salts, chloride salts, or combinations thereof.
  • the salts can be provided at a concentration of from greater than or equal to 1 millimolar (mM) to less than or equal to 500 mM.
  • the components of the elution buffer 118 may be provided at a concentration suitable to stabilize the pH of the elution buffer to a desired level.
  • elution buffer components may be vitrified to the substrate 106 in place of being included in the liquid fraction.
  • assay reagents that are not vitrified to the substrate 106 and/or collection surface 104 may be included in the elution buffer 118.
  • the elution buffer 118 includes 1 part CCLR (25 mM TRIS, 2 mM DTT, 2 mM EDTA, 10% Glycerol, 1% Triton) and 5 parts buffer (20 mM TRIS-HC1, 0.4 M urea, 10% glycerol) in 4.5 mM MgSO4, 25 mM EPPS.
  • the elution buffer 118 does not include TRIS, but instead includes EPPS in place of the TRIS.
  • the elution buffer 118 does not include EPPS, but instead includes TRIS in place of the EPPS.
  • the elution buffer has a pH of about 6.5 to about 8.0, including about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8.0, or any range having endpoints by any two of the aforementioned values.
  • the sample collection device 100 can be removed from the housing 110 to collect a biological sample from the surface of a sampling site. After the collection surface 104 contacts the surface, the sample collection device 100 is inserted into the housing. As shown in FIG. 1A, the sample collection device 100 may be inserted into the housing 110 at a first position. Optionally, a distal end 107 of the collection surface 104 is inserted into the housing 110, such as into the first chamber 112. Optionally, the distal end 107 is positioned adjacent to the internal partition 116. In the first position, the plunger 108 extends at least partially beyond a top edge of the housing 110.
  • the sample collection device 100 may move to a second position within the housing 110, optionally by engaging a second ring or portion of a plunger, and/or by rupturing the internal partition 116, as shown in FIG. IB.
  • the distal end 107 contacts and penetrates the internal partition 116.
  • the sample collection surface 104 and the substrate 106 are positioned in the second chamber 114.
  • the elution buffer 118 contacts the collection surface 104 and the substrate 106, facilitating detection of the analyte.
  • the plunger 108 is positioned entirely or substantially within the housing 110 in the second position.
  • the substrate 106 may be disposed in the second chamber 114.
  • the elution buffer 118 is disposed in the first chamber 112 to separate the elution buffer 118 from the substrate 106 until the internal partition 116 is ruptured.
  • the sample collection device 100 may move to a second position within the housing 110 by rupturing the internal partition 116.
  • the collection surface 104 may remain in the first chamber 112 while the elution buffer 118 that has interacted with the sample may move to the second chamber 114 to contact the substrate 106.
  • rupturing the internal partition 116 allows the elution buffer 118 to contact the collection surface 104 and/or the substrate 106. Upon contact with the elution buffer 118, the vitrified material is rehydrated and the components vitrified on the substrate 106 are released into the liquid and able to interact with the biological sample to facilitate the detection of the target analyte.
  • the device and methods herein may be used to detect the presence or absence of one or more agents or organisms.
  • An agent is optionally a biological agent that includes, but is not limited to proteins (e.g. antibodies, blood proteins, intracellular proteins, membrane proteins, etc.), RNA, DNA, lipid, or other.
  • a material to be detected is an organism or portion thereof, optionally a bacteria, virus, fungi, or other organism. In some embodiments, the organism is a bacteria.
  • detection of bacteria may be performed by collecting a sample on the collection surface that includes therein a vitrified detergent (e.g. Triton-X 100). The rehydration of the vitrified detergent allows the detergent to lyse the bacterial cells in the biological sample thereby releasing intracellular analytes, such as ATP.
  • a vitrified detergent e.g. Triton-X 100
  • detection of the analyte relies on an enzyme/substrate reaction.
  • the enzyme/substrate reaction emits detectable light proportional to the amount of the target analyte in the biological sample.
  • the enzyme and/or the substrate may be vitrified to the collection surface 104 and/or the substrate 106.
  • the enzyme and the substrate are vitrified to the substrate 106.
  • the enzyme/substrate combination is luciferase/luciferin.
  • the emitted light is measured.
  • the emitted light is measured using a luminometer. The measured values may then be compared to a standard curve to quantify the amount of the analyte in the biological sample.
  • a device includes an elongate shaft under a collection surface.
  • E. coli is grown in culture to confluence and serially diluted to about O.lxlO 6 cells/ml.
  • a sample of this cellular solution is coated onto a microscope slide and dried. These dried samples are used for the following studies demonstrating that the swab is functional to collect and detect cells as well as that vitrification of material into the a substrate or a collection surface is functional to provide materials for successful detection of cells.
  • the use of a swab for the detection of E. coli dried onto a surface is demonstrated.
  • a device in the form of a swab with a collection surface is contacted to the dried material on the surface of the microscope slide. This is then placed into a housing containing Biofix Buffer + 10% BSA, 0.875 pg luciferase (4.16 pg/mL), 139 pg luciferin (2.5 mM), 9 pg DTT (0.333 mM), 0.035 pg Triton-XlOO (0.2%) and 2 mM EDTA.
  • a sample of the eluted material is then subjected to analysis for the presence or absence of ATP by luminescence either following 20 minutes of incubation of the collection surface in the solution or within 1 minute of contact with the elution solution (Lysis Master Mix fast).
  • Comparative results are illustrated in FIG. 4.
  • the swab is readily able to detect the presence of the E. coli nearly identical to control. Allowing lysis of the cells to occur for 20 minutes still allows excellent detection of the organism, but the 20-minute incubation time reduces the luminescence in the system likely due to degradation of the luminescent signal over the incubation time.
  • a CMV solution is made containing Biofix Buffer + 10% BSA, 0.875 pg luciferase (4.16 pg/mL), 139 pg luciferin (2.5 mM), 9 pg DTT (0.333 mM), 0.035 pg Triton-XlOO (0.2%) and with or without 2 mM EDTA.
  • the substrate is subjected to vitrification. This substrate is then placed in an elution buffer containing 4.5 mM Mg acetate, 25 mM TRIS, 2% glycerol at pH 7.2 and the vitrified materials are dissolved into the elution solution.
  • a liquid sample is created with the identical amounts of materials in the vitrified material and used as a nonvitrified liquid control.
  • a detection device as above is then dipped in either the liquid control or the vitrified/reconstituted CMV materials, swabbed to the dry slide surface then analyzed for the presence or absence of free ATP from the lysed cells. Results are illustrated in Table 1.
  • Vitrified substrate included Biofix Buffer + 10% BSA, 0.875 pg luciferase (4.16 pg/mL), 139 pg luciferin (2.5 mM), 9 pg DTT (0.333 mM), 0.035 pg Triton-XlOO (0.2%) and with or without 2 mM EDTA.
  • the substrates were then contacted to a bacterial containing slide and placed in elution buffer in separate tubes with the control tube in which the clean substrate was included further including the same materials vitrified into the test substrate so that the test solutions were of identical composition.
  • the materials were then analyzed for the presence or absence of ATP to detect bacteria. The presence of ATP was detected in both samples by luciferin luminescence.
  • a sample device is formed substantially as illustrated in FIG. 1A.
  • 175 pL of a vitrification mixture containing Biofix Buffer + 10% BSA, 0.875 pg luciferase (4.16 pg/mL), 139 pg luciferin (2.5 mM), 9 pg DTT (0.333 mM), 0.035 pg Triton- XlOO (0.2%) and with or without 2 mM EDTA is coated and vitrified according to the teachings of US Patent No. 10,433,540.
  • This substrate is placed either under a collection surface, adjacent thereto, or otherwise in sufficient proximity to allow fluidic connection between the two within a housing.
  • the present disclosure relates to a detection apparatus comprising: a sample collection device comprising: a collection surface; and an elongate shaft, coupled to a proximal end of the collection surface; a substrate fluidly coupled to said collection surface, wherein said substrate comprises at least one detection agent vitrified thereto.
  • a detection apparatus comprising: a sample collection device comprising: a collection surface; and an elongate shaft, coupled to a proximal end of the collection surface; a substrate fluidly coupled to said collection surface, wherein said substrate comprises at least one detection agent vitrified thereto.
  • the present disclosure relates to a detection apparatus wherein the substrate is disposed on the sample collection device.
  • the present disclosure relates to a detection apparatus wherein the substrate is disposed on the elongate shaft.
  • the present disclosure relates to a detection apparatus wherein the substrate is a layer disposed underneath the collection surface.
  • the present disclosure relates to a detection apparatus further comprising a housing comprising a first chamber, said housing configured to accept said elongate shaft within the first chamber.
  • the present disclosure relates to a detection apparatus further comprising a second chamber fluidly coupled to said first chamber.
  • the present disclosure relates to a detection apparatus further comprising an internal partition between said first chamber and said second chamber.
  • the present disclosure relates to a detection apparatus wherein said internal partition comprises a rupturable material.
  • the present disclosure relates to a detection apparatus wherein said substrate is connected to said elongate shaft proximal to said collection surface.
  • the present disclosure relates to a detection apparatus wherein said substrate is proximal to said second chamber relative to said rupture disk.
  • the present disclosure relates to a detection apparatus further comprising a plunger connected to said elongate shaft distal from said collection surface.
  • the present disclosure relates to a detection apparatus wherein said plunger is configured to forceably associate with a wall of said housing.
  • the present disclosure relates to a detection apparatus wherein said plunger comprises one or more gaskets, wherein said one or more gaskets form a seal between the plunger and said housing.
  • the present disclosure relates to a detection apparatus comprising two or more gaskets.
  • the present disclosure relates to a detection apparatus further comprising a stop on or attached to said plunger, said stop distal from said collection surface.
  • the present disclosure relates to a detection apparatus further comprising an elution buffer.
  • the present disclosure relates to a detection apparatus wherein said elution buffer is housed in said first chamber. [0100] In an eighteenth aspect, alone or in combination with any other aspect provided herein, the present disclosure relates to a detection apparatus wherein said elution buffer is housed in said second chamber.
  • the present disclosure relates to a detection apparatus wherein said elution buffer is separated from said substrate by said internal partition.
  • the present disclosure relates to a detection apparatus wherein said elution buffer is separated from said second chamber by said internal partition.
  • the present disclosure relates to a detection apparatus, wherein the detection agent is a colorimetric agent, fluorescent label, a luminescent label, a radioisotope label, or a combination thereof.
  • the present disclosure relates to a detection apparatus further comprising a lysis agent.
  • the present disclosure relates to a detection apparatus, wherein the lysis agent is vitrified to the scaffold.
  • the present disclosure relates to a detection apparatus wherein the lysis agent is in said elution buffer.
  • the present disclosure relates to a method for detecting an organism using a detection device the method comprising collecting a sample to be tested for the organism on the collection surface; contacting the substrate with elution buffer, wherein the elution buffer rehydrates the vitrified detection agent, whereby the organism, if present in the sample, reacts with the detection agent; and detecting the presence or absence of the organism by the presence or absence of a detectable signal.
  • the present disclosure relates to a method for detecting an organism further comprising inserting the sample collection device into the sample chamber prior to the contacting.
  • the present disclosure relates to a method for detecting an organism further comprising rupturing the internal partition so as to contact the substrate with the elution buffer.
  • the present disclosure relates to a method for detecting an organism wherein the sample and the substrate are contacted with the elution buffer simultaneously.
  • the present disclosure relates to a method for detecting an organism wherein the detectable signal comprises a fluorescent signal or a color change.
  • the present disclosure relates to a method for detecting an organism wherein the presence or absence of the organism is detected in about 10 minutes or less.
  • the present disclosure relates to a method for detecting an organism wherein the presence or absence of the organism is detected in about 5 minutes or less.
  • 0 substantiallyO and 0 aboutO may be utilized herein to represent the inherent degree of uncertainty that may be attributed to any quantitative comparison, value, measurement, or other representation. These terms are also utilized herein to represent the degree by which a quantitative representation may vary from a stated reference without resulting in a change in the basic function of the subject matter at issue.
  • 0 substantiallyO is used herein also to represent the degree by which a quantitative representation may vary from a stated reference without resulting in a change in the basic function of the subject matter at issue.
  • first component is described as 0 comprising ⁇ or 0 includingO a second component
  • first component 0 consistsO or 0 consists essentially ofD the second component.
  • 0 consisting essentially offl is used in this disclosure to refer to quantitative values that do not materially affect the basic and novel characteristic(s) of the disclosure.
  • any two quantitative values assigned to a property or measurement may constitute a range of that property or measurement, and all combinations of ranges formed from all stated quantitative values of a given property or measurement are contemplated in this disclosure.
  • Patents, publications, and applications mentioned in the specification are indicative of the levels of those skilled in the art to which the invention pertains. These patents, publications, and applications are incorporated herein by reference to the same extent as if each individual patent, publication, or application was specifically and individually incorporated herein by reference.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Hematology (AREA)
  • Clinical Laboratory Science (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Physics & Mathematics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biochemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne des dispositifs et des procédés de détection d'un organisme microbien qui comprennent un appareil de détection avec un dispositif de collecte d'échantillon. Le dispositif de collecte d'échantillon comprend généralement une surface de collecte et une tige allongée, couplée à une extrémité proximale de la surface de collecte et un substrat couplé de manière fluidique à ladite surface de collecte, ledit substrat comprenant au moins un agent de détection vitrifié à celui-ci.
PCT/US2024/046881 2023-09-15 2024-09-16 Dispositif et procédés de détection d'un organisme Pending WO2025059638A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202363582974P 2023-09-15 2023-09-15
US63/582,974 2023-09-15

Publications (1)

Publication Number Publication Date
WO2025059638A1 true WO2025059638A1 (fr) 2025-03-20

Family

ID=95022152

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2024/046881 Pending WO2025059638A1 (fr) 2023-09-15 2024-09-16 Dispositif et procédés de détection d'un organisme

Country Status (1)

Country Link
WO (1) WO2025059638A1 (fr)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023278815A1 (fr) * 2021-07-01 2023-01-05 Upkara, Inc. Dispositif de vitrification et d'administration d'agents bioactifs vitrifiés
US20230012231A1 (en) * 2019-12-04 2023-01-12 Diag-Nose Medical Pty Ltd A method and apparatus for respiratory secretion collection and analysis

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20230012231A1 (en) * 2019-12-04 2023-01-12 Diag-Nose Medical Pty Ltd A method and apparatus for respiratory secretion collection and analysis
WO2023278815A1 (fr) * 2021-07-01 2023-01-05 Upkara, Inc. Dispositif de vitrification et d'administration d'agents bioactifs vitrifiés

Similar Documents

Publication Publication Date Title
US10845369B2 (en) Sampling devices and methods of use
US20110256531A1 (en) Biodetection articles
US20120100531A1 (en) Coated substrates comprising a cell extractant and biodetection methods thereof
WO2010129726A1 (fr) Articles à matrice comprenant un agent d'extraction cellulaire et procédés de biodétection associés
CN101918531B (zh) 环境取样制品和方法
US9284593B2 (en) Live bioload detection using microparticles
He et al. Laser-patterned paper-based sensors for rapid point-of-care detection and antibiotic-resistance testing of bacterial infections
US6881554B2 (en) Apparatus and methods for chemiluminescent assays
US8647508B2 (en) System and method for processing samples
JP2017535255A (ja) 滅菌剤耐性モジュレータを有する生物学的滅菌インジケータ
CA2805486C (fr) Procede pour relier des resultats d'analyses diagnostiques rapides au point d'intervention a des procedes en laboratoire
JP2005502363A (ja) 酵素アッセイのためのラテラルフローテストフォーマット
AU5350100A (en) A method for providing long term stability to cell for diagnostic testing
US20120094281A1 (en) Articles with shell structures including a cell extractant and biodetection methods thereof
AU2018203078A1 (en) Substrates and methods for collection, stabilization and elution of biomolecules
WO2025059638A1 (fr) Dispositif et procédés de détection d'un organisme
CN107109467A (zh) 含有荧光素的基底和包括基底的监测装置
EP1106683A2 (fr) Procédé de stabilisation à long terme de cellules pour des tests diagnostiques
WO2011017325A1 (fr) Dispositifs d'échantillonnage et procédés d'utilisation
WO1999032601A1 (fr) Dispositifs de dosage a disques et procede d'utilisation
US20180321202A1 (en) Methods and devices for detecting methanol poisoning using formate oxidase
EP2643471A1 (fr) Procédés et compositions pour détecter une activité biologique
EP3404418A2 (fr) Bande de diagnostic permettant de déterminer la quantité de sarcosine, de créatinine et de peroxyde d'hydrogène dans un échantillon biologique ou environnemental
WO2003050556A2 (fr) Dispositif et procede d'analyse
CZ30831U1 (cs) Diagnostický proužek pro stanovení množství sarkosinu, kreatininu a peroxidu vodíku v biologickém nebo environmentálním vzorku

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 24866496

Country of ref document: EP

Kind code of ref document: A1