WO2000055627A1 - Biopuce en plastique peu fluorescente reutilisable - Google Patents

Biopuce en plastique peu fluorescente reutilisable Download PDF

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Publication number
WO2000055627A1
WO2000055627A1 PCT/US2000/006866 US0006866W WO0055627A1 WO 2000055627 A1 WO2000055627 A1 WO 2000055627A1 US 0006866 W US0006866 W US 0006866W WO 0055627 A1 WO0055627 A1 WO 0055627A1
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Prior art keywords
biochip
biological binding
target analyte
nucleic acid
target
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PCT/US2000/006866
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English (en)
Inventor
Herbert L. Heyneker
Raymond Samaha
Kim Ha
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Eos Biotechnology, Inc.
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Publication date
Application filed by Eos Biotechnology, Inc. filed Critical Eos Biotechnology, Inc.
Priority to AU37500/00A priority Critical patent/AU3750000A/en
Publication of WO2000055627A1 publication Critical patent/WO2000055627A1/fr

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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
    • G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
    • G01N33/545—Synthetic resin

Definitions

  • the invention relates to novel biochips for the detection of target analytes.
  • the biochips comprise an array of ligands attached to a non-fluorescent acrylic that finds use in fluorescence detection and other methodologies.
  • One approach to detect ligand/target analyte interactions utilizes an array of ligands attached to a solid support.
  • Supports for ligand arrays have included porous membranes, such as, nitrocellulose or nylon. Detection methods using porous membranes are limited, owing to autofluorescence and scattering, to radioactive, chemiluminescent, and colorimetric techniques (Ross et al. 1992. In Techniques for the analysis of complex genomes (ed. Rakesh
  • ligand arrays have been produced on glass surfaces which can be mass produced, certaom of which are not autofluorescent and, therefore, are compatible with fluorescent detection methods. Fluorescent detection is the method of choice when using microarrays because, unlike most radioactive and chemiluminescent signals, fluorescent signals do not disperse and therefore allow for very dense ligand spacing in the array. Furthermore, glass surfaces when appropriately treated with silane or polylysine exhibit reduced nonspecific binding of target analytes, resulting in lower background than is typically encountered with porous membranes (Shalon et al. 1996. 6:639-645).
  • silane or polylysine coated slides will not readily withstand harsh conditions necessary to reduce autofluorescence and/or re-use due to the lability of the silane or polylysine coatings to harsh conditions, such as, elevated temperature and/or non-neutral pH, which are required for stripping the arrays of their target analytes after the hybridization and analysis steps.
  • non-autofluorescent solid support that is an alternative to glass that is a suitable for the construction of biochips that can be employed in high-sensitivity, fluorescence detection and other methodologies and that also are reusable.
  • the invention provides a biochip comprising a biological binding ligand immobilized to a non-fluorescent plastic support.
  • the invention provides a method of making a biochip comprising a biological binding ligand immobilized to a non-fluorescent plastic support.
  • the invention provides a method of detecting a target analyte in a sample comprising the steps of contacting a sample with a biochip of the invention under conditions that permit binding of the target analyte to at least one biological binding ligand attached to the biochip and detecting the presence of the target analyte.
  • the invention provides a method of stripping the bound target analyte from the biological binding ligand of the biochip and reuse of the biochip and binding ligand for further detection of a target analyte.
  • the invention provdes a kit comprising a biochip and at least one reagent.
  • the invention provides novel biochips comprising a substantially non-fluorescent plastic support containing an immobilized array of biological binding ligands.
  • the low background fluorescence of this plastic support in contrast to other plastics, permits the use of the biochip in fluorescence-based methodologies.
  • the novel biochip permits rigorous washing in order to reduce autofluorescence, thereby enhancing the signal to noise ratio.
  • the present invention provides biochips comprising a plurality of biological binding ligands immobilized onto a substantially non-fluorescent plastic support.
  • substantially nonfluorescent or “non-fluorescent” herein generally is meant from a low grade or dull fluorescence to completely non-fluorescent, preferably the fluorescence is as low as glass.
  • the fluorescent light if emitted from the support, does not interfere or compete with or mask the detection of the fluorescent signal of a ligand/target analyte binding pair assay complex.
  • the solid support emits very little light at the wavelength of the fluorescent label used in the assay, or, alternatively, the wavelength at which the solid support does fluorescence is sufficiently different from the emission wavelength of the label so as not to interfere with the detection of the label. Accordingly, solid support/label combinations are preferred.
  • the emission due to the solid support is only a small percentage of the label signal, i.e. preferably less than about 20% of the signal, with less than about 15% being preferred, and less than about 10 being especially preferred. This can be determined by taking a reading in an area different from that occupied by the labelled target analyte to give the background fluorescence.
  • the solid support is non- fluorescent, i.e. transparent.
  • plastics comprise plasticizers, UV protectants, and other additives that can cause background fluorescence. Accordingly, preferred embodiments utilize plastics that do not comprise these components.
  • the substantially non-fluorescent solid support is preferably a transparent plastic, such as acrylic or polypentene, and is generally smooth, can have any shape in cross-section and can be rigid or flexible.
  • the solid-support as described herein are depicted as a flat surface, which is only one of the possible conformations of the solid support and is for schematic purposes only.
  • the solid support may be a disc, square, sphere, circle, foam of closed-cells, etc.
  • the substrate is preferably flat but may take on a variety of alternative surface configurations. For example, the substrate may contain raised or depressed regions.
  • the conformation of the solid support will vary with the detection method used.
  • a flat planar solid-support may be preferred for optical detection methods, or when arrays of biological binding ligands are made, thus requiring addressable locations for both synthesis and detection.
  • the solid support may be in the form of a tube, with the biological binding ligands bound to the inner surface. This allows a maximum of surface area containing the biological binding ligands to be exposed to a small volume of sample.
  • the solid support is rigid or soft.
  • a soft support is easily manipulated to assume a variety of configurations.
  • the solid support is easily manipulated, cut or reshaped before or after arrayed.
  • plastic has an advantage over glass in that it will not shatter.
  • plasic supports can be re- positioned under or relative to a fixed detector.
  • the solid support also does not react adversely to chemicals and conditions used in the immobilization of the ligand array such that its physical, chemical properties, and opitcal properties are adversely altered.
  • the solid support also does not react adversely to chemicals and conditions used in the formation, detection, or removal of ligand/target analyte complexes. Therefore, the solid support can be manipulated as described below for the immobilization of the binding ligands for the construction of biochips that find use in a number of assays and techniques as described below.
  • the solid support is resistant to temperatures preferably of from about 0°C to about 100°C, more preferably from about 10°C to about 90°C, and even more preferably from about 20°C to about 80°C.
  • the solid support also is resistant to pH preferably from about 1 to about 14, and more preferably of about 5 to about 14.
  • the resistance of the chips to high concentrations of base i.e. 1 M NaOH, confers a number of benefits, including the ability to completely remove all non-covalently bound biomolecules.
  • the solid support is resistant to the chemicals and reagents used in either the generation of the biochip or the assay itself.
  • resistant and grammatical equivalents herein is meant that the optical, physical, and chemical properties of the solid support are not substantially altered in the construction, storage, or use of the biochip in such a way as to interfere or inhibit the immobilization of the ligand array or the detection and analysis of the target analyte.
  • the solid support is made of commercial grade close tolerance, UVT cast acrylic, available from Glasflex, (Glasflex, 4 Stirling Road, Stirling, New Jersey 07980). Cast acrylic is preferable, as extruded acrylic presumably comprises plasticizers and other components that may cause background fluorescence.
  • a potential solid support candidate is tested by scanning an area of the material at the wavelength of interest (i.e. the wavelength at which the biomolecule label fluoresces) and comparing it to a scan of the same area of a glass substrate, with those materials showing similar or less fluorescence being "substantially non-fluorescent" as defined herein.
  • the solid support is not made of substances having a substantial background fluoresence, such as, polymethylpentene (Westlake: TPX), polycarbonate (Westlake: Zelux), polyetherimide (Westlake: Ultem), polyphenylene oxide (Westlake: Noryl) , polyvinylidene fluoride (Westlake: Kynar), acetal (Westlake: Pomalux), Hytrel (Westlake), Ultraform (Westlake), lucite (ICI), clear acrylic (AIN plastic) , black acrylic (Allen extruders), clear acrylite (AIN), polypropylene (Sigma slides), extruded clear acrylic, black polystyrene, polycarbonate ZX (Cyro Industries), cell cast acrylite GP (Cyro Industries), impact modified acrylic colorless HP (Cyro Industries), continuously manufactured acrylic (Cyro Industries: Acrylite).
  • TPX polymethylpentene
  • Polycarbonate Westlake: Zelux
  • polyetherimide Westlake: Ultem
  • biochip or grammatical equivalents herein is meant a composition used to detect at least one target analyte.
  • target analyte or “analyte” or grammatical equivalents herein is meant any molecule, compound, particle, cell or substance to be detected.
  • target analytes preferably bind to at least one biological binding ligand.
  • a large number of analytes may be detected using the present methods; basically, any target analyte for which a biological binding ligand, described herein, may be made may be detected using the methods of the invention.
  • the biochip comprises a plurality of biological binding ligands.
  • plural and grammatical equivalents herein is meant at least two biological binding ligands.
  • biological binding ligand or grammatical equivalents herein is meant a compound that is used to probe for the presence of the target analyte, and that will bind to the analyte.
  • array and grammatical equivalents herein is meant a plurality of biological binding ligands in an array format; the size of the array will depend on the composition and end use of the array.
  • Arrays containing from about 2 different biological binding ligands to many millions can be made, with very large arrays being possible.
  • the array will comprise from two to as many as about 100,000 (all densities are per square cm) or more, depending on biological binding ligand, the size of the solid support, as well as the end use of the array, thus very high density, high density, moderate density, low density and very low density arrays may be made.
  • Preferred ranges for high density arrays are from about 10K to about 125K.
  • Moderate density arrays range from about IK to about 10K being preferred
  • Low density arrays are generally less than IK, with from about 0.1K to about IK being preferred.
  • Very low density arrays are less than 0.1K.
  • the biological binding ligands of the invention may not be in array format; that is, for some embodiments, biochips comprising a single ligand may be made as well.
  • multiple solid supports may be used, either of different or identical compositions. Thus for example, large arrays may comprise a plurality of smaller solid supports.
  • the composition of the biological binding ligand will depend on the composition of the target analyte.
  • Biological binding ligands for a wide variety of analytes are known or can be readily found using known techniques.
  • the biological binding ligands include proteins (particularly including antibodies or fragments thereof (FAbs, etc.)), small molecules, peptide "aptamers", or peptidomimmetic structures.
  • the binding ligand generally comprises traditional metal ion ligands or chelators.
  • Preferred biological binding ligand proteins include peptides.
  • suitable binding ligands include substrates and inhibitors.
  • Antigen-antibody pairs, receptor-ligands, and carbohydrates and their binding partners are also suitable analyte-binding ligand pairs.
  • the binding ligand may be nucleic acid, when nucleic acid binding proteins are the targets; alternatively, as is generally described in U.S. Patents 5,270,163, 5,475,096, 5,567,588, 5,595,877, 5,637,459, 5,683,867,5,705,337, and related patents, hereby incorporated by reference, nucleic acid "aptamers" can be developed for binding to virtually any target analyte.
  • the biological binding ligand is a nucleic acid.
  • Suitable analytes include organic and inorganic molecules, including biomolecules.
  • the analyte may be an environmental pollutant (including pesticides, insecticides, toxins, etc.); a chemical (including solvents, polymers, organic materials, etc.); therapeutic molecules (including therapeutic and abused drugs, antibiotics, etc.); biomolecules (including hormones, cytokines, proteins, lipids, carbohydrates, cellular membrane antigens and receptors (neural, hormonal, nutrient, and cell surface receptors) or their ligands, etc); whole cells (including procaryotic (such as pathogenic bacteria) and eukaryotic cells, including mammalian tumor cells); viruses (including retroviruses, herpesviruses, adenoviruses, lentiviruses, etc.); and spores; etc.; nucleic acids; proteins (including enzymes, antibodies, antigens, growth factors, cytokines, etc); therapeutic and abused drugs; cells; viruses; protozoa; and other infectious agents.
  • environmental pollutant including pesticides, insecticides,
  • the target analyte is a nucleic acid, i.e. a nucleic acid target sequence.
  • target sequence or grammatical equivalents herein means a nucleic acid sequence on a single strand of nucleic acid.
  • the target sequence may be chemically synthesized nucleic acid, such as, an oligonucleotide or a portion of a gene, a regulatory sequence, genomic DNA, cDNA, RNA including mRNA and rRNA, or others. It may be any length, with the understanding that longer sequences are more specific.
  • the complementary target sequence may take many forms. For example, it may be contained within a larger nucleic acid sequence, i.e.
  • probes are made to hybridize to target sequences to determine the presence or absence of the target sequence in a sample.
  • the target sequence may also be comprised of different target domains, for example, a first and second target domain which may be adjacent or separated.
  • first and second are not meant to confer an orientation of the sequences with respect to the 5'-3' orientation of the entire target sequence. For example, assuming a 5'-3' orientation of the complementary target sequence, the first target domain may be located either 5' to the second domain, or 3' to the second domain.
  • the target analyte is a protein.
  • protein herein is meant at least two covalently attached amino acids, which includes proteins, polypeptides, oligopeptides and peptides.
  • the protein may be made up of naturally occurring amino acids and peptide bonds, or synthetic peptidomimetic structures.
  • amino acid or “peptide residue”, as used herein means both naturally occurring and synthetic amino acids. For example, homo-phenylalanine, citrulline and norleucine are considered amino acids for the purposes of the invention.
  • Amino acid also includes imino acid residues such as proline and hydroxyproline.
  • the side chains may be in either the (R) or the (S) or L configuration. In the preferred embodiment, the amino acids are in the (S) or L-configuration. If non- naturally occurring side chains are used, non-amino acid substituents may be used, for example to prevent or retard in vivo degradations.
  • Suitable protein target analytes include, but are not limited to, (1) immunoglobulins, particularly IgEs, IgGs and IgMs, and particularly therapeutically or diagnostically relevant antibodies, including but not limited to, for example, antibodies to human albumin, apolipoproteins (including apolipoprotein E), human chorionic gonadotropin, cortisol, ⁇ - fetoprotein, thyroxin, thyroid stimulating hormone (TSH), antithrombin, antibodies to pharmaceuticals (including antiepileptic drugs (phenytoin, primidone, carbariezepin, ethosuximide, valproic acid, and phenobarbitol), cardioactive drugs (digoxin, lidocaine, procainamide, and disopyramide), bronchodilators ( theophylline), antibiotics
  • immunoglobulins particularly IgEs, IgGs and IgMs
  • therapeutically or diagnostically relevant antibodies including but not limited to, for example, antibodies to human album
  • viruses including orthomyxoviruses, (e.g. influenza virus), paramyxoviruses (e.g respiratory syncytial virus, mumps virus, measles virus), adenoviruses, rhinoviruses, coronaviruses, reoviruses, togaviruses (e.g. rubella virus), flaviviruses (e.g. yellow fever virus), parvoviruses, poxviruses (e.g. influenza virus), paramyxoviruses (e.g respiratory syncytial virus, mumps virus, measles virus), adenoviruses, rhinoviruses, coronaviruses, reoviruses, togaviruses (e.g. rubella virus), flaviviruses (e.g. yellow fever virus), parvoviruses, poxviruses (e.g.
  • orthomyxoviruses e.g. influenza virus
  • paramyxoviruses e.g
  • variola virus vaccinia virus
  • enteroviruses e.g. poliovirus, coxsackievirus
  • hepatitis viruses including A, B and C
  • herpesviruses e.g. Herpes simplex virus, varicella-zoster virus, cytomegalovirus, Epstein-Barr virus
  • rotaviruses Norwalk viruses
  • bunyavirus e.g., hantaan virus
  • arenavirus rhabdovirus
  • retroviruses including HIV, HTLV-I and -II
  • papovaviruses e.g.
  • bacteria including a wide variety of pathogenic and non-pathogenic prokaryotes of interest including Bacillus; Vibrio, e.g. V. cholerae; Escherichia, e.g. Enterotoxigenic E. coli, Shigella, e.g. S. dysenteriae; Salmonella, e.g. S. typhi; Mycobacterium e.g. M. tuberculosis, M. leprae; Clostridium, e.g. C. botulinum, C. tetani, C. difficile, C.perfringens;
  • Cornyebacterium e.g. C. diphtheriae; Streptococcus, S. pyogenes, S. pneumoniae; Staphylococcus, e.g. S. aureus; Haemophilus, e.g. H. influenzae; Neisseria, e.g. N. meningitidis, N. gonorrhoeae; Yersinia, e.g. Y. pestis, Pseudomonas, e.g. P. aeruginosa, P. putida; Chlamydia, e.g. C. trachomatis; Bordetella, e.g. B.
  • Treponema e.g. T. palladium
  • Rickettsia e.g. R. prowazekii, R. ricketsii and the like
  • protozoa including
  • Giardia e.g. G. lamblia, Entamoeba, E. histolytica
  • Plasmodia e.g. R. vivax, P. falciparum
  • Leishmania e.g., L. donovanii, E braziliensis
  • Trypanosoma e.g. T.
  • enzymes including but not limited to, enzymes used as indicators of or treatment for heart disease, including creatine kinase, lactate dehydrogenase, aspartate amino transferase, troponin T, myoglobin, fibrinogen, cholesterol, triglycerides, thrombin, tissue plasminogen activator (tPA); pancreatic disease indicators including amylase, lipase, chymotrypsin and trypsin; liver function enzymes, proteins, disease indicators including cholinesterase, bilirubin, and alkaline phosphatase; aldolase, prostatic acid phosphatase, terminal deoxynucleotidyl transferase, and bacterial and viral enzymes such as HIV protease; (3) hormones and cytokines (many of which serve as ligands for cellular receptors) such as erythropoietin (EPO), thrombopoietin (T)
  • the target analyte is a carbohydrate.
  • Suitable carbohydrate analytes include but are not limited to, markers for breast cancer (CA15-3, CA 549, CA 27.29), mucin-like carcinoma associated antigen (MCA), ovarian cancer (CA125), pancreatic cancer (DE-PAN-2), and colorectal and pancreatic cancer (CA 19, CA 50, CA242).
  • Other suitable carbohydrate analytes include, for example, carbohydrate moieties which bind to lectins.
  • any of the target analytes for which antibodies may be detected may be detected directly as well; that is, detection of virus, bacterial cells, protozoa and other microbes, therapeutic and abused drugs, etc., may be done directly.
  • the target analyte is a metal.
  • Suitable metal analytes include metal ions, particularly heavy and/or toxic metals, including but not limited to, aluminum, arsenic, cadmium, selenium, cobalt, copper, chromium, lead, silver and nickel.
  • the target analyte is a nucleic acid target sequence and the biological binding ligand is a nucleic acid probe.
  • nucleic acid and grammatical equivalents herein is meant two or more nucleotides joined together.
  • the nucleic acid analyte can be DNA, RNA, or cDNA, and can be naturally occurring or synthetic as well as analogs thereof.
  • the nucleic acid can have any combination of natural and synthetic bases, including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine, isoguanine, halogenated bases, etc.
  • nucleic acid analogs that may have alternate backbones, comprising, for example, phosphoramide (Beaucage et al., Tetrahedron 49(10):1925 (1993) and references therein; Letsinger, J. Org. Chem. 35:3800 (1970); Sblul et al, Eur. J. Biochem. 81 :579 (1977); Letsinger et al., Nucl.
  • the nucleic acid probes of the present invention are designed to be complementary to a target sequence (either the target sequence of the sample or to other probe sequences, as is described below, for example in sandwich assays), such that hybridization of the target sequence and the probes of the present invention occurs.
  • a target sequence either the target sequence of the sample or to other probe sequences, as is described below, for example in sandwich assays
  • this complementarity need not be perfect; there may be any number of base pair mismatches which will interfere with hybridization between the target sequence and the single stranded nucleic acids of the present invention. However, if the number of mutations is so great that no hybridization can occur under even the least stringent of hybridization conditions, the sequence is not a complementary target sequence.
  • nucleic acid probe is generally single or double stranded but can be partially single and partially double stranded. The strandedness of the probe is dictated by the structure, composition, and properties of the target sequence.
  • a nucleic acid probe of the present invention will generally contain phosphodiester bonds, although in some cases, as outlined above, nucleic acid analogs are included that may have alternate backbones or synthetic bases. As will be appreciated by those in the art, all of these nucleic acid analogs may find use in the present invention. In addition, mixtures of naturally occurring nucleic acids and nucleic acid analogs can be made.
  • nucleic acids When nucleic acids are used as the biological binding ligands for binding of a target analyte, the nucleic acid probes range from about 6 to about 150 basepairs long, with from about 10 to about 100 base pairs being preferred, and from about 12-15 to about 75 bp being particularly preferred and 50 bp being the most preferred. In some embodiments, much longer nucleic acids can be used, up to hundreds of basepairs, for example, partial or full length cDNAs.
  • a protein biological binding ligand of the present invention is generally a protein, oligopeptide and peptide, derivatives and analogs, including proteins containing non-naturally occurring amino acids and amino acid analogs, and peptidomimetic structures, as described above. As discussed below, when the protein is used as a binding ligand, it may be desirable to utilize protein analogs to retard degradation by sample components or contaminants.
  • proteins When proteins are used as the biological binding ligands for binding of a target analyte, the proteins range from about 5 to about 200 amino acids long, with from about 10 to about 100 amino acids being preferred. In some embodiments, much longer proteins can be used, up to hundreds of amino acids.
  • the binding of the target analyte to the biological binding ligand is specific, and the biological binding ligand is part of a binding pair.
  • specifically bind herein is meant that the ligand binds the analyte, with specificity sufficient to differentiate between the analyte and other components or contaminants of the test sample.
  • binding which is not highly specific; for example, the systems may use different binding ligands, for example an array of different ligands, and detection of any particular analyte is via its "signature" of binding to a panel of binding ligands. This finds particular utility in the detection of chemical analytes.
  • the binding should be sufficient to remain bound under the conditions of the assay, including wash steps to remove non-specific binding.
  • the disassociation constants of the analyte to the biological binding ligand will be less than about 10 "4 -10 "6 M “1 , with less than about 10 "5 to 10 "!0 M “1 being preferred, less than about 10 "7 -10 “9 M “1 being particularly preferred and 10 "10 -10 " " M "1 being most preferred.
  • the biochips are made as follows.
  • the method of immobilizing the ligand arrays of the solid-supports of the present invention may vary. Preferred methods are outlined herein and are known in the art.
  • the biological binding ligands are immobilized to a substantially non-fluorescent plastic solid support.
  • immobilized and grammatical equivalents herein is meant the association or binding between the biological binding ligand and the solid support is sufficient to be stable under the conditions of target analyte binding, washing, analysis, and removal as outlined below.
  • the binding can be covalent or non-covalent.
  • non-covalent binding and grammatical equivalents herein is meant one or more of either electrostatic, hydrophilic, and hydrophobic interactions. Included in non-covalent binding is the covalent attachment of a molecule, such as, streptavidin to the support and the non-covalent binding of biotinylated biological binding ligand to the streptavidin.
  • covalent binding and grammatical equivalents herein is meant that the two moieties, the solid support and the biological binding ligand, are attached by at least one bond, including sigma bonds, pi bonds and coordination bonds. Covalent bonds can be formed directly between the biological binding ligand and the solid support or can be formed by a cross linker or by inclusion of a specific reactive group on either the solid support or the biological binding ligand or both molecules. Immobilization may also involve a combination of covalent and non-covalent interactions.
  • the biological binding ligands can be attached to the biochip in a wide variety of ways, as will be appreciated by those in the art.
  • the biological binding partner may be either synthesized on the chip, or can be synthesized first and attached later.
  • the surface of the biochip and the biological binding ligand may be derivatized with chemical functional groups for subsequent attachment of the two.
  • the biochip is derivatized with a chemical function group including, for example, amino groups, carboxy groups, oxo groups, thiol groups, and N-oxysuccinimide.
  • the binding ligands can be attached using functional groups on the binding ligands.
  • proteins and nucleic acids containing amino groups can be attached to modified surfaces, as described below or, for example using linkers as are known in the art; for example, homo-or hetero-bifunctional linkers as are well known (see 1994 Pierce Chemical Company catalog, technical section on cross-linkers, pages 155-200, incorporated herein by reference).
  • linkers may be used as is described below.
  • the biological binding ligands are oligonucleotides, i.e. nucleic acid probes.
  • the nucleic acids can either be synthesized first, with subsequent attachment to the biochip, or can be directly synthesized on the biochip.
  • the oligonucleotides are synthesized as is known in the art, and then attached to the surface of the solid support. As will be appreciated by those skilled in the art, either the 5 ' or 3' terminus may be attached to the solid support, or attachment may be via an internal nucleoside.
  • Methods of modifying the surface of the solid support include, for example, photochemical coupling, plasma treatment, chemical etchants, and chemical grafting.
  • composition of the surface and the method of attachment is as described in U.S. Patent Nos. 5,427,779; 4,973,493; 4,979,959; 5,002,582; 5,217,492; 5,258,041 and 5,263,992, and references cited therein, all of which are hereby expressly incorporated by reference.
  • coupling of oligonucleotides to the reactive surface of the solid-support can proceed by a number of methods.
  • photochemical coupling can proceed in one of two ways: a) the oligonucleotide is derivatized with a photoreactive group, followed by attachment to the surface; or b) the surface is first treated with a photoreactive group, followed by application of the oligonucleotide.
  • the activating agent comprises a N-oxysuccinimide group (NOS) linked to a photoactive group, that upon exposure to light generates a free radical which then inserts into carbon-carbon bonds of the surface, which is put on the surface first.
  • NOS N-oxysuccinimide group
  • aminated oligonucleotides can be attached to aldehyde surfaces (reductive chemistry) or can be attached to carboxy surfaces via carbodiimide mediated condensation.
  • Phosphorothioated oligos can be attached, for example, to bromoacetyl surfaces via a nucleophyhc substitution or can be attached to a maleimide surface via the -SH moiety.
  • Phosphorylated oligos can be attached for example to an amine surface by carbodiimide mediate condensation.
  • the immobilization to the solid support may be very strong, yet non-covalent.
  • biotinylated oligonucleotides can be made, which bind to surfaces covalently coated with streptavidin, resulting in attachment.
  • the biological binding ligand is immobilized to the solid support that is coated by an antibody.
  • the biological binding ligand is immobilized to the solid support that is coated by a polyamino acid, for example, polylysine.
  • the oligonucleotides may be synthesized on the surface, as is known in the art. Preferred methods are outlined herein and are known in the art; see WO 95/251 16; WO 95/35505; U.S. Patent Nos. 5,700,637 and 5,445,934; and references cited within, all of which are expressly incorporated by reference.
  • nucleic acids can be synthesized in situ, using well known photolithographic techniques, such as those described in U.S. Patent No. 5,445,934 and related applications and publications; these methods of attachment form the basis of the Affimetrix GeneChipTM technology.
  • Linker moieties may also be used to immobilize the biological binding ligand to the solid support.
  • linker moieties or grammatical equivalents, herein is meant molecules which serve to immobilize the biological binding ligand at a distance from the solid support.
  • Linker moieties have a first and a second end. In one embodiment, branched linker moieties are preferred. The first end of the linker moiety is used to covalently attach the linker moiety to the solid support. The second end is used for attachment to the biological binding ligand; for example, preferred linkers include, but are not limited to, alkyl groups (including substituted alkyl groups and alkyl groups containing heteroatom moieties), with short alkyl groups being preferred.
  • Preferred functional groups include but are not limited to esters, amide, amine, aldehyde, bromoacetyl, carboxy, oxo, thiol, N-oxysuccinimide, maleimide, epoxy groups and ethylene glycol and derivatives.
  • the binding ligands can be attached to the surface.
  • proteins and nucleic acids containing amino groups can be attached to modified surfaces, as described herein or, for example using linkers as are known in the art; for example, homo-or hetero-bifunctional linkers as are well known (see 1994 Pierce Chemical Company catalog, technical section on cross-linkers, pages 155-200, incorporated herein by reference).
  • linkers as are known in the art; for example, homo-or hetero-bifunctional linkers as are well known (see 1994 Pierce Chemical Company catalog, technical section on cross-linkers, pages 155-200, incorporated herein by reference).
  • binding ligands including proteins, lectins, nucleic acids, small organic molecules, carbohydrates, etc. can
  • compositions of the invention are useful in a wide variety of applications.
  • the compositions of the invention find use in the detection of target analytes in test samples.
  • the compositions of the present invention may be used in a variety of research, clinical, quality control, or field testing settings and other known applications (see for example Chetverin et al., Bio/Technology, Vol. 12, November 1994, ppl034-1099, (1994)).
  • the target analyte is prepared using known techniques.
  • the sample may be treated to lyse the cells, using known lysis buffers, electroporation, etc., with purification and/or amplification such as PCR occuring as needed, as will be appreciated by those in the art.
  • the target analyte is labeled with, for example, a fluorescent, a chemiluminescent, a chemical, or a radioactive signal, to provide a means of detecting the target analyte' s specific binding to a biological binding ligand.
  • fluorescent labels include Cy-3 and Cy-5.
  • the label also can be an enzyme, such as, alkaline phosphatase or horseradish peroxidase, which when provided with an appropriate substrate produces a product that can be detected.
  • the label can be a labeled compound or small molecule, such as an enzyme inhibitor, that binds but is not catalyzed or altered by the enzyme.
  • the label also can be a moiety or compound, such as, an epitope tag or biotin which specifically binds to streptavidin.
  • an epitope tag or biotin which specifically binds to streptavidin.
  • the streptavidin is labeled as described above, thereby, providing a detectable signal for the bound target analyte.
  • unbound labeled streptavidin is removed prior to analysis.
  • the target analyte binds to a biological binding ligand, unbound contaminants are removed, and the bound target analyte is detected using second ligand of the target analyte that is labeled, for example, labeled antibody, nucleic acid or other compound that specifically binds the target analyte.
  • the biochips are used to detect or quantify the presence of target nucleic acid sequences.
  • These assays can be direct hybridization assays or can comprise "sandwich assays", which include the use of multiple probes, as is generally outlined in U.S.
  • the target nucleic acid is prepared as outlined above, and then added to the biochip comprising a plurality of nucleic acid probes, under conditions that allow the formation of an assay complex (in the case of nucleic acid, this can also be referred to as a hybridization complex).
  • hybridization conditions including high, moderate and low stringency conditions; see for example Maniatis et al., Molecular Cloning:
  • Tm thermal melting point
  • Stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes (e.g. 10 to 50 nucleotides) and at least about 60 °C for long probes (e.g. greater than 50 nucleotides).
  • Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.
  • the hybridization conditions may also vary when a non-ionic backbone, i.e. PNA is used, as is known in the art.
  • cross-linking agents may be added after target binding to cross-link, i.e. covalently attach, the two strands of the hybridization complex.
  • the assays are generally run under stringency conditions which allows formation of the label probe hybridization complex only in the presence of target.
  • Stringency can be controlled by altering a step parameter that is a thermodynamic variable, including, but not limited to, temperature, formamide concentration, salt concentration, chaotropic salt concentration pH, organic solvent concentration, etc.
  • reaction may be accomplished in a variety of ways, as will be appreciated by those in the art. Components of the reaction may be added simultaneously, or sequentially, in any order, with preferred embodiments outlined below.
  • the reaction may include a variety of other reagents may be included in the assays. These include reagents like salts, buffers, neutral proteins, e.g. albumin, detergents, etc which may be used to facilitate optimal hybridization and detection, and/or reduce non-specific or background interactions. Also reagents that otherwise improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, anti-microbial agents, etc., may be used, depending on the sample preparation methods and purity of the target.
  • biochips are used in genetic diagnosis.
  • biochips can be made using the techniques disclosed herein to detect target sequences such as the gene for nonpolyposis colon cancer, the BRCA1 breast cancer gene, P53 and mutants thereof, which is a gene associated with a variety of cancers, the Apo E4 gene that indicates a greater risk of Alzheimer's disease, allowing for easy presymptomatic screening of patients, mutations in the cystic fibrosis gene, or any of the others well known in the art.
  • the present invention also finds use as a methodology for the detection of mutations or mismatches in target nucleic acid sequences.
  • recent focus has been on the analysis of the relationship between genetic variation and phenotype by making use of polymorphic DNA markers.
  • Previous work utilized short tandem repeats (STRs) as polymorphic positional markers; however, recent focus is on the use of single nucleotide polymorphisms (SNPs), which occur at an average frequency of more than 1 per kilobase in human genomic DNA.
  • SNPs single nucleotide polymorphisms
  • microbial detection such as for viruses and bacteria
  • probes are designed to detect target sequences from a variety of bacteria and viruses.
  • current blood-screening techniques rely on the detection of anti-HIV antibodies.
  • the methods disclosed herein allow for direct screening of clinical samples to detect HIV nucleic acid sequences, particularly highly conserved HIV sequences. In addition, this allows direct monitoring of circulating virus within a patient as an improved method of assessing the efficacy of anti-viral therapies.
  • viruses associated with leukemia HTLV-I and HTLV-II
  • Bacterial infections such as tuberculosis, chlamydia, and other sexually transmitted diseases, may also be detected, for example using ribosomal RNA (rRNA) as the target sequences.
  • rRNA ribosomal RNA
  • the biochips of the invention find use in the screening of water and food samples for toxic bacteria and parasites.
  • samples may be treated to lyse the bacteria or parasites to release their nucleic acid (particularly rRNA or DNA), and then probes designed to recognize bacterial strains, including, but not limited to, such pathogenic strains as, Salmonella, Campylobacter, Vibrio cholerae, Leishmania, enterotoxic strains of E coli, and Legionnaire's disease bacteria.
  • bioremediation strategies may be evaluated using the compositions of the invention.
  • the biochips are used for forensic "DNA fingerprinting" to match crime-scene DNA against samples taken from victims and suspects.
  • the biological binding ligands in an array are used for sequencing by hybridization.
  • compositions of the invention are useful to detect successful gene amplification in PCR, thus allowing successful PCR reactions to be an indication of the presence or absence of a target sequence.
  • PCR may be used in this manner in several ways.
  • the PCR reaction is done as is known in the art, and then added to a biochip of the invention.
  • PCR product is labeled to provide a means to detect the PCR product when bound to a biological binding ligand.
  • the PCR label can be bound to one or more of the oligonucleotide primers or can be incorporated into the PCR product during polymerization. The label also can be detected directly or indirectly.
  • label herein is generally meant a substance that directly emits or produces a detectable signal, such as, an enzyme, a fluorophore, a chemiluminescent compound, a radioactivity isotope and the like.
  • the label is a ligand or moiety, such as, biotin, that is specifically recognized and bound by a second substance or compound that emits a detectable signal.
  • the bottom of the plate was also labeled with the date and plate number.
  • the plate was mixed on a multitube vortexer and was covered in foil and stored in a cold box (4°C) when arraying on the same day. Alternatively, the plates were stored in a -20°C freezer until use.
  • the biochips were individually dated and numbered.
  • the array area of the biochip also is marked to indicate the placement of a coverslip used for binding to a target analyte.
  • N- oxysuccinamide (NOS) was added to the biochips at Surmodics (9924 West 74th Street, Eden Prairie, Minnesota 55344-3523) as outlined above prior to oligonucleotide transfer. Transfer and arraying of the oligonucleotide from the multiwell plates to the biochips was performed using a XYZ stage (Norgren Systems) using pins purchased from Telechem International (San Jose, CA).
  • the transferred oligonucleotides were coupled to the biochips by incubation at 37°C at high humidity (up to 100% humidity) for 2 hours or by incubation at 25°C at 78% humidity for at least 12 hours. Unbound NOS groups were blocked with ethanolamine (0.5 M Tris, 1 N ethanolamine, pH 9) by: i) vigorously dunking biochips in ethanolamine; ii) incubating biochips at 60°C for one hour in a black slide box; and iii) placing biochips on an orbital shaker for 15 minutes.
  • ethanolamine 0.5 M Tris, 1 N ethanolamine, pH 9
  • the biochips were rinsed three times in deionized H 2 O; stripped with 1 N NaOH in black slide boxes at 70°C for 1 hour; washed four times in deionized H 2 O; and spun dry in racks at 1000 RPM (1 lOXg). Slides were stored in slide boxes until needed.

Abstract

L'invention concerne des biopuces réutilisables servant à détecter des analytes voulus. Les biopuces comportent un groupement de ligands fixé sur un support acrylique non fluorescent, utile dans la détection par fluorescence et dans d'autres procédés.
PCT/US2000/006866 1999-03-15 2000-03-15 Biopuce en plastique peu fluorescente reutilisable WO2000055627A1 (fr)

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AU37500/00A AU3750000A (en) 1999-03-15 2000-03-15 Reusable low fluorescent plastic biochip

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US27021499A 1999-03-15 1999-03-15
US09/270,214 1999-03-15

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FR2838737A1 (fr) * 2002-04-23 2003-10-24 Centre Nat Rech Scient Supports solides fonctionnalises par des dendrimeres phosphores, leur procede de preparation et applications
US6908678B2 (en) 2002-01-18 2005-06-21 Advanced Gene Technology, Corp. Plastic slides for the fabrication of biochips
US6911339B2 (en) 2001-04-09 2005-06-28 David P. Dumas Transparent polymer support for organic synthesis
US6967074B2 (en) * 2000-11-08 2005-11-22 Surface Logix, Inc. Methods of detecting immobilized biomolecules
EP2301576A1 (fr) 2004-03-29 2011-03-30 Abbott Biotherapeutics Corp. Utilisation thérapeutique d'anticorps anti-CS1
EP2371391A1 (fr) 2003-05-08 2011-10-05 Abbott Biotherapeutics Corp. Utilisation thérapeutique d'anticorps anti-CS1

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EP0294105A2 (fr) * 1987-06-05 1988-12-07 Pall Corporation Polyamide non-fluorescente et non-réflective pour utilisation dans des épreuves diagnostiques
WO1995030774A1 (fr) * 1994-05-05 1995-11-16 Beckman Instruments, Inc. Groupements repetes d'oligonucleotides
EP0882980A1 (fr) * 1997-06-06 1998-12-09 Commissariat A L'energie Atomique Traitement de surface d'un substrat limitant sa fluorescence naturelle
DE19739119A1 (de) * 1997-09-06 1999-03-11 Univ Schiller Jena Mikrotiterplatte

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EP0063810A1 (fr) * 1981-04-29 1982-11-03 Ciba-Geigy Ag Dispositif et trousses pour les analyses immunologiques
EP0294105A2 (fr) * 1987-06-05 1988-12-07 Pall Corporation Polyamide non-fluorescente et non-réflective pour utilisation dans des épreuves diagnostiques
WO1995030774A1 (fr) * 1994-05-05 1995-11-16 Beckman Instruments, Inc. Groupements repetes d'oligonucleotides
EP0882980A1 (fr) * 1997-06-06 1998-12-09 Commissariat A L'energie Atomique Traitement de surface d'un substrat limitant sa fluorescence naturelle
DE19739119A1 (de) * 1997-09-06 1999-03-11 Univ Schiller Jena Mikrotiterplatte

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6967074B2 (en) * 2000-11-08 2005-11-22 Surface Logix, Inc. Methods of detecting immobilized biomolecules
US6911339B2 (en) 2001-04-09 2005-06-28 David P. Dumas Transparent polymer support for organic synthesis
US6908678B2 (en) 2002-01-18 2005-06-21 Advanced Gene Technology, Corp. Plastic slides for the fabrication of biochips
FR2838737A1 (fr) * 2002-04-23 2003-10-24 Centre Nat Rech Scient Supports solides fonctionnalises par des dendrimeres phosphores, leur procede de preparation et applications
WO2003091304A2 (fr) * 2002-04-23 2003-11-06 Centre National De La Recherche Scientifique Supports solides fonctionnalises par des dendrimeres phosphores, leur procede de preparation et applications
WO2003091304A3 (fr) * 2002-04-23 2004-04-15 Centre Nat Rech Scient Supports solides fonctionnalises par des dendrimeres phosphores, leur procede de preparation et applications
US7517538B2 (en) 2002-04-23 2009-04-14 Centre National De La Recherche Scientifique Solid supports functionalised with phosphorus dendrimers, method for preparing same and uses thereof
US7658946B2 (en) 2002-04-23 2010-02-09 Centre National De La Recherche Scientifique Solid supports functionalized with phosphorus-containing dendrimers, process for preparing them and uses thereof
EP2371391A1 (fr) 2003-05-08 2011-10-05 Abbott Biotherapeutics Corp. Utilisation thérapeutique d'anticorps anti-CS1
EP2853272A1 (fr) 2003-05-08 2015-04-01 AbbVie Biotherapeutics Inc. Utilisation thérapeutique d'anticorps anti-CS1
EP3275463A1 (fr) 2003-05-08 2018-01-31 AbbVie Biotherapeutics Inc. Utilisation thérapeutique d'anticorps anti-cs1
EP2301576A1 (fr) 2004-03-29 2011-03-30 Abbott Biotherapeutics Corp. Utilisation thérapeutique d'anticorps anti-CS1

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