WO2020084620A1 - Means and method for point-of-care analysis of liquid samples - Google Patents

Means and method for point-of-care analysis of liquid samples Download PDF

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Publication number
WO2020084620A1
WO2020084620A1 PCT/IL2019/051150 IL2019051150W WO2020084620A1 WO 2020084620 A1 WO2020084620 A1 WO 2020084620A1 IL 2019051150 W IL2019051150 W IL 2019051150W WO 2020084620 A1 WO2020084620 A1 WO 2020084620A1
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WIPO (PCT)
Prior art keywords
layer
sensor
analyte
membrane
preventative
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Ceased
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PCT/IL2019/051150
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French (fr)
Inventor
Elztov EVGENI
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.)
Israel Ministry of Agriculture and Rural Development
Agricultural Research Organization of Israel Ministry of Agriculture
Original Assignee
Israel Ministry of Agriculture and Rural Development
Agricultural Research Organization of Israel Ministry of Agriculture
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Application filed by Israel Ministry of Agriculture and Rural Development, Agricultural Research Organization of Israel Ministry of Agriculture filed Critical Israel Ministry of Agriculture and Rural Development
Priority to CN201980081642.XA priority Critical patent/CN113272647A/en
Priority to EP19876402.9A priority patent/EP3870973A4/en
Priority to US17/288,542 priority patent/US20220034879A1/en
Publication of WO2020084620A1 publication Critical patent/WO2020084620A1/en
Priority to IL282583A priority patent/IL282583A/en
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54386—Analytical elements
    • G01N33/54387—Immunochromatographic test strips
    • G01N33/54391—Immunochromatographic test strips based on vertical flow
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • G01N2021/7756—Sensor type
    • G01N2021/7759—Dipstick; Test strip
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • G01N2021/7769—Measurement method of reaction-produced change in sensor
    • G01N2021/7786—Fluorescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/76—Chemiluminescence; Bioluminescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2470/00—Immunochemical assays or immunoassays characterised by the reaction format or reaction type
    • G01N2470/10—Competitive assay format
    • G01N2470/12—Displacement or release-type competition
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses

Definitions

  • the field of the invention is system for point-of-care analysis of samples.
  • the infestation or contamination by pathogens, pollutions or toxins in water or food is amajor cause of desease and sickness around the world. This is a particular problem in in the developing world where there is a lack of technology and budget for tracking the contamination of water sources and of the food chain.
  • the WHO estimates that 1.5 million deaths a year are caused by waterborne diseases, over half due to safe water supply, sanitation and hygiene (WHO: Burden of disease and cost-effectiveness estimates, 2014), while foodborne illness was responsible for 420,000 deaths in 2010 (WHO estimates of the global burden of foodborne diseases, 2015).
  • the present invention proposes a point-of-care sensor that provides a simple, portable and cost efficient solution, and to provide rapid and localized analysis of markers, pathogens, pollutions, toxins or vectors for the outbreak of infectious diseases.
  • Biomarker can be charectorized as proteins, metabolites, antibodies, peptides, hormones, lipids etc in addition to whole cells. Alternativly the analytes could be fragments of the origional biomarker created by fragmentation, desintgration, deterioration, decay etc.
  • LFA Lateral flow immunoassay
  • WO201897796 describes a device to determine or quantify the presence of an analyte molecule, virus or cell of interest in a sample. However '796 contains the addional steps of prepering a conjugated analyte-reporter molecule.
  • US7300802B2 describes a biosensor for point-of-care testing (POCT) whose detection sensitivity is improved by introducing successive cross-flow procedure for immune reaction and enzymatic reaction to a membrane strip chromatographic assay system.
  • POCT point-of-care testing
  • the biosensor described in ' 802 must be operated by a skilled perfetional due to the use of multiple systems.
  • the layers are arranged from a-to-e and are operatively arranged as a specific analyte signaling channel.
  • the layers are arranged from a-to-e and are operatively arranged as a specific analyte signaling channel.
  • sample layer is constructed from a porous, absorbent and nonreactive membrane.
  • the membrane is selected from a group comprising: cellulose acetate membrane, nitrocellulose membrane, cellulose ester membrane, polysulfone (PS) membrane, polyether sulfone (PES) membrane, polyacrilonitrile (PAN) membrane, polyamide membrane, polyimide membrane, polyethylene and polypropylene (PE and PP) membrane, polytetrafluoroethylene (PTFE) membrane, polyvinylidene fluoride (PVDF) membrane, polyvinylchloride (PVC) membrane and fiberglass paper membrane.
  • PS polysulfone
  • PES polyether sulfone
  • PAN polyacrilonitrile
  • PAN polyamide membrane
  • PE and PP polyamide membrane
  • PE and PP polyimide membrane
  • PE and PP polytetrafluoroethylene
  • PVDF polyvinylidene fluoride
  • PVC polyvinylchloride
  • reaction pad contains at least one kind of immobilized analyte-effector complex.
  • the analytic-effector complex comprising of: a. at least one analyte; and b. at least one effector, the effector specific to the composition of the preventative pad; wherein the analyte and effector are reversibly or irreversibly connected.
  • the reporter compound is selected from a group of compounds consisting of: dyes, pigments, electrochemical active compounds, enzymes, flourophores, chemiluminescent molecules and radionuclides. It is another object of the present invention to provide the aforementioned sensor, wherein the reporter compound is blocked from crossing the unaffected preventative layer due to size, electric or magnetic charge, hydrophobicity or lipophobicity.
  • preventative layer consists of at least one compound that is affected by the effector.
  • preventative layer is comprised of material that can be digested by an enzyme, the material selected from a group, the group consisting of sugars, hydrogels, peptides, lipids and polymers.
  • It is an object of the present invention to provide a sensor for the rapid onsite identification of analytes comprising: a. at least one reversibly immobilized analyte-effector complex; b. at least one reporter compound;
  • the preventative layer is positioned between the analyte and the reporter and between the absorption layer and is configures as a specific analyte signaling channel.
  • preventative pad consists of at least one compound that can be altered by interacting with the effector. It is another object of the present invention to provide the aforementioned sensor, wherein the preventative layer is comprised of material that can be digested by an enzyme, the material selected from a group of organic compounds, the group consisting of sugars, hydrogels, peptides, lipids and polymers.
  • It is the object of the present invention to provide method for analyzing a sample comprising steps of: a. obtaining a sample as a solution;
  • the preventative layer is positioned between the analyte and the reporter and the detector; wherein the preventative layer is positioned between the analyte and the reporter and between the absorption layer; c. loading sample solution; and d. reading analysis result.
  • the membrane is selected from a group comprising: cellulose acetate membrane, nitrocellulose membrane, cellulose ester membrane, polysulfone (PS) membrane, polyether sulfone (PES) membrane, polyacrilonitrile (PAN) membrane, polyamide membrane, polyimide membrane, polyethylene and polypropylene (PE and PP) membrane, polytetrafluoroethylene (PTFE) membrane, polyvinylidene fluoride (PVDF) membrane, polyvinylchloride (PVC) membrane and fiberglass paper membrane.
  • PS polysulfone
  • PES polyether sulfone
  • PAN polyacrilonitrile
  • PAN polyamide membrane
  • PE and PP polyamide membrane
  • PE and PP polyimide membrane
  • PE and PP polytetrafluoroethylene
  • PVDF polyvinylidene fluoride
  • PVC polyvinylchloride
  • reaction pad contains at least one kind of immobilized analyte-effector complex.
  • the reporter compound is selected from a group of compounds consisting of: pigments, dyes, electrochemical active compounds, enzymes, flourophores, chemiluminescent molecules and radionuclides. It is another object of the present invention to provide the aforementioned method, wherein preventative pad consists of at least one compound that can be altered, by interacting with the effector.
  • preventative layer is comprised of at least one material that can be digested by an enzyme, the material selected from a group, the group consisting of sugars, hydrogels, peptides, lipids and polymers.
  • Figure 1 - illustrates the schematic structure of the sensor.
  • Figure 2 - illustrates the schematic activity of the sensor.
  • Figure 3 - illustrates the schematic activity of the sensor.
  • Figure 4 - demonstrates the effect of the deposition processes on the uniformity of the gelatin layer
  • Figure 5 - demonstrates the effect of the gelatin concentration on the solution preventing properties.
  • Figure 6 - demonstrates that an increase in the gelatin concentration decreases the capability of the enzyme in the solution diffused through the preventative layer
  • the analyte can be any substance for which there exists at least one naturally occurring or synthetic specific binding partner.
  • the analyte can include a protein or protein fragment, a polypeptide peptide or peptide fragment, an amino acid, a DNA fragment, a RNA fragment, a small molecule, a bacterium, natural ligands, virus particles (virions), a virus or metabolites of or antibodies to or biomimetic of any of the above substances.
  • the analyte can be a polutent or can serve as a pesticide or a toxin.
  • the identified analytes could be only segments of the origional analyte, cased by fragmentation, desintgration, deterioration, deacy, oxidation etc. Fragmentation can be caused by exposure to the eviroment or as part of the sample preperation procedure.
  • an“anti-analyte antibody” refers to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes or fragments of immunoglobulin genes. This term encompasses polyclonal antibodies, monoclonal antibodies, and fragments thereof, as well as molecules engineered from immunoglobulin gene sequences.
  • the anti-analyte antibody is specific to the analyte of interest.
  • an“anti-analyte capture antibody” is an anti-analyte antibody that captures the analyte of interest. Such antibodies are conveniently affixed to a solid phase, such as the membrane of the reaction layer.
  • reporter compound or “reporter molecule” (or simply “reporter”) as used herein, refers to molecules useful for detecting the presence, intensity or quantity of the analyte due to an interaction between the reporter compound and the absorption layer and/or a detector.
  • Molecules are detectable by spectroscopic, photochemical, biochemical, enzymatic, immunochemical, electrical, radiographic and optical means.
  • Optically detectable molecules can be detectable in the in the ultraviolet, visual or infrared spectrum, including compounds such as dyes or fluorescent labels.
  • Reporter compounds useful in the present invention also include any suitable molecule which may be conjugated to the analyte molecule without compromising the ability of the reporter molecule to be detected or the analyte to be bound to the anti-body.
  • the reporter is selected from the group consisting of a dye, a radionuclide, an enzyme and combinations thereof.
  • the dye can be either "small molecule” dye/fluors, or macromolecule dye/fluors (e.g. green fluorescent proteins and all variants thereof).
  • the dye may be a tandem fluorophore conjugate.
  • the dye may be a fluorescent semiconductor nanocrystal particle, a quantum dot, an electroactive molecule/dye or an upconversion nanocrystal.
  • the reporter compound is loaded on to the "reporter layer” or "reporter membrane".
  • the reporter layer can be formed by binding the reporter in a selective or non-selective manor.
  • the membrane is loaded by saturating the membrane with a reporter compound solution, and subsequently drying the loaded membrane, binding the reporter to the membrane in a non-selective manor. In this configuration, as the sample solution rehydrates the layer, the reporter interacts with liquid/solution and migrates along with the flow, to the "preventative layer".
  • the reporter molecule can be loaded onto another layer, up-stream from the preventative layer.
  • Preventative layer refers to a treated porous or semi-porous membrane or solid layer constructed from a material that does not permit the passage of reporter molecules unless it is altered by interacting with the effector.
  • the Preventative layer obstructs passage of the reporter compound due to chemical or physical properties of the reporter compound and the preventative layer.
  • the preventative layer is any biological or chemical substances that can be digested by enzymes (sugars, hydrogels, peptides, proteins, fats, plastic polymers, etc.) such as gelatin.
  • effector refers to a compound that can be bound to the analyte without affecting its activity.
  • the effector has the ability to interact with the preventative layer, thereby changing its physical or chemical properties in a way that enables passage of reporter compounds.
  • the effector can be an enzyme, a macromolecule or a small molecule.
  • the effector can be organic, inorganic or organometallic in composition.
  • the effector is an enzyme capable of digesting the preventative layer, opening pores large enough for the reporter compound to pass.
  • membrane refers to a natural or synthetic/artificial membrane.
  • synthetic membrane or “artificial membrane” refer to a man made membrane that is produced from organic material, such as polymers and liquids, as well as inorganic materials. A wide variety of synthetic membranes are well known in the art.
  • the membranes of the sample layer, the at least one conjugation layer, the at least one preventative layer and the absorption layer are independently selected from the group consisting of cellulose acetate membrane, nitrocellulose membrane, cellulose ester membrane, polysulfone (PS) membrane, polyether sulfone (PES) membrane, polyacrilonitrile (PAN) membrane, polyamide membrane, polyimide membrane, polyethylene and polypropylene (PE and PP) membrane, polytetrafluoroethylene (PTFE) membrane, polyvinylidene fluoride (PVDF) membrane, polyvinylchloride (PVC) membrane and fiberglass paper membrane.
  • PS polysulfone
  • PES polyether sulfone
  • PAN polyacrilonitrile
  • PAN polyamide membrane
  • polyimide membrane polyimide membrane
  • PE and PP polytetrafluoroethylene
  • PVDF polyvinylidene fluoride
  • PVC polyvinylchloride
  • the term “absorption membrane”, “absorption layer” or “absorption pad” refers to a treated membrane that specifically or non-specifically binds to or reacts with the reporter compound.
  • the absorption layer further comprises at least one substrate for a reporter molecule (or simply a reporter).
  • Reporter substrate is intended to include any substrate capable of interacting with the reporter. Preferably, the interaction between the reporter and the reporter substrate produces a qualitative or quantitative effect.
  • a “reporter substrate” as used herein is a substrate (or substrates) that can facilitate measurement of either the disappearance of a substrate or the appearance of a product in connection with a catalyzed reaction.
  • Reporter substrates can be free in solution or bound (or "tethered"), for example, to a surface, or to another molecule.
  • a reporter substrate can be labelled by any of a large variety of means including, for example, fluorophores (with or without one or more additional components, such as quenchers), radioactive labels, biotin (e.g. biotinylation) or chemiluminescent labels.
  • the reporter is horseradish peroxidase, the substrate is preferably luminol.
  • the absorption layer is part of a detector.
  • the absorption pad facilitates the interaction between the reporter compound and the detector. In one configuration this is performed by containing reporter substrates or by capturing free reporter compounds.
  • reporter refers to a devise that enables the measurement of reporter compounds that reach the absorption layer or interact with reporter substrates.
  • the measurement can be of the compound itself or of the interaction with the substrate. This interaction can be measured by spectroscopic, photochemical, biochemical, enzymatic, immunochemical, electrical, radiographic or optical means.
  • the detector is configured to utilize Chemometrics to analyze the signal (or signals) generated by the reporter- absorption interaction to detect and measure the amount of analyte present in the sample.
  • the object of this invention is a sensor for the on-site, real-time, fast and simple analyte detection.
  • FIG. 1 describing one non limiting embodiment of the invention, a liquid sample collected and loaded on the sample layer (11).
  • the sample solution hydrates the layers and the sample solution flows through to the reaction layer (12).
  • the analyte in the sample binds to the anti-analyte antibody, dislocating the bound analyte- effector complex.
  • the analyte-effector complex passes through the reporter layer to the preventative layer (13).
  • the effector interacts with the preventative layer (13), affecting the physical and/or chemical properties of the preventative layer. This change enables the reporter compounds to traverse the preventative layer and reach the absorption layer (14).
  • Figure 2 describing one non limiting embodiment of the invention, a liquid sample collected and loaded on the sample layer 21.
  • the sample solution hydrates the layers and the sample solution flows through to the reaction layer 22. If the sample solution contains the analyte, then the analyte in the sample binds to the anti analyte antibody, dislocating the bound analyte-effector complex. The analyte-effector complex then passes through the reporter layer to the preventative layer 23. The effector interacts with the preventative layer 23, affecting the physical and/or chemical properties of the preventative layer. This change enables the reporter compounds to traverse the preventative layer 24 and reach the absorption layer 25. The sensor will then return a positive result 26.
  • the analyte-effector complex remains bound 27 and cannot affect the physical and/or chemical properties of the preventative layer 28.
  • the reporter compound will not be able to cross the preventative layer 29 and the sensor will return a negative result 30.
  • Reporter compounds that traverse the preventative layer interact with the absorption layer to generate a signal.
  • This interaction can be specific, such as binding to a substrate, or non-specific, such as the accumulation of dyes.
  • This interaction generates a signal, such as the generation of a color due to the accumulation of dyes.
  • the signal is then measured by the detector the sensor can be configures to detect the presence of more than one analyte in a single sample by using one reaction layer loaded with multiple anti-analyte capture anti-body's or by using multiple reaction layers, each layer corresponding to a different anti-analyte capture antibody.
  • the detector and absorption layer can be configured to detect the presence of more than one reporter compound, enabling the system to detect the presents of multiple analytes in a single sample.
  • the signal generated by each reporter must be distinctive and must not impede the detectors ability to detect signals generated by other reporter compounds.
  • the detector can use chemometrics to detect the level of various analytes in the sample.
  • the senor is constricted of a number of layers, each layer placed on each other affording to the flow
  • the senor is constructed as a single strip.
  • the layers are arraigned end-to-end and are regions of one strip
  • the membranes are paper (cellulose)
  • the preventative layer is constructed from gelatin
  • the reporter compound is a dye
  • the effector is pepsin.
  • the liquid sample is collected and deposited on the sample pad. The sample traverses through a sample pad until it reaches the reaction pad.
  • the reaction pad contains immobilized allergen-pepsin complex (anti-analyte-enzyme complex) bound to an allergen antibody.
  • the free allergen in the sample binds to the anti-analyte antibody capture complex, releasing the analyte- pepsin complex.
  • the complex passes through and rehydrating the color layer and reaches the gelatin preventative layer.
  • the pepsin digests the gelatin, creating pores (one pore for each freed complex).
  • the dyes molecules pass through the pores in the gelatin and reach the absorption pad, coloring the absorption layer.
  • the color indicates the presence of an allergen in the food, alerting to a possible health hazard.
  • the membranes are paper (cellulose)
  • the preventative layer is constructed from gelatin
  • the reporter layer contains red dye
  • the effector is pepsin.
  • the sensor contains 7 reaction layers, each one specific for a different pathogen:
  • Each reaction layer contains a specific immobilized pathogen-pepsin complex (anti- analyte-enzyme complex) bound to a pathogen anti-body.
  • Each pathogen-pepsin complex is additionally linked to an additional reporter compound, creating a pathogen-pepsin- reporter complex.
  • Each additional reporter compound is a different florescent compound, each florescent compound emitting light at a distinctive spectrum.
  • the liquid sample is collected and deposited on the sample pad.
  • the sample traverses through a sample pad until it reaches the specific reaction pad.
  • the specific reaction pad contains immobilized pathogen-pepsin-reporter complex bound to the pathogen antibody.
  • the free pathogen in the sample binds to the anti-analyte antibody capture complex, releasing the pathogen-pepsin-reporter complex.
  • the complex passes through and rehydrating the color layer and reaches the gelatine preventative layer.
  • the pepsin than digests the gelatin, creating pores (one pore for each freed complex).
  • the dyes molecules pass through the pores in the gelatin and reach the absorption pad, coloring the absorption pad.
  • the absorption layer is then loaded into a spectroscopic detector can then be used to identify the specific pathogen in the samples.
  • a colored layer warns about a possible water contamination and the use of a detector detects the specific pathogens present in the water source.
  • the membranes are paper (cellulose)
  • the preventative layer is constructed from gelatin
  • the reporter compound is a dye
  • the effector is pepsin.
  • the reported compound is dissolved in the sample collection solution.
  • a dose of the sample collection solution of deposited on the surface of the reactor comprises a suitable solvent and the dye (the reported compound).
  • the sample layer is dunked in the solution that is on the reactor surface.
  • the sample traverses through a sample pad until it reaches the reaction pad.
  • the reaction pad contains immobilized analyte-pepsin complex (anti-analyte-enzyme complex) bound to an anti-analyte antibody.
  • the free analyte in the sample binds to the anti-analyte antibody capture complex, releasing the analyte-pepsin complex.
  • the complex reaches the gelatin preventative layer.
  • the pepsin digests the gelatin, creating pores (one pore for each freed complex).
  • the dyes molecules pass through the pores in the gelatin and reach the absorption pad, coloring the absorption layer.
  • Bromelain is enzyme from pineapples that degrades gelatin, thus testing the stopping capabilities of the gelatin layers (with clear solution) and also determining effect of various gelatin concentrations on enzymatic reaction of the positive (+) solution (the solution containing the enzyme).
  • Figure 5 demonstrates that all solutions containing the Bromelain enzyme diffused through preventing layer, regardless of the gelatin concentration. This demonstrates the ability of the Bromelain enzyme to penetrate the preventative layer at any used gelatin concentrations. In the enzyme negative samples (tested with clear water), only layers with 5% gelatin (w/v) and higher showed the ability to stop the solution. This indicates the specificity of the diffusion/degradation mechanisms, where systems with a preventative layer containing at least 5% (w/v) gelatin will allow the passage of enzyme containing solutions while stopping negative samples and will pass solutions.
  • Figure 6 demonstrates that an increase in the gelatin concentration in the preventative layer lowers the ability of the Bromelain enzyme in the solution to enable diffuse of the colored solution through preventative layer. While both tested concentrations prevent false-negative results, a preventative layer containing 12.5 pg/mL gelatin were enough to prevent solutions containing two active units to diffuse or pass through preventative layer.
  • the negative samples show that a preventative layer containing 5% (w/v) gelatin prevents the negative solution (without enzyme) diffusion through it, as a positive solution (containing the Bromelain enzyme) can disassemble the preventative layer and enable the passage of the solution, with as low an enzyme concentration as possible.
  • Figure 7 further demonstrates the potential of the present invention to generate a positive result when in the present of a solution containing the Bromelain enzyme (+).

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  • Health & Medical Sciences (AREA)
  • Immunology (AREA)
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Abstract

A sensor for the rapid, onsite identification of analytes characterized by: (a) a sample layer; (b) at least one reaction layer, interconnected with the sample layer; (c) at least one reporter layer, interconnected with the reaction payer; (d) at least one preventative layer, interconnected with the reporter layer; and (e) an absorption pad, interconnected with the preventative layer; with the layers arranged as a signaling channel. The sensor utilized an anlalyte -effector complex wherein said effector affects the preventative layer as such enables a reporter compund to cross the preventative layer.

Description

TITLE
Means and method for point-of-care analysis of liquid samples.
FIELD OF THE INVENTION
The field of the invention is system for point-of-care analysis of samples.
BACKGROUND
The infestation or contamination by pathogens, pollutions or toxins in water or food is amajor cause of desease and sickness around the world. This is a particular problem in in the developing world where there is a lack of technology and budget for tracking the contamination of water sources and of the food chain. The WHO estimates that 1.5 million deaths a year are caused by waterborne diseases, over half due to safe water supply, sanitation and hygiene (WHO: Burden of disease and cost-effectiveness estimates, 2014), while foodborne illness was responsible for 420,000 deaths in 2010 (WHO estimates of the global burden of foodborne diseases, 2015).
Current technologies, such as GS-MS, Mass spectrometry or HPLC are expensive, immobile and need highly trained operators.
The present invention proposes a point-of-care sensor that provides a simple, portable and cost efficient solution, and to provide rapid and localized analysis of markers, pathogens, pollutions, toxins or vectors for the outbreak of infectious diseases. Biomarker can be charectorized as proteins, metabolites, antibodies, peptides, hormones, lipids etc in addition to whole cells. Alternativly the analytes could be fragments of the origional biomarker created by fragmentation, desintgration, deterioration, decay etc.
There exists a number of methods identify pathogens pollutions or toxins, such as cell plate culture, immunoassays, and nucleic acid related tests. However, these methods have various disadvantages such as low sensitivity, high price, assay complexity, requirement of a lab environment, and more. The adaptation of these methods for field use has proven to be challenging. Two types of devices have reached the necessary requirements to enable wide spread consumer use. One is the biosensor-based glucometer, and the other is the pregnancy test.
Lateral flow immunoassay (LFA) is currently the technology with the most potential to meet the challenge.
A number of advances have been made in developing cost-effective and rapid bacterial testing based on the lateral flow technology, with sensors for Escherichia coli, Listeria , Salmonella and Streptococcus being developed. However, low sensitivity and low specificity to the target analyte limit their use. ELISA based technologies (e.g. chemo luminescence, electrochemistry and, colorimetry) provide higher sensitivity and specificity, however they are more complicated and time consuming.
WO201897796 describes a device to determine or quantify the presence of an analyte molecule, virus or cell of interest in a sample. However '796 contains the addional steps of prepering a conjugated analyte-reporter molecule.
US7300802B2 describes a biosensor for point-of-care testing (POCT) whose detection sensitivity is improved by introducing successive cross-flow procedure for immune reaction and enzymatic reaction to a membrane strip chromatographic assay system. However the biosensor described in ' 802 must be operated by a skilled perfetional due to the use of multiple systems.
Therfore their exists an unmet need for a rapid, onsite, simple and low-cost system for the detection of contaminated -i-food and water sources-k
SUMMARY
It is an object of the present invention to provide a sensor for the rapid, onsite identification of analytes characterized by: a. a sample layer;
b. at least one reaction layer, interconnected with the sample layer: c. at least one reporter layer, interconnected with the reaction payer; d. at least one preventative layer, interconnected with the reporter layer; and e. an absorption pad, interconnected with the preventative layer;
wherein the layers are arranged from a-to-e and are operatively arranged as a specific analyte signaling channel.
It is another object of the present invention to provide the aforementioned sensor, comprising a. a sample layer;
b. at least one reporter layer, interconnected with the sample layer; c. at least one reaction layer, interconnected with the reporter layer; d. at least one preventative layer, interconnected with the reaction layer; and e. an absorption layer, interconnected with the preventative layer; wherein the layers are arranged from a-to-e and are operatively arranged as a specific analyte signaling channel.
It is another object of the present invention to provide the aforementioned, wherein the sample layer is constructed from a porous, absorbent and nonreactive membrane.
It is another object of the present invention to provide the aforementioned sensor, wherein the membrane is selected from a group comprising: cellulose acetate membrane, nitrocellulose membrane, cellulose ester membrane, polysulfone (PS) membrane, polyether sulfone (PES) membrane, polyacrilonitrile (PAN) membrane, polyamide membrane, polyimide membrane, polyethylene and polypropylene (PE and PP) membrane, polytetrafluoroethylene (PTFE) membrane, polyvinylidene fluoride (PVDF) membrane, polyvinylchloride (PVC) membrane and fiberglass paper membrane.
It is another object of the present invention to provide the aforementioned sensor, wherein the reaction pad contains at least one kind of immobilized analyte-effector complex.
It is another object of the present invention to provide the aforementioned sensor, wherein the analytic-effector complex comprising of: a. at least one analyte; and b. at least one effector, the effector specific to the composition of the preventative pad; wherein the analyte and effector are reversibly or irreversibly connected.
It is another object of the present invention to provide the aforementioned sensor, wherein the immobilized analyte-effector complex is reversibly bound to a specific anti analyte antibody, the antibody is specific for the analyte.
It is another object of the present invention to provide the aforementioned sensor, wherein the analyte-effector complex is released from the antibody through specific competitive or non-competitive binding of the free analyte from the sample to the anti body, immobilized to the reaction layer.
It is another object of the present invention to provide the aforementioned sensor, wherein the immobilized analyte-effector complex additionally comprises at least one reporter compound.
It is another object of the present invention to provide the aforementioned sensor, wherein the anti-analyte antibody is irreversibly bound to the reaction pad.
It is another object of the present invention to provide the aforementioned sensor, wherein the reporter layer is loaded with bound or unbound reporter compounds.
It is another object of the present invention to provide the aforementioned sensor, wherein the reporter compound characterized by being: a. incapable of crossing through undigested, whole preventative layer;
b. capable of crossing through pores created in preventative layer; and c. specific to the absorption pad or the detector.
It is another object of the present invention to provide the aforementioned sensor, wherein the reporter compound is selected from a group of compounds consisting of: dyes, pigments, electrochemical active compounds, enzymes, flourophores, chemiluminescent molecules and radionuclides. It is another object of the present invention to provide the aforementioned sensor, wherein the reporter compound is blocked from crossing the unaffected preventative layer due to size, electric or magnetic charge, hydrophobicity or lipophobicity.
It is another object of the present invention to provide the aforementioned sensor, wherein preventative layer consists of at least one compound that is affected by the effector.
It is another object of the present invention to provide the aforementioned sensor, wherein the preventative layer is comprised of material that can be digested by an enzyme, the material selected from a group, the group consisting of sugars, hydrogels, peptides, lipids and polymers.
It is another object of the present invention to provide the aforementioned sensor, wherein the absorption pad selectively or non-selectively reacts or binds with the reporter compound to generate a signal.
It is another object of the present invention to provide the aforementioned sensor, wherein the signal is identified visually or measured by a detector.
It is another object of the present invention to provide the aforementioned sensor, wherein the detector is based on spectroscopic, photochemical, biochemical, enzymatic, immunochemical, electrical, radiographic and optical means.
It is another object of the present invention to provide the aforementioned sensor, wherein the detector communicates results to a computer system or network.
It is an object of the present invention to provide a sensor for the rapid onsite identification of analytes comprising: a. at least one reversibly immobilized analyte-effector complex; b. at least one reporter compound;
c. at least one preventative layer; and
d. at least one absorption layer; wherein the reporter compound and the analyte are not bound to each other; wherein the preventative layer is positioned between the analyte and the reporter and between the absorption layer and is configures as a specific analyte signaling channel.
It is another object of the present invention to provide the aforementioned sensor, wherein the analyte is bound to an effector to create an analyte-effector complex.
It is another object of the present invention to provide the aforementioned sensor, wherein the effector is an enzyme.
It is another object of the present invention to provide the aforementioned sensor, wherein the analyte-effector complex is immobilized by being reversibly bound to an anti-analyte antibody, the anti-analyte antibody is specific for the analyte.
It is another object of the present invention to provide the aforementioned sensor, wherein the analyte-effector complex is released from the antibody through specific competitive or non-competitive binding of the free analyte from the sample to the anti body, immobilized to the reaction layer.
It is another object of the present invention to provide the aforementioned sensor, wherein the reporter compound is characterized by being: a. incapable of flowing/traversing/passing/crossing through un-affected preventative layer; and
b. capable of flowing/traversing/passing/crossing through affected preventative layer.
It is another object of the present invention to provide the aforementioned sensor, wherein the reporter compound is selected from a group of compounds consisting of: pigments, dyes, electrochemical active compounds, enzymes, flourophores, chemiluminescent molecules and radionuclides.
It is another object of the present invention to provide the aforementioned sensor, wherein preventative pad consists of at least one compound that can be altered by interacting with the effector. It is another object of the present invention to provide the aforementioned sensor, wherein the preventative layer is comprised of material that can be digested by an enzyme, the material selected from a group of organic compounds, the group consisting of sugars, hydrogels, peptides, lipids and polymers.
It is another object of the present invention to provide the aforementioned sensor, wherein the absorption pad selectively or non-selectively reacts and/or binds with the reporter compound to generate a signal.
It is another object of the present invention to provide the aforementioned sensor, wherein the signal is identified visually or measured by a detector.
It is another object of the present invention to provide the aforementioned sensor, wherein the detector is based on spectroscopic, photochemical, biochemical, enzymatic, immunochemical, electrical, radiographic and optical means.
It is another object of the present invention to provide the aforementioned sensor, wherein the detector communicates results to a computer system or network.
It is the object of the present invention to provide method for analyzing a sample comprising steps of: a. obtaining a sample as a solution;
b. obtaining a sensor, the sensor comprising;
i. at least one reversibly immobilized analyte-effector complex; ii. at least one reporter compound;
iii. at least one preventative layer; and
iv. at least one absorption pad; wherein the reporter compound and the analyte are not bound to each other; wherein the preventative layer is positioned between the analyte and the reporter and the detector; wherein the preventative layer is positioned between the analyte and the reporter and between the absorption layer; c. loading sample solution; and d. reading analysis result.
It is another object of the present invention to provide the aforementioned method, wherein the sample layer is constructed from a porous, absorbent and nonreactive membrane.
It is another object of the present invention to provide the aforementioned method, wherein the membrane is selected from a group comprising: cellulose acetate membrane, nitrocellulose membrane, cellulose ester membrane, polysulfone (PS) membrane, polyether sulfone (PES) membrane, polyacrilonitrile (PAN) membrane, polyamide membrane, polyimide membrane, polyethylene and polypropylene (PE and PP) membrane, polytetrafluoroethylene (PTFE) membrane, polyvinylidene fluoride (PVDF) membrane, polyvinylchloride (PVC) membrane and fiberglass paper membrane.
It is another object of the present invention to provide the aforementioned method, wherein the reaction pad contains at least one kind of immobilized analyte-effector complex.
It is another object of the present invention to provide the aforementioned method, wherein the analyte-effector complex is released from the antibody through specific competitive or non-competitive binding of the free analyte from the sample to the anti body, immobilized to the reaction layer.
It is another object of the present invention to provide the aforementioned method, wherein the reporter compound is characterized by being: a. incapable of crossing un-affected preventative layer; and
b. capable of crossing affected preventative layer; and
c. specific to the absorption pad or the detector.
It is another object of the present invention to provide the aforementioned method, wherein the reporter compound is selected from a group of compounds consisting of: pigments, dyes, electrochemical active compounds, enzymes, flourophores, chemiluminescent molecules and radionuclides. It is another object of the present invention to provide the aforementioned method, wherein preventative pad consists of at least one compound that can be altered, by interacting with the effector.
It is another object of the present invention to provide the aforementioned method, wherein the preventative layer is comprised of at least one material that can be digested by an enzyme, the material selected from a group, the group consisting of sugars, hydrogels, peptides, lipids and polymers.
It is another object of the present invention to provide the aforementioned method, wherein the absorption pad selectively or non-selectively reacts and/or binds with the reporter compound to generate a signal.
It is another object of the present invention to provide the aforementioned method, wherein the signal is identified visually or measured by a detector.
It is another object of the present invention to provide the aforementioned method, wherein the detector is based on spectroscopic, photochemical, biochemical, enzymatic, immunochemical, electrical, radiographic and optical means.
It is another object of the present invention to provide the aforementioned method, wherein the detector communicates results to a computer system or network.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 - illustrates the schematic structure of the sensor.
Figure 2 - illustrates the schematic activity of the sensor.
Figure 3 - illustrates the schematic activity of the sensor.
Figure 4 - demonstrates the effect of the deposition processes on the uniformity of the gelatin layer
Figure 5 - demonstrates the effect of the gelatin concentration on the solution preventing properties. Figure 6 - demonstrates that an increase in the gelatin concentration decreases the capability of the enzyme in the solution diffused through the preventative layer
Figure 7 - demonstrates the proof of concept of the present invention
DETAILED DESCRIPTION OF THE INVENTION
In this application, the term“Analyte” or“analyte of interest” as used herein, refers to the substance to be detected, which may be present in the liquid sample. The analyte can be any substance for which there exists at least one naturally occurring or synthetic specific binding partner. The analyte can include a protein or protein fragment, a polypeptide peptide or peptide fragment, an amino acid, a DNA fragment, a RNA fragment, a small molecule, a bacterium, natural ligands, virus particles (virions), a virus or metabolites of or antibodies to or biomimetic of any of the above substances. The analyte can be a polutent or can serve as a pesticide or a toxin. In some configurations, the identified analytes could be only segments of the origional analyte, cased by fragmentation, desintgration, deterioration, deacy, oxidation etc. Fragmentation can be caused by exposure to the eviroment or as part of the sample preperation procedure.
In this application, the term an“anti-analyte antibody” refers to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes or fragments of immunoglobulin genes. This term encompasses polyclonal antibodies, monoclonal antibodies, and fragments thereof, as well as molecules engineered from immunoglobulin gene sequences. The anti-analyte antibody is specific to the analyte of interest. The term an“anti-analyte capture antibody” is an anti-analyte antibody that captures the analyte of interest. Such antibodies are conveniently affixed to a solid phase, such as the membrane of the reaction layer.
In this application, the term "Reporter compound" or "reporter molecule" (or simply "reporter") as used herein, refers to molecules useful for detecting the presence, intensity or quantity of the analyte due to an interaction between the reporter compound and the absorption layer and/or a detector. Molecules are detectable by spectroscopic, photochemical, biochemical, enzymatic, immunochemical, electrical, radiographic and optical means. Optically detectable molecules can be detectable in the in the ultraviolet, visual or infrared spectrum, including compounds such as dyes or fluorescent labels.
"Reporter compounds" useful in the present invention also include any suitable molecule which may be conjugated to the analyte molecule without compromising the ability of the reporter molecule to be detected or the analyte to be bound to the anti-body.
In preferred embodiments, the reporter is selected from the group consisting of a dye, a radionuclide, an enzyme and combinations thereof.
The dye can be either "small molecule" dye/fluors, or macromolecule dye/fluors (e.g. green fluorescent proteins and all variants thereof). The dye may be a tandem fluorophore conjugate. In various embodiments, the dye may be a fluorescent semiconductor nanocrystal particle, a quantum dot, an electroactive molecule/dye or an upconversion nanocrystal.
In one preferred embodiment, the reporter compound is loaded on to the "reporter layer" or "reporter membrane". The reporter layer can be formed by binding the reporter in a selective or non-selective manor. In one preferred configuration, the membrane is loaded by saturating the membrane with a reporter compound solution, and subsequently drying the loaded membrane, binding the reporter to the membrane in a non-selective manor. In this configuration, as the sample solution rehydrates the layer, the reporter interacts with liquid/solution and migrates along with the flow, to the "preventative layer". Alternatively, the reporter molecule can be loaded onto another layer, up-stream from the preventative layer.
The term "Preventative layer", "preventative pad" or " preventative membrane" refers to a treated porous or semi-porous membrane or solid layer constructed from a material that does not permit the passage of reporter molecules unless it is altered by interacting with the effector. The Preventative layer obstructs passage of the reporter compound due to chemical or physical properties of the reporter compound and the preventative layer. In a preferred configuration, the preventative layer is any biological or chemical substances that can be digested by enzymes (sugars, hydrogels, peptides, proteins, fats, plastic polymers, etc.) such as gelatin.
The term "effector" (alternatively "effector compound" or" effector molecule") refers to a compound that can be bound to the analyte without affecting its activity. The effector has the ability to interact with the preventative layer, thereby changing its physical or chemical properties in a way that enables passage of reporter compounds. The effector can be an enzyme, a macromolecule or a small molecule. The effector can be organic, inorganic or organometallic in composition. In one preferred embodiment, the effector is an enzyme capable of digesting the preventative layer, opening pores large enough for the reporter compound to pass.
As used herein, the term "membrane" refers to a natural or synthetic/artificial membrane. The terms "synthetic membrane" or "artificial membrane" refer to a man made membrane that is produced from organic material, such as polymers and liquids, as well as inorganic materials. A wide variety of synthetic membranes are well known in the art. In various embodiments, the membranes of the sample layer, the at least one conjugation layer, the at least one preventative layer and the absorption layer are independently selected from the group consisting of cellulose acetate membrane, nitrocellulose membrane, cellulose ester membrane, polysulfone (PS) membrane, polyether sulfone (PES) membrane, polyacrilonitrile (PAN) membrane, polyamide membrane, polyimide membrane, polyethylene and polypropylene (PE and PP) membrane, polytetrafluoroethylene (PTFE) membrane, polyvinylidene fluoride (PVDF) membrane, polyvinylchloride (PVC) membrane and fiberglass paper membrane.
The term "absorption membrane", "absorption layer" or "absorption pad" refers to a treated membrane that specifically or non-specifically binds to or reacts with the reporter compound. In various embodiments, the absorption layer further comprises at least one substrate for a reporter molecule (or simply a reporter). Reporter substrate, as used herein, is intended to include any substrate capable of interacting with the reporter. Preferably, the interaction between the reporter and the reporter substrate produces a qualitative or quantitative effect. A "reporter substrate" as used herein is a substrate (or substrates) that can facilitate measurement of either the disappearance of a substrate or the appearance of a product in connection with a catalyzed reaction. Reporter substrates can be free in solution or bound (or "tethered"), for example, to a surface, or to another molecule. A reporter substrate can be labelled by any of a large variety of means including, for example, fluorophores (with or without one or more additional components, such as quenchers), radioactive labels, biotin (e.g. biotinylation) or chemiluminescent labels. In case the reporter is horseradish peroxidase, the substrate is preferably luminol.
In some configurations, the absorption layer is part of a detector. In this configuration the absorption pad facilitates the interaction between the reporter compound and the detector. In one configuration this is performed by containing reporter substrates or by capturing free reporter compounds.
The term "detector' refers to a devise that enables the measurement of reporter compounds that reach the absorption layer or interact with reporter substrates. The measurement can be of the compound itself or of the interaction with the substrate. This interaction can be measured by spectroscopic, photochemical, biochemical, enzymatic, immunochemical, electrical, radiographic or optical means. The detector is configured to utilize Chemometrics to analyze the signal (or signals) generated by the reporter- absorption interaction to detect and measure the amount of analyte present in the sample.
The object of this invention is a sensor for the on-site, real-time, fast and simple analyte detection.
Reference is made to Figure 1, describing one non limiting embodiment of the invention, a liquid sample collected and loaded on the sample layer (11). The sample solution hydrates the layers and the sample solution flows through to the reaction layer (12). The analyte in the sample binds to the anti-analyte antibody, dislocating the bound analyte- effector complex. The analyte-effector complex passes through the reporter layer to the preventative layer (13). The effector interacts with the preventative layer (13), affecting the physical and/or chemical properties of the preventative layer. This change enables the reporter compounds to traverse the preventative layer and reach the absorption layer (14). Reference is made to Figure 2, describing one non limiting embodiment of the invention, a liquid sample collected and loaded on the sample layer 21. The sample solution hydrates the layers and the sample solution flows through to the reaction layer 22. If the sample solution contains the analyte, then the analyte in the sample binds to the anti analyte antibody, dislocating the bound analyte-effector complex. The analyte-effector complex then passes through the reporter layer to the preventative layer 23. The effector interacts with the preventative layer 23, affecting the physical and/or chemical properties of the preventative layer. This change enables the reporter compounds to traverse the preventative layer 24 and reach the absorption layer 25. The sensor will then return a positive result 26.
If the sample does not contain the analyte, then the analyte-effector complex remains bound 27 and cannot affect the physical and/or chemical properties of the preventative layer 28. The reporter compound will not be able to cross the preventative layer 29 and the sensor will return a negative result 30.
Reference is made to Figure 3, describing the current invention in two instances: o 31 A sample containing the analyte -> where the preventative layer interacts with the affector -> enabling the reporter compound to reach the absorption layer.
o 32 A sample not containing the analyte -> preventative layer is not affected.
Reporter compounds that traverse the preventative layer interact with the absorption layer to generate a signal. This interaction can be specific, such as binding to a substrate, or non-specific, such as the accumulation of dyes. This interaction generates a signal, such as the generation of a color due to the accumulation of dyes. In some configurations the signal is then measured by the detector the sensor can be configures to detect the presence of more than one analyte in a single sample by using one reaction layer loaded with multiple anti-analyte capture anti-body's or by using multiple reaction layers, each layer corresponding to a different anti-analyte capture antibody. The detector and absorption layer can be configured to detect the presence of more than one reporter compound, enabling the system to detect the presents of multiple analytes in a single sample. In this configuration, the signal generated by each reporter must be distinctive and must not impede the detectors ability to detect signals generated by other reporter compounds. In this configuration the detector can use chemometrics to detect the level of various analytes in the sample.
In another non limiting embodiment of the invention the sensor is constricted of a number of layers, each layer placed on each other affording to the flow
In another non limiting embodiment of the invention, the sensor is constructed as a single strip. In this embodiment the layers are arraigned end-to-end and are regions of one strip
In this approach, an absorption cellulose membrane served as solid bedding support, onto which different assay components are immobilized onto the various layers.
Example 1
Herein is described a sensor for detecting allergens in food. In this example the membranes are paper (cellulose), the preventative layer is constructed from gelatin, the reporter compound is a dye and the effector is pepsin. In the first step, the liquid sample is collected and deposited on the sample pad. The sample traverses through a sample pad until it reaches the reaction pad. The reaction pad contains immobilized allergen-pepsin complex (anti-analyte-enzyme complex) bound to an allergen antibody. The free allergen in the sample binds to the anti-analyte antibody capture complex, releasing the analyte- pepsin complex. The complex passes through and rehydrating the color layer and reaches the gelatin preventative layer. The pepsin digests the gelatin, creating pores (one pore for each freed complex). The dyes molecules pass through the pores in the gelatin and reach the absorption pad, coloring the absorption layer.
In this configuration the color indicates the presence of an allergen in the food, alerting to a possible health hazard.
Example 2
Herein is described a sensor for detecting waterborne pathogens. In this example the membranes are paper (cellulose), the preventative layer is constructed from gelatin, the reporter layer contains red dye and the effector is pepsin. In this configuration the sensor contains 7 reaction layers, each one specific for a different pathogen:
• Cryptosporidium
• Giardia
• Shigella
• E. Coli 0l57:H7
• Legionella
• Campylobacter
• Salmonella
Each reaction layer contains a specific immobilized pathogen-pepsin complex (anti- analyte-enzyme complex) bound to a pathogen anti-body. Each pathogen-pepsin complex is additionally linked to an additional reporter compound, creating a pathogen-pepsin- reporter complex. Each additional reporter compound is a different florescent compound, each florescent compound emitting light at a distinctive spectrum.
In the first step, the liquid sample is collected and deposited on the sample pad. The sample traverses through a sample pad until it reaches the specific reaction pad. The specific reaction pad contains immobilized pathogen-pepsin-reporter complex bound to the pathogen antibody. The free pathogen in the sample binds to the anti-analyte antibody capture complex, releasing the pathogen-pepsin-reporter complex. The complex passes through and rehydrating the color layer and reaches the gelatine preventative layer. The pepsin than digests the gelatin, creating pores (one pore for each freed complex). The dyes molecules pass through the pores in the gelatin and reach the absorption pad, coloring the absorption pad. The absorption layer is then loaded into a spectroscopic detector can then be used to identify the specific pathogen in the samples.
In this configuration a colored layer warns about a possible water contamination and the use of a detector detects the specific pathogens present in the water source.
Example 3
Herein is described a method for validating cleaning in place of reactors. In this example the membranes are paper (cellulose), the preventative layer is constructed from gelatin, the reporter compound is a dye and the effector is pepsin. In this example the reported compound is dissolved in the sample collection solution.
In the first step, a dose of the sample collection solution of deposited on the surface of the reactor. The sample collection solution comprises a suitable solvent and the dye (the reported compound). The sample layer is dunked in the solution that is on the reactor surface. The sample traverses through a sample pad until it reaches the reaction pad. The reaction pad contains immobilized analyte-pepsin complex (anti-analyte-enzyme complex) bound to an anti-analyte antibody. The free analyte in the sample binds to the anti-analyte antibody capture complex, releasing the analyte-pepsin complex. The complex reaches the gelatin preventative layer. The pepsin digests the gelatin, creating pores (one pore for each freed complex). The dyes molecules pass through the pores in the gelatin and reach the absorption pad, coloring the absorption layer.
In this configuration the color indicates that the reactor has not been sufficiently cleaned.
Example 4
Herein is demonstrated the bio-physical effects of the method.
Reference is made to Figure 4, demonstrating the effect of the deposition processes on the uniformity of the gelatin layer, so as to determine the constant diffusion time gelatin layer supposed to be uniform. To determinate effect of the drying protocol on the uniformity of the gelatin layer formation, two different drying modes were tested:
In the first (Figure 4A), 250 pl of 5%(w/v) gelatin solution was placed above the 4x4 cm Kim wipes paper and drying on the flat surface at room temperature.
In the second, 4x4 cm Kim wipes paper was dipped in 1 mL 5%(w/v) gelatin solution, squeezed (to remove liquid excesses) and dried in the air stretched out.
Uniformity of the gelatin layer is a critical issue in the sensor development procedure, as a uniform layer not only will allow determination constant passing time through this stopping layer in the sensor, but also will be disassembled by the same enzyme concentrations during evaluation. Reference is made to Figure 5, demonstrating the effect of gelatin concentration on the ability of the preventative layer to prevent/stop/regulate the passage of the solution. Prevention layers with different gelatin concentrations were created as described in the previous section (for figure 4). The prevention layers were then placed above the absorption layer and 25 pl of colored solution with (+) and without (-) enzyme bromelain (250 units/mL) were placed above it. Bromelain is enzyme from pineapples that degrades gelatin, thus testing the stopping capabilities of the gelatin layers (with clear solution) and also determining effect of various gelatin concentrations on enzymatic reaction of the positive (+) solution (the solution containing the enzyme). Figure 5 demonstrates that all solutions containing the Bromelain enzyme diffused through preventing layer, regardless of the gelatin concentration. This demonstrates the ability of the Bromelain enzyme to penetrate the preventative layer at any used gelatin concentrations. In the enzyme negative samples (tested with clear water), only layers with 5% gelatin (w/v) and higher showed the ability to stop the solution. This indicates the specificity of the diffusion/degradation mechanisms, where systems with a preventative layer containing at least 5% (w/v) gelatin will allow the passage of enzyme containing solutions while stopping negative samples and will pass solutions.
Reference is made to Figure 6, showing the determining effect of the gelatin concentration on its preventing/stopping properties. For this step, preventative layers with 5% (w/v) gelatin concentration where generated as described in the figure 4 and placed above absorption pad. To test enzyme passing capability, different bromelain concentrations were prepared in colored solution and placed above preventative layers. As the enzyme degrades the gelatin in the preventative layer, the colored solution passes through to the absorption layer and colored it. This step not will show minimum enzyme concentration that may pass stopping layer and effect of gelatin concentrations on these passing capabilities.
Figure 6 demonstrates that an increase in the gelatin concentration in the preventative layer lowers the ability of the Bromelain enzyme in the solution to enable diffuse of the colored solution through preventative layer. While both tested concentrations prevent false-negative results, a preventative layer containing 12.5 pg/mL gelatin were enough to prevent solutions containing two active units to diffuse or pass through preventative layer. The negative samples show that a preventative layer containing 5% (w/v) gelatin prevents the negative solution (without enzyme) diffusion through it, as a positive solution (containing the Bromelain enzyme) can disassemble the preventative layer and enable the passage of the solution, with as low an enzyme concentration as possible.
Figure 7 further demonstrates the potential of the present invention to generate a positive result when in the present of a solution containing the Bromelain enzyme (+).

Claims

1. A sensor for the rapid, onsite identification of analytes characterized by:
a. a sample layer;
b. at least one reaction layer, interconnected with said sample layer: c. at least one reporter layer, interconnected with said reaction payer;
d. at least one preventative layer, interconnected with said reporter layer; and e. an absorption pad, interconnected with said preventative layer; wherein said layers are arranged from a-to-e and are operatively arranged as a specific analyte signaling channel.
2. A sensor for the rapid, onsite identification of analytes characterized by a sample layer;
a. at least one reporter layer, interconnected with said sample layer; b. at least one reaction layer, interconnected with said reporter layer; c. at least one preventative layer, interconnected with said reaction layer; and d. an absorption layer, interconnected with said preventative layer; wherein said layers are arranged from a-to-e and are operatively arranged as a specific analyte signaling channel.
3. The sensor of claim 1 or claim 2, wherein said sample layer is constructed from a porous, absorbent and nonreactive membrane.
4. The sensor of claim 3, wherein said membrane is selected from a group comprising: cellulose acetate membrane, nitrocellulose membrane, cellulose ester membrane, polysulfone (PS) membrane, polyether sulfone (PES) membrane, polyacrilonitrile (PAN) membrane, polyamide membrane, polyimide membrane, polyethylene and polypropylene (PE and PP) membrane, polytetrafluoroethylene (PTFE) membrane, polyvinylidene fluoride (PVDF) membrane, polyvinylchloride (PVC) membrane and fiberglass paper membrane.
5. The sensor of claim 1 or claim 2, wherein said reaction pad contains at least one kind of immobilized analyte-effector complex.
6. The sensor of claim 5, wherein said analytic-effector complex comprising of: a. at least one analyte; and b. at least one effector, said effector specific to composition of said preventative pad; wherein said analyte and effector are reversibly or irreversibly connected.
7. The sensor of claim 6, wherein said effector is an enzyme.
8. The sensor of claim 6, wherein said immobilized analyte-effector complex additionally comprises at least one reporter compound.
9. The sensor of claim 5, wherein said immobilized analyte-effector complex is reversibly bound to a specific anti-analyte antibody, said antibody is specific for said analyte.
10. The sensor of claim 9, wherein said analyte-effector complex is released from said antibody through specific competitive or non-competitive binding of said free analyte from said sample to said anti-body, immobilized to said reaction layer.
11. The sensor of claim 7, wherein said anti-analyte antibody is irreversibly bound to said reaction pad.
12. The sensor of claim 1 or claim 2, wherein said reporter layer is loaded with bound or unbound reporter compounds.
13. The sensor of claim 8 or claim 12, wherein said reporter compound characterized by being:
a. incapable of crossing unaffected preventative layer;
b. capable of crossing affected preventative layer; and
c. specific to said absorption pad or said detector.
14. The sensor of claim 13, wherein said reporter compound is selected from a group of compounds consisting of: dyes, pigments, electrochemical active compounds, enzymes, flourophores, chemiluminescent molecules and radionuclides.
15. The sensor of claim 13, wherein said reporter compound is blocked from crossing said unaffected preventative layer due to size, electric or magnetic charge, hydrophobicity or lipophobicity.
16. The sensor of claim 1 or claim 2, wherein preventative layer consists of at least one compound that is affected by said effector.
17. The sensor of claim 16, wherein said preventative layer is comprised of at least one material that can be digested by an enzyme, said material selected from a group or organic compounds, said group consisting of sugars, hydrogels, peptides, lipids and polymers.
18. The sensor of claim 1 or claim 2, wherein said absorption pad selectively or non- selectively reacts or binds with said reporter compound to generate a signal.
19. The sensor of claim 18, wherein said signal is identified visually or measured by a detector.
20. The sensor of claim 19, wherein detector is based on spectroscopic, photochemical, biochemical, enzymatic, immunochemical, electrical, radiographic and optical means.
21. The sensor of claim 19, wherein said detector communicates results to a computer system or network.
22. A sensor for the rapid onsite identification of analytes comprising:
a. at least one reversibly immobilized analyte-effector complex;
b. at least one reporter compound;
c. at least one preventative layer; and
d. at least one absorption layer; wherein at least one of said reporter compounds and said analyte are not bound to each other; and said preventative layer is positioned between said analyte and said reporter and between said absorption layer and is configures as a specific analyte signaling channel.
23. The sensor of claim 22, wherein said analyte is bound to an effector to create an analyte-effector complex.
24. The sensor of claim 23, wherein said analytic-effector complex comprising of: a. at least one analyte; and
b. at least one effector, said effector specific to composition of said preventative pad; wherein said analyte and effector are reversibly or irreversibly connected.
25. The sensor of claim 23, wherein said effector is an enzyme.
26. The sensor of claim 23, wherein said analyte-effector complex is immobilized by being reversibly bound to an anti-analyte antibody, said anti-analyte antibody is specific for said analyte.
27. The sensor of claim 26, wherein said analyte-effector complex is released from said antibody through specific competitive or non-competitive binding of said free analyte from said sample to said anti-body, immobilized to said reaction layer.
28. The sensor of claim 22, wherein said reporter compound is characterized by being:
a. incapable of crossing un-affected preventative layer; and
b. capable of crossing affected preventative layer; and
c. specific to said absorption pad or said detector.
29. The sensor of claim 28, wherein said reporter compound is selected from a group of compounds consisting of: pigments, dyes, electrochemical active compounds, enzymes, flourophores, chemiluminescent molecules and radionuclides.
30. The sensor of claim 22, wherein preventative pad consists of at least one compound that can be altered, by interacting with said effector.
31. The sensor of claim 30, wherein said preventative layer is comprised of material that can be digested by an enzyme, said material selected from a group, said group consisting of sugars, hydrogels, peptides, lipids and polymers.
32. The sensor of claim 22, wherein said absorption pad selectively or non-selectively reacts and/or binds with said reporter compound to generate a signal.
33. The sensor of claim 32, wherein said signal is identified visually or measured by a detector.
34. The sensor of claim 31, wherein detector is based on spectroscopic, photochemical, biochemical, enzymatic, immunochemical, electrical, radiographic and optical means.
35. The sensor of claim 31, wherein said detector communicates results to a computer system or network.
36. A method for analyzing a sample comprising steps of:
a. obtaining a sample as a solution;
b. obtaining a sensor, said sensor comprising;
i. at least one reversibly immobilized analyte-effector complex; ii. at least one reporter compound;
iii. at least one preventative layer; and
iv. at least one absorption layer; wherein at least of said reporter compounds and said analyte are not bound to each other; wherein said preventative layer is positioned between said analyte and said reporter and between said absorption; c. loading sample solution; and
d. reading analysis result.
37. The method of claim 36, wherein said sample layer is constructed from a porous, absorbent and nonreactive membrane.
38. The sensor of claim 37, wherein said membrane is selected from a group comprising: cellulose acetate membrane, nitrocellulose membrane, cellulose ester membrane, polysulfone (PS) membrane, polyether sulfone (PES) membrane, polyacrilonitrile (PAN) membrane, polyamide membrane, polyimide membrane, polyethylene and polypropylene (PE and PP) membrane, polytetrafluoroethylene (PTFE) membrane, polyvinylidene fluoride (PVDF) membrane, polyvinylchloride (PVC) membrane and fiberglass paper membrane.
39. The method of claim 36, wherein said reaction pad contains at least one kind of immobilized analyte-effector complex.
40. The sensor of claim 39, wherein said analyte-effector complex is released from said antibody through specific competitive or non-competitive binding of said analyte from said sample to the anti-body, immobilized to said reaction layer.
41. The method of claim 36, wherein said reporter compound is characterized by being:
a. incapable of crossing un-affected preventative layer; and
b. capable of crossing affected preventative layer; and
c. specific to said absorption pad or said detector.
42. The method of claim 41 , wherein said reporter compound is selected from a group of compounds consisting of: pigments, dyes, electrochemical active compounds, enzymes, flourophores, chemiluminescent molecules and radionuclides.
43. The method of claim 36, wherein preventative pad consists of at least one compound that can be altered, by interacting with said effector.
44. The method of claim 43, wherein said preventative layer is comprised of material that can be digested by an enzyme, said material selected from a group, said group consisting of sugars, hydrogels, peptides, lipids and polymers.
45. The method of claim 36, wherein said absorption pad selectively or non- selectively reacts and/or binds with said reporter compound to generate a signal.
46. The method of claim 45, wherein said signal is identified visually or measured by a detector.
47. The method of claim 46, wherein detector is based on spectroscopic, photochemical, biochemical, enzymatic, immunochemical, electrical, radiographic and optical means.
48. The method of claim 46, wherein said detector communicates results to a computer system or network.
PCT/IL2019/051150 2018-10-25 2019-10-24 Means and method for point-of-care analysis of liquid samples Ceased WO2020084620A1 (en)

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