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WO2015109255A1 - Dosages à flux latéral au moyen de dendrimères d'adn - Google Patents

Dosages à flux latéral au moyen de dendrimères d'adn Download PDF

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Publication number
WO2015109255A1
WO2015109255A1 PCT/US2015/011853 US2015011853W WO2015109255A1 WO 2015109255 A1 WO2015109255 A1 WO 2015109255A1 US 2015011853 W US2015011853 W US 2015011853W WO 2015109255 A1 WO2015109255 A1 WO 2015109255A1
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Prior art keywords
test
dna dendrimer
test device
label
binding pair
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Inventor
Louis J. CASTA
James M. Kadushin
Anthony CLEMENTE
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Genisphere LLC
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Genisphere LLC
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54386—Analytical elements
    • G01N33/54387—Immunochromatographic test strips
    • G01N33/54388—Immunochromatographic test strips based on lateral flow
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • 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
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2458/00—Labels used in chemical analysis of biological material
    • G01N2458/10—Oligonucleotides as tagging agents for labelling antibodies

Definitions

  • the present invention relates to novel lateral flow devices and kits using DNA dendrimers and one or more labels, and the methods for detecting an analyte using the lateral flow devices and kits.
  • the 3DNA ® DNA dendrimer is a proprietary dendritic molecule comprised solely of DNA.
  • dendrimers are complex, highly branched molecules built from interconnected natural or synthetic monomeric subunits.
  • a DNA dendrimer is constructed from DNA monomers, each of which is made from two DNA strands that share a region of sequence complementarity located in the central portion of each strand. Monomers are combined during the manufacturing process to prepare DNA dendrimers of different sizes and shapes.
  • chemical "spot welds” are sometimes added to the growing assembly during the process using UV light via the intercalation and activation of psoralen cross-linkers.
  • Dendrimers have been historically purified according to their size and molecular weight on denaturing sucrose gradients after ultracentrifugation.
  • DNA dendrimers have previously been used in membrane based assays, specifically for the detection of nucleic acids and proteins non-covalently immobilized to various membrane substrates, including nitrocellulose and nylon. These assays typically required the use of dendrimers specifically derivatized to contain targeting or binding moieties specific for the target analyte, and typically required several hours to overnight for optimal binding. Improvement of sensitivity from signal amplification ranged from 5 to 500 fold over the non-dendrimer version of the assay.
  • DNA dendrimers have also been shown to be useful as signal amplifiers in a number of other applications, including nucleic acid (DNA/RNA) microarrays, ELISAs, ELOSAs, bead based immunoassays, protein arrays and other similar assays. These assays are all characterized by the immobilization of the analyte or target material to a substrate either directly via a non-covalent or a covalent binding process, or indirectly via the binding to a previously immobilized ligand, which generally required several steps prior to or during the assay process. DNA dendrimers containing up to hundreds of label moieties would then bind either directly or indirectly to the analyte via a targeting device which would
  • the present disclosure provides for a test device for detecting an analyte in a liquid sample, which device comprises a porous matrix that comprises a test zone on said porous matrix, said test zone comprising a test reagent that binds to an analyte or another binding reagent that binds to said analyte, or is an analyte or an analyte analog that competes with an analyte in said sample for binding to a binding reagent for said analyte, wherein a liquid sample flows laterally along said test device and passes said test zone to form a detectable signal to indicate presence, absence and/or amount of said analyte in said liquid sample, the formation of said detectable signal requires the use of a detectable label and a DNA dendrimer, said DNA dendrimer comprises a first label or is configured to comprise a first label, and: 1) said DNA dendrimer is configured to bind to said test reagent via a polyvalent
  • the present disclosure provides for a method for detecting an analyte in a liquid sample, which method comprises: a) contacting a liquid sample with the above test device, wherein the liquid sample is applied to a site of the test device upstream of the test zone; b) transporting an analyte, if present in the liquid sample, a detectable label and a DNA dendrimer to the test zone; and c) assessing the presence, absence, and/or amount of a signal generated by the detectable label at the test zone to determining the presence, absence and/or amount of the analyte in the liquid sample.
  • kits for detecting an analyte in a liquid sample comprising: a) a porous matrix that comprises a test zone on said porous matrix, said test zone comprising a test reagent that binds to an analyte or another binding reagent that binds to said analyte, or is an analyte or an analyte analog that competes with an analyte in said sample for binding to a binding reagent for said analyte; and b) at least one of: 1) a detectable label; 2) a DNA dendrimer; 3) a test reagent; 4) a polyvalent linker that comprises a second label; and/or 5) a second DNA dendrimer, wherein said detectable label, DNA dendrimer, test reagent, polyvalent linker and second DNA dendrimer are described in connection with the above test device.
  • the principles of the present test devices, kits and methods can be applied, or can be adapted to apply, to the lateral flow test devices and assays known in the art.
  • the principles of the present test devices and methods can be applied, or can be adapted to apply, to the lateral flow test devices and assays disclosed and/or claimed in the U.S. patent Nos. 3,641,235, 3,959,078, 3,966,897, 4,094,647, 4,168,146, 4,299,916,
  • Figure 1A illustrates exemplary signal amplification via dendrimer binding to a gold conjugate: Version 1 - biotin dendrimer captured by an anti-biotin gold particle.
  • Figure IB exemplary "dry-down" schematic for Version 1 for a linear unidirectional lateral flow assay format.
  • Figure 2A illustrates exemplary signal amplification via dendrimer binding to a gold conjugate: Version 2 - biotin dendrimer captured by an anti-analyte/anti-biotin gold particle.
  • Figure 2B illustrates exemplary "dry-down" schematic for Version 2 for a linear unidirectional lateral flow assay format.
  • Figure 3A illustrates exemplary signal amplification via dendrimer binding to a gold conjugate: Version 3 - biotin/FITC dendrimer captured by an anti-analyte/anti-biotin gold particle.
  • Figure 3B illustrates exemplary "dry-down" schematic for Version 3 for a linear unidirectional lateral flow assay format.
  • Figure 4A illustrates exemplary signal amplification via dendrimer binding to a gold conjugate: Version 4 - biotin dendrimer as both primary and secondary amplifiers.
  • Figure 4B illustrates exemplary "dry-down" schematic for Version 4 for a linear
  • Figure 5A illustrates an exemplary signal amplification via dendrimer binding to a gold conjugate: Version 5 - FITC dendrimer captured by an anti-analyte/anti-FITC gold particle in an LF assay with a biotinylated analyte binding capture antibody binding to avidin/streptavidin test line.
  • Figure 5B illustrates exemplary "dry-down" schematic for Version 5 for a linear unidirectional lateral flow assay format.
  • Figure 6A illustrates exemplary signal amplification via dendrimer binding to a gold conjugate: Version 6 - FITC dendrimer as both primary and secondary amplifiers.
  • Figure 6B illustrates exemplary "dry-down" schematic for Version 6 for a linear
  • Figure 7A illustrates an exemplary, signal amplification via dendrimer binding to a gold conjugate: Version 7 - FITC dendrimer captured by an anti-biotin/anti-FITC gold particle bound to a biotinylated detection antibody in an LF assay.
  • Figure 7B illustrates exemplary "dry-down" schematic for Version 7 for a linear unidirectional lateral flow assay format.
  • Figure 8A illustrates exemplary signal amplification via dendrimer binding to a gold conjugate: Version 8 - FITC dendrimer captured by an anti-FITC gold particle bound to a FITC labeled detection antibody in an LF assay.
  • Figure 8B illustrates exemplary
  • FIG. 9A illustrates exemplary signal amplification via dendrimer binding to a gold conjugate: Version 9 - FITC dendrimer captured by an anti-analyte/anti-FITC gold particle.
  • Figure 9B illustrates exemplary "dry-down" schematic for Version 9 for a linear unidirectional lateral flow assay format.
  • Figure 10A illustrates exemplary signal amplification via dendrimer binding to a gold conjugate: Version 10 - biotin dendrimer captured by an anti-biotin/anti-FITC gold particle bound to a FITC labeled detection antibody in an LF assay.
  • Figure 10B illustrates exemplary "dry-down" schematic for Version 10 for a linear unidirectional lateral flow assay format.
  • binding reagent refers to any substance that binds to target or analyte with desired affinity and/or specificity.
  • Non-limiting examples of the binding reagent include cells, cellular organelles, viruses, particles, microparticles, molecules, or an aggregate or complex thereof, or an aggregate or complex of molecules.
  • Exemplary binding reagents can be an amino acid, a peptide, a protein, e.g., an antibody or receptor, a nucleoside, a nucleotide, an oligonucleotide, a nucleic acid, e.g., DNA or RNA, a vitamin, a
  • an "antibody” is an immunoglobulin molecule capable of specific binding to a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., through at least one antigen recognition site, located in the variable region of the immunoglobulin molecule, and can be an immunoglobulin of any class, e.g., IgG, IgM, IgA, IgD and IgE.
  • IgY which is the major antibody type in avian species such as chicken, is also included within the definition.
  • the term encompasses not only intact polyclonal or monoclonal antibodies, but also fragments thereof (such as Fab, Fab', F(ab')2, Fv), single chain (ScFv), mutants thereof, naturally occurring variants, fusion proteins comprising an antibody portion with an antigen recognition site of the required specificity, humanized antibodies, chimeric antibodies, and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site of the required specificity.
  • monoclonal antibody refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the antibodies comprising the population are identical except for possible naturally occurring mutations that are present in minor amounts. As used herein, a “monoclonal antibody” further refers to functional fragments of monoclonal antibodies.
  • the term "antigen" refers to a target molecule that is specifically bound by an antibody through its antigen recognition site.
  • the antigen may be monovalent or polyvalent, i.e., it may have one or more epitopes recognized by one or more antibodies.
  • Examples of kinds of antigens that can be recognized by antibodies include polypeptides, oligosaccharides, glycoproteins, polynucleotides, lipids, etc.
  • the term “specifically binds” refers to the specificity of a binding reagent, e.g., an antibody, such that it preferentially binds to a defined analyte or target.
  • binding reagents Recognition by a binding reagent or an antibody of a particular analyte or target in the presence of other potential interfering substance(s) is one characteristic of such binding.
  • a binding reagent that specifically binds to an analyte avoids binding to other interfering moiety or moieties in the sample to be tested.
  • the term "avoids binding” refers to the specificity of particular binding reagents, e.g., antibodies or antibody fragments. Binding reagents, antibodies or antibody fragments that avoid binding to a particular moiety generally contain a specificity such that a large percentage of the particular moiety would not be bound by such binding reagents, antibodies or antibody fragments.
  • This percentage generally lies within the acceptable cross reactivity percentage with interfering moieties of assays utilizing the binding reagents or antibodies directed to detecting a specific target.
  • the binding reagents, antibodies or antibody fragments of the present disclosure avoid binding greater than about 90% of an interfering moiety, although higher percentages are clearly
  • binding reagents, antibodies or antibody fragments of the present disclosure avoid binding about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, and about 99% or more of an interfering moiety. Less occasionally, binding reagents, antibodies or antibody fragments of the present disclosure avoid binding greater than about 70%, or greater than about 75%, or greater than about 80%, or greater than about 85% of an interfering moiety.
  • mammal refers to any of the mammalian class of species.
  • the term "mammal,” as used herein, refers to humans, human subjects or human patients.
  • the term “subject” is not limited to a specific species or sample type.
  • the term “subject” may refer to a patient, and frequently a human patient. However, this term is not limited to humans and thus encompasses a variety of mammalian species.
  • sample refers to anything which may contain an analyte for which an analyte assay is desired.
  • the sample may be a biological sample, such as a biological fluid or a biological tissue.
  • biological fluids include urine, blood, plasma, serum, saliva, semen, stool, sputum, cerebral spinal fluid, tears, mucus, amniotic fluid or the like.
  • Biological tissues are aggregate of cells, usually of a particular kind together with their intercellular substance that form one of the structural materials of a human, animal, plant, bacterial, fungal or viral structure, including connective, epithelium, muscle and nerve tissues. Examples of biological tissues also include organs, tumors, lymph nodes, arteries and individual cell(s).
  • isolated refers to material removed from its original environment, and is altered from its natural state.
  • an isolated polypeptide could be coupled to a carrier, and still be “isolated” because that polypeptide is not in its original environment.
  • high-throughput screening refers to processes that test a large number of samples, such as samples of diverse chemical structures against disease targets to identify "hits" (see, e.g., Broach, et al., High throughput screening for drug discovery, Nature, 384: 14-16 (1996); Janzen, et al., High throughput screening as a discovery tool in the pharmaceutical industry, Lab Robotics Automation: 8261-265 (1996); Fernandes, P.B., Letter from the society president, /. Biomol. Screening, 2: 1 (1997); Burbaum, et al., New technologies for high-throughput screening, Curr. Opin. Chem. Biol., 7:72-78 (1997)).
  • HTS operations are highly automated and computerized to handle sample preparation, assay procedures and the subsequent processing of large volumes of data.
  • polypeptide oligopeptide
  • peptide protein
  • polymers of amino acids of any length e.g., at least 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 1,000 or more amino acids.
  • the polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids.
  • the terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component.
  • polypeptides containing one or more analogs of an amino acid including, for example, unnatural amino acids, etc.
  • polynucleotide oligonucleotide
  • nucleic acid oligonucleotide
  • nucleic acid molecule a polymeric form of nucleotides of any length, e.g. , at least 8, 9, 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 1,000 or more nucleotides, and may comprise ribonucleotides, deoxyribonucleotides, analogs thereof, or mixtures thereof.
  • RNA triple-, double- and single- stranded ribonucleic acid
  • DNA triple-, double- and single- stranded deoxyribonucleic acid
  • RNA triple-, double- and single- stranded ribonucleic acid
  • DNA triple-, double- and single- stranded deoxyribonucleic acid
  • RNA triple-, double- and single- stranded ribonucleic acid
  • It also includes modified, for example by alkylation, and/or by capping, and unmodified forms of the polynucleotide.
  • polynucleotide examples include polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), including tRNA, rRNA, hRNA, and mRNA, whether spliced or unspliced, any other type of polynucleotide which is an N- or C-glycoside of a purine or pyrimidine base, and other polymers containing normucleotidic backbones, for example, polyamide ⁇ e.g. , peptide nucleic acids (“PNAs”)) and polymorpholino
  • PNAs peptide nucleic acids
  • nucleobases in a configuration which allows for base pairing and base stacking, such as is found in DNA and RNA.
  • these terms include, for example, 3'-deoxy-2',5'-DNA, oligodeoxyribonucleotide N3' to P5' phosphoramidates, 2'-0-alkyl-substituted RNA, hybrids between DNA and RNA or between PNAs and DNA or RNA, and also include known types of modifications, for example, labels, alkylation, "caps," substitution of one or more of the nucleotides with an analog, intemucleotide modifications such as, for example, those with uncharged linkages ⁇ e.g. , methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), with negatively charged linkages ⁇ e.g. , phosphorothioates,
  • aminoalkylphosphoramidates, aminoalkylphosphotriesters those containing pendant moieties, such as, for example, proteins (including enzymes ⁇ e.g. nucleases), toxins, antibodies, signal peptides, poly-L-lysine, etc.), those with intercalators ⁇ e.g. , acridine, psoralen, etc.), those containing chelates (of, e.g. , metals, radioactive metals, boron, oxidative metals, etc.), those containing alkylators, those with modified linkages (e.g. , alpha anomeric nucleic acids, etc.), as well as unmodified forms of the polynucleotide or oligonucleotide.
  • proteins including enzymes ⁇ e.g. nucleases), toxins, antibodies, signal peptides, poly-L-lysine, etc.
  • intercalators ⁇ e.g. , acridine, psoral
  • nucleoside and nucleotide will include those moieties which contain not only the known purine and pyrimidine bases, but also other heterocyclic bases which have been modified. Such modifications include methylated purines or pyrimidines, acylated purines or pyrimidines, or other heterocycles. Modified nucleosides or nucleotides can also include modifications on the sugar moiety, e.g. , wherein one or more of the hydroxyl groups are replaced with halogen, aliphatic groups, or are functionalized as ethers, amines, or the like.
  • the term “nucleotidic unit” is intended to encompass nucleosides and nucleotides.
  • Nucleic acid probe and “probe” are used interchangeably and refer to a structure comprising a polynucleotide, as defined above, that contains a nucleic acid sequence that can bind to a corresponding target.
  • the polynucleotide regions of probes may be composed of DNA, and/or RNA, and/or synthetic nucleotide analogs.
  • complementary or matched means that two nucleic acid sequences have at least 50% sequence identity. Preferably, the two nucleic acid sequences have at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of sequence identity. “Complementary or matched” also means that two nucleic acid sequences can hybridize under low, middle and/or high stringency condition(s).
  • substantially complementary or substantially matched means that two nucleic acid sequences have at least 90% sequence identity. Preferably, the two nucleic acid sequences have at least 95%, 96%, 97%, 98%, 99% or 100% of sequence identity. Alternatively, “substantially complementary or substantially matched” means that two nucleic acid sequences can hybridize under high stringency condition(s). [0040] In general, the stability of a hybrid is a function of the ion concentration and temperature. Typically, a hybridization reaction is performed under conditions of lower stringency, followed by washes of varying, but higher, stringency.
  • Moderately stringent hybridization refers to conditions that permit a nucleic acid molecule such as a probe to bind a complementary nucleic acid molecule.
  • the hybridized nucleic acid molecules generally have at least 60% identity, including for example at least any of 70%, 75%, 80%, 85%, 90%, or 95% identity.
  • Moderately stringent conditions are conditions equivalent to hybridization in 50% formamide, 5x Denhardt' s solution, 5x SSPE, 0.2% SDS at 42°C, followed by washing in 0.2x SSPE, 0.2% SDS, at 42°C.
  • High stringency conditions can be provided, for example, by hybridization in 50% formamide, 5x Denhardt' s solution, 5x SSPE, 0.2% SDS at 42°C, followed by washing in 0. lx SSPE, and 0.1% SDS at 65°C.
  • Low stringency hybridization refers to conditions equivalent to hybridization in 10% formamide, 5x
  • Denhardt' s solution 6x SSPE, 0.2% SDS at 22°C, followed by washing in lx SSPE, 0.2% SDS, at 37°C.
  • Denhardt' s solution contains 1% Ficoll, 1% polyvinylpyrolidone, and 1% bovine serum albumin (BSA).
  • 20x SSPE sodium chloride, sodium phosphate, ethylene diamide tetraacetic acid (EDTA) contains 3M sodium chloride, 0.2M sodium phosphate, and 0.025 M EDTA.
  • biological sample refers to any sample obtained from a living or viral source or other source of macromolecules and biomolecules, and includes any cell type or tissue of a subject from which nucleic acid or protein or other macromolecule can be obtained.
  • the biological sample can be a sample obtained directly from a biological source or a sample that is processed.
  • isolated nucleic acids that are amplified constitute a biological sample.
  • Biological samples include, but are not limited to, body fluids, such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine and sweat, tissue and organ samples from animals and plants and processed samples derived therefrom. Also included are soil and water samples and other environmental samples, viruses, bacteria, fungi, algae, protozoa and components thereof.
  • the angle between the at least two non-parallel different flow paths is at about 130, 125, 120, 115, 110, 105, 100, 95, 94, 93, 92 or 91 degrees, or the at least two
  • the present disclosure provides for a test device for detecting an analyte in a liquid sample, which device comprises a porous matrix that comprises a test zone on said porous matrix, said test zone comprising a test reagent that binds to an analyte or another binding reagent that binds to said analyte, or is an analyte or an analyte analog that competes with an analyte in said sample for binding to a binding reagent for said analyte, wherein a liquid sample flows laterally along said test device and passes said test zone to form a detectable signal to indicate presence, absence and/or amount of said analyte in said liquid sample, the formation of said detectable signal requires the use of a detectable label and a DNA dendrimer, said DNA dendrimer comprises a first label or is configured to comprise a first label, and: 1) said DNA dendrimer is configured to bind to said test reagent via a polyvalent
  • the DNA dendrimer can be configured to bind to the test reagent via a polyvalent linker that comprises a second label.
  • the DNA dendrimer can be configured to bind to the polyvalent in any suitable manner.
  • the test reagent can also be configured to bind to the polyvalent in any suitable manner.
  • the DNA dendrimer can be configured to bind to the polyvalent linker via a first binding pair, and/or the test reagent can be configured to bind to the polyvalent linker via a second binding pair.
  • the DNA dendrimer can be configured to bind to the first label via a third binding pair.
  • any suitable binding pair(s) can be used.
  • at least two of the first binding pair, the second binding pair and the third binding pair can be the same binding pair.
  • the first binding pair, the second binding pair and the third binding pair can be different binding pairs.
  • the first binding pair, the second binding pair and/or the third binding pair can be selected from the group consisting of an antigen/antibody pair, a ligand/receptor pair, a biotin/biotin-binding protein pair, and a polynucleotide/polynucleotide pair.
  • Any suitable biotin-binding protein can be used.
  • the biotin-binding protein can be avidin or streptavidin. Any suitable
  • the antigen/antibody pair can be used.
  • the antigen/antibody pair can be a fluorescein isothiocyanate (FITC)/anti- FITC antibody pair.
  • FITC fluorescein isothiocyanate
  • the first label and the second label can be any suitable label(s).
  • the first label and the second label can be different labels.
  • the first label and the second label can be the same label.
  • a substance can be dried on a portion of the matrix upstream from the test zone, the dried substance being capable of being moved by a liquid sample and/or a further liquid to the test zone and/or a control zone to generate a detectable signal, the dried substance being at least one of: 1) the DNA dendrimer comprising or not comprising the first label; 2) the test reagent; and/or 3) the polyvalent linker.
  • the DNA dendrimer, the test reagent and/or the polyvalent linker can be dried on a portion of the matrix upstream from the test zone in any suitable manner.
  • the DNA dendrimer, the test reagent and/or the polyvalent linker can be dried on a single flow path.
  • each of the DNA dendrimer, the test reagent and the polyvalent linker alone can be dried on a single flow path.
  • two of the DNA dendrimer, the test reagent and the polyvalent linker alone can be dried on a single flow path.
  • all three of the DNA dendrimer, the test reagent and the polyvalent linker alone can be dried on a single flow path.
  • the DNA dendrimer, the test reagent and/or the polyvalent linker can also be dried on at least two non-parallel different flow paths that intersect at a location upstream from the test zone.
  • Any suitable lateral flow devices with non-parallel different flow paths can be used. See e.g., US 7189522, US 7879597, EP 1856503, WO 2006/099191 and WO 2006/098804.
  • the at least two non-parallel different flow paths can have any suitable angle between the different flow paths.
  • the angle between the different flow paths can be about 5-25 degrees, 25-50 degrees, 50-75 degrees, or 75-90 degrees.
  • the at least two non-parallel different flow paths are substantially perpendicular or perpendicular to each other.
  • the DNA dendrimer, the test reagent and/or the polyvalent linker can be dried on at least two non-parallel different flow paths in any suitable manner.
  • at least 1, 2 or all 3 of the DNA dendrimer, the test reagent and/or the polyvalent linker can be dried on the at least two non-parallel different flow paths.
  • one of the DNA dendrimer, the test reagent and/or the polyvalent linker can be dried on one of the two non-parallel different flow paths.
  • two of the DNA dendrimer, the test reagent and/or the polyvalent linker can be dried on at least two non-parallel different flow paths.
  • the DNA dendrimer can be can be dried on one of the two non-parallel different flow paths, and the test reagent or the polyvalent linker can be dried on other flow path.
  • the test reagent can be can be dried on one of the two non-parallel different flow paths, and the DNA dendrimer or the polyvalent linker can be dried on other flow path.
  • the polyvalent linker can be dried on one of the two non-parallel different flow paths, and the DNA dendrimer or the test reagent can be dried on other flow path.
  • the DNA dendrimer, the test reagent and the polyvalent linker can be dried on at least two non-parallel different flow paths.
  • the DNA dendrimer can be can be dried on one of the two non-parallel different flow paths, and the test reagent and the polyvalent linker can be dried on other flow path.
  • the test reagent can be can be dried on one of the two non-parallel different flow paths, and the DNA dendrimer and the polyvalent linker can be dried on other flow path.
  • the polyvalent linker can be dried on one of the two non-parallel different flow paths, and the DNA dendrimer and the test reagent can be dried on other flow path.
  • a substance can be dried on a portion of the matrix upstream from the test zone, the dried substance being capable of being moved by a liquid sample and/or a further liquid to the test zone and/or a control zone to generate a detectable signal, the dried substance being at least one of: 1) a conjugate comprising the first label and the second member of the third binding pair; 2) the DNA dendrimer linked to the first member of the first binding pair, and the first member of the third binding pair; 3) the test reagent linked to the first member of the second binding pair; and/or 4) the polyvalent linker linked to the second member of the first binding pair, and second member of the second binding pair.
  • the conjugate in 1) and the DNA dendrimer in 2) are dried on a first flow path
  • the test reagent in 3) and the polyvalent linker in 4) are dried on a different second flow path, and the first flow path and the second flow path intersect at a location upstream from the test zone.
  • the first label and the second label are the same label
  • the first binding pair, the second binding pair and the third binding pair are the same binding pair. Any suitable binding pairs can be used.
  • the first binding pair, the second binding pair and the third binding pair can be a
  • biotin/biotin-binding protein pair or a fluorescein isothiocyanate (FITC)/anti- FITC antibody pair.
  • FITC fluorescein isothiocyanate
  • the first label and the second label can be the same label, and the conjugate in 1) and the polyvalent linker in 4) can be the identical moiety.
  • the conjugate (or the polyvalent linker), the DNA dendrimer and the test reagent can be dried on a single flow path in any suitable order.
  • the order can be: 1) the conjugate (or the polyvalent linker), the DNA dendrimer and the test reagent; 2) the DNA dendrimer, the test reagent and the conjugate (or the polyvalent linker); or 3) the test reagent, the conjugate (or the polyvalent linker) and the DNA dendrimer.
  • Any suitable binding pairs can be used.
  • the first binding pair, the second binding pair and the third binding pair can be a biotin/biotin-binding protein pair or a fluorescein isothiocyanate (FITC)/anti- FITC antibody pair.
  • the first label and the second label can be the same label
  • the first binding pair and the third binding pair can be the same binding pair
  • the first binding pair (or the third binding pair) is different from the second binding pair.
  • the first binding pair and the third binding pair can be a fluorescein isothiocyanate (FITC)/anti- FITC antibody pair
  • the second binding pair can be a biotin/biotin-binding protein pair
  • the first binding pair and the third binding pair can be a biotin/biotin-binding protein pair
  • the second binding pair can be a fluorescein isothiocyanate (FITC)/anti- FITC antibody pair.
  • the DNA dendrimer, the test reagent and a mixture of the conjugate and the polyvalent linker are dried on a single flow path in any suitable order.
  • the order can be: 1) the mixture of the conjugate and the polyvalent linker, the DNA dendrimer and the test reagent; 2) the DNA dendrimer, the test reagent, and the mixture of the conjugate and the polyvalent linker; or 3) the test reagent, the mixture of the conjugate and the polyvalent linker and the DNA dendrimer.
  • the DNA dendrimer can be configured to bind to the test reagent that comprises or is configured to comprise a second label.
  • the DNA dendrimer can be configured to bind to the test reagent or the first label in any suitable manner.
  • the DNA dendrimer can be configured to bind to the test reagent via a fourth binding pair, and/or the DNA dendrimer can be configured to bind to the first label via a fifth binding pair.
  • the fourth binding pair and the fifth binding pair can be the same binding pair or different binding pairs. Any suitable binding pairs can be used.
  • the fourth binding pair and/or the fifth binding pair can be selected from the group consisting of an antigen/antibody pair, a ligand/receptor pair, a biotin/biotin-binding protein pair, and a polynucleotide/polynucleotide pair.
  • Any suitable biotin-binding protein can be used.
  • the biotin-binding protein can be avidin or streptavidin.
  • Any suitable antigen/antibody pair can be used.
  • the antigen/antibody pair can be a fluorescein isothiocyanate (FITC)/anti- FITC antibody pair.
  • FITC fluorescein isothiocyanate
  • Any suitable labels can be used.
  • the first label and the second label can be different labels. In another example, the first label and the second label can be the same label.
  • a substance can be dried on a portion of the matrix upstream from the test zone, the dried substance being capable of being moved by a liquid sample and/or a further liquid to the test zone and/or a control zone to generate a detectable signal, the dried substance being at least one of: 1) the DNA dendrimer comprising or not comprising the first label; and/or 2) the test reagent.
  • the DNA dendrimer and/or the test reagent can be dried on a single flow path.
  • the DNA dendrimer and/or the test reagent can also be dried on at least two non-parallel different flow paths that intersect at a location upstream from the test zone. The at least two non-parallel different flow paths can be substantially perpendicular or perpendicular to each other.
  • a substance can be dried on a portion of the matrix upstream from the test zone, the dried substance being capable of being moved by a liquid sample and/or a further liquid to the test zone and/or a control zone to generate a detectable signal, the dried substance being at least one of: 1) a conjugate comprising the first label and the second member of the fifth binding pair; 2) the DNA dendrimer linked to the first member of the fourth binding pair, and the first member of the fifth binding pair; and/or 3) the test reagent linked to the second member of the fourth binding pair.
  • the conjugate, the DNA dendrimer and/or the test reagent can be dried on flow path(s) in any suable manner.
  • the conjugate in 1) and the DNA dendrimer in 2) can be dried on a first flow path, and the test reagent in 3) can be dried on a different second flow path, and the first flow path and the second flow path intersect at a location upstream from the test zone.
  • the first label and the second label can be the same label
  • the fourth binding pair and the fifth binding pair can be the same binding pair.
  • Any suitable binding pair can be used.
  • the fourth binding pair and/or the fifth binding pair can be a biotin/biotin-binding protein pair or a fluorescein isothiocyanate (FITC)/anti- FITC antibody pair.
  • the first label and the second label can be the same label
  • the DNA dendrimer and the test reagent can be dried on a single flow path.
  • the conjugate can be further dried on the same flow path on which the DNA dendrimer and the test reagent are dried.
  • the conjugate, the DNA dendrimer and the test reagent can be dried on the same flow path in any suitable order.
  • the order can be: 1) the conjugate, the DNA dendrimer and the test reagent; 2) the DNA dendrimer, the test reagent, and the conjugate; or 3) the test reagent, the conjugate, and the DNA dendrimer.
  • the first label and the second label can be the same label
  • the fourth binding pair and the fifth binding pair can be different binding pairs.
  • Any suitable binding pair can be used.
  • one of the fourth binding pair and/or the fifth binding pair can be a biotin/biotin-binding protein pair
  • the other binding pair can be a fluorescein isothiocyanate (FITC)/anti- FITC antibody pair.
  • the conjugate and the DNA dendrimer can be dried on a first flow path, and the test reagent can be dried on a different second flow path, and the first flow path and the second flow path intersect at a location upstream from the test zone.
  • the conjugate, the DNA dendrimer and the test reagent can be dried on a single flow path in any suitable order.
  • the order can be: 1) the conjugate, the DNA dendrimer and the test reagent; 2) the DNA dendrimer, the test reagent, and the conjugate; or 3) the test reagent, the conjugate, and the DNA dendrimer.
  • the DNA dendrimer can be configured to bind to the test reagent and bind to a second DNA dendrimer via a polyvalent linker, e.g., a polyvalent linker that comprises a second label, the second DNA dendrimer can comprise a first label or can be configured to comprise a first label.
  • the DNA dendrimer can be configured to bind to the test reagent and the polyvalent linker in any suitable manner and the second DNA dendrimer can be configured to bind to the polyvalent linker.
  • the DNA dendrimer is configured to bind to the test reagent via a sixth binding pair
  • the DNA dendrimer is configured to bind to the polyvalent linker via a seventh binding pair
  • the second DNA dendrimer is configured to bind to the polyvalent linker via an eighth binding pair.
  • Any suitable binding pair can be used.
  • the sixth binding pair, seventh binding pair and the eighth binding pair can be the same binding pair.
  • at least two of the sixth binding pair, seventh binding pair and the eighth binding pair can be different binding pairs.
  • the sixth binding pair, the seventh binding pair and/or the eighth binding pair can be selected from the group consisting of an antigen/antibody pair, a ligand/receptor pair, a biotin/biotin-binding protein pair, and a polynucleotide/polynucleotide pair.
  • Any suitable biotin-binding protein can be used.
  • the biotin-binding protein can be avidin or streptavidin.
  • Any suitable antigen/antibody pair can be used.
  • the sixth binding pair, the seventh binding pair and/or the eighth binding pair can be selected from the group consisting of an antigen/antibody pair, a ligand/receptor pair, a biotin/biotin-binding protein pair, and a polynucleotide/polynucleotide pair.
  • Any suitable biotin-binding protein can be used.
  • the biotin-binding protein can be avidin or streptavidin.
  • antigen/antibody pair can be a fluorescein isothiocyanate (FITC)/anti- FITC antibody pair.
  • FITC fluorescein isothiocyanate
  • the seventh binding pair and the eighth binding pair can be the same binding pair, and the seventh binding pair and the eighth binding pair can be different from the sixth binding pair.
  • the seventh binding pair and the eighth binding pair can be an antigen/antibody pair or a biotin/biotin-binding protein pair
  • the sixth binding pair can be a polynucleotide/polynucleotide pair.
  • Any suitable label can be used.
  • the first label and the second label can be different labels.
  • the first label and the second label can be the same label.
  • a substance can be dried on a portion of the matrix upstream from the test zone, the dried substance being capable of being moved by a liquid sample and/or a further liquid to the test zone and/or a control zone to generate a detectable signal, the dried substance being at least one of: 1) the DNA dendrimer comprising or not comprising the first label; 2) the second DNA dendrimer comprising or not comprising the first label; 3) the test reagent; 4) the polyvalent linker, and/or 5) a conjugate comprising the first label.
  • the DNA dendrimer, the second DNA dendrimer, the test reagent, the polyvalent linker and/or the conjugate can be dried on a single flow path.
  • the DNA dendrimer, the second DNA dendrimer, the test reagent, the polyvalent linker and/or the conjugate can be dried on at least two non-parallel different flow paths that intersect at a location upstream from the test zone.
  • the at least two non-parallel different flow paths can be substantially perpendicular or perpendicular to each other.
  • the first label and the second label can be the same label, and the DNA dendrimer can be configured to bind to the test reagent via a sixth binding pair, the DNA dendrimer can be configured to bind to the polyvalent linker via a seventh binding pair, and/or the second DNA dendrimer can be configured to bind to the polyvalent linker via an eighth binding pair.
  • the seventh binding pair and the eighth binding pair can be the same binding pair, and the polyvalent linker in 4) and the conjugate in 5) can be the identical moiety.
  • the second DNA dendrimer and the polyvalent linker (or the conjugate) can be dried on at least two non-parallel different flow paths that intersect at a location upstream from the test zone. The at least two non-parallel different flow paths can be substantially perpendicular or perpendicular to each other.
  • the second DNA dendrimer and the polyvalent linker (or the conjugate) can be dried on a single flow path in any suitable order. For example, from upstream to
  • the order can be: 1) the second DNA dendrimer and the polyvalent linker (or the conjugate); or 2) the polyvalent linker (or the conjugate) and the second DNA dendrimer.
  • the present devices can be used for detecting any suitable analyte.
  • the analyte is not a polynucleotide.
  • the analyte is a polypeptide or a small molecule
  • the test reagent is an antibody that binds to the polypeptide or small molecule.
  • the analyte is a polynucleotide
  • the test reagent is another polynucleotide that is substantially complementary to the analyte polynucleotide.
  • the DNA dendrimer and/or the second DNA dendrimer can comprise from about 400 to about 80,000, preferably from about 4,000 to about 80,000, from about 60,000 to about 80,000, DNA nucleotides.
  • the DNA dendrimer and/or the second DNA dendrimer can comprise from about 400 to about 1,000, about 1,000 to about 5,000, about 5,000 to about 10,000, about 10,000 to about 20,000, about 20,000 to about 30,000, 30,000 to about 40,000, about 40,000 to about 50,000, 50,000 to about 60,000, about 60,000 to about 70,000, or 70,000 to about 80,000 DNA nucleotides.
  • the present devices can be used in any suitable assay format, e.g. , a sandwich or competitive assay.
  • the test device is to be used in a sandwich assay for the analyte and wherein the test reagent at the test zone binds, and preferably specifically binds, to the analyte, and a second binding reagent that binds, and preferably specifically binds, to the analyte is used.
  • the second binding reagent can comprise a polynucleotide that is substantially complementary to a polynucleotide that is a component of a DNA dendrimer.
  • the second binding reagent can also comprise a member of a binding pair that can bind to the corresponding member of the binding pair that is part of the DNA dendrimer.
  • the test device is to be used in a sandwich assay for the analyte and wherein the test reagent at the test zone binds, and preferably specifically binds, to the analyte, a second binding reagent that binds to another binding reagent that binds, and preferably specifically binds, to an analyte is used.
  • the second binding reagent comprises a
  • the second binding reagent can also comprise a member of a binding pair that can bind to the corresponding member of the binding pair that is part of the DNA dendrimer.
  • the test device is to be used in a competitive assay for the analyte and wherein the test reagent at the test zone is an analyte or an analyte analog, and a second binding reagent that binds, and preferably specifically binds, to the analyte is used.
  • the second binding reagent can comprise a polynucleotide that is substantially
  • the second binding reagent can also comprise a member of a binding pair that can bind to the
  • the test device is to be used in a competitive assay for the analyte and wherein the test reagent at the test zone is an analyte or an analyte analog, and a second binding reagent that binds to another binding reagent that binds, and preferably specifically binds, to an analyte is used.
  • the second binding reagent can comprise a polynucleotide that is substantially complementary to a polynucleotide that is a component of a DNA dendrimer.
  • the second binding reagent can also comprise a member of a binding pair that can bind to the corresponding member of the binding pair that is part of the DNA dendrimer.
  • the analyte or an analyte analog at the test zone competes with an analyte in the sample for binding to the binding reagent that is bound to the second binding reagent.
  • the test reagent can be any suitable substance.
  • the test reagent can be an inorganic molecule, an organic molecule or a complex thereof.
  • Exemplary organic molecules or moieties include an amino acid, a peptide, a protein, a nucleoside, a nucleotide, an oligonucleotide, a nucleic acid, a vitamin, a monosaccharide, an oligosaccharide, a carbohydrate, a lipid and a complex thereof.
  • the analytes and the various reagents used in connection with the present devices can be any suitable substances.
  • the analyte, analyte analog, test reagent and/or binding reagent is an inorganic molecule, an organic molecule or a complex thereof.
  • Exemplary organic molecules include an amino acid, a peptide, a protein, a nucleoside, a nucleotide, an oligonucleotide, a nucleic acid, a vitamin, a monosaccharide, an oligosaccharide, a
  • the analyte is a polypeptide, a small molecule or an antigen
  • the test reagent and/or binding reagent that binds to the analyte is an antibody that binds, and preferably specifically binds, to the polypeptide, small molecule or an antigen.
  • the analyte is a polynucleotide
  • the test reagent and/or binding reagent that binds to the analyte is another polynucleotide that is substantially complementary to the analyte polynucleotide.
  • the analyte is a polynucleotide
  • the test reagent and/or binding reagent that binds to the analyte is a non-polynucleotide moiety, e.g. , a polypeptide, an antibody or a receptor that binds to the analyte polynucleotide.
  • the matrix can comprise or be made of any suitable material.
  • the matrix comprises nitrocellulose, glass fiber, polypropylene, polyethylene (preferably of very high molecular weight), polyvinylidene flouride, ethylene vinylacetate, acrylonitrile and/or polytetrafluoro-ethylene.
  • the matrix can also be made from paper or other cellulosic materials.
  • the matrix comprises or is made of nitrocellulose or glass fiber.
  • the matrix can also be in any suitable form or shape. In some embodiments, the matrix is in the form a strip or a circle. The matrix can also comprise or be made of any suitable number of element. In some embodiments, the matrix is a single element or comprises multiple elements.
  • test devices can comprise any suitable additional elements.
  • the test device can further comprise a sample application element upstream from and in fluid communication with the matrix.
  • the test device can further comprise a liquid absorption element downstream from and in fluid communication with the matrix.
  • the test device can further comprise a control zone comprising means for indicating proper flow of the liquid sample and/or a valid test result.
  • at least a portion of the matrix is supported by a solid backing.
  • the entire matrix is supported by a solid backing.
  • the various substances or reagents used in connection with the present devices can be dried on the test devices before use.
  • a substance is dried on a portion of the matrix upstream from the test zone, the dried substance being capable of being moved by a liquid sample and/or a further liquid to the test zone and/or a control zone to generate a detectable signal.
  • two substances are dried on a portion of the matrix upstream from the test zone, the dried substances being capable of being moved by a liquid sample and/or a further liquid to the test zone and/or a control zone to generate a detectable signal.
  • three or more substances are dried on a portion of the matrix upstream from the test zone, the dried substances being capable of being moved by a liquid sample and/or a further liquid to the test zone and/or a control zone to generate a detectable signal.
  • the above substance(s) can be dried on any suitable location of the test devices.
  • the substance(s) is dried on a conjugate element that is upstream from the test zone.
  • the substance(s) is located downstream from a sample application place on the test device.
  • the substance(s) is located upstream from a sample application place on the test device.
  • the substance(s) is dried in the presence of a material that: a) stabilizes the dried substance(s); b) facilitates solubilization or resuspension of the dried substance(s) in a liquid; and/or c) facilitates mobility of the dried substance(s).
  • a material that: a) stabilizes the dried substance(s); b) facilitates solubilization or resuspension of the dried substance(s) in a liquid; and/or c) facilitates mobility of the dried substance(s).
  • a material can be a protein, a peptide, a polysaccharide, a sugar, a polymer, a gelatin and a detergent. See e.g., U.S. patent Nos. 5,120,643 and 6,187,598.
  • the detectable label is a soluble label, e.g., a soluble enzyme or fluorescent label.
  • the detectable label is a particle label.
  • the particle label can be a visible or a non- visible particle label.
  • the visible particle label is selected from the group consisting of a gold particle, a latex particle, a Q-Dot, a carbon nanotube, a silver particle and a silver coated particle.
  • the non-visible particle label is a fluorescent particle.
  • a detectable label can also be a moiety that effects a change in mass and/or charge.
  • a DNA dendrimer can effect a change in mass and/or charge.
  • a detectable label can be a DNA dendrimer, e.g., the DNA dendrimer itself or an additional DNA dendrimer.
  • a sample liquid alone is used to transport the analyte and/or the substance(s) to the test zone.
  • a developing liquid is used to transport the analyte and/or the substance(s) to the test zone.
  • the test device can further comprise a housing that covers at least a portion of the test device, wherein the housing comprises a sample application port to allow sample application upstream from or to the test zone and an optic opening around the test zone to allow signal detection at the test zone and/or the control zone.
  • the housing covers the entire test device.
  • at least a portion of the sample receiving portion of the matrix or the sample application element is not covered by the housing and a sample is applied to the portion of the sample receiving portion of the matrix or the sample application element outside the housing and then transported to the test zone.
  • One copy of the DNA dendrimer and/or the second DNA dendrimer can comprise or can be configured to bind to any suitable number of the detectable label and/or the test reagent.
  • one copy of the DNA dendrimer and/or the second DNA dendrimer comprises or is configured to bind to from about 10 to about 1,500, preferably from about 40 to about 1,500, from about 900 to about 1,100, copies of the detectable label(s), e.g., about 10 to about 50, about 50 to about 100, about 100 to about 200, about 200 to about 300, about 300 to about 400, about 400 to about 500, about 500 to about 600, about 600 to about 700, about 700 to about 800, about 800 to about 900, about 900 to about 1,000, about 1,000 to about 1,100, about 1,100 to about 1,200, about 1,200 to about 1,300, about 1,300 to about 1,400, or about 1,400 to about 1,500, copies of the detectable label(s).
  • one copy of the DNA dendrimer and/or the second DNA dendrimer comprises or is configured to bind to from about 10 to about 1,500, preferably from about 40 to about 1,500, from about 900 to about 1,100, copies of the test reagent(s), e.g., about 10 to about 50, about 50 to about 100, about 100 to about 200, about 200 to about 300, about 300 to about 400, about 400 to about 500, about 500 to about 600, about 600 to about 700, about 700 to about 800, about 800 to about 900, about 900 to about 1,000, about 1,000 to about 1,100, about 1,100 to about 1,200, about 1,200 to about 1,300, about 1,300 to about 1,400, or about 1,400 to about 1,500, copies of the test reagent(s).
  • the test reagent(s) e.g., about 10 to about 50, about 50 to about 100, about 100 to about 200, about 200 to about 300, about 300 to about 400, about 400 to about 500, about 500 to about 600, about 600 to about 700, about 700
  • one copy of the DNA dendrimer and/or the second DNA dendrimer comprises or is configured to bind to from about 10 to about 1,500, preferably from about 40 to about 1,500, from about 900 to about 1,100, copies of the detectable label(s) and the test reagent(s), e.g., about 10 to about 50, about 50 to about 100, about 100 to about 200, about 200 to about 300, about 300 to about 400, about 400 to about 500, about 500 to about 600, about 600 to about 700, about 700 to about 800, about 800 to about 900, about 900 to about 1,000, about 1,000 to about 1,100, about 1,100 to about 1,200, about 1,200 to about 1,300, about 1,300 to about 1,400, or about 1,400 to about 1,500, copies of the detectable label(s) and the test reagent(s).
  • the detectable label(s) and the test reagent(s) e.g., about 10 to about 50, about 50 to about 100, about 100 to about 200, about 200 to about 300, about 300
  • one copy of the DNA dendrimer and/or the second DNA dendrimer comprises or is configured to bind to from about 10 to about 1,500, preferably from about 40 to about 1,500 or from about 900 to about 1,100, the detectable label molecules or particles.
  • any suitable DNA dendrimer can be used.
  • the DNA dendrimers disclosed and/or claimed in the U.S. patent Nos. 5,175,270, 5,487,973, 6,046,038, 6,072,043, 6,110,687, 6,117,631 and 6,274,723 Bl can be used.
  • the DNA dendrimers can comprise any suitable number of layers.
  • the DNA dendrimer comprises a one-layer, a two-layer, a three-layer or a four-layer structure.
  • the DNA dendrimer and/or the second DNA dendrimer, before the test device is used, can be dried on a location on the test device upstream from the test zone, and at least a suitable percentage of the dried DNA dendrimer and/or the second DNA dendrimer can be capable of being moved to the test zone by a liquid.
  • the DNA dendrimer and/or the second DNA dendrimer, before the test device is used is dried on a location on the test device upstream from the test zone, and at least about 5%, 10%, 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100 % of the dried DNA dendrimer and/or the second DNA dendrimer is capable of being moved to the test zone by a liquid.
  • the DNA dendrimer and/or the second DNA dendrimer can be dried on a location on the test device in the presence of a material that: a) stabilizes the dried DNA dendrimer and/or the second DNA dendrimer; b) facilitates solubilization or resuspension of the dried DNA dendrimer and/or the second DNA dendrimer in a liquid; and/or c) facilitates mobility of the dried DNA dendrimer and/or the second DNA dendrimer.
  • a material can be used.
  • the material can be a protein, a peptide, a polysaccharide, a sugar, a polymer, a gelatin, a detergent, or a combination thereof.
  • Any suitable sugar can be used.
  • Exemplary sugar can be trehalose and/or sucrose.
  • the liquid can comprise a material that: a) stabilizes the dried DNA dendrimer and/or the second DNA dendrimer; b) facilitates solubilization or resuspension of the dried DNA dendrimer and/or the second DNA dendrimer in the liquid; and/or c) facilitates mobility of the dried DNA dendrimer and/or the second DNA dendrimer.
  • a material can be used.
  • the material can be a protein, a peptide, a polysaccharide, a sugar, a polymer, a gelatin, a detergent, or a combination thereof.
  • Any suitable protein can be used.
  • Exemplary protein can be BSA.
  • Any suitable detergent can be used.
  • Exemplary detergent can be Tween-20.
  • the liquid can have any suitable volume.
  • the liquid has a volume of at least about 20 ⁇ , 25 ⁇ , 30 ⁇ , 35 ⁇ , 40 ⁇ , 45 ⁇ , 50 ⁇ , 55 ⁇ , 60 ⁇ , 65 ⁇ , 70 ⁇ , 75 ⁇ , 80 ⁇ , 85 ⁇ , 90 ⁇ , 95 ⁇ , 100 ⁇ , 110 ⁇ , 120 ⁇ , 150 ⁇ , 200 ⁇ , 300 ⁇ , 400 ⁇ , 500 ⁇ , or a volume larger than 500 ⁇ .
  • the DNA dendrimer and/or the second DNA dendrimer can comprise: a) an one-layer DNA dendrimer that comprises from about 1-2, 2-4 or 4-8 of the detectable label(s) and/or the test reagent(s); b) a two-layer DNA dendrimer that comprises from about 1-4, 4-8 or 8-18 of the detectable label(s) and/or the test reagent(s); c) a three-layer DNA dendrimer that comprises from about 2-6, 6-10, 10-18, 18-28, 28-38, 38-50 or more than 50 of the detectable label(s) and/or the test reagent(s); and/or d) a four-layer DNA dendrimer that comprises from about 15-25, 25-45, 45-60, 60-80, 85-105, 105-130 or more than 130 of the detectable label(s) and/or the test reagent(s). [0085] In some embodiments, the present disclosure provides for a
  • kits for detecting an analyte in a liquid sample comprising: a) a porous matrix that comprises a test zone on said porous matrix, said test zone comprising a test reagent that binds to an analyte or another binding reagent that binds to said analyte, or is an analyte or an analyte analog that competes with an analyte in said sample for binding to a binding reagent for said analyte; and b) at least 1, 2, 3, 4 or all of: 1) a detectable label; 2) a DNA dendrimer; 3) a test reagent; 4) a
  • polyvalent linker that comprises a second label; and/or 5) a second DNA dendrimer, wherein said detectable label, DNA dendrimer, test reagent, polyvalent linker and second DNA dendrimer are described in connection with the test devices above.
  • the detectable label, DNA dendrimer, test reagent, polyvalent linker and second DNA dendrimer can be placed at any suitable location(s) in the kit. In some embodiments, at least 1, 2, 3, 4 or all of the detectable label, DNA dendrimer, test reagent, polyvalent linker and second DNA dendrimer can be dried on the porous matrix upstream from the test zone. In other embodiments, at least 1, 2, 3, 4 or all of the detectable label, DNA dendrimer, test reagent, polyvalent linker and second DNA dendrimer can be contained in a container.
  • the container can be placed at any suitable location(s) in the kit.
  • the container can be a part of the test device.
  • the container can be separate from the test device.
  • the container is configured to be in fluid
  • the present kit can comprise any of the test devices of claims 1-115.
  • the present kit can comprise any suitable number of the test devices, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more of the test devices.
  • the present disclosure provides for a method for detecting an analyte in a liquid sample, which method comprises: a) contacting a liquid sample with the test device or kit described in the above Section B, wherein the liquid sample is applied to a site of the test device upstream of the test zone; b) transporting an analyte, if present in the liquid sample, a detectable label and a DNA dendrimer to the test zone; and c) assessing the presence, absence, and/or amount of a signal generated by the detectable label at the test zone to determining the presence, absence and/or amount of the analyte in the liquid sample.
  • the present methods can be used in any suitable assay formats.
  • the DNA dendrimer(s), the various test reagent(s) and/or the detectable label(s) can be premixed with a sample liquid and/or developing liquid, and then the mixture is applied to the test device to initiate an assay.
  • one or more of the DNA dendrimer(s), the various test reagent(s) and/or the detectable label(s) can be dried on a suitable location of the test device, and a sample liquid and/or developing liquid is applied to the test device to initiate an assay.
  • the liquid sample and at least one of: 1) the detectable label, e.g., the first label; 2) the DNA dendrimer; 3) the test reagent; 4) the polyvalent linker that comprises a second label; and/or 5) the second DNA dendrimer are premixed to form a mixture and the mixture is applied to the test device.
  • the liquid sample and at least two of: 1) the detectable label, e.g., the first label; 2) the DNA dendrimer; 3) the test reagent; 4) the polyvalent linker that comprises a second label; and/or 5) the second DNA dendrimer are premixed to form a mixture and the mixture is applied to the test device.
  • the liquid sample and at least 3, 4 or all of: 1) the detectable label, e.g., the first label; 2) the DNA dendrimer; 3) the test reagent; 4) the polyvalent linker that comprises a second label; and/or 5) the second DNA dendrimer are premixed to form a mixture and the mixture is applied to the test device.
  • the present methods can be conducted in a liquid comprising a surfactant or detergent, e.g., Tween-20.
  • the method is conducted in a liquid comprising from about 0.001% (v/v) to about 5% (v/v), preferably, from about 0.001% (v/v) to about 0.01% (v/v), from about 0.01% (v/v) to about 0.5% (v/v), or at about 0.01% (v/v) or less Tween-20.
  • the present methods can also be conducted in a liquid comprising a polymer, e.g., dextran sulfate.
  • the method is conducted in a liquid comprising from about 0.1% (v/v) to about 5% (v/v), preferably from about 0.5% (v/v) to about 1% (v/v), dextran sulfate.
  • a substance can be dried on a portion of the test device upstream from the test zone, the dried substance can be solubilized or resuspended, and transported to the test zone and/or a control zone to generate a detectable signal.
  • a substance is dried on a portion of the test device upstream from the test zone, the dried substance is solubilized or resuspended, and transported to the test zone and/or a control zone to generate a detectable signal, the dried substance being at least 1, 2, 3, 4 or all of: 1) the detectable label; 2) the DNA dendrimer; 3) the test reagent; 4) the polyvalent linker that comprises a second label; and/or 5) the second DNA dendrimer.
  • a substance is dried on a portion of the test device upstream from the test zone, the dried substance is solubilized or resuspended, and transported to the test zone and/or a control zone to generate a detectable signal, the dried substance being at least 1, 2, 3 or all of: 1) the first label; 2) the DNA dendrimer; 3) the test reagent; and/or 4) the polyvalent linker that comprises a second label.
  • a substance is dried on a portion of the test device upstream from the test zone, the dried substance is solubilized or resuspended, and transported to the test zone and/or a control zone to generate a detectable signal, the dried substance being at least 1, 2 or all of: 1) the first label; 2) the DNA dendrimer; and/or 3) the test reagent that comprises a second label.
  • a substance is dried on a portion of the test device upstream from the test zone, the dried substance is solubilized or resuspended, and
  • the dried substance being at least 1, 2, 3, 4 or all of: 1) the first label; 2) the DNA dendrimer; 3) the test reagent; 4) the polyvalent linker that comprises a second label; and/or 5) the second DNA dendrimer.
  • the substance can be dried on any suitable location of the test device.
  • the dried substance is located downstream from the sample application site, and the dried substance is solubilized or resuspended, and transported to the test zone and/or a control zone by the liquid sample.
  • the dried substance is located upstream from the sample application site, and the dried substance is solubilized or resuspended, and transported to the test zone and/or a control zone by another liquid.
  • the dried substance is solubilized or resuspended, and transported to the test zone and/or a control zone by the liquid sample alone.
  • the analyte and/or dried substance is solubilized or resuspended, and transported to the test zone and/or a control zone by another liquid. In yet other embodiments, the analyte and/or dried substance is solubilized or resuspended, and transported to the test zone and/or a control zone by a mixture of a sample liquid and another liquid.
  • the present methods can be used to assess an analyte in any suitable sample.
  • the present methods can be used to assess an analyte in a body fluid sample, e.g., a whole blood, a serum, a plasma and a urine sample.
  • the present methods can be used to assess an analyte in a sample derived from a biological, a forensics, a food, a biowarfare, or an environmental source.
  • the present methods can be used for any suitable purpose.
  • the present methods can be used to quantify or semi-quantify the amount of an analyte in a liquid sample.
  • the present methods can be used to detect multiple analytes in a liquid sample.
  • the present methods can be used to quantify or semi-quantify the amounts of the multiple analytes in the liquid sample.
  • the present methods can be used to assess any suitable analyte.
  • the present methods can be used to assess an analyte selected from the group consisting of a cell, a virus and a molecule.
  • the signal generated by the detectable label at the test zone can be enhanced relative to a signal generated by the detectable label at the test zone using a DNA dendrimer comprising a smaller number of the detectable label(s) and/or the test reagent(s).
  • the signal generated by the detectable label at the test zone is enhanced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, or 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 folds or higher relative to a signal generated by the detectable label at the test zone using a DNA dendrimer comprising a smaller number of the detectable label(s) and/or the test reagent(s).
  • the signal generated by the detectable label at the test zone can be enhanced relative to a signal generated by the detectable label at the test zone without using a DNA dendrimer.
  • the signal generated by the detectable label at the test zone is enhanced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, or 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 folds or higher relative to a signal generated by the detectable label at the test zone without using a DNA dendrimer.
  • the present methods are automated and/or are used in high throughput format.
  • Figure 1A illustrates exemplary signal amplification via dendrimer binding to a gold conjugate: Version 1 - biotin dendrimer captured by an anti-biotin gold particle.
  • a sandwich assay using a capture binder, e.g., a capture antibody, and a detection binder, e.g., a detection antibody, is illustrated.
  • the capture antibody is immobilized in a test zone of the test device.
  • the detection antibody is biotinylated.
  • a capture binder e.g., a capture antibody
  • detection binder e.g., a detection antibody
  • antibody/analyte/detection antibody sandwich is formed at the test zone.
  • a gold particle containing anti-biotin antibodies binds to the biotinylated detection antibody to generate a detectable signal at the test zone.
  • a DNA dendrimer containing biotin moieties on the dendrimer "core” and on extended “arms” is also used. Multiple anti-biotin-gold
  • nanoparticles bind to biotin labels on the DNA dendrimer to form a DNA dendrimer/gold nanoparticles conjugate.
  • the conjugate via the biotin moieties, binds to the anti-biotin antibodies in the gold particle, which in turn, binds to the biotinylated detection antibody in the sandwich to enhance the detectable signal at the test zone.
  • Figure IB illustrates exemplary "dry-down" schematic for Version 1 for a linear unidirectional lateral flow assay format.
  • the biotinylated detection antibody, the biotinylated DNA dendrimer and the anti-biotin gold conjugate are dried on a conjugate pad, upstream from the test line and the control line.
  • Layout A from upstream to downstream, the order of the dried down substances is the anti-biotin gold conjugate, the biotinylated DNA dendrimer and the biotinylated detection antibody.
  • the order of the dried down substances is the biotinylated detection antibody, the anti-biotin gold conjugate, and the biotinylated DNA dendrimer.
  • a sample liquid and/or a further liquid, e.g., a developing fluid are added to the sample pad to move the dried biotinylated detection antibody, the biotinylated DNA dendrimer and the anti-biotin gold conjugate to the test line and the control line (in this case located on a nitrocellulose membrane) to generate a detectable signal at the test line (if the analyte is present the sample liquid) and the control line (if the assay is properly conducted).
  • the Version 1 can also be used with an exemplary lateral flow device with dual-flow-paths, e.g., two non-parallel different flow paths that are substantially
  • the biotinylated detection antibody and the anti-biotin gold conjugate are dried on a first flow path (Strip 1) upstream from the junction between the two flow paths and the biotinylated DNA dendrimer and the anti-biotin gold conjugate are dried on a second flow path (Strip 2) upstream from the junction between the two flow paths.
  • the junction between the two flow paths is upstream from the test line and the control line located on the Strip 2.
  • a sample liquid and/or a further liquid are added to both the Strip 1 and Strip 2 to move the dried biotinylated detection antibody, the biotinylated DNA dendrimer and the anti-biotin gold conjugate to the junction between the Strip 1 and Strip 2, and eventually to the test line and the control line (in this case located on the Strip 2) to generate a detectable signal at the test line (if the analyte is present the sample liquid) and the control line (if the assay is properly conducted).
  • Figure 2A illustrates exemplary signal amplification via dendrimer binding to a gold conjugate: Version 2 - biotin dendrimer captured by an anti-analyte/anti-biotin gold particle.
  • a sandwich assay using a capture binder, e.g., a capture antibody, and a detection binder, e.g., a detection antibody, is illustrated.
  • the capture antibody is immobilized in a test zone of the test device.
  • the detection antibody is contained in a gold particle containing both anti-analyte antibodies and anti-biotin antibodies capable of binding to both analyte and biotinylated dendrimer.
  • a capture antibody/analyte/detection antibody sandwich is formed at the test zone to generate a detectable signal at the test zone.
  • a DNA dendrimer containing biotin moieties on the dendrimer "core” and on extended “arms” is also used.
  • Multiple anti-biotin-gold nanoparticles bind to biotin labels on the DNA dendrimer to form a DNA dendrimer/gold nanoparticles conjugate.
  • the conjugate via the biotin moieties, binds to the anti-biotin antibodies in the gold particle in the sandwich to enhance the detectable signal at the test zone.
  • Figure 2B illustrates exemplary "dry-down" schematic for Version 2 for a linear unidirectional lateral flow assay format.
  • the gold particle containing both anti-analyte antibodies and anti-biotin antibodies and the biotinylated DNA dendrimer, and optionally the anti-biotin gold conjugate are dried on a conjugate pad, upstream from the test line and the control line.
  • a sample liquid and/or a further liquid are added to the sample pad to move the dried the gold particle containing both anti-analyte antibodies and anti-biotin antibodies and the biotinylated DNA dendrimer, and optionally the anti-biotin gold conjugate, to the test line and the control line (in this case located on a nitrocellulose membrane) to generate a detectable signal at the test line (if the analyte is present the sample liquid) and the control line (if the assay is properly conducted).
  • a further liquid e.g., a developing fluid
  • the Version 2 can also be used with an exemplary lateral flow device with dual-flow-paths, e.g., two non-parallel different flow paths that are substantially
  • the gold particle containing both anti-analyte antibodies and anti-biotin antibodies is dried on a first flow path (Strip 1) upstream from the junction between the two flow paths and the biotinylated DNA dendrimer and the gold particle containing both anti-analyte antibodies and anti-biotin antibodies, and optionally, the anti-biotin gold conjugate, are dried on a second flow path (Strip 2) upstream from the junction between the two flow paths.
  • the junction between the two flow paths is upstream from the test line and the control line located on the Strip 2.
  • a sample liquid and/or a further liquid are added to both the Strip 1 and Strip 2 to move the dried the gold particle containing both anti-analyte antibodies and anti-biotin antibodies, biotinylated DNA dendrimer, and optionally, anti-biotin gold conjugate, to the junction between the Strip 1 and Strip 2, and eventually to the test line and the control line (in this case located on the Strip 2) to generate a detectable signal at the test line (if the analyte is present the sample liquid) and the control line (if the assay is properly conducted).
  • a further liquid e.g., a developing fluid
  • Figure 3A illustrates exemplary signal amplification via dendrimer binding to a gold conjugate: Version 3 - biotin/FITC dendrimer captured by an anti-analyte/anti-biotin gold particle.
  • a sandwich assay using a capture binder, e.g., a capture antibody, and a detection binder, e.g., a detection antibody, is illustrated.
  • the capture antibody is immobilized in a test zone of the test device.
  • the detection antibody is contained in a gold particle containing both anti-analyte antibodies and anti-biotin antibodies capable of binding to both analyte and biotinylated dendrimer.
  • a capture antibody/analyte/detection antibody sandwich is formed at the test zone to generate a detectable signal at the test zone.
  • a DNA dendrimer containing FITC moieties on the dendrimer "core” and biotin moieties on extended “arms” is also used.
  • Multiple anti-FITC-gold nanoparticles bind to FITC labels on the DNA dendrimer to form a DNA dendrimer/gold nanoparticles conjugate.
  • the conjugate, through the biotinylated DNA dendrimer binds to the gold particle containing both anti-analyte antibodies and anti-biotin antibodies in the sandwich to enhance the detectable signal at the test zone.
  • Figure 3B illustrates exemplary "dry-down" schematic for Version 3 for a linear unidirectional lateral flow assay format.
  • the gold particle containing both anti-analyte antibodies and anti-biotin antibodies, the biotinylated DNA dendrimer that also contains FITC and the anti-FITC gold conjugate are dried on a conjugate pad, upstream from the test line and the control line.
  • a sample liquid and/or a further liquid e.g.
  • a developing fluid are added to the sample pad to move the dried the gold particle containing both anti-analyte antibodies and anti-biotin antibodies, the biotinylated DNA dendrimer that also contains FITC and the anti-FITC gold conjugate to the test line and the control line (in this case located on a nitrocellulose membrane) to generate a detectable signal at the test line (if the analyte is present the sample liquid) and the control line (if the assay is properly conducted).
  • the Version 3 can also be used with an exemplary lateral flow device with dual-flow-paths, e.g. , two non-parallel different flow paths that are substantially
  • the gold particle containing both anti-analyte antibodies and anti-biotin antibodies is dried on a first flow path (Strip 1) upstream from the junction between the two flow paths and the biotinylated DNA dendrimer that also contains FITC and the anti- FITC gold conjugate are dried on a second flow path (Strip 2) upstream from the junction between the two flow paths.
  • Strip 1 first flow path
  • Strip 2 second flow path
  • the junction between the two flow paths is upstream from the test line and the control line located on the Strip 2.
  • a sample liquid and/or a further liquid e.g.
  • FIG. 4A illustrates exemplary signal amplification via dendrimer binding to a gold conjugate: Version 4 - biotin dendrimer as both primary and secondary amplifiers.
  • a sandwich assay using a capture binder, e.g. , a capture antibody, and a detection binder, e.g. , a detection antibody, is illustrated.
  • the capture antibody is immobilized in a test zone of the test device.
  • the detection antibody is linked to an oligonucleotide to form an antibody-oligo conjugate.
  • a capture antibody/analyte/detection antibody sandwich is formed at the test zone.
  • primary DNA dendrimer complementary to the oligonucleotide in the antibody-oligo conjugate
  • secondary DNA dendrimer biotinylated DNA dendrimer that does not contain another oligonucleotide complementary to the oligonucleotide in the antibody-oligo conjugate
  • anti-biotin gold conjugates are also used.
  • the anti-biotin gold conjugates bind to the biotinylated primary DNA dendrimer that binds to the antibody-oligo conjugate in the sandwich to generate a detectable signal at the test zone.
  • the primary DNA dendrimer, through the anti-biotin gold conjugates, can be further linked to additional secondary DNA dendrimer(s), which in turn, is or are linked to additional anti-biotin gold conjugates to further enhance the detectable signal at the test zone.
  • Figure 4B illustrates exemplary "dry-down" schematic for Version 4 for a linear unidirectional lateral flow assay format.
  • the anti-biotin gold conjugate and secondary DNA dendrimer are dried on a conjugate pad, upstream from the test line and the control line.
  • a sample liquid and/or a further liquid e.g.
  • a developing fluid, the antibody-oligo conjugate and the primary DNA dendrimer are premixed and added to the sample pad to move the dried anti-biotin gold conjugate and secondary DNA dendrimer to the test line and the control line (in this case located on a nitrocellulose membrane) to generate a detectable signal at the test line (if the analyte is present the sample liquid) and the control line (if the assay is properly conducted).
  • the Version 4 can also be used with an exemplary lateral flow device with dual-flow-paths, e.g., two non-parallel different flow paths that are substantially
  • the anti-biotin gold conjugate is dried on a first flow path (Strip 1) upstream from the junction between the two flow paths and the anti-biotin gold conjugate and the secondary DNA dendrimer are dried on a second flow path (Strip 2) upstream from the junction between the two flow paths.
  • the junction between the two flow paths is upstream from the test line and the control line located on the Strip 2.
  • a sample liquid and/or a further liquid e.g., a developing fluid
  • the antibody-oligo conjugate and the primary DNA dendrimer are premixed and added to the Strip 1
  • a further liquid is added to Strip 2 to move the dried anti-biotin gold conjugate and secondary DNA dendrimer to the junction between the Strip 1 and Strip 2, and eventually to the test line and the control line (in this case located on the Strip 2) to generate a detectable signal at the test line (if the analyte is present the sample liquid) and the control line (if the assay is properly conducted).
  • Figure 5 A illustrates an exemplary signal amplification via dendrimer binding to a gold conjugate: Version 5 - FITC dendrimer captured by an anti-analyte/anti-FITC gold particle in an LF assay with a biotinylated analyte binding capture antibody binding to avidin/streptavidin test line.
  • a sandwich assay using a capture binder, e.g., a capture antibody, and a detection binder, e.g., a detection antibody, is illustrated.
  • the capture antibody is biotinylated and is configured to be immobilized in a test zone of the test device via binding to avidin/streptavidin on test line.
  • the detection antibody is contained in a gold particle containing both anti-analyte antibodies and anti-FITC antibodies capable of binding to both analyte and a DNA dendrimer that contains FITC.
  • a capture binder e.g., a capture antibody
  • antibody/analyte/detection antibody sandwich is formed at the test zone to generate a detectable signal at the test zone.
  • a DNA dendrimer containing FITC moieties on the dendrimer "core” and on extended “arms” is also used.
  • nanoparticles bind to FITC moieties on the DNA dendrimer to form a DNA dendrimer/gold nanoparticles conjugate.
  • the conjugate binds to the gold particle containing both anti-analyte antibodies and anti-FITC antibodies in the sandwich to enhance the detectable signal at the test zone.
  • Figure 5B illustrates exemplary "dry-down" schematic for Version 5 for a linear unidirectional lateral flow assay format.
  • the gold particle containing both anti-analyte antibodies and anti-FITC antibodies, the DNA dendrimer that contains FITC and the anti-FITC gold conjugate are dried on a conjugate pad, upstream from the test line and the control line.
  • a sample liquid and/or a further liquid, e.g., a developing fluid, and the biotinylated capture antibody are added to the sample pad to move the dried gold particle containing both anti-analyte antibodies and anti-FITC antibodies, DNA dendrimer that contains FITC and anti-FITC gold conjugate to the test line and the control line (in this case located on a nitrocellulose membrane) to generate a detectable signal at the test line (if the analyte is present the sample liquid) and the control line (if the assay is properly conducted).
  • the Version 5 can also be used with an exemplary lateral flow device with dual-flow-paths, e.g., two non-parallel different flow paths that are substantially
  • the gold particle containing both anti-analyte antibodies and anti-FITC antibodies is dried on a first flow path (Strip 1) upstream from the junction between the two flow paths and the DNA dendrimer that contains FITC and the anti-FITC gold conjugate are dried on a second flow path (Strip 2) upstream from the junction between the two flow paths.
  • the junction between the two flow paths is upstream from the test line and the control line located on the Strip 2.
  • a sample liquid and/or a further liquid e.g., a developing fluid, and the biotinylated capture antibody are added to the Strip 1 to move the dried gold particle containing both anti-analyte antibodies and anti- FITC antibodies, and a sample liquid and/or a further liquid is added to the Strip 2 to move the dried DNA dendrimer that contains FITC and the anti-FITC gold conjugate, to the junction between the Strip 1 and Strip 2, and eventually to the test line and the control line (in this case located on the Strip 2) to generate a detectable signal at the test line (if the analyte is present the sample liquid) and the control line (if the assay is properly conducted).
  • a sample liquid and/or a further liquid e.g., a developing fluid, and the biotinylated capture antibody
  • Figure 6 A illustrates exemplary signal amplification via dendrimer binding to a gold conjugate: Version 6 - FITC dendrimer as both primary and secondary amplifiers.
  • a sandwich assay using a capture binder, e.g., a capture antibody, and a detection binder, e.g., a detection antibody, is illustrated.
  • the capture antibody is immobilized in a test zone of the test device.
  • the detection antibody is linked to an oligonucleotide to form an antibody-oligo conjugate.
  • a capture antibody/analyte/detection antibody sandwich is formed at the test zone.
  • DNA dendrimer complementary to the oligonucleotide in the antibody-oligo conjugate (primary DNA dendrimer), a DNA dendrimer that contains FITC but does not contain another
  • oligonucleotide complementary to the oligonucleotide in the antibody-oligo conjugate secondary DNA dendrimer
  • anti-FITC gold conjugates are also used.
  • the primary DNA dendrimer, through the anti-FITC gold conjugates can be further linked to additional secondary DNA dendrimer(s), which in turn, can be linked to additional anti-FITC gold conjugates to enhance the detectable signal at the test zone.
  • Figure 6B illustrates exemplary "dry-down" schematic for Version 6 for a linear unidirectional lateral flow assay format.
  • the anti-FITC gold conjugate and secondary DNA dendrimer are dried on a conjugate pad, upstream from the test line and the control line.
  • a sample liquid and/or a further liquid, e.g., a developing fluid, the antibody-oligo conjugate and the primary DNA dendrimer are premixed and added to the sample pad to move the dried anti-FITC gold conjugate and secondary DNA dendrimer to the test line and the control line (in this case located on a nitrocellulose membrane) to generate a detectable signal at the test line (if the analyte is present the sample liquid) and the control line (if the assay is properly conducted).
  • the Version 6 can also be used with an exemplary lateral flow device with dual-flow-paths, e.g., two non-parallel different flow paths that are substantially
  • the anti-FITC gold conjugate is dried on a first flow path (Strip 1) upstream from the junction between the two flow paths and the anti-FITC gold conjugate and the secondary DNA dendrimer are dried on a second flow path (Strip 2) upstream from the junction between the two flow paths.
  • the junction between the two flow paths is upstream from the test line and the control line located on the Strip 2.
  • a sample liquid and/or a further liquid e.g., a developing fluid
  • the antibody-oligo conjugate and the primary DNA dendrimer are premixed and added to the Strip 1 and a further liquid is added to Strip 2 to move the dried anti-FITC gold conjugate and secondary DNA dendrimer to the junction between the Strip 1 and Strip 2, and eventually to the test line and the control line (in this case located on the Strip 2) to generate a detectable signal at the test line (if the analyte is present the sample liquid) and the control line (if the assay is properly conducted).
  • Figure 7A illustrates an exemplary, signal amplification via dendrimer binding to a gold conjugate: Version 7 - FITC dendrimer captured by an anti-biotin/anti-FITC gold particle bound to a biotinylated detection antibody in an LF assay.
  • a sandwich assay using a capture binder, e.g., a capture antibody, and a detection binder, e.g., a detection antibody, is illustrated.
  • the capture antibody is immobilized in a test zone of the test device.
  • the detection antibody is biotinylated.
  • a capture antibody/analyte/detection antibody sandwich is formed at the test zone.
  • a gold particle containing anti-biotin antibodies and anti-FITC antibodies binds to the biotinylated detection antibody to generate a detectable signal at the test zone.
  • a DNA dendrimer containing FITC moieties on the dendrimer "core” and on extended “arms” is also used.
  • Multiple anti-FITC-gold nanoparticles bind to FITC labels on the DNA dendrimer to form a DNA dendrimer/gold nanoparticles conjugate.
  • the conjugate, via the FITC labels binds to the anti-FITC antibodies in the gold particle, which in turn, via the anti-biotin antibodies, binds to the biotinylated detection antibody in the sandwich to enhance the detectable signal at the test zone.
  • FIG. 7B illustrates exemplary "dry-down" schematic for Version 7 for a linear unidirectional lateral flow assay format.
  • the biotinylated detection antibody, the DNA dendrimer containing FITC moieties and the gold particle containing anti-biotin antibodies and anti-FITC antibodies are dried on a conjugate pad, upstream from the test line and the control line.
  • Layout A from upstream to downstream, the order of the dried down substances is the gold particle containing anti-biotin antibodies and anti-FITC antibodies, the DNA dendrimer containing FITC moieties and the biotinylated detection antibody.
  • the order of the dried down substances is the biotinylated detection antibody, the gold particle containing anti-biotin antibodies and anti-FITC antibodies and the DNA dendrimer containing FITC moieties.
  • a sample liquid and/or a further liquid, e.g., a developing fluid are added to the sample pad to move the dried biotinylated detection antibody, DNA dendrimer containing FITC moieties and gold particle containing anti-biotin antibodies and anti-FITC antibodies to the test line and the control line (in this case located on a nitrocellulose membrane) to generate a detectable signal at the test line (if the analyte is present the sample liquid) and the control line (if the assay is properly conducted).
  • the Version 7 can also be used with an exemplary lateral flow device with dual-flow-paths, e.g., two non-parallel different flow paths that are substantially
  • the biotinylated detection antibody and the gold particle containing anti-biotin antibodies and anti-FITC antibodies are dried on a first flow path (Strip 1) upstream from the junction between the two flow paths and the DNA dendrimer containing FITC moieties and the anti-FITC-gold nanoparticles are dried on a second flow path (Strip 2) upstream from the junction between the two flow paths.
  • Strip 1 first flow path
  • Strip 2 second flow path
  • a sample liquid and/or a further liquid are added to both the Strip 1 and Strip 2 to move the dried biotinylated detection antibody, gold particle containing anti-biotin antibodies and anti-FITC antibodies, DNA dendrimer containing FITC moieties and anti-FITC-gold nanoparticles to the junction between the Strip 1 and Strip 2, and eventually to the test line and the control line (in this case located on the Strip 2) to generate a detectable signal at the test line (if the analyte is present the sample liquid) and the control line (if the assay is properly conducted).
  • a further liquid e.g., a developing fluid
  • Figure 8A illustrates exemplary signal amplification via dendrimer binding to a gold conjugate: Version 8 - FITC dendrimer captured by an anti-FITC gold particle bound to a FITC labeled detection antibody in an LF assay.
  • a sandwich assay using a capture binder, e.g., a capture antibody, and a detection binder, e.g., a detection antibody, is illustrated.
  • the capture antibody is immobilized in a test zone of the test device.
  • the detection antibody is labeled with FITC.
  • a capture antibody/analyte/detection antibody sandwich is formed at the test zone.
  • a gold particle containing anti-FITC antibodies binds to the FITC labeled detection antibody to generate a detectable signal at the test zone.
  • a DNA dendrimer containing FITC moieties on the dendrimer "core” and on extended “arms” is also used.
  • Multiple anti-FITC-gold nanoparticles bind to FITC labels on the DNA dendrimer to form a DNA dendrimer/gold nanoparticles conjugate.
  • the conjugate via the FITC labels, binds to the anti-FITC antibodies in the gold particle, which in turn binds to the FITC labeled detection antibody in the sandwich to enhance the detectable signal at the test zone.
  • Figure 8B illustrates exemplary "dry-down" schematic for Version 8 for a linear unidirectional lateral flow assay format.
  • the FITC labeled detection antibody, anti-FITC gold conjugate and FITC labeled DNA dendrimer are dried on a conjugate pad, upstream from the test line and the control line.
  • Layout A from upstream to downstream, the order of the dried down substances is the anti-FITC gold conjugate, the FITC labeled DNA dendrimer and the FITC labeled detection antibody.
  • Layout B from upstream to downstream, the order of the dried down substances is the FITC labeled detection antibody, the anti-FITC gold conjugate and the FITC labeled DNA dendrimer.
  • a sample liquid and/or a further liquid are added to the sample pad to move the dried the FITC labeled detection antibody, the anti-FITC gold conjugate and the FITC labeled DNA dendrimer to the test line and the control line (in this case located on a nitrocellulose membrane) to generate a detectable signal at the test line (if the analyte is present the sample liquid) and the control line (if the assay is properly conducted).
  • a further liquid e.g., a developing fluid
  • the Version 8 can also be used with an exemplary lateral flow device with dual-flow-paths, e.g., two non-parallel different flow paths that are substantially
  • the FITC labeled detection antibody and the anti-FITC gold conjugate are dried on a first flow path (Strip 1) upstream from the junction between the two flow paths and the FITC labeled DNA dendrimer and the anti-FITC gold conjugate are dried on a second flow path (Strip 2) upstream from the junction between the two flow paths.
  • the junction between the two flow paths is upstream from the test line and the control line located on the Strip 2.
  • a sample liquid and/or a further liquid are added to both the Strip 1 and Strip 2 to move the dried FITC labeled detection antibody, anti-FITC gold conjugate and FITC labeled DNA dendrimer to the junction between the Strip 1 and Strip 2, and eventually to the test line and the control line (in this case located on the Strip 2) to generate a detectable signal at the test line (if the analyte is present the sample liquid) and the control line (if the assay is properly conducted).
  • Figure 9A illustrates exemplary signal amplification via dendrimer binding to a gold conjugate: Version 9 - FITC dendrimer captured by an anti-analyte/anti-FITC gold particle.
  • a sandwich assay using a capture binder, e.g., a capture antibody, and a detection binder, e.g., a detection antibody, is illustrated.
  • the capture antibody is immobilized in a test zone of the test device.
  • the detection antibody is contained in a gold particle containing both anti-analyte antibodies and anti- FITC antibodies capable of binding to both analyte and FITC labelled dendrimer.
  • a capture antibody/analyte/detection antibody sandwich is formed at the test zone to generate a detectable signal at the test zone.
  • a DNA dendrimer containing FITC moieties on the dendrimer "core” and on extended “arms” is also used.
  • Multiple anti-FITC-gold nanoparticles bind to FITC labels on the DNA dendrimer to form a DNA dendrimer/gold nanoparticles conjugate.
  • the conjugate binds to the gold particle containing both anti-analyte antibodies and anti-FITC antibodies in the sandwich to enhance the detectable signal at the test zone.
  • Figure 9B illustrates exemplary "dry-down" schematic for Version 9 for a linear unidirectional lateral flow assay format.
  • the FITC labeled DNA dendrimer and the gold particle containing both anti-analyte antibodies and anti-FITC antibodies, and optionally the anti-FITC gold conjugate are dried on a conjugate pad, upstream from the test line and the control line.
  • a sample liquid and/or a further liquid are added to the sample pad to move the dried FITC labeled DNA dendrimer and gold particle containing both anti-analyte antibodies and anti-FITC antibodies, and optionally anti-FITC gold conjugate, to the test line and the control line (in this case located on a nitrocellulose membrane) to generate a detectable signal at the test line (if the analyte is present the sample liquid) and the control line (if the assay is properly conducted).
  • the Version 9 can also be used with an exemplary lateral flow device with dual-flow-paths, e.g., two non-parallel different flow paths that are substantially
  • the gold particle containing both anti-analyte antibodies and anti-FITC antibodies is dried on a first flow path (Strip 1) upstream from the junction between the two flow paths and the FITC labeled DNA dendrimer and the gold particle containing both anti-analyte antibodies and anti-FITC antibodies, and optionally the anti-FITC gold conjugate, are dried on a second flow path (Strip 2) upstream from the junction between the two flow paths.
  • Strip 1 first flow path
  • strip 2 second flow path upstream from the junction between the two flow paths.
  • the junction between the two flow paths is upstream from the test line and the control line located on the Strip 2.
  • a sample liquid and/or a further liquid are added to both the Strip 1 and Strip 2 to move the dried FITC labeled DNA dendrimer and the gold particle containing both anti-analyte antibodies and anti-FITC antibodies, and optionally the anti-FITC gold conjugate, to the junction between the Strip 1 and Strip 2, and eventually to the test line and the control line (in this case located on the Strip 2) to generate a detectable signal at the test line (if the analyte is present the sample liquid) and the control line (if the assay is properly conducted).
  • a further liquid e.g., a developing fluid
  • FIG. 10A illustrates exemplary signal amplification via dendrimer binding to a gold conjugate: Version 10 - biotin dendrimer captured by an anti-biotin/anti-FITC gold particle bound to a FITC labeled detection antibody in an LF assay.
  • a sandwich assay using a capture binder, e.g., a capture antibody, and a detection binder, e.g., a detection antibody, is illustrated.
  • the capture antibody is immobilized in a test zone of the test device.
  • the detection antibody is labeled with FITC.
  • a capture antibody/analyte/detection antibody sandwich is formed at the test zone.
  • a gold particle containing anti-biotin antibodies and anti-FITC antibodies binds to the FITC labeled detection antibody to generate a detectable signal at the test zone.
  • a DNA dendrimer containing biotin moieties on the dendrimer "core” and on extended “arms” is also used. Multiple anti-biotin-gold
  • nanoparticles bind to biotin labels on the DNA dendrimer to form a DNA dendrimer/gold nanoparticles conjugate.
  • the conjugate via the biotin labels, binds to the anti-biotin antibodies in the gold particle, which in turn, via the anti-FITC antibodies, binds to the FITC labeled detection antibody in the sandwich to enhance the detectable signal at the test zone.
  • Figure 10B illustrates exemplary "dry-down" schematic for Version 10 for a linear unidirectional lateral flow assay format.
  • the FITC labeled detection antibody, gold particle containing anti-biotin antibodies and anti-FITC antibodies, and the biotinylated DNA dendrimer are dried on a conjugate pad, upstream from the test line and the control line.
  • Layout A from upstream to downstream, the order of the dried down substances is the gold particle containing anti-biotin antibodies and anti-FITC antibodies, the biotinylated DNA dendrimer and the FITC labeled detection antibody.
  • the order of the dried down substances is the FITC labeled detection antibody, the gold particle containing anti-biotin antibodies and anti-FITC antibodies, and the biotinylated DNA dendrimer.
  • a sample liquid and/or a further liquid, e.g., a developing fluid are added to the sample pad to move the dried FITC labeled detection antibody, the gold particle containing anti-biotin antibodies and anti-FITC antibodies, and the biotinylated DNA dendrimer to the test line and the control line (in this case located on a nitrocellulose membrane) to generate a detectable signal at the test line (if the analyte is present the sample liquid) and the control line (if the assay is properly conducted).
  • the Version 10 can also be used with an exemplary lateral flow device with dual-flow-paths, e.g., two non-parallel different flow paths that are substantially
  • the FITC labeled detection antibody and the gold particle containing anti-biotin antibodies and anti-FITC antibodies are dried on a first flow path (Strip 1) upstream from the junction between the two flow paths and the biotinylated DNA dendrimer and the anti-biotin-gold nanoparticles are dried on a second flow path (Strip 2) upstream from the junction between the two flow paths.
  • Strip 1 first flow path
  • Strip 2 second flow path
  • a sample liquid and/or a further liquid are added to both the Strip 1 and Strip 2 to move the dried FITC labeled detection antibody, gold particle containing anti-biotin antibodies and anti-FITC antibodies, the biotinylated DNA dendrimer and the anti-biotin-gold nanoparticles to the junction between the Strip 1 and Strip 2, and eventually to the test line and the control line (in this case located on the Strip 2) to generate a detectable signal at the test line (if the analyte is present the sample liquid) and the control line (if the assay is properly conducted).
  • a further liquid e.g., a developing fluid

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Abstract

La présente invention concerne de nouveaux dispositifs à flux latéral et des kits utilisant des dendrimères d'ADN et une ou plusieurs étiquettes, ainsi que les procédés pour la détection d'un analyte à l'aide des dispositifs à flux latéral et des kits.
PCT/US2015/011853 2014-01-16 2015-01-16 Dosages à flux latéral au moyen de dendrimères d'adn Ceased WO2015109255A1 (fr)

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Cited By (5)

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CN105486854A (zh) * 2015-09-29 2016-04-13 南方医科大学 一种信号放大方法及相关装置
WO2017196139A1 (fr) * 2016-05-13 2017-11-16 광주과학기술원 Kit de diagnostic
WO2019071051A1 (fr) * 2017-10-04 2019-04-11 The Broad Institute, Inc. Diagnostics basés sur un système effecteur crispr
WO2019245744A1 (fr) 2018-06-18 2019-12-26 Becton, Dickinson And Company Systèmes, dispositifs et procédés permettant d'amplifier des signaux d'un dosage à écoulement latéral
WO2022258829A1 (fr) * 2021-06-11 2022-12-15 Ludwig-Maximilians-Universität München Nanostructure de marquage pour amplification de signal dans des dosages immunologiques et dosages immunologiques faisant appel à la nanostructure de marquage

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US20020165179A1 (en) * 2000-05-12 2002-11-07 Baker James R. Multifunctional nanodevice platform
US20100136614A1 (en) * 2005-10-18 2010-06-03 Dan Luo Dendrimer-like modular delivery vector
US20110160090A1 (en) * 2008-05-05 2011-06-30 Los Alamos National Laboratory Nanocrystal-Based Lateral Flow Microarrays and Low-Voltage Signal Detection Systems

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020165179A1 (en) * 2000-05-12 2002-11-07 Baker James R. Multifunctional nanodevice platform
US20100136614A1 (en) * 2005-10-18 2010-06-03 Dan Luo Dendrimer-like modular delivery vector
US20110160090A1 (en) * 2008-05-05 2011-06-30 Los Alamos National Laboratory Nanocrystal-Based Lateral Flow Microarrays and Low-Voltage Signal Detection Systems

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105486854A (zh) * 2015-09-29 2016-04-13 南方医科大学 一种信号放大方法及相关装置
CN105486854B (zh) * 2015-09-29 2017-12-05 南方医科大学 一种信号放大方法及相关装置
WO2017196139A1 (fr) * 2016-05-13 2017-11-16 광주과학기술원 Kit de diagnostic
WO2019071051A1 (fr) * 2017-10-04 2019-04-11 The Broad Institute, Inc. Diagnostics basés sur un système effecteur crispr
CN111630162A (zh) * 2017-10-04 2020-09-04 博德研究所 基于crispr效应系统的诊断
US11633732B2 (en) 2017-10-04 2023-04-25 The Broad Institute, Inc. CRISPR effector system based diagnostics
WO2019245744A1 (fr) 2018-06-18 2019-12-26 Becton, Dickinson And Company Systèmes, dispositifs et procédés permettant d'amplifier des signaux d'un dosage à écoulement latéral
JP2021529940A (ja) * 2018-06-18 2021-11-04 ベクトン・ディキンソン・アンド・カンパニーＢｅｃｔｏｎ， Ｄｉｃｋｉｎｓｏｎ Ａｎｄ Ｃｏｍｐａｎｙ 側方流動アッセイのシグナルを増幅させるシステム、装置および方法
EP3807618A4 (fr) * 2018-06-18 2022-01-19 Becton, Dickinson and Company Systèmes, dispositifs et procédés permettant d'amplifier des signaux d'un dosage à écoulement latéral
JP7451431B2 (ja) 2018-06-18 2024-03-18 ベクトン・ディキンソン・アンド・カンパニー 側方流動アッセイのシグナルを増幅させるシステム、装置および方法
WO2022258829A1 (fr) * 2021-06-11 2022-12-15 Ludwig-Maximilians-Universität München Nanostructure de marquage pour amplification de signal dans des dosages immunologiques et dosages immunologiques faisant appel à la nanostructure de marquage

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