EP2245135A2 - Dispositifs d'analyse de petites molécules et de protéines à partir de polymères à empreintes moléculaires - Google Patents

Dispositifs d'analyse de petites molécules et de protéines à partir de polymères à empreintes moléculaires

Info

Publication number
EP2245135A2
EP2245135A2 EP08868717A EP08868717A EP2245135A2 EP 2245135 A2 EP2245135 A2 EP 2245135A2 EP 08868717 A EP08868717 A EP 08868717A EP 08868717 A EP08868717 A EP 08868717A EP 2245135 A2 EP2245135 A2 EP 2245135A2
Authority
EP
European Patent Office
Prior art keywords
binding
conjugate
analyte
reporter
sample
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.)
Withdrawn
Application number
EP08868717A
Other languages
German (de)
English (en)
Other versions
EP2245135A4 (fr
Inventor
Raphael Levi
Ido Margalit
Yarden Dloomy
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.)
Infigo Diagnostics Ltd
Original Assignee
Infigo Diagnostics Ltd
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 Infigo Diagnostics Ltd filed Critical Infigo Diagnostics Ltd
Publication of EP2245135A2 publication Critical patent/EP2245135A2/fr
Publication of EP2245135A4 publication Critical patent/EP2245135A4/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • 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
    • G01N33/54306—Solid-phase reaction mechanisms
    • 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
    • G01N33/54388—Immunochromatographic test strips based on lateral flow
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2600/00—Assays involving molecular imprinted polymers/polymers created around a molecular template

Definitions

  • the present invention relates to the field of diagnostics, and more particularly to flow through or lateral flow devices based on the use of molecularly imprinted polymers for measurement of analyte levels in a liquid sample, and methods and kits for use thereof.
  • analytes such as protein related analytes
  • detection and measurement of protein molecules and protein containing organisms such as bacteria and viruses involves immunoassays that employ target specific antibodies. Rapid, real-time, and simple analysis of various analytes and protein related molecules of interest in liquid samples is performed today using sophisticated instruments and antibody based devices.
  • Antibodies are used in several areas, such as therapy, immunoaffinity purification and particularly in immunoassays. Antibodies are produced by immunization of animals with the respective antigen, leading to polyclonal antibodies, or by using fused cells, allowing the obtained cell lines to produce monoclonal antibodies.
  • Non-biological antibody mimics artificial antibodies
  • Use of such systems could reduce the need for animal sources.
  • antibody mimics can be obtained against molecules against which it is generally considered to be difficult or impossible to raise antibodies, such as immunosuppressive agents, short peptides or other small molecules.
  • Such non-biological systems, prepared by chemical methods would also be much more stable than natural antibodies, allowing repeated use, performance at higher temperatures, easy sterilization and increased batch to batch reproducibility.
  • a molecular imprint polymer is a polymer which is prepared by polymerizing functional monomers around a template or "print” molecule, which is then removed from the polymer by extraction or other means so that the polymer selectively absorbs the template or print molecule upon re-exposure to the print molecule.
  • ionic molecular images of polypeptides have been created by mixing a matrix material with the intact polypeptide chain to be bound by the molecular image (U.S. Pat. No. 5,756,717).
  • Molecular imprints of cytochrome c, hemoglobin and myoglobin, respectively, have been prepared by polymerizing acrylamide in the presence of each intact protein (U.S. Pat. No. 5,814,223).
  • An imprint of horse myoglobin selectively bound horse myoglobin from a mixture of proteins including whale myoglobin (U.S. Pat. No. 5,814,223).
  • Molecular imprinting methods may involve "epitope imprinting” (Rachkov and Minoura, 2000, J. Chromatogr A.;889, 111-1 18) wherein key epitopes are identified on the surface of the protein and the MIP is prepared with the linear peptide representing this epitope as target.
  • MIP-based sensors are as follows:
  • Yan et al. (U.S. Pat. No. 5,587,273) describe sensors employing molecularly imprinted film and measuring the capacitance or the light characteristics of the film after the exposing step or analyzing the film spectroscopically.
  • MIP-based devices for detecting, analyzing and quantifying macromolecules are disclosed by Huang (U.S. Pat. No. 6,680,210). Detection is performed by dissociating the analyte molecules from the polymer after the binding and then analyzing them.
  • Williams et al. (U.S. Pat. 6,807,842) disclose a molecular recognition sensor system for detecting the presence and concentration of an analyte including a resistive sensor having a semiconductive polymer film which swells when exposed to the analyte.
  • Kroeger et al. (G.B. Pat. No. 2,337,332) disclose an electrode that has a surface modified with an imprinted synthetic polymer that specifically recognizes, binds and concentrates the analyte, in close proximity to the electrode surface. Either the bound analyte itself or an electrochemically active derivative pre-incubated with the electrode or added to the sample (competition/displacement assay) is quantified electrochemically (for example by differential pulse or square wave voltametry) directly at the electrode surface.
  • Penelle U.S. Pat. No. 6,890,486) discloses an hexachlorobenzene MIP-based sensor by combining MIP techniques with quartz crystal microbalance (QCM).
  • Green et al. (U.S. Pat. No. 6,638,498) disclose devices utilizing MIPs with specific binding capacity for particular bile acids and/or salts, such as DCA and CDCA. The detection is performed by displacement of radioactive-labeled CDCA by
  • CDCA in the sample.
  • a fluorescent derivative of cholic acid is used as the assay substance.
  • Lawrence et al. (U.S. Pat. No. 6,833,274) disclose a Cortisol fiber optic sensor using a cortisol-imprinted polymer and displacement of a cortisol-fluorescent chromophore conjugate.
  • Schwartz et al. U.S. Pat. No. 6,967,103 disclose an explosive detector utilizing an array of MIP-coated, bifurcated fiber optic cables. Individual sensor fiber assemblies, each with a calibrated airflow, are used to expose the fibers to the target molecule. The detector energizes a dedicated excitation light source for each fiber and, through a detector comprising a filter and photodiode, simultaneously reads and processes the intensity of the resulting fluorescence that is indicative of the concentration of the target molecules.
  • PCT application WO 07/002237 describes a method of analysing a liquid sample comprising contacting the sample with an MIP.
  • the background art does not disclose the use of MIPs in a flow through or lateral flow device. Furthermore, the background art methods involve cumbersome sample preparation, and require use of sophisticated auxiliary analytical instrumentation by trained personnel.
  • the present invention in various embodiments thereof, provides devices, methods and kits for rapid and simple determination of target molecules, including small molecules, polypeptides, proteins, cells and infectious disease agents in liquid samples that are capable of real-time measurement of these entities in fluid samples that are highly selective, highly sensitive, simple to operate, low cost, and portable.
  • the devices, methods and kits also provide, in at least some embodiments, the use of MIPs in a flow through or lateral flow device.
  • the devices, methods and kits are suitable for use by untrained personnel without the need for uncommon and complicated to operate equipment. When the kits of the invention optionally require auxiliary equipment, it is preferably portable and hand held, relatively cheap, and simple to use.
  • the present invention provides a rapid and simple to use assay device, method and kit for determination of target polypeptides, proteins, cells and infectious disease agents in liquid samples, designed for use in the home, clinic, doctor's office, hospital bedside, factory or field.
  • the assay devices achieve greater sensitivity than conventional rapid test assays, without compromising specificity, leading to stronger and/or more stable visual signals than those produced by conventional devices, easier interpretation of results, and reduced occurrence of indeterminate results.
  • the devices may be used for detecting the target analytes in a variety of biological environmental and industrial samples in a short amount of time without the need for complicated sample preparation procedures, and thus are suitable for use by untrained personnel even in field conditions.
  • the present invention combines the advantages of the lateral and flow through devices (rapid, relatively cheap, and simple to use) with the advantages of MIPs for diagnostics (specific, controlled production, and very stable) with a novel double displacement/competition approach.
  • the devices of the invention will, in at least some embodiments, provide a "real-time" measurement, i.e., a relatively rapid detection time, preferably of about 15 minutes more preferably less than 15 minutes.
  • a "real-time" measurement i.e., a relatively rapid detection time, preferably of about 15 minutes more preferably less than 15 minutes.
  • auxiliary instrumentation such as readers, scanners, etc. may be used to interpret the results.
  • a target-specific molecular imprinted polymer is used to detect the presence of the target analyte in a sample based on binding of the analyte by the MIP. According to some embodiments of the present invention, there is provided a method of detecting at least one analyte in a liquid sample.
  • the method comprises providing a diagnostic device comprising a molecular imprinted polymer having analyte-specif ⁇ c binding sites fixed to a solid support, wherein the liquid sample is movable along a flow path along or through the solid support, and a sample application area for applying the liquid sample to said device; applying the liquid sample to the sample application area and allowing the sample to flow along or through the solid support and contact the molecular imprinted polymer; and detecting binding of said analyte to said molecular imprinted polymer.
  • a diagnostic device for detecting at least one analyte in a liquid sample, the device comprising a molecular imprinted polymer having analyte-specif ⁇ c binding sites fixed to a solid support, wherein the liquid sample is movable along a flow path along the solid support; a sample application area for applying the liquid sample to the device and bringing it in contact with the molecular imprinted polymer; and detection zone for detecting binding of said analyte to said molecular imprinted polymer.
  • the device comprises a lateral flow device, a flow through device, or a device combining lateral flow and flow through elements, wherein the liquid sample flows from the sample application area along or through the solid support components of the device along a flow path of the device.
  • the analyte displaces an analyte analog:reporter conjugate releasably bound to the molecular imprinted polymer, wherein displacement of the analyte analog:reporter conjugate indicates the presence of the analyte in the sample.
  • a releasable analyte analog:reporter conjugate is bound to the molecular imprinted polymer, wherein the affinity of the analyte for binding sites of the molecular imprinted polymer is at least equal to the affinity of the analyte analog:reporter conjugate for analyte-specif ⁇ c binding sites of the molecular imprinted polymer.
  • the device optionally further comprises a results zone comprising an analyte analog:reporter conjugate binding element immobilized to the solid support on the flow path of the sample, and the reporter - conjugate binding element is capable of binding displaced analyte analog :reporter conjugate.
  • Displacement of the analyte analog:reporter conjugate is optioanlly proportional to a concentration of the analyte in the liquid sample, such that detecting binding of the analyte to the molecular imprinted polymer comprises detecting binding of the displaced analyte :reporter conjugate to the analyte analog:reporter conjugate binding element.
  • the analyte displaces a first binding agent:analyte conjugate releasably bound to said molecular imprinted polymer, wherein the displaced first binding agent:analyte conjugate displaces a second binding agenfcreporter conjugate releasably bound to the solid support, wherein displacement of the second binding agentreporter conjugate indicates the presence of the analyte in the sample.
  • a releasable first binding agent:analyte conjugate is bound to the molecular imprinted polymer, wherein the affinity of the analyte for the binding sites of the molecular imprinted polymer is at least equal to the affinity of the first binding agent:analyte conjugate for the analyte-specific binding sites of the molecular imprinted polymer.
  • the device optionally further comprises a reporter-conjugate binding zone, comprising a reporter-conjugate binding element fixed to the solid support on the flow path of the liquid sample, the reporter-conjugate binding element having binding sites with a detectable, releasable, second binding agent reporter conjugate attached thereto.
  • a reporter-conjugate binding zone comprising a reporter-conjugate binding element fixed to the solid support on the flow path of the liquid sample, the reporter-conjugate binding element having binding sites with a detectable, releasable, second binding agent reporter conjugate attached thereto.
  • the affinity of the first binding agent:analyte conjugate for analyte-specific binding sites of the reporter conjugate binding element is at least equal to the affinity of the second binding agentreporter conjugate for binding sites of the reporte ⁇ conjugate binding element.
  • Binding of the first binding agentanalyte conjugate displaces the second binding agent:reporter conjugate, and displacement of the second binding agentreporter conjugate is optionally and preferably proportional to a concentration of the analyte in the liquid sample.
  • the device optionally further comprises a second binding agent: reporter conjugate binding element immobilized to the solid support on the flow path of the sample, the reporter conjugate binding element being capable of binding the displaced second binding agentreporter conjugate.
  • Detecting binding of the analyte to the molecular imprinted polymer optionally comprises detecting binding of the second binding agentreporter conjugate to the analyte analog:reporter conjugate binding element.
  • the method or device of the present invention comprises an analyte analog reporter conjugate, wherein the analyte competes with the analyte analog: reporter conjugate for analyte-specific binding sites of the molecular imprinted polymer, wherein the presence of unbound analyte analog:reporter conjugate is detected and indicates the presence of the analyte in the sample.
  • the method or device of the present invention comprises an analyte analog:reporter conjugate, wherein the affinity of the analyte for the analyte-specific binding sites of the molecular imprinted polymer is at least equal to the affinity of the analyte analog:reporter conjugate for analyte-specific binding sites of the molecular imprinted polymer.
  • the analyte and analyte analog: reporter conjugate compete for binding sites of the molecular imprinted polymer.
  • the device preferably further comprises a results zone comprising an analyte analog:reporter conjugate binding element bound to the solid support, the reporter conjugate binding element being capable of binding unbound analyte analog reporter conjugate and providing a detectable signal that indicates the concentration of the analyte in the liquid sample.
  • the method or device of the present invention comprises a first binding agent:analyte conjugate, a second binding agentreporter conjugate, and a second binding agentreceptor conjugate binding element.
  • the analyte competes with the first binding agentanalyte conjugate competes for analyte-specific binding sites of the molecular imprinted polymer, such that in the presence of the analyte, at least a portion of said first binding agentanalyte conjugate is unbound.
  • the unbound first binding agentanalyte conjugate then competes with the second binding agentreporter conjugate for binding to the second binding agent:receptor conjugate binding element.
  • the method or device of the present invention comprises a first binding agent: analyte conjugate, wherein the affinity of the analyte for analyte-specific binding sites of the molecular imprinted polymer is at least equal to the affinity of the first binding agentanalyte conjugate for analyte- specific binding sites of the molecular imprinted polymer, wherein upon contacting said molecular imprinted polymer with the analyte in the liquid sample, the analyte and the first binding agentanalyte conjugate analyte compete for analyte-specific binding sites of the molecular imprinted polymer. Unbound first binding agent:analyte conjugate flows in a flow path of the liquid sample.
  • the device optionally further ⁇ comprises a second binding agent:reporter application area, comprising a second binding agentreporter conjugate in a dry state; a reporter conjugate binding zone downstream, comprising a reporter-conjugate binding element fixed to the solid support on the flow path of the liquid sample.
  • the affinity of the first binding agentanalyte conjugate to the reporter-conjugate binding element is at least equal to the affinity of the second binding agent:reporter conjugate to the binding sites.
  • the second binding agent:reporter conjugate and the unbound first binding agentanalyte conjugate compete for binding to the reporter- conjugate binding element, and unbound second binding agentreporter conjugate flows downstream in the flow path of the liquid sample.
  • the device optionally further comprises a results zone comprising a second binding agentreporter conjugate binding element bound to the solid support, the reporter-conjugate binding element being capable of binding unbound second binding agentreporter conjugate and providing a detectable signal that indicates the concentration of the analyte in the liquid sample.
  • the method or device of the present invention further comprises a first binding agentanalyte analog, wherein unbound first binding agentanalyte analog is produced by at least one of competition with the analyte for binding sites of the molecular imprinted polymer and displacement by said analyte from the molecular imprinted polymer.
  • the device optionally further comprises a second binding agentreporter conjugate binding element, wherein an unbound second binding agentreporter conjugate is produced by at least one of competition with the first binding agentanalyte conjuage and displacement by the first binding agent:analyte conjugate, wherein the presence of unbound second binding agentreporter conjugate indicates the presence of the analyte in the sample.
  • the first binding agentanalyte analog is provided either in the sample application area, or bound to the molecular imprinted polymer.
  • the reporter conjugate binding element is optionally fixed to the solid support on the flow path of the liquid sample.
  • the second binding agentreporter conjugate is provided either in a reporter conjugate application area or releasably bound to the reporter conjugate binding element.
  • the device preferably further comprises a results zone comprising a second binding agentreporter conjugate binding element bound to the solid support, the reporter-conjugate binding element being capable of binding unbound second binding agent: reporter conjugate and providing a detectable signal that indicates the concentration of the analyte in the liquid sample.
  • At least one of the reporter conjugate or the binding agent conjugate comprises biotin, and the binding element is selected from the group consisting of avidin, NeutrAvidin, and Extravidin.
  • the reporter-conjugate-binding element is immobilized horizontally to the sample liquid flow path on the solid support and unbound reporter conjugate saturates the binding sites of the reporter-conjugate binding element.
  • the results are optionally visualized as an advancing column, having a length proportional to an amount of the analyte in the sample.
  • the device further comprising at least one of a positive control zone; a reference zone; an absorbent zone downstream of the results zone on the flow path of the liquid sample, the absorbent zone comprising absorbent material capable of absorbing the liquid sample; and a housing including at least one window aligned with at least one of the results zone, reference zone, and control zone to allow observation of test results on the device.
  • the intensity of a signal in the results zone is compared with the intensity of a signal in the reference zone, the device being arranged and constructed to provide a positive result when the analyte is present at or above a threshold level, to indicate the concentration of the analyte in the sample.
  • the device further comprises reference zone, comprisings at least one discrete band of a binding element comprising a known quantity of analyte analog:reporter conjugate, wherein the presence or absence of the target analyte in the sample is determined by comparison of the intensity of the signal from the reference zone with the intensity of the signal from the results zone.
  • the device is devoid of a reference zone
  • the results zone further comprises a scale parallel to the immobilized reporter- conjugate binding element calibrated to correspond to the analyte concentration in the liquid sample being analyzed, such that the presence and concentration of analyte in the liquid is determined by the area covered by the reporter-conjugate along and through the reporter-conjugate binding element in the results zone.
  • the sample is selected from the group consisting of biological fluids, environment samples, food stuffs, toxins, industrial samples and byproducts of industrial production procedures.
  • the analyte is selected from the group consisting of a cell, an organism, a small molecule, a protein, a hormone, an enzyme, a biomarker, metabolites of biomarkers, metabolites of drugs, a drug, a drug metabolite a drug-protein conjugate, a drug metabolite-protein conjugate, a vitamin, a drug of abuse, a natural or synthetic toxin, a chemical or biological warfare agent, antibodies to a drug, antibodies to infectious agents, an environmental pollutant, an immunoglobulin, a lymphokine, a cytokine, a soluble cancer antigen, a growth factor, a neurotransmitter, a molecule indicating the safety or quality of a foodstuff, a process chemical, a byproduct of a production process, a pesticide, an insecticide, a herbicide, a fertilizer, a surfactant, an adhesive, and an agent used in the manufacture of food, industrial agents or chemical products.
  • the analyte is selected from the group consisting of MMA, homocysteine, troponin sub-units I or T, CKMB, LDH and GOT/SGOT, citrozine, Acetaminophen, Carbamazepine, Chloramphenicol, Digitoxin, Digoxin, Gentamicin, Kanamycin, Lidocaine, Lithium, Methotrexate, Phenobarbital, Propranolol, Quinidine, Theophylline, BNP, FSH, cumadine, vitamin K, aflatoxins, ricin, 2,4-Dichlorophenoxyacetic acid (2-4,D), atrazine, Alachlor, cyanazine, metolachlor, simazine, epinephrine, amphetamine, TSH, free T3 T4, PSA, steroids, estradiol, progesterone, testosterone, estrogen, Arsenic, Cyanide, Strychnine, Avian influenza virus, HIV virus
  • CoIi Neisseria, Helicobacter, Haemophilus, Mycobacterium, Staphylococcus, Pertussis, Salmonella, Shigella, Streptococcus, Treponema, Tetanus, Yershinia. staphylococcal enterotoxin, staphylococcal enterotoxin B, ricin, botulinum toxin, mycotoxin, tetanus toxin, cholera toxin, trichothecene mycotoxins, antibodies ⁇ specific to infectious disease agents and auto-antibodies related to auto-immune diseases, cancerous cells, virally infected cells, viruses, bacterial cells and fungal cells.
  • the releasable first binding agent is selected from the group consisting of biotin, a biotin analog, a biotin derivative, an antigen, Protein A and Protein G, cellulose binding protein, hormones, toxins, lipids, fatty acids, nucleic acids, glycoconjugates, lectins, substrates and ligands.
  • the releasable second binding agent is selected from the group consisting of biotin, a biotin analog, a biotin derivative, HABA, DTB an antigen, Protein A and Protein G, cellulose binding protein, liposomes, hormones, toxins, lipids, fatty acids, nucleic acids, glycoconjugates, lectins, substrates and ligands or their analogs.
  • the reporter is selected from the group consisting of HABA, dyes, fluorescers, fluorescent dyes, radiolabels, magnetic particles, metallic particles, semiconductor particles, quantum dots, colored particles, fluorescent particles, metal salts, enzyme substrates, enzymes, chemiluminescers, photosensitizers and suspendable particles.
  • the first binding agent and the second binding agent are identical or different and each are selected from the group consisting of avidin, streptavidin, NeutrAvidin, ExtrAvidin compounds having high specific affinity to biotin, membranes, receptors, immunoglobulins, cellulose, enzymes, lectins, glycoconjugates, complementary nucleic acids and hydrophobic sites having high affinity to their respective binding partners.
  • the solid support is selected from the group consisting of a porous material, a porous membrane, a granular material, and an absorbent material.
  • the molecular imprint polymer comprises a polymer selected from the group consisting of a polymer polymerized from one or more monomers cross-linked with a cross-linker in the presence of the target analyte or a target analyte-conjugate and a porogen, said polymer having a capacity for selectively binding the target analyte and its conjugate; a polymer polymerized in the presence of MMA, said polymer having a capacity for selectively binding MMA; a polymer polymerized in the presence of the synthetic peptide comprising amino acids LSCKCGKCNTDY, or a peptide representing an epitope of the beta subunit of TSH in a porogen, said polymer having a capacity for selectively binding TSH.
  • a method of detecting TSH present in a liquid sample comprising the steps of contacting the sample the device of the present invention, detecting, in the results zone, the amount of reporter conjugate bound to the reporter-conjugate binding element, wherein the amount of the reporter-conjugate is indicative of the presence or amount of TSH in the sample; and diagnosing a condition of hypothyroidism or hyperthyroidism
  • a kit comprising the device of the present invention; optionally, one or more reagents or compositions for extracting or processing the sample to elute the analyte; optionally, one or more diluents; and optionally, instructions for practicing a method of detecting and determining the presence, absence or concentration of a target analyte in a liquid sample.
  • detecting of the analyte comprises use of a auxiliary detector devices, wherein the detector devices are selected from the group consisting of fluorescent sensors, magnetic sensors, chemiluminesense sensors, densitometer, image analyzers, and radioactivity sensors.
  • the sample is pretreated to modify the character of the sample.
  • the pretreatment is selected from the group consisting of dilution, filtration, distillation, concentration, inactivation of interfering components, and the addition of reagents, or combinations thereof.
  • the device comprises at least one porous pad positioned in one or more of the sample application zone, results zone, reference zone, control zone or said absorbent zone.
  • the pad is pretreated to facilitate or control the flow rate of the sample through the device.
  • FIG. IA is a side view illustration of a first embodiment of a lateral flow device with competition or single displacement and vertical visualization as described in Example 1.
  • FIG. IB is a side view illustration of lateral flow device with competition or single displacement and horizontal visualization as described in Example 2.
  • FIG. 2A is a side view illustration of lateral flow device with competition/double displacement and vertical visualization as described in Example 3.
  • FIG. 2B is a side view illustration of lateral flow device with competition/double displacement and horizontal visualization as described in Example 4.
  • FIG. 3 A is a side view illustration of flow through device with competition or single displacement and vertical visualization as described in Example 5.
  • FIG. 3 B is a side view illustration of flow through device with competition or single displacement and horizontal visualization as described in Example 6.
  • FIG. 4A is a side view illustration of flow through device with competition/double displacement and vertical visualization as described in Example 7.
  • FIG. 4B is a side view illustration of flow through device with competition/double displacement and horizontal visualization as described in
  • FIG. 5 is a side view illustration of a lateral flow as described in Example 9
  • the present invention provides diagnostic methods, devices, and kits for determining the presence or absence or concentration of at least one analyte in a liquid sample.
  • a diagnostic device for detecting at least one analyte in a liquid sample, the device comprising at least one solid support, to which is bound a molecular imprinted polymer (MIP) having analyte-specific binding sites; a sample application area; and a detection zone.
  • MIP molecular imprinted polymer
  • the MIP is bound to the solid support either by physical means or by chemical bonding (immobilization) of the object to the support.
  • the liquid sample is moveable along a flow path along or through the solid support, such as, for example by capillary action or by any type of flow.
  • the liquid sample is applied to the sample application area, and brought into contact with the molecular imprinted polymer, which is downstream of the sample application area, on the flow path of the liquid sample.
  • Binding of the analyte to the molecular imprinted polymer is determined in the detection zone, which is preferably situated downstream of the location of the molecular imprinted polymer.
  • the detection zone provides a detectable signal that indicates the presence of the analyte in the liquid sample.
  • detection zone provides a detectable signal that indicates the presence of the analyte in the liquid sample.
  • detection involves the direct detection of the analyte in the sample, while alternatively (or additionally), such detection optionally involves indirect detection of the analyte in the sample, for example according to competition and/or displacement, or a combination thereof.
  • a method of detecting at least one analyte in a liquid sample comprising providing a diagnostic device for detecting at least one analyte in a liquid sample, the device comprising at least one solid support, to which is bound a molecular imprinted polymer having analyte-specif ⁇ c binding sites, a sample application area, and a detection zone; applying the liquid sample to the sample application area and detecting binding of the analyte to the molecular imprinted polymer.
  • analyte' refers to a molecule, group of molecules or compound of natural or synthetic origin sought to be detected or measured, or an analogue or derivative thereof, that is capable of binding specifically to at least one binding partner (e.g., MIP).
  • Analogues or derivatives may be used when they participate in an assay, as one member of a binding pair, in a manner that is substantially equivalent to that of the analyte itself.
  • the methods of this invention may be practiced with assays for virtually any analyte.
  • Analytes vary in size and may range from small molecules, polypeptide analytes, having less than 30 amino acids, to whole organisms, such as bacteria.
  • the target analyte may be any molecule, polypeptide, protein or infectious disease agent of interest. Any known analyte to which an appropriate analyte-specif ⁇ c MIP may be prepared may be easily detected and/or quantified using the disclosed methods and devices.
  • the methods and devices described herein can be used in the context of basic scientific research, the practice of medicine, including veterinary and dental medicine, forensic analysis, environmental protection monitoring and studies, industrial or chemical manufacturing, and the development and testing of pharmaceutical, food, and cosmetic products.
  • the analytes detected may include, but are not limited to, analytes selected from the group consisting of a bio-marker, pesticide, drug of abuse protein, a hormone, an enzyme, a biomarker, a natural or synthetic toxin, chemical warfare agent, biological warfare agent, antibodies to a drug, antibodies to infectious agents, an immunoglobulin, a lymphokine, a cytokine, a soluble cancer antigen, a growth factor and an infectious disease agent.
  • analytes selected from the group consisting of a bio-marker, pesticide, drug of abuse protein, a hormone, an enzyme, a biomarker, a natural or synthetic toxin, chemical warfare agent, biological warfare agent, antibodies to a drug, antibodies to infectious agents, an immunoglobulin, a lymphokine, a cytokine, a soluble cancer antigen, a growth factor and an infectious disease agent.
  • the analyte may comprise a biomarker (both for humans and for animals) selected from the group consisting of methylmalonic acid (MMA), homocysteine, creatinine, TSH, BNP, FSH, troponin, CK, CKMB, AST, GOT, LDH, Myoglobin, PSA, AST, ALT, ALKP, GGT and Bilirubin.
  • MMA methylmalonic acid
  • the analyte may comprise an infectious disease agent selected from the group consisting of Avian influenza, HIV virus, Hepatitis virus, Polio virus, Cytomegalovirus, Dengue, Ebola, Herpes virus, Rubeola, EBV, Rabies virus, Respiratory Syncytial Virus, Rotavirus, SARS virus, West Nile virus, Yellow fever virus, Campylobacter, Chlamydia, Cholera, Clostridium, Diphtheria, E. CoIi, Neisseria, Helicobacter, Haemophilus, Mycobacterium, Staphylococcus, Pertussis, Salmonella, Shigella, Streptococcus, Treponema, Tetanus and Yershinia.
  • an infectious disease agent selected from the group consisting of Avian influenza, HIV virus, Hepatitis virus, Polio virus, Cytomegalovirus, Dengue, Ebola, Herpes virus, Rubeola, EBV, Rabies virus, Respir
  • the analyte may comprise a toxin selected from the group consisting of staphylococcal enterotoxin, staphylococcal enterotoxin B, ricin, botulinum toxin, mycotoxin, tetanus toxin, cholera toxin and trichothecene mycotoxins.
  • the analyte may be selected from the group consisting of antibodies specific to infectious disease agents or autoantibodies related to auto-immune diseases.
  • the liquid sample of the present invention optionally comprises any liquid that might contain a specific target analyte. Such liquids include biological fluids, environment samples, foodstuffs, drugs, toxins, industrial samples and byproducts of industrial production procedures.
  • the biological fluid is optionally and preferably selected from the group consisting of body fluids, liquid obtained from breath, tissue homogenates, and process fluids.
  • the liquid sample may optionally comprise an environmental sample selected from the group consisting of liquids, such as water, oil, liquid waste, or liquid extracted from solids, such as liquid extracted from solid waste, soil and plants.
  • the sample can optionally be used as obtained directly from the source or following a pretreatment so as to modify its character. Pretreatment may involve, for example, separating plasma from blood, diluting viscous fluids, diluting the sample or the like. Methods of treatment can involve filtration, distillation, concentration, inactivation of interfering components, and the addition of reagents.
  • the sample may be centrifuged or filtered to remove particulate matter, or may be dissolved in or supplemented by a buffer or surfactants to provide a suitable medium in order to allow more efficient detection of the analyte.
  • Suitable buffers include any of those known to skilled artisans, such as a 1-1,000 mM solution of Tris (TRIZMA, Sigma Chemical Co., St. Louis, Mo.), or 1-1,000 mM TRIS (2-Amino-2-(hydroxy- methyl)-l, 3 -propanediol) or 0.05-0.5 % of the surfactant Polysorbate 20 (commercially also known as Tween 20).
  • Other buffers include phosphate buffered saline (PBS), citrate buffer, or bicarbonate buffer.
  • the present invention may also optionally be used to detect analytes that are initially contained within solid-phase samples. These analytes would simply be extracted and suspended or dissolved in liquid prior to analysis using appropriate reagents or compositions for extracting or processing the sample to elute the analyte.
  • the extraction process could be as simple as shaking a solid sample in a diluent or buffer such as those listed above, which could then be applied to the solid support.
  • equipment such as a manual press, mortar and pestle, homogenizer, juicer, mixer or food processor may be used to pre-treat or extract a sample to bring the target analyte into liquid form suitable for testing in the device.
  • Solid samples may include biological tissues (obtained, for example, in the process of performing a biopsy), soil, or foliage.
  • the analyte-specific molecular imprinted polymer may be prepared in accordance with any technique known to those skilled in the art. These methods include covalent imprinting (WuIIf G, 1982, Pure & Appl.Chem., 54, 2093-2102) whereby the monomers are covalently attached to the analyte and polymerized using a cross-linker. Subsequently, the analyte is cleaved from the polymer leaving analyte-specific binding cavities.
  • a non- covalent imprinting method such as disclosed by Mosbach (U.S. Pat.
  • the molecular imprint polymer comprises a polymer polymerized from monomers cross-linked with cross-linker in the presence of the target analyte and a porogen, the polymer having a capacity for selectively binding the target analyte.
  • a relevant method for proteins imprinting is the so called “epitope imprinting" (Rachkov and Minoura, 2000, J. Chromatogr A. ;889, 111-118) where key epitopes are identified on the surface of the protein and the MIP is prepared with the linear peptide representing this epitope as target.
  • the MIP may be prepared against the analyte or to any conjugate of the analyte with other molecules, providing that the MIP binds specifically both the analyte and its conjugate.
  • the device of the present invention the
  • MIP is provided in an MIP-conjugate zone, which is a zone on the device comprising analyte-specific molecular imprinted polymer ("MIP") possessing specific binding affinity for the tested analyte, either to the whole analyte, to a discrete portion of the analyte or to a peptide representing a specific epitope on the analyte, fixed to the solid support, either directly or indirectly.
  • MIP molecular imprinted polymer
  • the analyte-specific binding sites of the MIP are saturated with molecules of either a releasable analyte analogrreporter conjugate, or a releasable first binding agent:analyte conjugate.
  • the term 'analyte specific' refers to a binding reaction which is determinative of the presence of the analyte in the presence of a heterogeneous population of molecules.
  • the term 'analyte analog' refers to a modified analyte or any other molecule or combination thereof, that has structural similarity to the form of the analyte as potentially found in the sample, and which can bind to at least one analyte binding partner.
  • the analyte analog is a detectable analyte analog-reporter conjugate.
  • an analyte analog- conjugate may comprise the analyte conjugated to biotin or to any other reporter pair binding element, in which biotin is an example of such an element, forming one -half of the reporter pair.
  • the analyte analog can be MMA, a target protein or a peptide representing a discrete epitope on the tested analyte.
  • the term 'reporter' refers to any molecule, particle or composition that is capable of being attached to an analyte, analyte analog or binding partner that is detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, radio-activity detection or chemical means.
  • the reporter is selected from a large variety of available molecules and particles, according to the desired attributes of the analytical device.
  • Some examples of such reporters include: HABA, dyes, fluorescent dyes, radiolabels, magnetic particles, metallic particles, colored particles (such as gold and latex sols), fluorescent particles, metal sols, enzyme substrates and enzymes, chemiluminescent molecules, photosensitizers, semiconductor particles, quantum dots and suspendable particles.
  • the coupling of a compound (e.g., an analyte) to a reporter can be through covalent bonds, ionic bonds, hydrogen bonding, chelate formation, adsorption processes, hydrophobic interactions, hydrophylic interactions, electrostatic interactions and the like, or any combinations of these bonds and interactions and/or may involve a linking group.
  • the detectable reporter is optionally selected from the group consisting of HABA, dyes, fluoresces, fluorescent dyes, radiolabels, magnetic particles, metallic particles, colored particles, metal sols, enzyme substrates, enzymes, chemiluminescers, photosensitizers, suspendable particles, polymer particles, semiconductor particles, quantum dots.
  • the detectable reporter may be a visible substance, such as a colored latex bead, or it may participate in a reaction by which a colored product is produced.
  • the reaction product may be visible when viewed with the naked eye, or may be apparent, for example, when exposed to a specialized light source, such as ultraviolet light.
  • a specialized light source such as ultraviolet light.
  • the concentration of analyte in the sample is indicated by the amount of the detectable reporter which subsequently becomes associated with the results zone.
  • a detectable signal that indicates the concentration of the analyte is produced in the results zone.
  • the term 'analyte analog:reporter conjugate' refers to a conjugate used for detection of small molecules and when the "epitope imprinting" method is used in the production of the analyte specific MIP.
  • the synthetic peptide, or other synthetic or naturally obtained molecule which may represent a single molecule or group of such molecules, represents the analyte specific epitope or its analog and is conjugated to a reporter molecule, which may for example be selected from the group of molecules discussed below, such as gold sol.
  • the analyte analog:reporter conjugate has high binding affinity to the analyte-specific MIP, but lower than the affinity of the unmodified analyte.
  • conjugated molecules remain attached to the binding cavities of the analyte-specific MIP even in a moist state unless they are displaced, optionally and preferably in a dose-dependent manner, by the target analyte in the tested liquid sample.
  • the analyte analog:reporter conjugate When displaced from the polymer the analyte analog:reporter conjugate is freely mobile within the moist solid support.
  • the term 'analyte analog:reporter:binding partner conjugate' refers to a conjugate used when imprinting the whole polypeptide/protein or other molecule or group of molecules.
  • the analyte analog:reporter:binding partner conjugate has high binding affinity to the analyte-specific MIP, but lower than the affinity of the unmodified analyte.
  • the conjugated molecules remain attached to the binding cavities of the analyte-specific MIP even in a moist state unless they are displaced, in a dose-dependent manner, by the target analyte in the tested liquid sample.
  • the analyte analog:reporter:binding partner conjugate When displaced from the polymer the analyte analog:reporter:binding partner conjugate is freely mobile within the moist solid support.
  • the analyte analog:reporter:binding partner conjugate is conjugated simultaneously to reporter such as gold sol as well as to binding agents, such as biotin, that can be used to capture the displaced analyte analog:reporter:binding partner conjugate.
  • binding affinity refers to the strength of binding of one molecule to another. If a particular molecule binds to or specifically associates with another particular molecule, these two molecules are said to exhibit binding affinity for each other. Binding affinity is related to the association constant and dissociation constant for a pair of molecules, but it is not critical to the invention that these constants be measured or determined.
  • affinities as used herein to describe interactions between molecules of the described methods and devices are generally apparent affinities (unless otherwise specified) observed in empirical studies, which can be used to compare the relative strength with which one molecule (e.g., a MIP or other specific binding partner) will bind two other molecules (e.g., an analyte and an analyte-reporter conjugate).
  • the concepts of binding affinity, association constant, and dissociation constant are well known.
  • the analyte-specif ⁇ c binding sites of the MIP are unoccupied, and are available for binding by either the analyte or by a binding competitor.
  • the MIP is TSH-specif ⁇ c.
  • a MIP comprises a polymer polymerized from Methacrylic acid (MAA) as monomer, ethylene glycol dimethacrylate (EGDMA) as cross linker, 2,2P-azobis(2,4- dimethylvaleronitrile) (ABDV) as initiator and 3% water in acetonitrile as porogen, in the presence of the synthetic peptide corresponding to an epitope on the TSH ⁇ subunit, such as the amino acid residues at positions 95-112 of the TSH ⁇ subunit (LSCKCGKCNTDY).
  • the synthetic peptide may optionally be prepared on a conventional peptide synthesizer using standard procedures.
  • the solid support may comprise any porous material used in the flow devices industry and having a porous structure that facilitates liquid advancement along the device by capillary forces.
  • the solid support is selected from the group consisting of a porous material, a porous membrane, a granular material, and an absorbent material. These materials often possess also good binding capabilities for proteins.
  • Non-Protein Binding Nitrocellulose (High Protein Binding) Cellulose Acetate (Low Protein Binding) Glass Fiber Membranes (Non-Protein Binding), membranes made of Nylon, Polyvinylidene Fluoride (PVDF), poly Ether Sulfone (PES) or other membranes of this nature that are available or will be developed by companies specializing in diagnostic membranes manufacturing, such as Millipore Corporation, Pall Corporation and Whatman as well as novel micro- fluidic systems, such as the system disclosed in U.S. Pat. Application No. 20050042766.
  • Nitrocellulose High Protein Binding
  • Cellulose Acetate Low Protein Binding
  • Glass Fiber Membranes Non-Protein Binding
  • membranes made of Nylon Polyvinylidene Fluoride (PVDF), poly Ether Sulfone (PES) or other membranes of this nature that are available or will be developed by companies specializing in diagnostic membranes manufacturing, such as Millipore Corporation, Pall Corporation and Whatman
  • the devices may optionally comprise more than one solid support, whereby more than one analyte can be detected in a sample.
  • the sample application area comprises an absorbent pad made of bibulous, porous or fibrous material capable of absorbing liquid rapidly. Its porosity may be unidirectional (i.e., with pores or fibers running wholly or predominantly parallel to an axis of the flow) or multidirectional (i.e., omnidirectional, a sponge-like structure).
  • a porous plastic material such as polypropylene, polyethylene (preferably of very high molecular weight), polyvinylidene flouride, ethylene vinylacetate, acrylonitrile and polytetrafluoro-ethylene may be used.
  • Pre-treatment with a surface-active agent during manufacture may optionally be used to reduce any inherent hydrophobicity and improve the ability to take up and transport a moist sample rapidly and efficiently.
  • the material may also be made of paper or other cellulosic materials. The material connects an opening in the casing of the device with the solid support of the device.
  • a pad separating serum from whole blood such as described in U.S. Pat. No. 5,660,798, may be used with devices that test analytes in serum.
  • a sample applied to the sample application area is absorbed by the absorbent pad, flows freely by capillary forces and comes in contact with the solid support.
  • the volume of the sample that enters the device may be either random or controlled by a smart application pad that allows a precise pre-determined sample volume to enter the device (see for example U.S. Pat. No. 6,008,056).
  • the device of the present invention further comprises a casing.
  • the casing keeps the solid support in a suitable configuration in order to ensure correct functioning of the entire device.
  • the casing optionally comprises windows, preferably between 1 and 4 windows. More preferably, the casing comprises a results window that serves to allow observation of the result. Further preferably, the casing comprises a control window, which allows for viewing of a control reaction, e.g., to confirm adequate performance of the test. Also optionally, the casing comprises a third window or opening in the casing allows application of the liquid sample to the sample application area, at the sample window, either by direct placement of the device in the sample (allowing contact of the sample at the open window), or by application of the sample with a dropper or a similar device to the sample window. Additionally, the casing may optionally comprise a reference window to allow comparison of the intensity of the results in the results zone with the intensity of indicator bands in the reference zone.
  • the solid support further comprises a reference zone.
  • the reference zone is a zone where several lines of analyte analog-reporter binding element are immobilized to the solid support, having known amounts of the analyte analog-reporter conjugate bound to them and producing a distinctive signal with intensity proportional to the amount of the bound reporter.
  • the device may optionally further comprise a positive control zone comprising means for generating a positive control confirming the proper flow and binding of the analyte analog:reporter conjugate to the results zone to thereby determine that a test is working.
  • the devices of the invention may include one or more absorbent pads that are positioned to facilitate the flow of the analyte through the results zone, the reference zone, and/or the control zone.
  • the support may optionally include an absorbent zone comprising a pad of absorbent material in fluid communication with the solid support when the pad and solid support are wet, the pad having sufficient porosity and capacity to absorb excess liquid.
  • the device of the present invention may comprise either a lateral flow device, which is intended to include devices with a dipstick format, or a flow-through device, or a combination thereof.
  • the detection system may be either by single displacement or competition and direct monitoring of the conjugated reporter or by the double displacement or competition approach, as will be explained in further detail below.
  • the detection system may be either by single displacement or competition and direct monitoring of the conjugated reporter or by the double displacement or competition approach, as will be explained in further detail below.
  • optionally flow through or lateral flow may be implemented.
  • a single displacement diagnostic device for determining the presence, absence or concentration of a target analyte present in a liquid sample.
  • the device comprises an analyte analog:reporter conjugate releasably bound to a molecular imprinted polymer on a solid support; a sample application area for applying the sample to the device and bringing it in contact with the solid support; and a detection area.
  • the analyte has an affinity for the binding sites of the MIP which is equal or greater than that of the analyte analogrreporter conjugate, such that contact between the analyte and the MIP causes the analyte analog:reporter to be displaced.
  • the invention is directed to a method for detecting and determining the presence, absence or concentration of a target analyte present in a liquid sample by an assay comprising the steps of applying a sample suspected of containing the analyte to the sample application area of the single displacement diagnostic device of the invention and allowing the sample to flow along or through the solid support and contact the MIP -conjugate zone so that, if analyte is present in the sample, analyte binds to the binding sites of the MIP, displacing analyte analog:reporter conjugate which flows to the results zone where it is captured by the analyte analog:reporter conjugate binding element, producing a detectable signal that indicates the presence or amount of the analyte in the sample.
  • the detection area preferably comprises a results zone comprising an analyte analog:reporter conjugate binding element immobilized to the solid support, wherein the reporter conjugate binding element is capable of binding the displaced analyte analog:reporter conjugate.
  • analyte analog:reporter conjugate binding element refers to a zone on the device comprising element(s) that possess high affinity to the analyte analog: reporter conjugate molecule.
  • These elements may optionally comprise, but are not limited to, biotin binding elements (for example Avidin, StrepAvidin, NeutrAvidin and ExtrAvidin), reporter-specific antibodies, enzymes, substrates and MIP prepared specifically against the analyte analog:reporter conjugate.
  • the single displacement method comprises introducing a liquid sample which is suspected of containing the analyte to be tested for, into the sample application area, and permitting the sample to migrate along the support. If the analyte is present in the sample, it binds to the analyte-specific binding sites of the MIP, and displaces the analyte analog:reporter conjugate. The displaced reporter conjugate migrates along or through the solid support to the results zone, where it binds to the reporter conjugate binding element, providing a detectable signal.
  • the presence and/or intensity of the detectable signal is preferably at least related to the amount of analyte in the sample, whether quantitatively or qualitatively, although optionally a binary "yes/no" answer may be obtained; this is also optionally possible as for the other embodiments below.
  • the invention is directed to a double displacement diagnostic device for directly detecting and determining the presence, absence or concentration of a target analyte in a liquid sample.
  • the device comprises a solid support to which is bound an MIP, and a releasable first binding agent:analyte conjugate bound to the MIP; a sample application area; and a reporte ⁇ conjugate binding zone downstream of the sample application area.
  • the double displacement approach is designed to facilitate signal formation and interpretation and that may be applied in other apparatus of similar nature. This approach finds particular utilization with the well-established biotin-avidin system that offers very strong binding affinity together with a multitude of analogs and derivatives that may be used.
  • the double displacement approach is highly applicable when dealing with the "epitope imprinting" method, when the binding agent:analyte conjugate is a synthetic peptide representing an epitope on the target analyte, against which a specific MIP was prepared, conjugated to the binding agent, such as biotin.
  • This double displacement approach has several advantages over the common single displacement method when the reporter is directly bound to the target molecule.
  • biotin is a relatively small molecule, adding it to the target molecule should not interfere much with its interaction with the analyte-specific MIP.
  • the chemistry of biotin is well established and many commercial reagents are available for the modification of the target analytes.
  • the binding of biotin to the various biotin- binding elements is very rapid and strong, contributing to the overall performance of the devices. Since the reporter is not bound directly to the analyte, there is great flexibility in the choice of the reporter and the ability to conjugate it to the biotin- derivative.
  • Another advantage is that for devices for various analytes the invention employs the same signal formation method, most of the production is similar and only the specific MIP and the analyte conjugated to bio tin would need to be developed for every individual product. This makes the time to market of new products shorter and reduces development and production costs.
  • the binding agentanalyte conjugate may optionally be composed of the target protein to which both binding agent and reporter molecules are conjugated.
  • the conjugate may carry enough reporter molecules such that amplification might not be needed.
  • the term 'binding agentanalyte conjugate' refers to the target analyte, conjugated to a binding agent (preferably bio tin or a derivative of biotin) directly or via a spacer.
  • a binding agent preferably bio tin or a derivative of biotin
  • the binding agentanalyte conjugate can optionally be a peptide corresponding to an epitope on the analyte conjugated to a binding agent.
  • the binding agent has specific affinity to its corresponding binding partner. Where the binding agent is biotin and its derivatives, the corresponding binding partner or agent is Avidin, StrepAvidin, NeutrAvidin and similar biotin binding agents.
  • the first binding agentanalyte conjugate has high binding affinity to the analyte-specific MIP, but lower than the affinity of the unmodified analyte.
  • the first binding agent: analyte conjugate molecules remain attached to the binding sites of the analyte-specific MIP even in a moist state unless they are displaced, in a dose- dependent manner, by the target analyte in the tested liquid sample.
  • the first binding agent:analtye conjugate is freely mobile within the moist solid support.
  • the double displacement device further comprises a results zone, comprising a second binding agentreporter conjugate binding element bound to the solid support on the flow path of the sample.
  • the reporter-conjugate binding element has binding sites to which are attached a detectable, releasable, second binding agent:reporter conjugate.
  • binding element refers to a molecular structure able to specifically bind its respective binding partner with sufficient affinity. Neither the specific sequences nor the specific boundaries of such elements are critical, as long as binding activity is exhibited. Binding characteristics necessarily include a range of affinities, avidities and specificities, and combinations thereof, so long as binding activity is exhibited.
  • the binding agentreporter conjugate comprises any reporter- entity, as described above for the analyte analog: reporter conjugate, conjugated to a binding agent capable of specific binding to the same binding element as the binding agentanalyte conjugate.
  • the second binding agent possesses high affinity to this binding element, but lower than the affinity of the first binding agentanalyte conjugate that is employed in the device. Contact between the first binding agentanalyte conjugate and the binding element therefore causes displacement of the second binding agentreporter conjugate. Displacement of the second binding agentreporter conjugate is proportional to a concentration of the analyte in the liquid sample, such that the presence and/or intensity of the detectable signal is related to the amount of analyte in the sample.
  • biotin for example, is employed in the device as the binding agent in the binding agentanalyte conjugate
  • a derivative of biotin or other molecules such as HABA and DTB
  • biotin binding elements such as Avidin, StrepAvidin NeutrAvidin and ExtrAvidin
  • binding partner' or 'binding agent' refers to any molecule or composition capable of recognizing and binding to a specific structural aspect of another molecule or composition.
  • binding partners and corresponding molecule or composition include biotin/avidin, antigen/antibody, hapten/antibody, hormone/receptor, nucleic acid strand/complementary nucleic acid strand, substrate/enzyme, inhibitor/enzyme, protein A (or G)/immunoglobulins, carbohydrate/lectin, virus/cellular receptor and apoprotein/lipid.
  • the releasable first binding agent is selected from the group consisting of biotin, a biotin analog, a biotin derivative, an antigen, Protein A and Protein G, cellulose binding protein, hormones, toxins, lipids, fatty acids, complementary nucleic acid sequences, glycoconjugates, lectins, substrates and ligands.
  • the releasable second binding agent may be selected from the group consisting of a biotin analog, HABA, DTB, an antigen, Protein A and Protein G, cellulose binding protein, liposomes, hormones, toxins, lipids, fatty acids, complementary nucleic acids, glycoconjugates, lectins, substrates and ligands or their analogs, provided that said second binding agent has lower affinity to the reporter- conjugate binding element than said first binding agent.
  • Biotin analogs are identified in Advances in Protein Chemistry, edited by Anfinsen, Edsall and Richards, Academic Press (1975), pages 104-111.
  • the present invention further provide a diagnostic device wherein the sample application area comprises an analyte:analog conjugate, and the binding sites of the MIP are unoccupied.
  • the conjugate may be provided, for example, in a pad, such that the conjugate become free-flowing once hydrated by the sample.
  • the analyte analog:reporter conjugate competes with the analyte in the sample for the analyte-specific binding sites of the MIP.
  • the unbound conjugate then migrates and is detected as described above for the displaced conjugate.
  • the invention is directed to a method for detecting and determining the presence, absence or concentration of a target analyte present in a liquid sample by an assay comprising the steps of applying a sample suspected of containing the analyte to the sample application area of the competition type diagnostic device of the invention and allowing the sample to flow along or through the solid support and contact the MIP-conjugate zone so that, if analyte is present in the sample, analyte competes with the analyte analog:reporter conjugate for the binding sites of the MIP.
  • the non-bound analyte analog-.reporter conjugate flows to the results zone where it is captured by the analyte analog:reporter conjugate binding element, producing a detectable signal that indicates the presence or amount of the analyte in the sample.
  • Double competition
  • the present invention further provides a diagnostic device comprising a first binding agent:analyte conjugate, a second binding agentreporter conjugate, and a second binding agent:receptor conjugate binding element.
  • the first binding agentranalyte conjugate is optionally provided in the sample application area, and competes with the analyte for the analyte-specific binding sites of the MIP. Unbound first binding agentanalyte conjugate flows downstream, and competes with the second binding agentreporter conjugate for binding to the second binding agentreceptor conjugate binding element.
  • the invention is directed to a method for detecting and determining the presence, absence or concentration of a target analyte present in a liquid sample by an assay, the method comprising the steps of:
  • the invention is directed to a device comprising a solid support including a first component comprising a sample application pad impregnated with an analyte analog-conjugate comprising the analyte conjugated to biotin, optionally through a spacer (optionally, the analyte:analog conjugate may be impregnated in a dedicated reagent pad between the sample pad and the MIP); a second component comprising analyte-specific MIP, a third component containing a first biotin binding element saturated with reporter-conjugate comprising a biotin analog having lower affinity to the biotin binding element than biotin (such as desthiobiotin - DTB) conjugated to a reporter and a fourth component comprising a second biotin binding element
  • the invention is directed to a device comprising a solid support including a first component comprising an analyte-specif ⁇ c MIP saturated with an analyte analog-conjugate comprising the analyte conjugated to biotin, optionally through a spacer; a second component comprising a first biotin binding element saturated with reporter-conjugate comprising a biotin analog having lower affinity to the biotin binding element than biotin conjugated to a reporter, and a third component comprising a second biotin binding element.
  • the present invention may optionally further provide a diagnostic device and method comprising competition and displacement methods, in any order and combination.
  • the method may comprise a first step wherein a first binding agentanalyte conjugate bound to the MIP is displaced by the analyte; and a second step wherein the displaced conjugate competes with a second binding agentreporter conjugate for binding sites of a reporter conjugate binding element.
  • the first step may comprise competition between a first binding agentanalyte conjugate and the analyte for the binding sites of the MIP; and the second step may comprise displacement by the unbound analyte conjugate of a second binding agentreporter conjugate from a reporter conjugate binding element.
  • the method includes the step of comparing the intensity of the signal in the results zone with the intensity of the signal in a reference zone to determine the concentration of the analyte in the sample.
  • the results zone includes a scale parallel to the immobilized reporter-conjugate binding element calibrated to correspond to the analyte concentration in the liquid sample being analyzed and wherein the presence and concentration of analyte in the liquid is determined by the area covered by the reporter-conjugate along and through the reporter-conjugate binding element in the results zone.
  • the invention is directed to a method of detecting TSH present in a liquid sample, the method comprising the steps of: a) contacting the sample with a competition, single or double displacement device according to the invention, and b) detecting, in the results zone, the amount of reporter conjugate bound to the reporter-conjugate binding element, wherein the amount of the reporter-conjugate is indicative of the presence or amount of TSH in the sample; c) diagnosing the activity of the thyroid.
  • the method of detecting TSH may be used for identification of an underactive thyroid gland (hypothyroidism) that can cause symptoms such as weight gain, tiredness, dry skin, constipation, a feeling of being too cold, or frequent menstrual periods; or an overactive thyroid (hyperthyroidism) that can cause symptoms such as weight loss, rapid heart rate, nervousness, diarrhea, a feeling of being too hot, or irregular menstrual periods.
  • an underactive thyroid gland hyperthyroidism
  • an overactive thyroid hyperthyroidism
  • the devices of the invention may be packaged together in a diagnostic kit with any or all of the diluents or reagents for extracting or processing the sample to elute the analyte and to detect a given analyte.
  • the diagnostic kit further includes packaging material and instructions for performance of the quantitative analysis on at least one type of liquid sample.
  • the sensitivity of particular assays is a function of the relative affinity and concentration of the various reagents, the times during which particular reagents and analyte are in contact with each other and the intensity of the signal produced by the reporting system.
  • Those skilled in the art are familiar with methods for optimizing and characterizing the sensitivity and dose-response characteristics of conventional assays used in lateral flow and flow through devices.
  • the reporter-conjugate binding element any of a biotin-binding, or other reporter pair binding, element or analyte-reporter binding element
  • the reporter-conjugate binding element is placed vertical to the sample flow path to capture and concentrate as many as possible of the free-flowing reporter conjugate molecules, in a random order.
  • a reference window comprised of several indicator lines of binding element impregnated each with increasing precise amounts of the reporter conjugate. These lines will exhibit a range of color intensities, which are proportional to the known amount of the reporter conjugate bound to them. Comparison of the intensity of the signal in the results window with the intensity of the lines at the adjacent reference window enables good estimation of the amount of the analyte in the tested sample.
  • the horizontal visualization method a stripe of well-defined reporter-conjugate binding element is accurately placed, horizontally, at the path of the sample flow.
  • the device at this location should be manipulated in such a manner as to allow the flow of the liquid only through the area covered with the reporter-conjugate binding element and take measures to avoid sample flow around or below the binding element covered area. This can be achieved, for example, by physically cutting the porous material in such a way that only the binding element covered area remains in the flow path, or by using methods such as patterning of flow channels by photolithography [Martinez et al, 2007 Angwe. Chem. Int. 119, 1340-1342].
  • Pre-calibration of the area covered by known amounts of the reporter-conjugate will enable accurate determination of the analyte concentration according to the distance covered by the reporter-conjugate along the stripe of the binding element by incorporating in the device a calibrated scale next to the immobilized binding element that allows the user to determine the amount of the analyte in the sample.
  • the devices may comprise more than one solid support, whereby more than one analyte can be detected in a sample.
  • the first binding area of reporter-conjugate binding element and the second binding area of reporter-conjugate binding element may be identical or different and each are selected from the group consisting of avidin, streptavidin, NeutrAvidin, ExtrAvidin compounds having high specific affinity to biotin, membranes, receptors, immunoglobulins, cellulose, enzymes, lectins, glycoconjugates, complementary nucleic acids and hydrophobic sites having high affinity to their respective binding partners.
  • FIGS. 1-2 show four different embodiments of a lateral flow device of the present invention
  • FIGS. 3-4 show four different embodiments of a flow through device of the present invention
  • FIG. 5 show an embodiment of a device combining lateral flow and flow through elements, each of which will be described separately in Examples 1-9.
  • EXAMPLE I Embodiment of FIG. IA
  • FIG. IA is a side view illustration of a lateral flow device for detecting and determining the presence, absence or concentration of a target analyte present in a liquid sample.
  • the analytical test device comprises a hollow, solid casing 10 that contains a solid support 12 in the form of a porous test strip that serves as a carrier capable of conveying a liquid sample therethrough, the sample being movable along the solid support in the path of liquid flow by capillary action.
  • the solid support 12 has defined zones, including a sample application area comprising a sample application pad 14 for applying the sample to the device and bringing it in contact with the solid support 12.
  • the sample application pad 14 is located adjacent to an opening or window 16 in the casing 10 for applying the liquid sample.
  • competition type is employed the sample application pad 14 is impregnated with a releasable analyte analog:reporter conjugate in a dry state, alternatively a dedicated pad can be added.
  • the target analyte if present in the liquid sample, is carried from the sample application area to a MlP-conjugate zone downstream of the sample application area comprising an analyte-specific MIP 18 fixed to the solid support 12 on the flow path of the sample.
  • the MIP has analyte-specific binding sites saturated with a releasable analyte analog:reporter conjugate in a dry state, comprising the target analyte conjugated to a binding partner and to reporter molecules.
  • the affinity of the analyte to the binding sites of the analyte-specific MIP is greater than the affinity of the analyte analog:reporter conjugate to the binding sites of the analyte-specific MIP.
  • the analyte in the sample binds to the analyte-specific cavities of the MIP and thereby displaces the analyte analog-reporter conjugate which occupy these cavities in an amount directly proportional to the concentration of the specific analyte, causing the displaced analyte analog:reporter conjugate to flow downstream in the path of liquid flow.
  • the liquid sample dissolves the impregnated analyte analog:reporter conjugate and it is mixed with the analyte in the sample.
  • analyte analog:reporter conjugate proportional to the concentration of the specific analyte is left unbound and flow downstream in the path of liquid flow.
  • an analyte analog:reporter conjugate binding element that is the binding partner of the binding element bound to the analyte analogrreporter conjugate, 20 immobilized to the solid support 12 on the flow path of the sample downstream of the analyte-specific MIP 18.
  • An opening in the casing 10 is located above the analyte analog:reporter conjugate binding partner 20, comprising the results window 22 of the device.
  • the reporter conjugate binding partner 20 binds the unbound or displaced analyte analog:reporter conjugate displaced from the analyte-specific MIP 18 when a liquid sample containing the analyte flows in the flow path zone and provides a detectable signal in the results window 22 that indicates the presence or concentration of the analyte in the sample.
  • the three indicator bands comprise a reference zone for establishing a reference point in determining the presence or semi- quantification of an analyte in the tested sample.
  • a corresponding reference window 26 appears in the casing 10 above the reference bands 24a, 24b and 24c.
  • the reference zone may comprise at least one discrete band of binding element impregnated with a known quantity of the analyte analog: reporter conjugate in the case where the presence or absence of a target analyte in the sample is to be determined.
  • the reference zone may comprise at least two spaced indicator bands of binding element impregnated each with increasing quantities of the analyte analog:reporter conjugate and exhibiting a range of color intensities proportional to the amount of the analyte analog-.reporter conjugate bound to it, in the case where the amount of a target analyte in a sample is to be determined.
  • the user interprets the results, and determines the presence, absence or semi-quantification of the concentration of the analyte in the sample, by visually comparing the intensity of the signal in the results window 22 with the intensity of one or more bands in the reference window 26.
  • a control pad 32 Downstream of the reference bands 24a, 24b and 24c is a control pad 32 impregnated with analyte analog:reporter conjugate that is stationary in a dry state but becomes freely flowing when the solid support 12 is wetted with the liquid of the sample. Further downstream is another analyte analog:reporter conjugate binding partner 34 anchored to the solid support 12. There is a corresponding control window 30 in the casing above the analog:reporter conjugate binding partner 34 for viewing the results. The analyte analog: reporter conjugate that is released from the control pad 32 by the fluid from the sample reaches the binding partner 34 and become fixed to the solid support 12, producing a visual signal in the control window 30.
  • control pad and analyte analog:reporter conjugate binding element 34 together comprise a control zone for generating a positive control confirming the proper flow and binding of the analyte analog:reporter conjugate to the results zone to thereby determine that a test is working properly.
  • an absorbent pad 36 of absorbent material in fluid communication with the solid support 12 when the pad 36 and solid support 12 are wet.
  • the pad has sufficient porosity and capacity to absorb the surplus of the fluid and ensure continuous flow throughout the device.
  • the lateral flow device is the same as that described above in Example 1 , with similar numerals designating similar parts except that modifications are indicated with the reference numeral and the letter "a" affixed.
  • the modifications are as follows: There is no reference zone in the device of Fig. IB, thus, the casing 38 has fewer windows or openings, and has an enlarged results window 42. Results are read only from the results window 42 according to the distance that the analyte analog:reporter conjugate covered. The area covered by the immobilized analyte analog:reporter conjugate binding element 44 is enlarged with a scale 46 running parallel to it.
  • the scale 46 and the area covered by the immobilized analyte analog:reporter conjugate binding element 44 are co-calibrated to correspond to the analyte concentration in the liquid sample being analyzed.
  • the presence and concentration of analyte in the liquid is determined at the results window 42 by the area covered by the analyte analog:reporter-conjugate along and through the analyte: analog reporter-conjugate binding element 44.
  • the results of the control signal are viewed in control window 30a.
  • FIG. 2A is a side view illustration of a lateral flow device for detecting and determining the presence, absence or concentration of a target analyte present in a liquid sample.
  • the device comprises a hollow, solid casing 10b that contains a solid porous support 12b in the form of a test strip capable of conveying a liquid sample therethrough, the sample being movable along the solid support in the path of liquid flow by capillary action.
  • the solid support 12b has defined zones, including a sample application area comprising a sample application pad 14b for applying the sample to the device and bringing it in contact with the solid support 12b.
  • the sample application pad 14b is located adjacent to an opening or window 16b in the casing 10b for applying the liquid sample.
  • competition type the sample application pad 14b is impregnated with a releasable analyte analog:reporter conjugate in a dry state, alternatively a dedicated pad can be added
  • the target analyte if present in the liquid sample, is carried from the sample application pad 14b along and through a MIP-conjugate zone downstream of the sample application area 14b, the MIP-conjugate zone comprises an analyte-specific MIP 50 fixed to the solid support 12b on the flow path of the sample.
  • the MIP 50 has analyte-specific binding sites saturated with a releasable first binding agentanalyte conjugate, which is the target, conjugated to a binding element, in a dry state.
  • the affinity of the analyte to the binding sites of the analyte-specific MIP 50 is sufficiently greater than the affinity of the first binding agentanalyte conjugate to the binding sites of the analyte-specific MIP 50 to bring about the displacement of the firs binding agent:analyte conjugate from the analyte specific binding sites of the MIP 50 in the presence of the analyte.
  • the analyte in the sample binds to the analyte-specific cavities of the MIP and thereby displaces the first binding agentanalyte conjugate which occupy these cavities in an amount directly proportional to the concentration of the specific analyte, causing the displaced first binding agentanalyte conjugate to flow downstream in the path of liquid flow.
  • the liquid sample dissolves the impregnated analyte analog:reporter conjugate and it is mixed with the analyte in the sample.
  • the two molecules compete for the analyte-specific binding sites.
  • An amount of analyte analog:reporter conjugate, proportional to the concentration of the specific analyte is left unbound and flow downstream in the path of liquid flow
  • reporter-conjugate binding zone that comprises a first binding area of reporter-conjugate binding element 52 immobilized to the solid support 12b on the flow path of the sample downstream of the analyte-specific MIP 50, comprising reporter-conjugate binding element 52 with its binding sites saturated with a detectable, releasable second binding agentreporter conjugate in a dry state.
  • the affinity of the first binding agentanalyte conjugate to the binding sites of the reporter-conjugate binding element 52 is greater than the affinity of the second binding agentreporter conjugate to the binding sites of the reporter- conjugate binding element 52.
  • the first binding area of reporter-conjugate binding element 52 binds the first binding agentanaltye conjugate and displaces the second binding agentreporter conjugate in an amount directly proportional to the concentration of the specific analyte in the sample, causing the displaced second binding agentreporter conjugate to continue to migrate downstream in the path of liquid flow.
  • competition can be used and the second binding agentreporter conjugate can be dried on a pad, rehydrated by the sample liquid and compete with the first binding agentanalyte conjugate for the binding sites at the first binding area, in this case, there is no need for greater affinity of the first binding agentanalyte to the binding sites of the reporter-conjugate binding element 52. The excess of unbound second binding agentreporter conjugate will continue to migrate downstream in the path of liquid flow.
  • reporter-conjugate binding element 54 is immobilized to the solid support 12b on the flow path of the sample downstream of the first binding area of reporter-conjugate binding element 52.
  • An opening in the casing 10b is located above the second binding area of reporter-conjugate binding element 54, comprising the results window 22b of the device.
  • the second binding area of reporter-conjugate-binding element 54 binds the second binding agentreporter conjugate displaced from the first binding area of reporter-conjugate binding element 52 when a liquid sample containing the analyte flows in the flow path zone and provides a detectable signal that indicates the presence or concentration of the analyte in the sample.
  • the solid support 12b located further downstream on the solid support 12b are three discrete and non-overlapping indicator bands of the second binding agent:reporter conjugate binding element, wherein 56a represents a low concentration reference band, 56b represents a medium concentration reference band and 56c represents a high concentration reference band.
  • the three indicator bands comprise a reference zone for establishing a reference point in determining the presence or semi-quantification of an analyte in the tested sample, above which is a corresponding reference window 26b in casing 10b.
  • the user interprets the results, and determines the presence, absence or semi-quantification of the concentration of the analyte in the sample, by visually comparing the intensity of the signal in the results window 22b with the intensity of one or more bands in the reference window 26b.
  • a control pad 32b impregnated with second binding agentrreporter conjugate that is stationary in a dry state but becomes freely flowing when the solid support 12b is wetted with the liquid of the sample.
  • another reporter-conjugate binding element 56 anchored to the solid support 12b.
  • the second binding agent:reporter conjugate that is released from the control pad 32b by the fluid from the sample reaches the binding element 56 and become fixed to the solid support 12b, producing a visual signal, viewed in the control window 30b.
  • the control pad 32b and reporter-conjugate binding element 56 together comprise a control zone for generating a positive control.
  • an absorbent pad 36b made of absorbent material in fluid communication with the solid support 12b when the pad 36b and solid support 12b are wet.
  • the pad has sufficient porosity and capacity to absorb the surplus of the fluid and ensure continuous flow throughout the device.
  • a lateral flow device with competition/double displacement and horizontal visualization of the test results.
  • the lateral flow device is the same as that described above in Example 3, with similar numerals designating similar parts except that modifications are indicated with the reference numeral and the letter "a" affixed.
  • the modifications are as follows: There is no reference zone in the device of Fig. 2B. Results are read only from the results window 60 according to the distance that the second binding agent:reporter conjugate covered.
  • the second binding area of reporter-conjugate binding element 66 under the results window 60 in casing 38a is elongated and includes a scale 68 parallel to the immobilized second binding area of reporter-conjugate binding element 66 calibrated to correspond to the analyte concentration in the liquid sample being analyzed.
  • the presence and concentration of analyte in the liquid is determined by the area covered by the second binding agent:reporter-conjugate along the second binding area of reporter-conjugate binding element 66 as viewed in the results window 60.
  • EXAMPLE 5 Embodiment of FIG. 3 A There is described below the structure and operation of a flow through device with competition or single displacement and vertical visualization.
  • FIG. 3 A is a side view illustration of a flow through device for detecting and determining the presence, absence or concentration of a target analyte present in a liquid sample.
  • the device comprises a hollow, solid casing 80 containing defined layers or zones made of reagent-containing porous materials, arranged so that fluid that is applied to the top of the device flows vertically through the various layers of the device, from one layer to another, until the fluid contacts an absorbent material at the bottom of the device.
  • a sample application area comprising a sample application pad 82 for applying the sample to the device is located adjacent to an opening or window 84 in the casing 80 at the top of the device and leads to a MIP-conjugate zone comprising an analyte- specific MIP 86 having analyte-specific binding sites saturated with a releasable analyte analog:reporter conjugate in a dry state.
  • the analyte analog:reporter conjugate may comprise also polypeptide or protein corresponding to the target analyte, conjugated to both binding partner and reporter molecules.
  • the affinity of the analyte to the binding sites of the analyte-specific MIP 86 is greater than the affinity of the analyte analog:reporter conjugate to the binding sites of the analyte-specific MIP 86.
  • the application pad, or a dedicated reagent pad bellow it is impregnated with the analyte analog:reporter conjugate, while the MIP 86 have its binding sites free, in this case, difference in affinity to the MIP is not mandatory.
  • the target analyte if present in the liquid sample, migrates from the sample application pad 82 through the analyte-specific MIP 86.
  • the analyte in the sample binds to the analyte-specific cavities of the MIP and thereby displaces the analyte analog-reporter conjugate which occupy these cavities in an amount directly proportional to the concentration of the specific analyte in the sample, causing the displaced analyte analog: reporter conjugate to migrate further down through the device.
  • the liquid of the sample dissolves the analyte analog: reporter conjugate from the sample pad or the dedicated reagent pad and the analyte analog:reporter conjugate is mixed together with the analyte in the sample and flow together until the MIP zone 86 where they compete for the analyte-specific binding sites.
  • An amount of analyte analog:reporter conjugate, directly proportional to the concentration of the specific analyte in the sample is left unbound and migrate further down stream the device.
  • an analyte analog:reporter conjugate binding element 90 which specifically binds its binding partner conjugated to the analyte analog:reporter conjugate, fixated to the porous carrier or support 88 on the flow path of the sample downstream of the analyte-specific MIP 86.
  • a clear window in the solid casing 80 is located in front of the analyte analog:reporter conjugate binding element 90, comprising the results window 92 of the device.
  • the unbound analyte analog:reporter conjugate or the analyte analog:reporter conjugate displaced from the analyte-specific MIP 86 by the analyte migrate to the analyte analog:reporter conjugate binding element 90 where they are captured and provide a detectable signal, indicatin the presence or concentration of the analyte in the sample in the results window 92.
  • the next zone is a reference zone comprising three discrete and non-overlapping indicator bands of analyte analog:reporter conjugate binding element, separated by porous carrier zones 88, wherein 94a represents a low concentration reference band, 94b represents a medium concentration reference band and 94c represents a high concentration reference band.
  • a clear reference window 96 is located in front of the three indicator bands 94a, 94b and 94c. The user interprets the results by visually comparing the intensity of the signal in the results window 92 to the intensity of the bands in the reference window 96.
  • the reference zone is a porous carrier zone 88 followed by a control area 100 comprising a porous carrier impregnated with unbound analyte analog:reporter conjugate.
  • This conjugate 100 is stationary in the dry state but becomes freely flowing when the porous carrier is wetted with the liquid of the sample.
  • Another analyte analog:reporter conjugate binding element 102 is another analyte analog:reporter conjugate binding element 102.
  • a control window 104 is located in front of the reporter conjugate binding element 102.
  • an absorbent material 106 At the distal end of the device is an absorbent material 106 that absorbs the surplus of the fluid and ensures continuous flow throughout the device.
  • the flow through device is the same as that described above in Example 5, with similar numerals designating similar parts, with minor modifications as follows: There is no reference zone in the casing 108 of the device of Fig. 3B. Results are read only from the results window 114 according to the distance that the analyte analog:reporter conjugate covered. The analyte analog:reporter-co ⁇ jugate binding element 110 as well as the results window 114 are elongated. A scale 112 runs parallel to the immobilized analyte analog: reporter-conjugate binding element 110, both co-calibrated to correspond to the analyte concentration in the liquid sample being analyzed.
  • FIG. 4 A is a sectional side view illustration of a flow through device for detecting and determining the presence, absence or concentration of a target analyte present in a liquid sample using competition/double displacement.
  • the device comprises a hollow, solid casing 80b containing defined layers or zones made of reagent-containing porous materials, arranged so that fluid that is applied to the top of the device flows vertically through the various layers of the device, from one layer to another, until the fluid contacts an absorbent material at the bottom of the device.
  • a sample application pad 82b for applying the sample to the device is located adjacent to an opening or window 84b in the casing 80b at the top of the device and leads to a MIP-conjugate zone comprising an analyte-specific MIP 122 having analyte-specific binding sites saturated with a releasable first binding agentanalyte conjugate in a dry state.
  • the first binding agentanalyte conjugate may comprises a synthetic peptide corresponding to the epitope of the target that was imprinted, conjugated to a binding partner.
  • the affinity of the analyte to the binding sites of the analyte-specific MIP 122 is greater than the affinity of the first binding agentanalyte conjugate to the binding sites of the analyte-specific MIP 122.
  • the target analyte found in the liquid sample migrates from the sample application pad 82 through the
  • the MIP-conjugate zone where it binds to the analyte-specific cavities of the MIP 122 and thereby displaces the first binding agentanalyte conjugate which occupy these cavities in an amount directly proportional to the concentration of the specific analyte, causing the displaced first binding agentanalyte conjugate to migrate further down through the device.
  • the application pad 82b, or a dedicated reagent pad is impregnated with the releasable first binding agentanalyte conjugate.
  • the liquid of the sample dissolves the analyte analog:reporter conjugate from the sample pad or the dedicated reagent pad and the analyte analog:reporter conjugate is mixed together with the analyte in the sample and flow together until the MIP zone 122 where they compete for the analyte-specific binding sites, an amount of analyte analog:reporter conjugate, directly proportional to the concentration of the specific analyte in the sample is left unbound and migrate further down stream the device.
  • the next layer is a porous carrier or support 88b, followed by a layer of a reporter-conjugate binding zone comprising a first binding area of reporter-conjugate binding element 124 having binding sites saturated with a detectable, releasable second binding agent: reporter conjugate in a dry state.
  • This binding element has specific affinity to its binding partners conjugated both to the first binding agentanalyte as well as to the second binding agentreporter conjugate.
  • the affinity of the first binding agentanalyte conjugate to the binding sites of the reporter- conjugate binding element 124 is greater than the affinity of the to the binding sites of the reporter-conjugate binding element 124.
  • the first binding area of reporter- conjugate binding element 124 in the reporter conjugate binding zone binds the first binding agentanaltye conjugate and displaces the second binding agentreporter conjugate in an amount directly proportional to the concentration of the specific analyte in the sample, causing the displaced (displacement) or unbound (competition) second binding agentreporter conjugate to continue to migrate further down the device.
  • competition can be used and the second binding agent: reporter conjugate can be dried prior to the binding area of reporter-conjugate binding element 124, rehydrated by the sample liquid and compete with the first binding agentanalyte conjugate for the binding sites at the first binding area. In this case, there is no need for greater affinity of the first binding agentanalyte to the binding sites of the reporter-conjugate binding element 124. The excess of unbound second binding agentreporter conjugate will continue to migrate downstream in the path of liquid flow.
  • reporter-conjugate binding element 126 fixated to a porous carrier.
  • An opening in the casing 80b is located in front of reporter-conjugate binding element 126, comprising the results window 92b of the device.
  • the second binding area of reporter-conjugate-binding element 126 binds the unbound or displaced second binding agentreporter conjugate coming from the analyte-specific MIP 122 when a liquid sample containing the analyte migrates to reporter-conjugate binding element 126, and provides a detectable signal in the results window 92b that indicates the presence or concentration of the analyte in the sample.
  • the next zone is the reference zone comprising three discrete and non- overlapping indicator bands of second binding agentrreporter conjugate binding element, wherein 128a represents a low concentration reference band, 128b represents a medium concentration reference band and 128c represents a high concentration reference band.
  • a clear reference window 96b is located in front of the reference bands. The user interprets the results by visually comparing the intensity of the signal in the results window 92b to the intensity of the bands in the reference window 96b.
  • a control zone comprising a porous carrier 132 impregnated with unbound second binding agent: reporter conjugate.
  • This conjugate is stationary in the dry state but becomes freely flowing when the porous carrier is wetted with the liquid of the sample.
  • another reporter- conjugate binding element 134 visible at the control window 104b.
  • the second binding agent reporter conjugate which is released from the control pad 132 by the fluid from the sample reaches the reporter conjugate binding element 134 and is bound to the carrier, producing a visual signal at the control window 104b.
  • an absorbent material 106b At the distal end of the device is an absorbent material 106b that absorbs the surplus of the fluid and ensures continuous flow throughout the device.
  • a scale 112b runs parallel to the immobilized second binding agentrreporter conjugate binding element 136 co-calibrated to correspond to the analyte concentration in the liquid sample being analyzed.
  • the presence and concentration of analyte in the liquid is determined by the area covered by the second binding agentrreporter conjugate through the second binding agentrreporter conjugate binding element 136 that is viewed in the results window 114.
  • FIG. 5 is a side view illustration of a lateral flow device for detecting and determining the presence, absence or concentration of a target analyte present in a liquid sample.
  • the device comprises a hollow, solid casing 40 that contains designated porous pads; sample pad 14c, conjugate pad 70 impregnated with releasable first binding agentranalyte conjugate, MIP pad 72 comprises analyte-specific MIP and reagent reporter pad 74 impregnated with second binding agentrreporter conjugate, all stacked in close contact one on top of the other, pad 74 being in close contact with a solid porous support 12c.
  • the stacked pads and following test strip capable of conveying a liquid sample therethrough, the sample being movable through the pads and along the solid support in the path of liquid flow by gravity and capillary action.
  • the sample application pad 14c is located adjacent to an opening or window 16c in the casing 40 for applying the liquid sample.
  • the target analyte if present in the liquid sample, is carried from the sample application pad 14c through the conjugate pad 70 where the liquid sample dissolves the impregnated releasable first binding agentranalyte conjugate and it is mixed with the analyte in the sample.
  • first binding agentranalyte conjugate proportional to the concentration of the specific analyte in the sample is left unbound and flow downstream in the path of liquid flow.
  • the unbound first binding agentanalyte conjugate continues flowing with the sample liquid to the reporter reagent pad 74 where the liquid dissolves the second binding agent:reporter conjugate.
  • the liquid containing the unbound first binding agentanalyte conjugate and the second binding agentreporter conjugate flows through pad 74 and come into contact the solid porous support 12c, which convey the flow of the liquid further downstream to the first reporter-conjugate binding zone 54c comprising reporter-conjugate binding element immobilized to the solid support 12c.
  • the first binding agent:analyte conjugate and the second binding agentreporter conjugate competes for the binding sites of the first reporter-conjugate binding zone, living unbound second binding agentreporter conjugate.
  • the unbound second binding agentreporter conjugate to continue to migrate downstream in the path of liquid flow
  • reporter-conjugate binding element 60c immobilized to the solid support 12c on the flow path of the sample
  • the second binding area of reporter-conjugate binding element 66c under the results window 60c in casing 40 is elongated and includes a scale 68c parallel to the immobilized second binding area of reporter-conjugate binding element 66c calibrated to correspond to the analyte concentration in the liquid sample being analyzed.
  • the presence and concentration of analyte in the liquid is determined by the length of the area covered by the second binding agentrreporter- conjugate along the second binding area of reporter-conjugate binding element 66 as viewed in the results window 60.
  • reporter- conjugate binding element 64c there is a corresponding control window 30c in the casing 40 above reporter- conjugate binding element 64c for viewing the results.
  • the second binding agentrreporter conjugate that is released by the sample liquid from control pad 58c reaches the binding element64c and become fixed to the solid support 12c, producing a visual signal, viewed in the control window 30c.
  • the control pad 58c and reporter- conjugate binding element 64c together comprise a control zone for generating a positive control.
  • an absorbent pad 36c made of absorbent material in fluid communication with the solid support 12c. The pad has sufficient porosity and capacity to absorb the surplus of the fluid and ensure continuous flow throughout the device.
  • Porous, solid support used in the rapid diagnostic device of the invention is a membrane filter comprising a strongly adsorptive substance having a large surface area, such as PuraBindTM (Whatman,
  • TSH-specific MIP Preparation was in accordance with the methods set forth in the review by Yan and Row (Int. J. MoI. Sci. 2006, 7, 155-178) as follows: The functional monomer, Methacrylic acid (MAA) (Cat. No. 155721, Aldrich) is mixed with the target print molecule, in this case the synthetic peptide described above, together with the cross-linking monomer ethylene glycol dimethacrylate (EGDMA), (Cat. No. 33568-1, Aldrich) in 3% water in acetonitrile (Cat. No. 360457,Sigma-Aldrich) together with the initiator 2,2'-azobis(2,4- dimethylvaleronitrile) (Cat.
  • MAA Methacrylic acid
  • EGDMA cross-linking monomer ethylene glycol dimethacrylate
  • TSH peptide-biotin conjugate The synthetic peptide was conjugated to biotin using the PeptiTag - Biotin kit (BioSight, Israel).
  • E. Preparation of the MIP-Conjugate Zone The binding cavities of the TSH- specific MIP are first saturated with the TSH peptide-Conjugate (i.e., peptide-biotin conjugate) by incubation of the sieved TSH-specific MIP particles with a solution of the TSH peptide-biotin conjugate for 24 hours at 37°C followed by washing to remove the excess conjugate.
  • TSH peptide-Conjugate i.e., peptide-biotin conjugate
  • the washed MIP particles are then dried at 60 °C in an oven and packaged inside filter paper bags, manufactured by Filtech Fabrics Ltd, India, similar to those used as tea bags.
  • the bag containing the loaded MIP particles is attached to the nitrocellulose membrane at the MIP-conjugate zone by pressure- sensitive adhesive-coated films (Cat. No. ARcare® 8570, Adhesives Research, U.S.).
  • HABA 4-hydroxyazobenzene-2-carboxylic acid
  • BSA Cat. No. A3902, Sigma
  • the HABA is attached to BSA in accordance with the method described by Hofstetter et al., ⁇ Analytical Biochemistry (2000) 284, 354-366).
  • Gold sol is available from BioAssay Works MD, USA. Loading of HABA-BSA conjugate with gold sol is performed using the coupling method described by Horrisberger and Clerc (Histochemistry, 1985, 82, 219-223 A).
  • NeutrAvidinTM Biotin- Binding Protein (Pierce, USA) is preferably employed. This protein is an excellent alternative to other biotin-binding proteins, such as avidin or streptavidin, when nonspecific binding must be minimized. Its immobilization was performed by direct blotting of the NeutrAvidin to the nitrocellulose membrane. 20 ⁇ g/ml NeutrAvidin in 0.5M phosphate buffer, pH 7.2 with 0.5M NaCl, was incubated O.N. at room temperature and washed once with PBS and air dried. The prepared membrane was stored desiccated at room temperature until the final construction of the device.
  • TSH-reporter:binding partner conjugate (Biotin-TSH-Gold) is accomplished by attaching biotin to the TSH beta subunit using EZ-Link Sulfo-NHS- Biotin labeling kit .(Pierce, USA)
  • the TSH-Biotin conjugate is then coated with gold sol. The coating is done as described earlier for the coating of the BSA with the gold sol. When in free mobile form, this conjugate is captured by the immobilized NeutrAvidin, and a visual signal is obtained due to the gold particles covering the molecule.
  • TSH-reporter conjugate binding zone is comprised of a zone on the nitrocellulose solid support having NeutrAvidin immobilized to it (as described above for the biotin-binding element zones).
  • the reference zone is preferably comprised of five identical parallel bands of TSH-reporter conjugate binding zones. Each band is applied accurately with a solution of the biotin-TSH-Gold conjugate. Eventually, the five bands have 0.2, 1.0, 3.0, 5.0 and 7.5 mIU/L of the conjugate bound to them, respectively. By comparing the color intensity of band in the results window with the reference bands, the user may determine the range of the amount of TSH in the tested sample.
  • EXAMPLE 11 Assay For TSH Using A Lateral Flow Device (double displacement) There is described below the structure and operation of a particular displacement assay of the invention. Structure: A device as shown in FIG.
  • the device comprises a housing containing windows that exposes areas of the solid support for viewing.
  • the device includes a sample application area comprising a pad (LFl, Whatman, USA) to which a liquid sample of whole blood is introduced, bringing the sample fluid in contact with a solid support (test strip) of porous membrane (PuraBindTM, Whatman, USA).
  • the pad is designed to separate the plasma from the blood cells.
  • the solid support comprises a defined MIP-conjugate zone comprising TSH-specific MIP saturated with TSH-peptide-biotin conjugate.
  • a reporter-conjugate binding zone comprising NeutrAvidin impregnated with HABA conjugated to BSA coated with gold sol.
  • a results zone comprising immobilized NeutrAvidin with a scale to be used as an aid in quantifying the concentration of MMA in the sample, followed by a control pad comprising dried HABA-B SA-GOLD (reporter- conjugate) and a control zone comprising NeutrAvidin immobilized to the solid support. Operation To initiate the assay, a drop of whole blood is applied to the sample application area.
  • the plasma leaving the sample application area contacts the nitrocellulose membrane and flows along the device by capillary action to contact the TSH-specific MIP zone, which is impregnated with TSH-peptide-biotin conjugate.
  • the TSH present in the sample displaces molecules of TSH-peptide-biotin conjugate from the MIP, in an amount proportional to its concentration.
  • the displaced TSH-peptide-biotin conjugate molecules flow downstream in the fluid, reaching the reporter-conjugate binding zone that comprises HABA-BSA-GoId reporter-conjugate, immobilized to NeutrAvidin.
  • the biotin of the TSH-peptide-biotin conjugate displaces the reporter- conjugate from the NeutrAvidin, in an amount proportional to the amount of the displaced TSH-peptide-biotin conjugate, due to the higher affinity of biotin.
  • the displaced reporter-conjugate migrates further downstream until it comes in contact with the NeutrAvidin binding element at the results zone.
  • Parallel to the NeutrAvidin binding element is a reference scale titrated to allow interpretation of the amount of TSH in the sample by determining the distance traveled by the reporter- conjugate along the binding element at the results window. A visual signal is evident in the results zone allowing the user to determine the amount of TSH in the blood sample. The results are determined about 15 minutes after application of the sample, which is the time required for ensuring the proper functioning of all components of the device.
  • the fluid sample After passing the results zone, the fluid sample continues to move laterally across the control pad, bringing the reporter-conjugate namely HABA-BSA-GoId impregnated in the control pad into free flowing condition along the device until the NeutrAvidin band located in the control zone, where it is captured. A visual line forms across the entire control zone indicating that the assay has functioned properly. The excess liquid and reagents will continue to move laterally across the device and collect in the absorbent pad.
  • EXAMPLE 12 Assay For TSH Using A Diagnostic Flow Through Device (Double Displacement)
  • a device as shown in FIG. 4B is used to quantify TSH in liquid samples obtained from humans.
  • the device comprises a housing containing windows that exposes areas of the solid support for viewing.
  • the device includes a sample application area comprising a pad designed to separate the plasma from the blood cells (LFl, Whatman, USA) to which a liquid sample of whole blood is introduced, bringing the fluid sample in contact with a zone of porous solid support comprising unmodified beads (Sigmacell ® Cellulose, Type 50, Cat. No S5504 Sigma). Further downstream is a MlP-conjugate zone comprising packed TSH-specific
  • MIP particles saturated with TSH-peptide-biotin conjugate located in physical contact underneath the solid support zone.
  • a reporter-conjugate binding zone comprising packed cellulose biotin-immobilized sepharose beads (Cat. No VIT-H-4S, Affiland S.A. Belgium) coated with NeutrAvidin saturated with HABA-BSA-GOLD.
  • a results zone comprising packed biotin- immobilized sepharose beads coated with NeutrAvidin, separated by a zone of unmodified beads. The chromatographic flow of the molecules through the beads helps to form a distinct and concentrated zone of the analyte in the fluid.
  • a scale visible in a viewing window, to be used as an aid in quantifying the concentration of the TSH in the sample, followed by a control pad comprising dried HABA-BSA-GoId (reporter-conjugate) and a control zone comprising biotin-immobilized sepharose beads coated with NeutrAvidin.
  • Sample is introduced at the sample application area and following analysis of a sample in the device, a visual signal is evident in the results zone and in the control zones, allowing the user to determine the amount of TSH in the blood sample, as well as validating the proper performance of the device. Operation:
  • a drop of whole blood is applied to the sample application area at the top of the device.
  • the plasma leaving the sample application area flows along the device by capillary action to contact the TSH-specific MIP zone, which is impregnated with TSH-peptide-biotin conjugate.
  • the TSH present in the sample displaces molecules of TSH-peptide-biotin conjugate from the MIP, in an amount proportional to its concentration.
  • the displaced TSH-peptide-biotin conjugate molecules flow down the device in the fluid, reaching the reporter-conjugate binding zone.
  • the biotin of the TSH- peptide-biotin conjugate displaces the reporter conjugate from the NeutrAvidin, in an amount proportional to the amount of the displaced TSH-peptide-biotin conjugate due to the higher affinity of biotin.
  • the displaced reporter conjugate migrates down the device until it comes in contact with the NeutrAvidin coated cellulose beads at the results zone.
  • Parallel to the NeutrAvidin containing binding element is a reference scale, titrated to allow interpretation of the amount of the TSH in the sample by determining the distance traveled by the reporter-conjugate along the binding element at the results window. The results are determined about 15 minutes after the sample application, which is the time required to ensure the proper functioning of all components of the device.
  • the fluid sample continues to move down to the control pad bringing the un-bound reporter-conjugate, namely, HABA-B SA-GOLD impregnated in the control pad into free flowing condition down the device until the NeutrAvidin coated beads located at the control zone.
  • a visual line forms across the entire control zone indicating that the assay has functioned properly. The excess liquid and reagents will continue to move down the device and collect in the absorbent pad.
  • EXAMPLE 13 Fluorescent Displacement Assay Kit for the Determination of B-type naturietic peptide (BNP) Structure: This device comprises a portable detection unit.
  • BNP B-type naturietic peptide
  • the device includes a sample application area comprising a pad designed to separate the plasma from the blood cells (LFl, Whatman, USA) to which a liquid sample of whole blood is introduced, bringing the fluid sample in contact with the nitrocellulose membrane.
  • the MlP-conjugate zone comprises a BNP-peptide-specific MIP, saturated with BNP-peptide-biotin conjugate.
  • the reporter-conjugate is HABA-B S A-coated with fluorescent dye molecules (HABA- BSA-FLUROFOR).
  • the reporter molecule of the reporter-conjugate is the fluorescent dye Alexa Fluor 488 (Invitrogen, USA) and determination of the results is done by means of a handheld portable fluorescent assay reader (ESE; Stockach, Germany).
  • ESE handheld portable fluorescent assay reader
  • the device for detecting BNP is used to monitor the presence and concentration of the BNP in serum. Prior to application of the serum sample, the device is fitted into the reader which activates the instrument and brings it to a standby position (only proper fitting activates the instrument or else an error message appears).
  • the sample application triggers the instrument to perform a count-down that results in the measurement of the fluorescence intensity about 15 minutes after the sample application, which is the time required to ensure the proper function of all the components of the assay.
  • the BNP in the sample displaces the BNP-peptide-biotin from the BNP- " peptide-specific MIP and the displaced atrazine-biotin in turn displaces the HABA- BSA-fluor dye from the NeutrAvidin in the reporter-conjugate binding zone.
  • the reporter-conjugate migrates downstream until the result zone, where it is captured by the NeutrAvidin.
  • the amount of BNP in the sample is determined by the fluorescent reader by comparing the signal obtained from the sample to that of an internal calibration curve. The results are displayed on the LCD of the instrument in pg/ml (with a limit of detection of 10 pg/ml).
  • EXAMPLE 14 Assay For MMA Using A Lateral Flow Device (competition/displacement)
  • a device as shown in FIG. 2B is used to quantify MMA in liquid samples obtained from humans.
  • the device comprises a housing containing windows that exposes areas of the solid support for viewing.
  • the device includes a sample application area comprising a pad (LFl, Whatman, USA) impregnated with to which MMA-biotin conjugate, to which a liquid sample of whole blood is introduced, bringing the sample fluid in contact with a solid support (test strip) of porous membrane (PuraBindTM, Whatman, USA).
  • the pad is designed to separate the plasma from the blood cells.
  • the solid support comprises a defined MIP zone comprising MMA-specific MIP.
  • a reporter-conjugate binding zone comprising NeutrAvidin impregnated with desthiobiotin (DTB) attached to colored polystyrene beads (Kl-030 di(39457), Merck - Estapor, France).
  • DTB desthiobiotin
  • results zone comprising immobilized NeutrAvidin with a scale to be used as an aid in quantifying the concentration of MMA in the sample, followed by a control pad comprising dried DTB-beads (reporter-conjugate) and a control zone comprising NeutrAvidin immobilized to the solid support.
  • a drop of whole blood is applied to the sample application area and dissolves the impregnated MMA-biotin conjugate.
  • the plasma containing the conjugate leaves the sample application area contacts the nitrocellulose membrane and flows along the device by capillary action to contact the MMA- specific MIP zone.
  • the MMA present in the sample competes with the molecules of MMA-biotin conjugate for the MMA-specific binding sites of the MIP, and due to its superior affinity to these sites an amount of conjugate, proportional to its concentration in the sample is left unbound.
  • the unbound MMA-biotin conjugate molecules flow downstream in the fluid, reaching the reporter-conjugate binding zone that comprises DTB-beads reporter- conjugate, immobilized to NeutrAvidin.
  • the biotin of the MMA-biotin conjugate displaces the reporter- conjugate from the NeutrAvidin, in an amount proportional to the amount of the displaced MMA-biotin conjugate, due to the higher affinity of biotin.
  • the displaced reporter-conjugate migrates further downstream until it comes in contact with the NeutrAvidin binding element at the results zone.
  • Parallel to the NeutrAvidin binding element is a reference scale, titrated to allow interpretation of the amount of MMA in the sample by determining the distance traveled by the reporter-conjugate along the binding element at the results window. A visual signal is evident in the results zone allowing the user to determine the amount of MMA in the blood sample. The results are determined about 15 minutes after application of the sample, which is the time required to ensure the proper functioning of all components of the device.
  • the fluid sample After passing the results zone, the fluid sample continues to move laterally across the control pad, bringing the reporter-conjugate namely DTB-beads impregnated in the control pad into free flowing condition along the device until the NeutrAvidin band located in the control zone, where it is captured. A visual line forms across the entire control zone indicating that the assay has functioned properly. The excess liquid and reagents will continue to move laterally across the device and collect in the absorbent pad.

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Abstract

La présente invention a pour objet des dispositifs, des procédés et des kits pour la détermination rapide et simple de molécules cibles, comprenant de petites molécules, des polypeptides, des protéines, des cellules et des agents de maladies infectieuses dans des échantillons liquides, qui sont capables d'effectuer la mesure en temps réel de ces entités dans des échantillons de fluide et qui sont hautement sélectifs, hautement sensibles, simples à mettre en œuvre, peu coûteux et portatifs. Les dispositifs, les procédés et les kits permettent également, dans au moins certains modes de réalisation, l'utilisation de polymères à empreintes moléculaires (MIP) dans un dispositif à flux continu ou à flux latéral.
EP08868717A 2007-12-27 2008-12-28 Dispositifs d'analyse de petites molécules et de protéines à partir de polymères à empreintes moléculaires Withdrawn EP2245135A4 (fr)

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PCT/IL2008/001688 WO2009083975A2 (fr) 2007-12-27 2008-12-28 Dispositifs d'analyse de petites molécules et de protéines à partir de polymères à empreintes moléculaires

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US11885803B2 (en) 2015-09-04 2024-01-30 The Regents Of The University Of California Methods and devices for analyte collection, extraction, concentration, and detection for clinical applications
US11828755B2 (en) 2016-06-09 2023-11-28 The Regents Of The University Of California Biomarker concentration and signal amplification for use in paper-based immunoassays and a single platform for extracting, concentrating, and amplifying DNA
US11327075B2 (en) 2016-08-22 2022-05-10 The Regents Of The University Of California Hydrogel platform for aqueous two-phase concentration of a target to enhance its detection
US11209427B2 (en) 2017-03-27 2021-12-28 The Regents Of The University Of California Semi-quantitative lateral-flow immunoassay for the detection of CSF leaks
US11867693B2 (en) 2017-03-27 2024-01-09 The Regents Of The University Of California Semi-quantitative lateral-flow immunoassay for the detection of CSF leaks

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