WO2006083269A2 - Luminescent nanosensors - Google Patents

Luminescent nanosensors Download PDF

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Publication number
WO2006083269A2
WO2006083269A2 PCT/US2005/016818 US2005016818W WO2006083269A2 WO 2006083269 A2 WO2006083269 A2 WO 2006083269A2 US 2005016818 W US2005016818 W US 2005016818W WO 2006083269 A2 WO2006083269 A2 WO 2006083269A2
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Prior art keywords
nanoprobe
antibody
molecule
analyte
labeled
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PCT/US2005/016818
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French (fr)
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WO2006083269A3 (en
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Rakesh Mogul
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Florida Atlantic University
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y15/00—Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • 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
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/588—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with semiconductor nanocrystal label, e.g. quantum dots

Definitions

  • the invention relates to sensors and nanoparticles including biosensors and methods for molecular identification.
  • Screening can involve detection of harmful chemicals, bacteria and viruses. For example, there is a need for early medical diagnostics, genomics assays, proteomics analyses, drug discovery screening, and detection of biological and chemical warfare agents for homeland security and defense. [0003] Screening can also be used to identify the presence or absence of medical diseases and infectious pathogens. Regarding blood, the use of inexpensive screening analyses can allow the rapid detection and improved treatments of many illnesses. Rapid and effective medical screening tests can also reduce the cost of health care by preventing unnecessary and costly reactive medical treatment.
  • a critical factor in many diagnostics is the rapid, selective, and sensitive detection of biochemical substances, such as proteins, metabolites, nucleic acids, biological species or living systems, such as bacteria, virus or related components at ultra-trace levels in samples provided.
  • biochemical substances such as proteins, metabolites, nucleic acids, biological species or living systems, such as bacteria, virus or related components at ultra-trace levels in samples provided.
  • biological samples can include tissues, blood and other bodily fluids.
  • a nanoparticle composition comprising a plurality of biomolecules attached thereto, detects analytes or target molecules with specificity and sensitivity.
  • the nanoparticle composition or nanoprobe utilizes a biomolecule and nanoparticle assembly that serves as a scaffold for a fluorescence competition assay.
  • a nanoprobe for detection of molecular interactions comprises a nanoparticle and a detectably labeled biomolecule attached thereto.
  • the nanoparticle is an inorganic or organic nanoparticle.
  • the nanoparticle is a metal.
  • the metal is selected from the group consisting of: gold, silver, copper and platinum.
  • the nanoparticle is selected from the group consisting of : ZnS, ZnO, TiO 2 , AgI, AgBr, HgI 2 , PbS, PbSe, ZnTe, CdTe, In 2 S 3 , In 2 S 3 ,
  • the nanoparticle is about 5 nm up to 100 nm in diameter., preferably, the nanoparticle is about 5 nm to about 20 nm in diameter.
  • a plurality of detectably labeled biomolecules are attached to the nanoparticle.
  • the biomolecule can be an antibody, aptamer, hapten, oligonucleotide, protein or peptide.
  • the biomolecule is labeled with a detectable moiety.
  • the detectable moiety is a fluorescent molecule.
  • Preferred fluorescent molecules comprises at least one of: biotin, fluorescein (5- carboxyfluorescein-N-hydroxysuccinimide ester), rhodamine (5,6-tetramethyl rhodamine),
  • 6-carboxyfluorescein (6-FAM), 2',4',1,4,- tetrachlorofluorescein (TET), 2 l ,4',5 l ,7',l,4-hexachlorofluorescein (HEX), 2',7'-dimethoxy-
  • 6-carboxyfluorescein NORD
  • 2'-chloro-7'-phenyl-l,4-dichloro-6-carboxyfluorescein VIC
  • fluorescein isothiocyanate FITC
  • 5,6-carboxymethyl fluorescein Texas red
  • NBD 6-carboxyfluorescein
  • VIC 2'-chloro-7'-phenyl-l,4-dichloro-6-carboxyfluorescein
  • FITC fluorescein isothiocyanate
  • 56-carboxymethyl fluorescein Texas red
  • nitrobenz-2- oxa-l,3-diazol-4-yl NBD
  • coumarin dansyl chloride
  • rhodamine amino-methyl coumarin
  • AMCA Eosin
  • Erythrosin BODIPYTM
  • Cascade BlueTM Oregon GreenTM
  • pyrene lissamine
  • xanthenes acridines
  • oxazines phycoerythrin
  • quantum dyeTM fluorescent energy transfer dyes
  • thiazole orange-ethidium heterodimer thiazole orange-ethidium heterodimer
  • the detectable moiety comprises a donor and acceptor molecule.
  • the donor molecule is a fluorophore and the acceptor molecule is a quencher.
  • a quencher molecule comprises at least one of: rhodamine dyes, tetramethyl-6-carboxyrhodamine (TAMRA), tetrapropano-6- carboxyrhodamine (ROX), DABSYL, DABCYL, cyanine dyes, nitrothiazole blue (NTB), anthraquinone, malachite green, nitrothiazole, or nitroimidazole compounds.
  • the biomolecules are covalently attached to the nanoparticle.
  • the biomolecules are attached to the nanoparticles maleimide linkages, carbodiimide linkages, lysine linkages, amine-reactive organic donors or quenchers.
  • the biomolecule comprises a first molecule bound to a second molecule.
  • the first molecule is labeled with a fluorescent moiety and the second molecule is labeled with a quenching moiety. The first molecule and second molecule do not emit a fluorescent signal when the first molecule and second molecule are associated together.
  • a method of detecting specific molecular interactions comprises providing a nanoprobe comprising a differentially labeled antibody- hapten complex; contacting the nanoprobe with an analyte, binding of the analyte to the antibody releases the labeled hapten; resulting in a change in fluorescence; and, detecting specific molecular interactions between the nanoprobe and analyte.
  • the antibody is labeled with a fluorophore and the hapten is labeled with a fluorophore quenching molecule.
  • the antibody is labeled with a fluorophore quenching molecule and the hapten is labeled with a fluorophore.
  • the nanoprobe is in a quenched fluorescent state as compared to fluorescence emitted when the hapten is released. Binding of the analyte to a detectably labeled antibody is detected by fluorescence as compared to a baseline fluorescence of nanoprobe.
  • the hapten comprises protein, peptide, oligonucleotide or organic molecule.
  • antibody-analyte binding is determined by affinity of the antibody for the analyte and dissociation constants (K D ) between an antibody- hapten complexes and antibody-analyte complexes.
  • K D dissociation constants
  • either the antibody or the hapten are fluorescently labeled i.e. one is labeled with a donor molecule and the other is labeled with a quenching molecule.
  • the antibody is labeled with a donor molecule and the hapten is labeled with a quenching molecule.
  • fluorescent molecules include: biotin, fluorescein (5-carboxyfluorescein-N-hydroxysuccinimide ester), rhodamine (5,6-tetramethyl rhodamine), Cy3, Cy3.5, Cy5, Cy5.5 and Cy7.
  • 6-carboxyfluorescein (6-FAM), 2',4',1,4,- tetrachlorofluorescein (TET), 2',4',5',7',l,4-hexachlorofluorescein (HEX), 2',7'-dimethoxy- 4',5'-dichloro-6-carboxyrhodamine (JOE), 2'-chloro-5 l -fluoro-7',8'-fused phenyl- 1,4-dichloro- 6-carboxyfluorescein (NED), 2'-chloro-7'-phenyl-l,4-dichloro-6-carboxyfluorescein (VIC), fluorescein isothiocyanate (FITC), 5,6-carboxymethyl fluorescein, Texas red, nitrobenz-2- oxa-l,3-diazol-4-yl (NBD), coumarin, dansyl chloride, rhodamine,
  • quencher molecules includes: rhodamine dyes, tetramethyl-6- carboxyrhodamine (TAMRA), tetrapropano-6-carboxyrhodamine (ROX), DABSYL, DABCYL, cyanine dyes, nitrothiazole blue (NTB), anthraquinone, malachite green, nitrothiazole, or nitroimidazole compounds.
  • TAMRA tetramethyl-6- carboxyrhodamine
  • ROX tetrapropano-6-carboxyrhodamine
  • DABSYL DABCYL
  • cyanine dyes nitrothiazole blue (NTB), anthraquinone, malachite green, nitrothiazole, or nitroimidazole compounds.
  • each antibody or aptamer can include a reporter such as a molecular beacon that changes fluorescence properties upon target binding, i.e. from quenched state to fluorescence state.
  • a reporter such as a molecular beacon that changes fluorescence properties upon target binding, i.e. from quenched state to fluorescence state.
  • Each species of, for example, antibody can be labeled with a different fluorescent dye to allow simultaneous detection of multiple target molecules, e.g., one species might be labeled with fluorescein and another with rhodamine.
  • the fluorescence excitation wavelength (or spectrum) can be varied and/or the emission spectrum can be observed to simultaneously detect the presence of multiple targets.
  • the fluorescence measurement can be performed with a number of different instruments, including standard fluorescence spectrophotometers, or in a small volume using a high-intensity source, such a laser, high-efficiency light collection optics, such as a high- numeric aperture microscope objective, and a high-efficiency low-noise detector, such as photo-multiplier tube, a photodiode or a CCD camera.
  • a high-intensity source such a laser
  • high-efficiency light collection optics such as a high- numeric aperture microscope objective
  • a high-efficiency low-noise detector such as photo-multiplier tube, a photodiode or a CCD camera.
  • the method can further include a computer program that includes instructions for causing the processor to compare the measured fluorescence emission or excitation spectrum with the known spectrum of each of the individual dyes to quantitatively determine the concentration of each of the target molecules in the solution.
  • the new detection systems can include pattern recognition software.
  • the software compares the target molecule binding pattern corresponding to the unknown sample with binding patterns corresponding to known compounds. From these comparisons, the software can determine the composition of the sample, or deduce information about the source of the sample.
  • the systems can be used to detect the existence of characteristic compounds, or "molecular fingerprints," associated with certain chemicals or conditions. For example, the systems can be used for human drug testing by detecting the presence of metabolites of particular drugs.
  • the systems can also be used to infer the existence of a disease (e.g., cancer) by detecting the presence of compounds associated with the disease state, or for pollution monitoring by detecting compounds characteristic of the discharge of certain pollutants. Numerous other applications are also possible.
  • Other aspects of the invention are described infra.
  • Figure 1 is a schematic illustration showing the detection strategy utilizing metal nanoparticles as the donor and/or quencher components.
  • Figures 2A and 2B are a schematic illustration showing that the fluorescence signal may arise from either the labeled antibody or hapten subsequent to analyte detection. Potential arrangements, as shown, include labeling of the antibody and hapten with a luminescent quantum dot (the donor) and a gold nanoparticle (the quencher), respectively, and the reverse configuration. These assemblies yield either fluorescent antibody ( Figure 2A) or hapten ( Figure 2B) products.
  • Figure 2A luminescent quantum dot
  • Figure 2B hapten
  • the invention describes a self-assembling fluorescent nanometer-sized probe that structurally and functionally integrates an analyte-specific protein (such as an antibody) with nanoparticles. Detection of the target analyte is afforded through a fluorescence competition assay thereby allowing for a high sensitivity.
  • analyte-specific protein such as an antibody
  • metal nanoparticles provides excellent spectral properties, as cadmium nanoparticles are brightly luminescent and gold nanoparticles possess luminescence quenching properties.
  • biomolecule refers to proteins and peptides, antibodies, aptamers,
  • DNA DNA, RNA (including mRNA, rRNA, tRNA and tmRNA), nucleotides and nucleosides.
  • the biomolecule is used to detect a complementary biomolecule. Examples include antibodies that detect antigens, oligonucleotides that detect complimentary oligonucleotides, and ligands that detect receptors. Such probes are preferably immobilized on a microelectrode comprising a substrate.
  • aptamer or "selected nucleic acid binding species” shall include non-modified or chemically modified RNA or DNA.
  • the method of selection may be by, but is not limited to, affinity chromatography and the method of amplification by reverse transcription (RT) or polymerase chain reaction (PCR).
  • the term “signaling aptamer” shall include aptamers with reporter molecules, preferably a fluorescent dye, appended to a nanoparticle in such a way that upon conformational changes resulting from the aptamer's interaction with a ligand, the reporter molecules yields a differential signal, preferably a change in fluorescence intensity.
  • reporter molecules preferably a fluorescent dye
  • the terms “ligand,” “target,” and “bait” are used interchangeably throughout the specification and includes any molecule that binds to the aptamer.
  • a “nucleic acid” is a polymeric compound comprised of covalently linked subunits called nucleotides.
  • Nucleic acid includes polyribonucleic acid (RNA) and polydeoxyribonucleic acid (DNA), both of which maybe single-stranded or double-stranded.
  • DNA includes cDNA, genomic DNA, synthetic DNA, and semi-synthetic DNA. The sequence of nucleotides or nucleic acid sequence that encodes a protein is called the sense sequence.
  • fragment or segment as applied to a nucleic acid sequence, gene or polypeptide, will ordinarily be at least about 5 contiguous nucleic acid bases (for nucleic acid sequence or gene) or amino acids (for polypeptides), typically at least about 10 contiguous nucleic acid bases or amino acids, more typically at least about 20 contiguous nucleic acid bases or amino acids, usually at least about 30 contiguous nucleic acid bases or amino acids, preferably at least about 40 contiguous nucleic acid bases or amino acids, more preferably at least about 50 contiguous nucleic acid bases or amino acids, and even more preferably at least about 60 to 80 or more contiguous nucleic acid bases or amino acids in length.
  • “Overlapping fragments” as used herein, refer to contiguous nucleic acid or peptide fragments which begin at the amino terminal end of a nucleic acid or protein and end at the carboxy terminal end of the nucleic acid or protein. Each nucleic acid or peptide fragment has at least about one contiguous nucleic acid or amino acid position in common with the next nucleic acid or peptide fragment, more preferably at least about three contiguous nucleic acid bases or amino acid positions in common, most preferably at least about ten contiguous nucleic acid bases amino acid positions in common.
  • oligonucleotide includes linear or circular oligomers of natural and/or modified monomers or linkages, including deoxyribonucleosides, ribonucleosides, substituted and alpha-anomeric forms thereof, peptide nucleic acids (PNA), locked nucleic acids (LNA), phosphorthiorate, methylphosphonate, and the like. Oligonucleotides are capable of specifically binding to a target polynucleotide by way of a regular pattern of monomer-to-monomer interactions, such as Watson-Crick type of base pairing, Ho ⁇ gsteen or reverse Ho ⁇ gsteen types of base pairing, or the like. [0043] The oligonucleotide may be "chimeric", that is, composed of different regions.
  • chimeric compounds are oligonucleotides, which contain two or more chemical regions, for example, DNA region(s), RNA region(s), PNA region(s) etc. Each chemical region is made up of at least one monomer unit, i.e., a nucleotide in the case of an oligonucleotide compound. These oligonucleotides typically comprise at least one region wherein the oligonucleotide is modified in order to exhibit one or more desired properties.
  • the desired properties of the oligonucleotide include, but are not limited, for example, to increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid.
  • the chimeric oligonucleotides of the present invention can be formed as mixed structures of two or more oligonucleotides, modified oligonucleotides, oligonucleosides and/or oligonucleotide analogs.
  • the oligonucleotide can be composed of regions that can be linked in "register", that is, when the monomers are linked consecutively, as in native DNA, or linked via spacers.
  • the spacers are intended to constitute a covalent "bridge” between the regions and have in preferred cases a length not exceeding about 100 carbon atoms.
  • the spacers may carry different functionalities, for example, having positive or negative charge, carry special nucleic acid binding properties (intercalators, groove binders, toxins, fluorophores etc.), being lipophilic, inducing special secondary structures like, for example, alanine containing peptides that induce alpha-helices.
  • nucleobase covers naturally occurring nucleobases as well as non-naturally occurring nucleobases.
  • nucleobase includes not only the known purine and pyrimidine heterocycles, but also heterocyclic analogues and tautomers thereof.
  • nucleobases are adenine, guanine, thymine, cytosine, uracil, purine, xanthine, diaminopurine, 8-oxo-N 6 -methyladenine, 7-deazaxanthine, 7-deazaguanine, N ⁇ N ⁇ ethanocytosin, N 6 ,N 6 -ethano-2,6-diaminopurine, 5-methylcytosine, 5- (C 3 -C 6 )-alkynylcytosine, 5-fluorouracil, 5-bromouracil, pseudoisocytosine, 2-hydroxy-5- methyl-4-triazolopyridin, isocytosine, isoguanin, inosine and the "non-naturally occurring" nucleobases described in Benner et al., U.S.
  • nucleobase is intended to cover every and all of these examples as well as analogues and tautomers thereof.
  • Especially interesting nucleobases are adenine, guanine, thymine, cytosine, and uracil, which are considered as the naturally occurring nucleobases in relation to therapeutic and diagnostic application in humans.
  • nucleoside includes the natural nucleosides, including 2'-deoxy and 2'-hydroxyl forms, e.g., as described in Kornberg and Baker, DNA Replication, 2nd Ed. (Freeman, San Francisco, 1992).
  • nucleosides in reference to nucleosides includes synthetic nucleosides having modified base moieties and/or modified sugar moieties, e.g., described generally by Scheit, Nucleotide Analogs, John Wiley, New York, 1980; Freier & Altmann, Nucl. Acid.
  • Such analogs include synthetic nucleosides designed to enhance binding properties, e.g., duplex or triplex stability, specificity, or the like.
  • the term "stability" in reference to duplex or triplex formation generally designates how tightly an antisense oligonucleotide binds to its intended target sequence; more particularly, “stability” designates the free energy of formation of the duplex or triplex under physiological conditions. Melting temperature under a standard set of conditions, e.g., as described below, is a convenient measure of duplex and/or triplex stability.
  • oligonucleotides of the invention are selected that have melting temperatures of at least 45 0 C when measured in 100 niM NaCl, 0.1 mM EDTA and 10 mM phosphate buffer aqueous solution, pH 7.0 at a strand concentration of both the oligonucleotide and the target nucleic acid of 1.5 ⁇ M.
  • duplex or triplex formation will be substantially favored over the state in which the antigen and its target are dissociated.
  • a stable duplex or triplex may in some embodiments include mismatches between base pairs and/or among base triplets in the case of triplexes.
  • modified oligonucleotides, e.g. comprising LNA units of the invention form perfectly matched duplexes and/or triplexes with their target nucleic acids.
  • Antibody refers to a polypeptide ligand substantially encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof, which specifically binds and recognizes an epitope (e.g., an antigen).
  • the recognized immunoglobulin genes include the kappa and lambda light chain constant region genes, the alpha, gamma, delta, epsilon and mu heavy chain constant region genes, and the myriad immunoglobulin variable region genes.
  • Antibodies exist, e.g., as intact immunoglobulins or as a number of well characterized fragments produced by digestion with various peptidases. This includes, e.g., Fab' and F(ab)' 2 fragments.
  • antibody also includes antibody fragments either produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA methodologies. It also includes polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies, or single chain antibodies. "Fc" portion of an antibody refers to that portion of an immunoglobulin heavy chain that comprises one or more heavy chain constant region domains, CH 2 and CH 3 , but does not include the CH 1 and the heavy chain variable region.
  • Immunoassay is an assay that uses an antibody to specifically bind an antigen (e.g., a marker).
  • the immunoassay is characterized by the use of specific binding properties of a particular antibody to isolate, target, and/or quantify the antigen.
  • the phrase “specifically (or selectively) binds" to an antibody or “specifically (or selectively) immunoreactive with,” when referring to a protein or peptide, refers to a binding reaction that is determinative of the presence of the protein in a heterogeneous population of proteins and other biologies.
  • the specified antibodies bind to a particular protein at least two times the background and do not substantially bind in a significant amount to other proteins present in the sample.
  • Specific binding to an antibody under such conditions may require an antibody that is selected for its specificity for a particular protein.
  • antibodies raised to a desired antigen from specific species such as rat, mouse, or human can be selected to obtain only those polyclonal antibodies that are specifically immunoreactive with the desired antigen and not with other proteins, except for polymorphic variants and alleles. This selection may be achieved by subtracting out antibodies that cross-react with the desired antigen molecules from other species.
  • a variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein.
  • solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein (see, e.g., Harlow & Lane, Antibodies, A Laboratory Manual (1988), for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity).
  • a specific or selective reaction will be at least twice background signal or noise and more typically more than 10 to 100 times background.
  • the dissociation constant (K D ) is a useful measure to describe the strength of binding (or affinity) between receptors and their ligands.
  • K D of antibody+antigen is about 10 "7 to 10 " ⁇ M (i.e. 10 '7 M is equivalent to 0.1 mM or 10OnM; TCR+MHC/peptide is about 10 "6 M.
  • the relationship can be written as an equation: [0054] kl (rate of dissociation) AB ⁇ A+B [0055] k2 (rate of association) A+B ⁇ AB
  • sample is used herein in its broadest sense.
  • a sample comprising polypeptides, peptides, antibodies, polynucleotides, organic molecules, and the like, may comprise a bodily fluid; a soluble fraction of a cell preparation, or media in which cells were grown; a chromosome, an organelle, or membrane isolated or extracted from a cell; genomic DNA, RNA, or cDNA, polypeptides, or peptides in solution or bound to a substrate; a cell; a tissue; a tissue print; a fingerprint, skin or hair; environmental samples; and the like.
  • infectious agent refers to an organism wherein growth/multiplication leads to pathogenic events in humans or animals. Examples of such agents are: bacteria , fungi, protozoa and viruses.
  • cancer refers to all types of cancer or neoplasm or malignant tumors found in mammals, including, but not limited to: leukemias, lymphomas, melanomas, carcinomas and sarcomas.
  • Examples of cancers are cancer of the brain, breast, pancreas, cervix, colon, head & neck, kidney, lung, non-small cell lung, melanoma, mesothelioma, ovary, sarcoma, stomach, uterus and Medulloblastoma.
  • compositions and methods thereof lies both in the biochemical utility of the nanosensor and in its application towards medicinal and environmental diagnostics.
  • biosensors or probes which integrate biomolecules and metal nanoparticles, are designed to target nucleic acids (i.e. RNA and DNA) via specific binding events with a concomitant fluorescent detection of the targeted species.
  • nucleic acids i.e. RNA and DNA
  • very few of these nanoprobes are designed to target other important molecules such as proteins, biotoxins, and pollutants.
  • Detection of the target analyte is afforded through a fluorescence competition assay thus allowing for high sensitivity.
  • the versatility of this probe is fully realized when considering the availability of differing biomolecules such as antibodies (with specificities towards peptides, poisons and other organic molecules) and the ease of, for example, antibody production, m other words, the proposed nanoprobe can be customized towards the application of interest. Judicious selection of the antibody component, therefore, would allow for detection of cancer epitopes, chemical toxins, or environmental contaminants.
  • the underlying premise of the detection strategy relies on (1) an antibody based fluorescence competition assay and (2) a fluorescence resonance energy transfer utilizing metal nanoparticles (or organic molecules) as the donor and/or acceptor components.
  • This overall strategy is outlined in Figure 1, where the constructed nanoprobe or sensor contains an antibody-hapten complex containing two differentially labeled spectrally unique nanoparticles or organic donors/acceptors.
  • the target detection or analyte binding
  • the labeled hapten is released from the complex (the nanoprobe) thereby yielding a change in the distances between the donor and acceptor components. This change in distance manifests an increase in overall measured fluorescence of the sample, which therefore serves as a signal confirming the detection of analyte.
  • the fluorescence signal may arise from either the labeled antibody or hapten subsequent to analyte detection.
  • Potential arrangements include labeling of the antibody and hapten with a luminescent quantum dot (the donor) and a gold nanoparticle (the quencher), respectively, and the reverse configuration. These assemblies yield either fluorescent antibody ( Figure 2A) or hapten ( Figure 2B) products.
  • Figure 2A fluorescent antibody
  • Figure 2B hapten
  • the covalent binding of the nanoparticles (or organic donors and quenchers) to the antibody and hapten are accomplished using maleimide activated Qdots® (Quantum Dot Corp.), lysine reactive Nanogold® labeling reagents (Nanoprobes Corp.), or amine-reactive organic donors or quenchers (Molecular Probes).
  • Qdots® Quantum Dot Corp.
  • lysine reactive Nanogold® labeling reagents Napentalysine reactive Nanogold® labeling reagents
  • amine-reactive organic donors or quenchers Molecular Probes
  • the binding sites of the labeled antibody are then loaded with the labeled hapten, thus forming the fluorescently quenched nanosensor.
  • Target detection is then accomplished using a competitive binding assay between the free target analyte and the quenched hapten- antibody-gold nanoprobe.
  • covalent binding of the donor and acceptor components to the antibody and/or hapten will be accomplished using maleimide activated Qdots® (Quantum Dot Corp.), monomaleimido or NHS-Nanogold® labeling reagents (Nanoprobes Corp.), or amine-reactive organic fluorophores or quenchers (Molecular Probes).
  • the antibody or hapten will be modified to contain reactive thiol groups using 2- iminothiolane.
  • the self-assembly of the nanoprobe is then afforded by complexation of the labeled hapten to the labeled antibody.
  • the antibody-hapten complex containing differentially labeled donor and acceptor components (the nanoprobe) can then be used for detection of the target analyte ( Figure 1).
  • nanoprobe comprises a nanoparticle to which is attached a detectably labeled biomolecule, such as for example an antibody.
  • a second molecule is usually associated with the first molecule, as illustrated in Figure's 1 and 2.
  • the antibody is attached to the nanoparticle and is labeled with a fluorophore.
  • the second molecule is a hapten and is labeled with a quencher.
  • the antibody-hapten complex which is attached to the nanoparticle either by the antibody or, if desired, by the hapten are in a quenched fluorescent state, i.e. little or no fluorescent emissions are detected. If an analyte is provided and the antibody is specific for the analyte, the hapten dissociates from the antibody and is released into the surrounding medium. The fluorophore-quenching moieties, which were in spatial proximity to each other due to the antibody-hapten complex, become dissociated in the presence of analyte for which the antibody is specific for, resulting in the fluorescent emission. [0069] This strategy is applied to the detection of particular nucleic acid sequences, protein sequences or molecules such as those found in cancer epitopes or in environmental analysis.
  • a self-assembling luminescent nanoparticle probe that acts as a sensor towards particular peptide sequences, organic ligands, or nucleic acids.
  • detection method is the coupling of a fluorescence competition assay with the fluorescence quenching of a gold nanoparticle.
  • Figure 1 is a schematic illustration of the general method and mechanism of the nanoparticle probe.
  • the self-assembling nanoparticle comprises an inert metallic component such as for example, gold particles and gold colloids, CdSe, CdTe, or CdS nanocrsytals, and ceramic particles such as silica.
  • the biomolecule such as an antibody, may be physically adsorbed onto the particle; however, greater stability and longer shelf-life are obtained when the antibody is covalently attached. See for example J. L. Ortega- Vinuesa et al. J. Biomater. Sd. Polymer Edn., 12(4), 379-408 (2001).
  • Nanoparticles useful in the practice of the invention include metal (e.g., gold, silver, copper and platinum), semiconductor (e.g., CdSe, CdS, and CdS or CdSe coated with ZnS) and magnetic (e.g., ferromagnetite) colloidal materials.
  • Other nanoparticles useful in the practice of the invention include ZnS, ZnO, TiO 2 , AgI, AgBr, HgI 2 , PbS, PbSe, ZnTe, CdTe, In 2 S 3 , In 2 S 3 , Cd 3 P 2 , Cd 3 As 2 , InAs, and GaAs.
  • the size of the nanoparticles is preferably from about 5 nm to about 150 run (mean diameter), more preferably from about 5 to about 50 nm, most preferably from about 5 nm to about 20 nm.
  • the nanoparticles may also be rods.
  • Methods of making metal, semiconductor and magnetic nanoparticles are well- known in the art. See, e.g., Schmid, G. (ed.) Clusters and Colloids (VCH, Weinheim, 1994); Hayat, M. A. (ed.) Colloidal Gold: Principles, Methods, and Applications (Academic Press, San Diego, 1991); Massart, R., IEEE Transactions On Magnetics, 17, 1247 (1981); Ahmadi, T. S.
  • Suitable nanoparticles are also commercially available from, e.g., Ted Pella, Inc. (gold), Amersham Corporation (gold), Nanoprobes, Inc. (gold), and Quantum Dot Corporation (semiconductor). Suitable organic dyes are available from Molecular Probes, Inc.
  • nanoparticle containing probes Presently preferred for use in detecting biomolecules and nucleic acids are nanoparticle containing probes.
  • Gold nanoparticles are also presently preferred for use in nanofabrication for the same reasons given above and because of their stability, ease of imaging by electron microscopy, and well-characterized modification. Also preferred for use in nanofabrication are semiconductor nanoparticles because of their unique electronic and luminescent properties.
  • Particles having covalently bound antibodies are typically prepared by activation of the particles, followed by coupling of antibodies to the activated particles.
  • particles having amine reactive functionalities such as N-hydroxysuccinimide (NHS)
  • the antibody surface lysine residues will be used to coupling.
  • thiol reactive functionalities such as maleimides
  • native cysteines resulting from mild reduction of the antibody will be used for coupling.
  • reagents such as 2-iminothilane, will be used to introduce reactive thiol groups onto the antibody.
  • Carbodiimide couplers include, for example, N-ethyl- N'-(3-dimethyl-aminopropyl)carbodiimide (EDC); dicyclohexylcarbodiimide (DCC); and diisopropylcarbodiimide (DIC).
  • Antibodies for example IgG, can then be coupled to the particles by mixing the activated particles and the antibodies in an aqueous mixture, thereby forming sensitized particles.
  • the nanoprobe of the present invention comprises a labeled biomolecule, such as an antibody, and a labeled hapten.
  • a labeled biomolecule such as an antibody
  • the antibody releases the labeled hapten and undergoes a specific interaction with the analyte in the sample.
  • the release of the labeled hapten yields an increase in the fluorescence emission of the nanoprobe, which can then be monitored and correlated with the amount of the analyte in the sample.
  • Analyte or target molecule refers to the substance, or group of substances, whose presence or amount thereof in a liquid medium is to be determined including, but not limited to, any drug or drug derivative, hormone, protein antigen, oligonucleotide, hapten, or hapten-carrier complex.
  • An analyte analog is any substance, or group of substances, which behaves in a similar manner to the analyte, or in a manner conducive to achieving a desired assay result with respect to binding affinity and/or specificity of the antibody for the analyte including, but not limited to, derivatives, metabolites, and isomers thereof.
  • Antibody means a specific binding partner of the analyte and is meant to include any substance, or group of substances, which has a specific binding affinity for the analyte to the exclusion of other substances.
  • the term includes polyclonal antibodies, monoclonal antibodies and antibody fragments.
  • Haptens are substances, typically of low molecular weight, which are not capable of stimulating antibody formation, but which do react with antibodies. The latter are formed by coupling the hapten to a high molecular weight carrier and injecting this coupled product into humans or animals.
  • haptens include therapeutic drugs such as digoxin and theophylline; drugs of abuse such as morphine, lysergic acid diethylamide (LSD), and ⁇ 9- tetrahydrocannabinol (THC); antibiotics such as aminoglycosides and vancomycin; hormones such as estrogen and progesterone; vitamins such as vitamin B 12 and folic acid; thyroxin; histamine; serotonin; adrenaline and others.
  • therapeutic drugs such as digoxin and theophylline
  • drugs of abuse such as morphine, lysergic acid diethylamide (LSD), and ⁇ 9- tetrahydrocannabinol (THC)
  • antibiotics such as aminog
  • a carrier refers to an immunogenic substance, commonly a protein that can join with a hapten, thereby enabling the hapten to stimulate an immune response.
  • Carrier substances include proteins, glycoproteins, complex polysaccharides and nucleic acids that are recognized as foreign and thereby elicit an immunologic response from the host.
  • immunogen and immunogenic refer to substances capable of producing or generating an immune response in an organism.
  • a peptide is any compound formed by the linkage of two or more amino acids by amide (peptide) bonds, usually a polymer of ⁇ -amino acids in which the ⁇ -amino group of each amino acid residue (except the NH 2 -terminal) is linked to the ⁇ -carboxy group of the next residue in a linear chain.
  • the terms peptide, polypeptide and poly(amino acid) are used synonymously herein to refer to this class of compounds without restriction as to size. The largest members of this class are referred to as proteins.
  • a covalent bond is a chemical bond between two species, and may involve single bonds or multiple bonds.
  • the term "covalent” does not include hydrophobic/hydrophilic interactions, Hydrogen-bonding, van der Waals interactions, and ionic interactions.
  • Any sample that is suspected of containing the analyte can be analyzed by the method of the present invention.
  • the sample is typically an aqueous solution such as a body fluid from a host, for example, urine, whole blood, plasma, serum, saliva, semen, stool, sputum, cerebral spinal fluid, tears, mucus or the like, but preferably is urine, plasma or serum.
  • the sample can be pretreated if desired and can be prepared in any convenient medium that does not interfere with the assay.
  • Calibration material means any standard or reference material containing a known amount of the analyte to be measured. The sample suspected of containing the analyte and the calibration material are assayed under similar conditions. Analyte concentration is then calculated by comparing the results obtained for the unknown specimen with results obtained for the standard.
  • Particles which may be treated according to the present invention, include any type of particle and may be activated with succinimide esters.
  • Such particles include polymer particles including polystyrene and polymethylmethacrylate); gold particles including gold nanoparticles and gold colloids; luminescent semiconductor particles; and ceramic particles including silica, glass, and metal oxide particles. See for example C. R. Martin et al. Analytical Chemistry— News Sc Features, May 1, 1998, 322A-327A. These particles may be activated succinimide esters or maleimides directly, or they may be activated once their surfaces have been modified to contain carboxylate groups.
  • Carboxylate groups can be introduced to surfaces, for example by hydrolysis reactions, by treatment with a carboxylating reagent, or by formation of self-assembled monolayers (SAMs) containing carboxylate groups. See for example J. G. Chapman et al. J. Am. Chem. Soc, 122, 8303- 8304 (2000).
  • SAMs self-assembled monolayers
  • the amine or thiol compounds are applied to the sensitized particles in an excess amount to provide for maximum reaction of the succinimide ester or maleimide functionalities.
  • the particles are contacted with a solution containing at least 50 equivalents of the amine or thiol relative to the amount of reactive groups originally present on the particles before activation. More preferably, the particles are contacted with a solution containing at least 100 equivalents of the amine or thiol. Even more preferably, the particles are contacted with a solution containing at least 200 equivalents of the amine or thiol.
  • the particles are exposed to the contacting solution for a time sufficient to allow an acceptable amount of reaction. This time may be from a few minutes to several hours.
  • the particles After the particles have been treated by exposure to the amine or thiol, the particles are filtered from the reaction solution. These particles thus have a surface, which contains antibody, the reaction products of residual functionalities with the amine or thiol and, optionally, immobilized BSA.
  • the particles are then used in the fluorescence competition assay, thus allowing for high sensitivity.
  • the particles can be used in an immunoassay for the corresponding analyte of the antibody using standard immunoassay techniques.
  • Immunoassay mixtures using these particles may also contain a tertiary amine compound to reduce interference due to the presence of tertiary amine groups on the particle surface. Suitable tertiary amine compounds include triethanolamine (TEO).
  • buffers will normally be present in the assay medium, as well as stabilizers for the assay medium and the assay components.
  • additional proteins may be included, such as albumin; or surfactants may be included, particularly non-ionic surfactants and the like.
  • Suitable nanoparticles are also commercially available from, e.g., Ted Pella, Inc. (gold), Amersham Corporation (gold), Nanoprobes, Inc. (gold) and Quantum Dot Corporation.
  • the nanoparticles, the oligonucleotides or both are functionalized in order to attach the oligonucleotides to the nanoparticles.
  • Such methods are known in the art.
  • oligonucleotides functionalized with alkanethiols at their 3'-termini or 5'-termini readily attach to gold nanoparticles. See Whitesides, Proceedings of the Robert A. Welch Foundation 39th Conference On Chemical Research Nanophase Chemistry, Houston, Tex., pages 109-121 (1995). See also, Mucic et al. Chem. Commun.
  • 555-557 (1996) (describes a method of attaching 3' thiol DNA to flat gold surfaces; this method can be used to attach oligonucleotides to nanoparticles).
  • the alkanethiol method can also be used to attach oligonucleotides to other metal, semiconductor and magnetic colloids and to the other nanoparticles listed above.
  • Other functional groups for attaching oligonucleotides to solid surfaces include phosphorothioate groups (see, e.g., U.S. Pat. No. 5,472,881 for the binding of oligonucleotide-phosphorothioates to gold surfaces), substituted alkylsiloxanes (see, e.g.
  • Oligonucleotides terminated with a 5' thionucleoside or a 3' thionucleoside may also be used for attaching oligonucleotides to solid surfaces.
  • Each nanoparticle will have a plurality of oligonucleotides attached to it. As a result, each nanoparticle-oligonucleotide conjugate can bind to a plurality of oligonucleotides or nucleic acids having the complementary sequence.
  • Oligonucleotides of defined sequences are used for a variety of purposes in the practice of the invention. Methods of making oligonucleotides of a predetermined sequence are well-known. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd ed. 1989) and F. Eckstein (ed.) Oligonucleotides and Analogues, 1st Ed.
  • Oligoribonucleotides and oligodeoxyribonucleotides can also be prepared enzymatically.
  • the invention provides methods of detecting nucleic acids. Any type of nucleic acid may be detected, and the methods may be used, e.g., for the diagnosis of disease and in sequencing of nucleic acids.
  • nucleic acids that can be detected by the methods of the invention include genes (e.g., a gene associated with a particular disease), viral RNA and DNA, bacterial DNA, fungal DNA, cDNA, mRNA, RNA and DNA fragments, oligonucleotides, synthetic oligonucleotides, modified oligonucleotides, single-stranded and double-stranded nucleic acids, natural and synthetic nucleic acids, etc.
  • examples of the uses of the methods of detecting nucleic acids include: the diagnosis and/or monitoring of viral diseases (e.g., human immunodeficiency virus, hepatitis viruses, herpes viruses, cytomegalovirus, and Epstein-Barr virus), bacterial diseases (e.g., tuberculosis, Lyme disease, H.
  • viral diseases e.g., human immunodeficiency virus, hepatitis viruses, herpes viruses, cytomegalovirus, and Epstein-Barr virus
  • bacterial diseases e.g., tuberculosis, Lyme disease, H.
  • pylori Escherichia coli infections, Legionella infections, Mycoplasma infections, Salmonella infections
  • sexually transmitted diseases e.g., gonorrhea
  • inherited disorders e.g., cystic fibrosis, Duchene muscular dystrophy, phenylketonuria, sickle cell anemia
  • cancers e.g., genes associated with the development of cancer
  • the nucleic acid to be detected may be isolated by known methods, or may be detected directly in cells, tissue samples, biological fluids (e.g., saliva, urine, blood, serum), solutions containing PCR components, solutions containing large excesses of oligonucleotides or high molecular weight DNA, and other samples, as also known in the art. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd ed. 1989) and B. D. Hames and S. J. Higgins, Eds., Gene Probes 1 (IRL Press, New York, 1995). Methods of preparing nucleic acids for detection with hybridizing probes are well known in the art.
  • a nucleic acid is present in small amounts, it may be applied by methods known in the art. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd ed. 1989) and B. D. Hames and S. J. Higgins, Eds., Gene Probes 1 (IRL Press, New York, 1995).
  • Preferred is polymerase chain reaction (PCR) amplification.
  • One method according to the invention for detecting nucleic acid comprises contacting a nucleic acid with one or more types of nanoparticles having oligonucleotides attached thereto and are fluorescently labeled.
  • the nucleic acid to be detected has at least two portions. The lengths of these portions and the distance(s), if any, between them are chosen so that when the oligonucleotides on the nanoparticles hybridize to the nucleic acid, a detectable change in fluorescence occurs, i.e. quenching.
  • the portions of the nucleic acid to which the oligonucleotides on the nanoparticles are to bind must be chosen so that they contain sufficient unique sequence so that detection of the nucleic acid will be specific.
  • oligonucleotides Methods of labeling oligonucleotides with fluorescent molecules and measuring fluorescence are well known in the art. Suitable fluorescent molecules are also well known in the art and include, but not limited to: the fluoresceins, biotin, rhodamines and Texas Red. The oligonucleotides will be attached to the nanoparticles as described above.
  • FRET Fluorescence resonance energy transfer
  • Fluorescence resonance energy transfer occurs between the electronic excited states of two fluorophores when they are in sufficient proximity to each other, in which the excited-state energy of the donor fiuorophore is transferred to the acceptor fluorophore.
  • the result is a decrease in the lifetime and a quenching of fluorescence of the donor species and a concomitant increase in the fluorescence intensity of the acceptor species.
  • a fluorescent moiety is caused to be in close proximity to a quencher molecule.
  • Donor and acceptor molecules operate in a set wherein one or more acceptor molecules accepts energy from one or more donor molecules, or otherwise quenches signal from the donor molecule, when the donor and acceptor molecules are closely associated.
  • the donor and acceptor molecules are about 30 to about 200 A apart or about 10 to about 40 nucleotides apart.
  • Transfer of energy may occur through collision of the closely associated molecules of a set, or through a non-radiative process such as fluorescence resonance energy transfer (FRET).
  • FRET fluorescence resonance energy transfer
  • transfer of energy between donor and acceptor molecules requires that the molecules be close in space and that the emission spectrum of a donor have substantial overlap with the absorption spectrum of the acceptor (Yaron et al. Analytical Biochemistry, 95, 228-235 (1979), the teachings of which are incorporated herein by reference).
  • intramolecular energy transfer may occur between very closely associated donor and acceptor molecules (e.g., within 10 A) whether or not the emission spectrum of a donor molecule has a substantial overlap with the absorption spectrum of the acceptor molecule (Yaron et al.) This process is referred to as intramolecular collision since it is believed that quenching is caused by the direct contact of the donor and acceptor molecule (Yaron et al).
  • the efficiency of both collision and non-radiative transfer of energy between the donor and acceptor molecules is directly dependent on the proximity of the donor and acceptor molecules
  • formation and dissociation of the complexes of this invention can be monitored by measuring at least one physical property of at least one member of the set which is detectably different when the complex is formed, as compared with when the biomolecules and target/bait exist independently and unassociated.
  • the means of detection will involve measuring fluorescence of an acceptor fluorophore of a set or the fluorescence of the donor fluorophore in a set containing a fluorophore and quencher pair (e.g. a donor and acceptor).
  • the fluorescent molecules may interact with one another via hydrophobic interactions, thereby reducing the adverse effect of distance between the donor and acceptor fluorescent molecules.
  • fluorescence energy transfer can occur when the donor and acceptor fluorescent molecules are up to about 40 nucleotides away from each other.
  • any fluorophore may be used, including BODIPY, fluoroscein, fluorescein substitutes (Alexa Fluor dye, Oregon green dye), long wavelength dyes, and UV- excited fluorophores. These and additional fiuorophores are listed in Fluorescent and Luminescent Probes for Biological Activity. A Practical Guide to Technology for Quantitative Real-Time Analysis, Second Ed. W. T. Mason, ed. Academic Press (1999) (incorporated herein by reference).
  • a quencher is a molecule that absorbs the energy of the excited fluorophore. Close proximity of a fluorophore and a quencher allow for the energy to be transferred from the fluorophore to the quencher. By absorbing this energy, the quencher prevents the fluorophore from releasing the energy in the form of a photon, thereby preventing fluorescence. Fluorescent quenchers tend to be specific to fluorophores that emit at a specific wavelength range.
  • Fluorescent quenchers often involve fluorescence resonance energy transfer (FRET).
  • FRET fluorescence resonance energy transfer
  • the fluorescent quencher molecule is also a fluorophore.
  • close proximity of the fluorophore and fluorescent quencher is indicated by a decrease in fluorescence of the "fluorophore” and an increase in fluorescence of the fluorescent quencher.
  • Commonly used fluorescent fluorophore pairs include fluorescein/tetramethyhrhodamine, IAEDANS/fluorescein, fluorescein/fluorescein, and BODIPY FL/BODIPY FL.
  • the 3 '-end of the biomolecule is labeled with N, N 1 , N, N 1 -tetramethyl-6-carboxy rhodamine (TAMRA).
  • TAMRA tetramethyl-6-carboxy rhodamine
  • Donor and acceptor molecules suitable for FRET are well known in the art (see page 46 of R. P. Haugland, Handbook of Fluorescent Probes and Research Chemicals, 6th ed.; Molecular Probes, Oregon, the teachings of which are incorporated herein by reference).
  • the donor fluorescent molecule has a shorter excitation wavelength than the acceptor fluorescent molecule and the donor emission wavelength overlaps with the acceptor excitation wavelength, to allow transfer of energy from the donor to the acceptor.
  • Preferred fluorescent molecules are: biotin, fluorophores are fluorescein and derivatives thereof, such as 5-(2'-aminoethyl)-aminoapthalene-l -sulfonic acid (EDANS) and rhodamine and derivatives thereof such as Cy3, Cy5 and Texas Red.
  • EDANS 5-(2'-aminoethyl)-aminoapthalene-l -sulfonic acid
  • rhodamine derivatives thereof such as Cy3, Cy5 and Texas Red.
  • Suitable donor/acceptor pairs are, for example, fluorescein/tetramethyrhodamine, IAEDANS/fluorescein and ED ANS/D ABCYL.
  • the same fluorescent molecule is used for the donor and acceptor.
  • the wavelength used to excite the detection complexes is polarized. Unpolarized emission detected is indicative of FRET.
  • labeled, unlabeled and modified biomolecules such as antibodies, oligonucleotides, aptamers and the like, are readily available for the method of the present invention. They can be synthesized using commercially available instrumentation and reagents or they can be purchased from numerous commercial vendors of custom manufactured oligonucleotides.
  • antibodies are labeled with a fluorophore and a quencher to form intra-molecular FRET.
  • the folded conformations of the are stabilized by binding to their target molecules and produce a fluorescence signal change of the fluorophore induced by FRET when the antibody binds to its target.
  • the target-binding induced FRET cause between about 40% up to 100% fluorescence quenching.
  • FRET can be formed within an antibody even if the antibody lacks the necessary conformational changes accompanying the binding to the target molecules.
  • WO 99/27351 (incorporated herein in its entirety) describes a monolithic bioelectrical device comprising a bioreporter and an optical application specific integrated circuit (OASIC).
  • OASIC optical application specific integrated circuit
  • the device allows remote sampling for the presence of substances in solution.
  • the fluorescence may be measured by a number of different modes. Examples include fluorescence intensity, lifetime, and anisotropy in either steady state or kinetic rate change modes (Lakowicz, J. R. In Principles of Fluorescence Spectroscopy; 2nd ed.; Kluwer Academic/Plenum: New York, 1999).
  • an oligonucleotide or an assembly of oligonucleotides can also be labeled with at least one pair of resonantly interacting detection moieties.
  • a first detection moiety and a second detection moiety of a pair can each be linked to an oligonucleotide or an assembly of oligonucleotides flanking the cleavage recognition sequence, such that upon cleavage of the recognition sequence by the cleaving agent separation of these moieties occurs.
  • the detection moieties are selected such that at least one of these moieties is capable of producing a detectable signal when separated to a non-interacting distance from the other detection moiety.
  • Examples of resonantly interacting pairs of detection moieties which can be used include, but are not limited to, a fluorescer and a quencher and any other type of fluorescent resonant energy transfer (FRET) pairs (for reference, see for example “Fluoroscence resonance energy transfer” by Paul R. Selvin, 1995, Methods in Enzymol. VoI 246, Chap. 13, pp. 300; and "Handbook of fluorescent probes and research chemicals" by Richard P. Haugland, sixth ed. Molecular probes. Specific examples of molecules which can be used in fluorescent resonant energy transfer are listed hereinbelow.
  • FRET fluorescent resonant energy transfer
  • the optimal distance between a first and a second detection moieties of a pair when linked to a biomolecule such as antibody, aptamer, oligonucleotide, will be that distance wherein the emissions of the first moiety are absorbed by the second moiety.
  • This optimal distance varies with the specific moieties used, and is defined by Forster Radius.
  • Forster Radius (Ro) is the distance between a donor and acceptor that allows quenching of 50% of the excited donor molecules by the quencher. Ro may be defined for any given FRET pair, and may be used as the guideline for designing a FRET-labeled probe.
  • FAM which has an emission maximum of 525 nm
  • ROX ROX
  • R6G all of which have an excitation maximum of 514 nm
  • moieties which can be used include but are not limited to, A- acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid acridine and derivatives such as, acridine, acridine isothiocyanate, 5-(2'-aminoethyl)aminonaphthalene-l-sulfonic acid (EDANS), 4-amino-N-_13-vinylsulfonyl)phenyl!naphthalimide-3,5, disulfonate (Lucifer Yellow VS), N-(4-anilino-l-naphthyl)maleimide, anthranilamide and Brilliant Yellow; coumarin and derivatives such as, coumarin, 7-amino-4-methylcoumarin (AMC, Coumarin 120), 7-amino-4-trifluoromethylcouluarin (Coumaran 151), cyanosine 4',6-di
  • T. Brilliant Red 3B-A rhodamine and derivatives such as, 6-carboxy-X-rhodamine (ROX), 6- carboxyrhodamine (R6G), lissaniine rhodamine B sulfonyl chloride, rhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine X isothiocyanate, sulforhodamine B, sulforhodamine 101, sulforhodamine 101, (Texas Red), N,N,N',N'-tetramethyl-6- carboxyrhodamine (TAMRA), tetramethyl rhodamine, tetramethyl rhodamine isothiocyanate (TRITC), riboflavin, rosolic acid and terbium chelate derivatives.
  • ROX 6-carboxy-X-rhodamine
  • detection labels can be directly incorporated into the biomolecules.
  • a detection label is any molecule that can be associated with biomolecules, such as antibodies, directly or indirectly, and which results in a measurable, detectable signal, either directly or indirectly.
  • a label may be any moiety covalently attached to a biomolecule.
  • Many such labels for incorporation into antibodies, nucleic acids are known to those of skill in the art. Examples of detection labels suitable for use in the disclosed method are, fluorescent molecules, phosphorescent molecules and radioactive isotopes.
  • a preferred class of labels are detection labels, which may provide a signal for detection of the labeled antibodies by fluorescence, chemiluminescence, and electrochemical luminescence.
  • Fluorescent dyes useful for labeling antibodies include fluoresceins, rhodamines, cyanines, and metal porphyrin complexes.
  • Preferred fluorescein dyes include 6- carboxyfluorescein (6-FAM), 2', 4',1,4-tetrachlorofluorescein (TET), 2',4',5',7',1,4- hexachlorofluorescein (HEX), 2',7'-dimethoxy-4',5 l -dichloro ⁇ 6-carboxyrhodamm (JOE), T- chloro-5'-fluoro-7',8'-fused phenyl- l ⁇ -dichloro- ⁇ -caroxyflurescein (NED), 2'-chloro-7'- phenyl-l,4-dichloro-6-carboxyfluorescein (VIC), and (JODA).
  • the 5-carboxyl, and other regio-isomers may also have useful detection properties. Fluorescein, biotin and rhodamine dyes with 1,4-dichloro substituents are especially preferred.
  • Another preferred class of labels include quencher moieties.
  • the emission spectra of a quencher moiety overlaps with a proximal intramolecular or intermolecular fluorescent dye such that the fluorescence of the fluorescent dye is substantially diminished, or quenched, by fluorescence resonance energy transfer (FRET).
  • FRET fluorescence resonance energy transfer
  • Oligonucleotides which are intramolecularly labeled with both fluorescent dye and quencher moieties are useful in nucleic acid hybridization assays, e.g. the "TaqmanTM" exonuclease-cleavage PCR assay.
  • Particularly preferred quenchers include but are not limited to (i) rhodamine dyes selected from the group consisting of tetramethyl-6-carboxyrhodamine (TAMRA), tetrapropano-6-carboxyrhodamine (ROX), and (ii) DABSYL, DABCYL, cyanine dyes including nitrothiazole blue (NTB), anthraquinone, malachite green, nitrothiazole, and nitroimidazole compounds and the like.
  • TAMRA tetramethyl-6-carboxyrhodamine
  • ROX tetrapropano-6-carboxyrhodamine
  • DABSYL DABCYL
  • cyanine dyes including nitrothiazole blue (NTB), anthraquinone, malachite green, nitrothiazole, and nitroimidazole compounds and the like.
  • Preferred fluorescent labels are fluorescein (5-carboxyfluorescein-N- hydroxysuccinimide ester), rhodamine (5,6-tetramethyl rhodamine), and the cyanine dyes Cy3, Cy3.5, Cy5, Cy5.5 and Cy7.
  • the absorption and emission maxima, respectively, for these fluors are: FITC (490 nm; 520 nm), Cy3 (554 nm; 568 nm), Cy3.5 (581 run; 588 nm), Cy5 (652 nm: 672 nm), Cy5.5 (682 nm; 703 nm) and Cy7 (755 nm; 778 nm), thus allowing their simultaneous detection.
  • fluorescein dyes include 6- carboxyfluorescein (6-FAM), 2 l ,4',l,4,-tetrachlorofluorescein (TET), 2',4',5',7',1,4- hexachloro fluorescein (HEX), 2',7'-dimethoxy-4',5'-dichloro-6-carboxyrhodamine (JOE), T- chloro-5 l -fluoro-7',8'-fused phenyl-l,4-dichloro-6-carboxyfluorescein (NED), and 2'-chloro- 7'-phenyl-l,4-dichloro-6-carboxyfluorescein (VIC).
  • 6-FAM 6- carboxyfluorescein
  • TET 2 l ,4',l,4,-tetrachlorofluorescein
  • HEX 2',4',5',7',1,4- hexachlor
  • Fluorescent labels can be obtained from a variety of commercial sources, including Amersham Pharmacia Biotech, Piscataway, NJ. ; Molecular Probes, Eugene, Oreg.; and Research Organics, Cleveland, Ohio.
  • suitable fluorescent labels include fluorescein isothiocyanate (FITC), 5,6-carboxymethyl fluorescein, Texas red, nitrobenz-2-oxa-l,3-diazol-4-yl (NBD), coumarin, dansyl chloride, rhodamine, amino-methyl coumarin (AMCA), Eosin, Erythrosin, BODIPYTM, Cascade BlueTM, Oregon GreenTM, pyrene, lissamine, xanthenes, acridines, oxazines, phycoerythrin, macrocyclic chelates of lanthanide ions such as quantum dyeTM, fluorescent energy transfer dyes, such as thiazole orange-ethidium heterodimer, and the
  • Examples of other specific fluorescent labels include 3-Hydroxypyrene 5,8,10-Tri Sulfonic acid, 5-Hydroxy Tryptamine (5-HT), Acid Fuchsin, Alizarin Complexon, Alizarin Red, Allophycocyanin, Aminocoumarin, Anthroyl Stearate, Astrazon Brilliant Red 4G, Astrazon Orange R, Astrazon Red 6B, Astrazon Yellow 7 GLL, Atabrine, Auramine, Aurophosphine, Aurophosphine G, BAO 9 (Bisaminophenyloxadiazole), BCECF, Berberine Sulphate, Bisbenzamide, Blancophor FFG Solution, Blancophor SV, Bodipy Fl, Brilliant Sulphoflavin FF, Calcien Blue, Calcium Green, Calcofluor RW Solution, Calcofluor White, Calcophor White ABT Solution, Calcophor White Standard Solution, Carbostyryl, Cascade Yellow, Catecholamine, Chinacrine, Coriphosphine O, Coumarin
  • Additional labels of interest include those that provide for signal only when the antibody with which they are associated is specifically bound to a target molecule, where such labels include: "molecular beacons" as described in Tyagi & Kramer, Nature Biotechnology (1996) 14:303 and EP 0 070 685 Bl.
  • Other labels of interest include those described in U.S. Pat. No. 5,563,037; WO 97/17471 and WO 97/17076.
  • Labeled nucleotides are also a preferred form of detection label since they can be directly incorporated into the amplification products during synthesis.
  • nucleotide analogs such as BrdUrd (5-bromodeoxyuridine, Hoy and Schimke, Mutation Research 290:217-230 (1993)
  • aminoallyldeoxyuridine Henegariu et al, Nature Biotechnology 18:
  • Suitable fluorescence-labeled nucleotides are Fluorescein-isothiocyanate-dUTP, Cyanine-3-dUTP and Cyanine-5-dUTP (Yu et al, Nucleic Acids Res., 22:3226-3232 (1994)).
  • nanoparticles of the invention can be purchased commercially, such as Nanogold®, or manufactured according to methods known in the art. Extremely small and well defined structures, for example of inorganic particles, can be formed in association with a surface using self-assembly approaches. Nanoparticles with very uniform sizes are preferred components for forming self-assembled structures, although other functional compositions can be used. The nanoparticles are organized into a well defined structures using fabrication techniques that take advantage of molecular recognition characteristics of self-assembly approaches. Molecular recognition can involve various interactions, such as commingling, key-lock relationships and guest-host interactions. [00128] The deposition techniques are combined with localization techniques that constrain the resulting structures within isolated islands along the substrate surface.
  • the islands can be ordered or disordered arrays.
  • the organized structures or islands are suitable for binding of biomolecules, such as antibodies, peptides, nucleic acid molecules and the like.
  • Preferred nanoparticles can be produced by laser pyrolysis with or without additional processing.
  • the self-assembly approaches provide an alternative to traditional masking techniques and direct formation approaches for fabricating device structures.
  • Laser pyrolysis is an excellent approach for efficiently producing a wide range of nanoscale particles with a narrow distribution of average particle diameters.
  • laser pyrolysis can be used to produce a variety of inorganic particles, such as elemental metal particles, metal/silicon oxide particles, metal/silicon carbide particles, metal/silicon nitride particles and metal/silicon sulfide particles.
  • nanoparticles can be produced using a flame production apparatus such as the apparatus described in U.S. Pat. No. 5,447,708 to Helble et al., entitled “Apparatus for Producing Nanoscale Ceramic Particles," incorporated herein by reference.
  • nanoparticles can be produced with a thermal reaction chamber such as the apparatus described in U.S. Pat. No. 4,842,832 to Inoue et al., "Ultrafme Spherical Particles of Metal Oxide and a Method for the Production Thereof," incorporated herein by reference.
  • a basic feature of successful application of laser pyrolysis for the production of desirable inorganic nanoparticles is the generation of a reactant stream containing a metal/silicon precursor compound, a radiation absorber and, generally, a secondary reactant.
  • the secondary reactant can be a source of atoms, such as oxygen, required for the desired product or an oxidizing or reducing agent to drive a desired product formation.
  • a secondary reactant is not needed if the precursor decomposes to the desired product under intense light radiation.
  • the reactant stream is pyrolyzed by an intense light beam, generally a laser beam. As the reactant stream leaves the laser beam, the particles are rapidly quenched.
  • Nanoparticles produced by laser pyrolysis can be subjected to additional processing to alter the nature of the particles, such as the composition and/or the crystallinity.
  • the nanoparticles can be subjected to heat processing in a gas atmosphere prior to use. Under suitably mild conditions, heat processing is effective to modify the characteristics of the particles without destroying the nanoscale size or the narrow particle size distribution of the initial particles.
  • the powder is dispersed in a liquid or other fluid for use or for further processing.
  • particle dispersions have concentrations of nanoparticles no more than about 80 weight percent. Appropriate properties of the resulting dispersion may depend on the features of the self-assembly approach, as described below.
  • Preferred collections of inorganic nanoparticles have an average diameter less than a 100 nm and a very narrow distribution of primary particle diameters.
  • the nanoparticle is between about 1 to 50 nm in diameter, preferably, the nanoparticle is between about 1 to 20 nm in diameter.
  • the formation of the structures involves self-assembly approaches that generate well defined organized deposits of nanoparticles.
  • the self-assembly techniques can be used to directly form the deposits of nanoparticles.
  • the self-assembled structures can be produced using dispersions of nanoparticles and by manipulating the conditions on the surface of the material and in the solution to lead to the desired structure formation.
  • a linker is used to chemically bind on one end to the substrate surface and on the other end to the nanoparticle. Selective binding with the linker can be used to direct the self-assembly process.
  • Another alternative approach makes use of natural interactions, such as electrostatic and chemical interactions to help direct the self-assembly process.
  • the nanoparticles are deposited within miniature pores to localize the nanoparticles within the boundaries defined by the porous region. Miniature pores are found within certain materials, such as inorganic oxides or two dimensional organic crystals, or suitable pores can be formed, for example, by ion milling or chemical etching. Further details and additional self-assembly approaches are described below.
  • the reaction conditions determine the qualities of the particles produced by laser pyrolysis.
  • the reaction conditions for laser pyrolysis can be controlled relatively precisely in order to produce particles with desired properties.
  • the appropriate reaction conditions to produce a certain type of particles generally depend on the design of the particular apparatus. Increasing the laser power results in increased reaction temperatures in the reaction region as well as a faster quenching rate. A rapid quenching rate tends to favor production of high energy phases, which may not be obtained with processes near thermal equilibrium. Similarly, increasing the chamber pressure also tends to favor the production of higher energy structures. Also, increasing the concentration of a reactant, such as a reactant serving as an oxygen source, in the reactant stream favors the production of particles with increased amounts of atoms from the secondary reactant.
  • Reactant flow rate and velocity of the reactant gas stream are inversely related to particle size so that increasing the reactant gas flow rate or velocity tends to result in smaller particle sizes. Also, the growth dynamics of the particles have a significant influence on the size of the resulting particles. In other words, different forms of a product compound have a tendency to form different size particles from other phases under relatively similar conditions. Light intensity/laser power also influences particle size with increased light intensity favoring larger particle formation for lower melting materials and smaller particle formation for higher melting materials.
  • Metal/silicon precursor compounds can be delivered into the reaction chamber as a gas.
  • Appropriate metal/silicon precursor compounds for gaseous delivery generally include metal/silicon compounds with reasonable vapor pressures, i.e., vapor pressures sufficient to get desired amounts of precursor vapor in the reactant stream.
  • the vessel holding liquid or solid precursor compounds can be heated to increase the vapor pressure of the metal/silicon precursor, if desired.
  • a carrier gas can be bubbled through a liquid precursor to facilitate delivery of a desired amount of precursor vapor.
  • a carrier gas can be passed over the solid precursor to facilitate delivery of the precursor vapor.
  • a suitable container for heating and delivering a solid precursor to a laser pyrolysis apparatus is described below.
  • Solid precursors generally are heated to produce a sufficient vapor pressure.
  • the use of exclusively gas phase reactants is somewhat limiting with respect to the types of precursor compounds that can be used conveniently.
  • techniques have been developed to introduce aerosols containing reactant precursors into laser pyrolysis chambers.
  • solid precursor compounds can be delivered by dissolving the compounds in a solvent.
  • powdered precursor compounds can be dispersed in a liquid/solvent for aerosol delivery.
  • Liquid precursor compounds can be delivered as an aerosol from a neat liquid, a multiple liquid dispersion or a liquid solution.
  • Aerosol reactants can be used to obtain a significant reactant throughput.
  • a solvent/dispersant can be selected to achieve desired properties of the resulting solution/dispersion. Suitable solvents include water, methanol, ethanol, isopropyl alcohol, other organic solvents and mixtures thereof. The solvent should have a desired level of purity such that the resulting particles have a desired purity level. Some solvents, such as isopropyl alcohol, are significant absorbers of infrared light from a CO 2 laser such that no additional laser absorbing compound may be needed within the reactant stream if a CO 2 laser is used as a light source.
  • aerosol precursors are formed with a solvent present, the solvent preferably is rapidly evaporated by the light beam in the reaction chamber such that a gas phase reaction can take place.
  • the fundamental features of the laser pyrolysis reaction are unchanged by the presence of an aerosol. Nevertheless, the reaction conditions are affected by the presence of the aerosol.
  • a number of suitable solid, metal, metal/silicon precursor compounds can be delivered as an aerosol from solution.
  • the compounds are dissolved in a solution generally with a concentration greater than about 0.5 molar.
  • concentration the greater the concentration of precursor in the solution the greater the throughput of reactant through the reaction chamber.
  • the solution can become more viscous such that the aerosol may have droplets with larger sizes than desired.
  • selection of solution concentration can involve a balance of factors in the selection of a preferred solution concentration.
  • Preferred secondary reactants serving as oxygen source include, for example, O 2 , CO, CO 2 , O 3 and mixtures thereof.
  • Oxygen can be supplied as air.
  • T he secondary reactant compound should not react significantly with the metal/silicon precursor prior to entering the reaction zone since this generally would result in the formation of large particles.
  • Alternative secondary reactants can be selected based on the desired product particles and precursors.
  • Laser pyrolysis can be performed with a variety of optical frequencies. Preferred light sources operate in the infrared portion of the electromagnetic spectrum. CO 2 lasers are particularly preferred sources of light.
  • Infrared absorbers for inclusion in the reactant stream include, for example, C 2 H 4 , isopropyl alcohol, NH 3 , SF 6 , SiH 4 and O 3 .
  • O 3 can act as both an infrared absorber and as an oxygen source.
  • the radiation absorber such as the infrared absorber, absorbs energy from the radiation beam and distributes the energy to the other reactants to drive the pyrolysis.
  • the energy absorbed from the light beam increases the temperature at a tremendous rate, many times the rate that heat generally would be produced by exothermic reactions under controlled condition. While the process generally involves nonequilibrium conditions, the temperature can be described approximately based on the energy in the absorbing region.
  • the laser pyrolysis process is qualitatively different from the process in a combustion reactor where an energy source initiates a reaction, but the reaction is driven by energy given off by an exothermic reaction. Thus, while this light driven process is referred to as laser pyrolysis, it is not a thermal process even though traditional pyrolysis is a thermal process.
  • An inert shielding gas can be used to reduce the amount of reactant and product molecules contacting the reactant chamber components.
  • Inert gases can also be introduced into the reactant stream as a carrier gas and/or as a reaction moderator.
  • Appropriate inert shielding gases include, for example, Ar, He and N 2 .
  • An appropriate laser pyrolysis apparatus generally includes a reaction chamber isolated from the ambient environment. A reactant inlet connected to a reactant delivery apparatus produces a reactant stream through the reaction chamber. A laser beam path intersects the reactant stream at a reaction zone. The reactant/product stream continues after the reaction zone to an outlet, where the reactant/product stream exits the reaction chamber and passes into a collection apparatus.
  • the light source such as a laser, is located external to the reaction chamber, and the light beam enters the reaction chamber through an appropriate window.
  • the nanoparticles comprise synthetic polymer molecules.
  • the synthetic polymer molecules typically comprise linear or branched polymer chains, where the branched polymer can be a star polymer, a hyperbranched polymer, a graft polymer, a dendritic polymer or a combination thereof.
  • the synthetic polymer molecule can also be a block copolymer, where the crosslinkable groups are typically contained in at least one block of the polymer molecule.
  • the polymer molecules will typically have a number average molecular weight in the range of about 500 to 5,000,000, more typically within the range of about 10,000 to 500,000.
  • the molecular weight of the polymer molecules is typically selected so as to provide crosslinked particles that are approximately 2-100 nm in diameter. In one embodiment, the molecular weight is selected to provide crosslinked particles approximately 2-25 nm in diameter and in yet another embodiment, the molecular weight is selected so as to provide approximately 2-10 nm particles.
  • the synthetic polymer molecule has a backbone comprising monomer units such as ethylenically unsaturated polymerizable monomers, nitrogenous polymers, olefins, condensation monomers, ring-opening monomers including epoxides and norbornenes, esters, sulfones, lactides, lactones, carbonates, imides, arylenes, amides, propylene, ethers, urethanes, vinyl and vinyl derivatives, and organic polysilicas, non-limiting examples of which are described below.
  • monomer units such as ethylenically unsaturated polymerizable monomers, nitrogenous polymers, olefins, condensation monomers, ring-opening monomers including epoxides and norbornenes, esters, sulfones, lactides, lactones, carbonates, imides, arylenes, amides, propylene, ethers, urethanes, vinyl and vinyl derivatives, and organic poly
  • Exemplary ethylenically unsaturated polymerizable monomers include acrylic and methacrylic acids, esters and amides; alkyl acrylates (e.g., methyl acrylate, ethyl acrylate and butyl acrylate); aryl acrylates (e.g., benzyl acrylate); alkyl methacrylates; aryl methacrylates (e.g., methyl acrylate, methyl methacrylate, n-butyl acrylate, n-butyl methacrylate, t-butyl acrylate, t-butyl methacrylate, 2-ethylhexyl methacrylate, benzyl methacrylate and N-phenylacrylamide); alpha-olefins (e.g., ethylene and propylene); and combinations thereof.
  • alkyl acrylates e.g., methyl acrylate, ethyl acrylate and butyl acryl
  • Exemplary nitrogenous polymers include poly(acrylamide); poly(methacrylamide); N,N-dialkyl poly(acrylamide) (particularly wherein the nitrogen- bearing substituents are C 1 -C 12 alkyl); N,N-dialkyl poly(methacrylamide) (particularly wherein the nitrogen-bearing substituents are C 1 -C 12 alkyl); poly(alkoxylated polyamide) (e.g., N-methoxymethylated polyamide and hydroxyethylated polyamide); poly( ⁇ - caprolactam); polypropiolactam; polycapryllactam; polylauryllactam; poly(pyrrolidin-2-one); poly(vinylamine); poly(vinyl pyrrolidone); poly(2-vinylpyridine); poly(3-vinylpyridine); poly(4-vinylpyridine); poly(o-aminostyrene); poly(m-aminostyrene); poly(p-aminostyrene); polyo
  • Exemplary olefins are C 3-20 (generally C 3-15 ) cyclic olefin monomers such as ethylene, propylene, norbornene and tetracyclododecene.
  • Exemplary condensation monomers include dicarboxylic acids, their anhydrides and esters; aminocarboxylic acids and lactams; hydroxycarboxylic acids and lactones; diols, polyether diols and polyester diols; and diisocyanates; and combinations thereof.
  • Exemplary ring-opening monomers include those monomers that contain a 3- carbon carbocyclic ring or a 5-carbon heterocyclic ring (having up to 2 heteroatoms) and include epoxides and norbornenes.
  • Exemplary imides include those polyamides formed by imidization of a poly(amic acid ester) which is formed from a dianhydride selected from the group consisting of pyrometallic dianhydride, benzophenone dianhydride and 9,9-bis-(trifluoromethyl) xanthenetetracarboxylic dianhydride; and a diamine selected from the group consisting of p- phenylene diamine, 4,4'-diamino-diphenyl ether, l,3-bis(p-aminophenoxy) benzene and 2,2- bis[4-aminophenyl]hexa-fluoropropane.
  • a dianhydride selected from the group consisting of pyrometallic dianhydride, benzophenone dianhydride and 9,9-bis-(trifluoromethyl) xanthenetetracarboxylic dianhydride
  • a diamine selected from the group consisting of p- phenylene di
  • Exemplary arylenes include phenylenes, phenylquinoxalines, arylene ethers and combinations thereof.
  • Exemplary vinyl and vinyl derivatives include vinyl acetate, vinyl bromide, vinylidene chloride, butylacrylate unsubstituted styrene and styrene substituted with one or two lower alkyl, halogen or hydroxyl groups (e.g., styrene derivatives such as 4-vinyltoluene, 4-vinylphenol, ⁇ -methylstyrene, 2,5-dimethylstyrene, 4-t-butylstyrene and 2-chlorostyrene); and combinations thereof.
  • Exemplary organic polysilicas include silsesquioxanes (polymeric silicate materials of the type (RSiO 1 ⁇ ) n where R is an organic substituent); alkoxy silanes (particularly, partially condensed alkoxysilanes, e.g., partially condensed by controlled hydrolysis of tetraethoxysilane having an Mn of about 500 to 20,000); organically modified silicates having the composition RSiO 3 and R 2 SiO 2 wherein R is an organic substituents; and orthosilicates (particularly, partially condensed orthosilicates having the composition SiOR 4 ); and combinations thereof.
  • silsesquioxanes polymeric silicate materials of the type (RSiO 1 ⁇ ) n where R is an organic substituent
  • alkoxy silanes particularly, partially condensed alkoxysilanes, e.g., partially condensed by controlled hydrolysis of tetraethoxysilane having an Mn of about 500 to 20,000
  • the synthetic polymer molecules used in the methods of the invention can have a plurality of crosslinkable groups that are-inert until-activated, but which when activated undergo a rapid and irreversible intramolecular crosslinking reaction.
  • the crosslinking groups In order to perform well in the pseudo-high dilution methods of the invention, the crosslinking groups must react at a rapid rate, the crosslinking chemistry must be irreversible and the resulting coupled structure must be unreactive under the conditions required for crosslinking. Accordingly, the crosslinking groups are referred to as "crosslinkable" since they are inert until activated, but which when activated undergo an irreversible intramolecular crosslinking reaction.
  • crosslinkable groups that are suitable for use in the instant invention and they are typically covalently bound to one or more monomer units within a given polymer molecule. They can be directly bound to the monomers or indirectly bound, such as through a linking group.
  • the crosslinkable groups can be thermally activatible; photolytically activatible; activatible with ultraviolet radiation, ionizing radiation, or electron beam radiation; or activatible by a chemical activating agent.
  • the number of crosslinkable groups on the polymer molecules can be is selected to provide a crosslinked particle of suitable size, with the number of crosslinkable groups being inversely related to the particle size since a larger number of groups will provide for more intramolecular crosslinking and thus a smaller particle.
  • the number of crosslinkable groups can be selected so as to provide particles that are approximately 2-100 nm in diameter.
  • the desired particle diameter may be within the range of 2-25 run, or about 2-10 urn, and the number of crosslinkable groups can be selected accordingly.
  • the crosslinking density on the polymer molecules can be selected so as to provide the desired particle diameter, for example, within the range of about 2-100 nm, about 2-25 nm or about 2-10 nm.
  • Exemplary crosslinkable groups include by way of illustration and not limitation, acryloyl, lower alkyl-substituted acryloyl, vinyl, substituted vinyl, cyclic ether, cyclic ester, activated ester, cycloalkenyl, acid halide, amino, alcohol, phenol, carboxylic acid, diacetylene, unsubstituted and substituted acetylene groups (e.g., optionally substituted with one or more alkyl, aryl, ester; acid or amide groups), eonophiles, dienophiles and substituted and unsubstituted bicyclo[4.2.0]octa-l,3,5-trienyl groups.
  • a particularly suitable crosslinkable group is the benzocyclobutene functionality and its substituted derivatives (especially oxy substituted), a group which has found wide use as a thermally cross-linkable group and in the formulation of thermosetting materials.
  • Some embodiments of the methods of the invention utilize solvents and there are numerous solvents that are well suited for use in the invention.
  • the solvent is inert with respect to the polymer molecules and the produced crosslinked particles.
  • High boiling point solvents can be used. These include, by way of illustration and not limitation, benzyl ether; N-cyclohexylpyrrolidinone; N-methylpyrrolidone; dimethylacetamide; dimethylphenyl urea; N,N-dimethyltrimethylene urea; butyl acetate; 2- ethoxyethanol; cyclopentanone; cyclohexanone; ⁇ -butyrolactone; lactate esters such as ethyl lactate; ethoxyethylpropionate; alkylene glycol alkyl ether esters such as propylene glycol methyl ether acetate; alkylene glycol alkyl ethers such as propylene glycol methyl ether and propylene glycol n-propyl ether; alkylene glycol monoalkyl esters such as methyl cellosolve, butyl acetate, 2-ethoxyethanol, and ethyl 3-ethoxyprop
  • a coupling agent in the methods of the invention and there are numerous suitable coupling agents that are known in the art. These include, by way of illustration and not limitation, esters, dihaloalkanes such as 1,2-dibromoethane, iodine, bis(bromomethyl)benzene, silicon tetrachloride and tin tetrachloride, di(isopropenyl)benzene and divinyl benzene, alkyltrichlorosilanes and dialkyldichlorosilanes. Selection of the appropriate coupling agent will be determined by the monomers used and/or the nature of the polymer being synthesized as is well known in the art.
  • the incorporation of various chemical moieties allows for the preparation of tailored nanoparticles.
  • the chemical moiety can be attached to the preformed polymer molecule or it can be attached to the particle during its formation. Li the latter case, the activation step can be conducted in the presence of a chemical moiety so that the chemical moiety is incorporated into the crosslinked particle.
  • the crosslinked particle can have at least one functional group on its backbone so that the chemical moiety is covalently attached to the crosslinked particle at the functional group.
  • Such chemical moieties include, by way of illustration, pharmaceutical agents, catalysts, functional groups, surfactants, sensor groups and photoresponsive units.
  • the polymer molecule can be prepared by first preparing a short carboxy- functionalized polystyrene block, which is then used to initiate the polymerization of a mixture of styrene and vinylbenzocyclobutene. The resulting polymer can then be used as a starting material in the preparation of crosslinked nanoparticles, in which a single carboxy functional group and linear block are now attached.
  • the versatility inherent in the synthesis again allows the length of the linear block, nature of the repeat units and the number of functional groups to be easily varied.
  • Exemplary pharmaceutical agents include antibodies, aptamers,peptides, DNA oligomers, lipids, enzymes, carbohydrates and aminoglycosides.
  • Exemplary catalysts include metals, acids, bases, oxidizing and reducing agents and chelating groups.
  • Exemplary functional groups include acids, esters, alcohols, phenols, amines, thiols, amides, imines, nitriles, ethers, acetylenes, alkenes and heterocyclics.
  • the nanoparticle surface is coated with a lipophilic material and the tether is anchored into the coating through a hydrophobic moiety such as one or more aliphatic hydrocarbon chains.
  • the particles themselves can be described generally as nanoparticles having an inert core surrounded by a coating to which any desired materials can be coupled. In the agent of the invention, these materials can include a chelate containing a paramagnetic ion.
  • the inert core can be a vegetable, animal or mineral oil, but is preferably a fluorocarbon compound—perfluorinated or otherwise rendered additionally inert.
  • Mineral oils include petroleum derived oils such as paraffin oil and the like. Vegetable oils include, for example, linseed, safflower, soybean, castor, cottonseed, palm and coconut oils. Animal oils include tallow, lard, fish oils, and the like. Many oils are triglycerides.
  • Fluorinated liquids are particularly useful as cores. These include straight chain, branched chain, and cyclic hydrocarbons, preferably perfluorinated. Some satisfactorily fluorinated, preferably perfluorinated organic compounds useful in the particles of the invention themselves contain functional groups. However, perfluorinated hydrocarbons are preferred.
  • the nanoparticle core may comprise a mixture of such fluorinated materials.
  • the inert core has a boiling point of above 20 0 C, more preferably above 3O 0 C, still more preferably above 5O 0 C, and still more preferably above about 9O 0 C
  • the perfluoro compounds that are particularly useful in the above-described nanoparticle aspect of the invention include partially or substantially or completely fluorinated compounds. Chlorinated, brominated or iodinated forms may also be used. A detailed list of compounds useful as nanoparticle cores is included below.
  • the coating on the nanoparticles in this aspect is provided with a lipid/surfactant coating that will serve to anchor the desired moieties to the nanoparticle itself. If an emulsion is to be formed, the coating typically should include a surfactant. Typically, the coating will contain lecithin type compounds which contain both polar and non-polar portions as well as additional agents such as cholesterol.
  • lipid surfactants such as natural or synthetic phospholipids, but also fatty acids, cholesterols, lysolipids, sphingomyelins, tocopherols, glucolipids, stearylamines, cardiolipins, a lipid with ether or ester linked fatty acids, polymerized lipids, and lipid conjugated polyethylene glycol.
  • Other surfactants are commercially available. The foregoing may be mixed with anionic and cationic surfactants.
  • Fluorochemical surfactants may also be used.
  • perfluorinated alcohol phosphate esters and their salts include perfluorinated alcohol phosphate esters and their salts; perfluorinated sulfonamide alcohol phosphate esters and their salts; perfluorinated alkyl sulfonamide alkylene quaternary ammonium salts; N 5 N- (carboxyl-substituted lower alkyl) perfluorinated alkyl sulfonamides; and mixtures thereof.
  • perfluorinated means that the surfactant contains at least one perfluorinated alkyl group.
  • the lipids/surfactants are used in a total amount of 0.01-5% by weight of the nanoparticles, preferably 0.1-1% by weight.
  • lipid/surfactant encapsulated emulsions can be formulated with cationic lipids in the surfactant layer that facilitate the adhesion of nucleic acid material to particle surfaces.
  • Cationic lipids include DOTMA, N-[l-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammoium chloride; DOTAP, 1,2- dioleoyloxy-3-(trimethylammonio)propane; and DOTB, l,2-dioleoyl-3-(4'-trimethyl- ammonio)butanoyl-sn-glycerol may be used.
  • DOTMA N-[l-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammoium chloride
  • DOTAP 1,2- dioleoyloxy-3-(trimethylammonio)propane
  • DOTB l,2-dioleoyl-3-(4'-trimethyl- ammonio)butanoyl-sn-glycerol may be used.
  • the molar ratio of cationic lipid to non-cationic lipid in the lipid/surfactant monolayer may be, for example, 1 : 1000 to 2: 1, preferably, between 2:1 to 1:10, more preferably in the range between 1:1 to 1:2.5 and most preferably 1:1 (ratio of mole amount cationic lipid to mole amount non-cationic lipid, e.g., DPPC).
  • a wide variety of lipids may comprise the non-cationic lipid component of the emulsion surfactant, particularly dipalmitoylphosphatidylcholine, dipahnitoylphosphatidyl- ethanolamine or dioleoylphosphatidylethanolamine in addition to those previously described.
  • lipids bearing cationic polymers such as polyamines, e.g., spermine or polylysine or polyarginine may also be included in the lipid surfactant and afford binding of a negatively charged therapeutic, such as genetic material or analogues there of, to the outside of the emulsion particles.
  • the particles may be liposomal particles.
  • the literature describing various types of liposomes is vast and well known to practitioners.
  • the liposomes themselves are comprised of lipid moieties, the above-described lipids and surfactants are applicable in the description of moieties contained in the liposomes themselves.
  • These lipophilic components can be used to couple to the chelating agent in a manner similar to that described above with respect to the coating on the nanoparticles having an inert core.
  • Micelles are composed of similar materials, and this approach to coupling desired materials, and in particular, the chelating agents applies to them as well. Solid forms of lipids may also be used.
  • proteins or other polymers can be used to form the particulate carrier. These materials can form an inert core to which a lipophilic coating is applied, or the chelating agent can be coupled directly to the polymeric material through techniques employed, for example, in binding affinity reagents to particulate solid supports.
  • particles formed from proteins can be coupled to tether molecules containing carboxylic acid and/or amino groups through dehydration reactions mediated, for example, by carbodiimides.
  • Sulfur-containing proteins can be coupled through maleimide linkages to other organic molecules which contain tethers to which the chelating agent is bound.
  • the method of coupling so that an offset is obtained between the dentate portion of the chelating agent and the surface of the particle will be apparent to the ordinarily skilled practitioner.
  • the particles can be coupled through the spacer to a chelator in which a transition metal is disposed.
  • Typical chelators include porphyrins, ethylenediaminetetraacetic acid (EDTA), diethylenetriamine-N,N,N',N",N"- ⁇ entaacetate (DTPA), 1 ,4,10,13-tetraoxa-7,16- diazacyclooctadecane-7 (ODDA), 16-diacetate, N-2-(azol-l(2)-yl)ethyliminodiacetic acids, l,4,7,10-tetraazacyclododecane-N,N l ,N",N'"-tetraacetic acid (DOTA), l,7,13-triaza-4,10,16- trioxacyclo-octadecane-N,N',N"-triacetate (TTTA), tetraethylene glycols, 1,5, 9- triazacyclododecane
  • Examples of typical core components include, but not limited to: perfluorocarbon compounds which may be employed are perfluorotributylamine (FC47), perfluorodecalin (PP5), perfluoromethyldecalin (PP9), perfluorooctylbromide, perfluorotetrahydrofuran (FC80), perfluroether (PID), [(CF 3 ).sub.2 CFOCF 2 (CF 2 ) 2 CF 2 OCF(CF 3 ) 2 ]perfluoroether (PIID) [(CF 3 ) 2 CFOCF 2 (CF 2 ) 6 CF 2 OCF(CF 3 ) 2 ], perfluoroethe ⁇ olymer (Fomblin Y/01), perfluorododecane, perfluorobicyclo[4.3.0.] nonane, perfluorotritrimethylbicyclohexane, perfluorotripropylamine, perfluoroisopropyl cycl
  • Chlorinated perfluorocarbons such as chloroadamantane and chloromethyladamantane as described in U.S. Pat. No. 4,686,024 may be used. Such compounds are described, for example in U.S. Pat. Nos. 3,962,439; 3,493,581, 4,110,474, 4,186,253; 4,187,252; 4,252,824; 4,423,077; 4,443,480; 4,534,978 and 4,542,147.
  • Commercially available surfactants are Pluronic F-68, HamposylTM L30 (W.R.
  • egg yolk phospholipids alkylphosphoryl choline or alkylglycerolphosphoryl choline surfactants, and specific examples of these such as 1,2- dioctylglycero-3-phosphoryl choline, l,2-ditetradecylglycero-3-phosphoryl choline, 1,2- dihexadecylglycero-3-phosphoryl choline, l,2-dioctadecylglycero-3-phosphorylcholine, 1- hexadecyl-2-tetradecylglycero-3 -phosphoryl choline, 1 -octadecyl-2-tetradecylglycero-3 - phosphoryl choline, l-tetradecyl-2-octadecylglycero-3-phosphoryl choline, l-hexadecyl-2- octadecylglycero-3-phosphoryl choline,
  • Suitable perfluorinated alcohol phosphate esters include the free acids of the diethanolamine salts of mono- and bis(lH,lH,2H,2H-perfluoroalkyl)phosphates.
  • the phosphate salts available under the trade name "Zonyl RP" (E.I. Dupont de Nemours and Co., Wilmington, Del.), are converted to the corresponding free acids by known methods.
  • Suitable perfluorinated sulfonamide alcohol phosphate esters are described in U.S. Pat. No. 3,094,547.
  • Suitable perfluorinated sulfonamide alcohol phosphate esters and salts of these include perfluoro-n-octyl-N-ethylsulfonarnidoethyl phosphate, bis(perfluoro-n-octyl-N- ethylsulfonamidoethyl)phosphate, the ammonium salt of bis(perfluoro-n-octyl-N- ethylsulfonamidoethyl)phosphate,bis(perfluoro-decy l-N-ethylsulfonamidoethyl)-phosphate and bis(perfluorohexyl-N ethylsulfonamidoethyl)-phosphate.
  • the preferred formulations use phosphatidylcholine, derivatized-phosphatidylethanolamine and cholesterol as the aqueous surfactant.
  • Suitable paramagnetic metals include a lanthanide element of atomic numbers 58-70 or a transition metal of atomic numbers 21-29, 42 or 44, i.e., for example, scandium, titanium, vanadium, chromium, manganese, iron, cobalt, nickel, copper, molybdenum, ruthenium, cerium, praseodymium, neodymium, promethium, samarium, europium, gadolinium, terbium, dysprosium, holmium, erbium, thulium, and ytterbium, most preferably Gd(III), Mn(II), iron, europium and/or dysprosium.
  • a lanthanide element of atomic numbers 58-70 or a transition metal of atomic numbers 21-29, 42 or 44 i.e., for example, scandium, titanium, vanadium, chromium, manganese, iron, cobalt, nickel,
  • the chelating moiety can be coupled to the particle through a spacer or tether which may be an aliphatic chain, a peptide, a polyethylene glycol polymer, or any suitable spacing molecule.
  • One end of the spacer is bound, preferably covalently, to the dentate portion of the chelating agent; the other is anchored to the particle.
  • the coupling to the particle can be covalent or the spacer may be anchored through ionic bonding, hydrogen bonding or van der Waals forces.
  • particularly preferred anchoring moieties are the hydrocarbon side chains of phosphatides or other di-substituted glycerol derivatives.
  • the particles will be coupled to at least 10,000 chelators and/or paramagnetic ions, preferably 20,000 chelators and/or paramagnetic ions, more preferably 50,000 chelators and/or paramagnetic ions, more preferably at least 70,000 chelators and/or paramagnetic ions and more preferably at least 100,000 chelators and/or paramagnetic ions.
  • the particles may also be coupled to ligands for targeting and/or biologically active molecules. It is possible also to include among the components coupled to the particles bearing the chelated paramagnetic ion, radionuclides for use in treatment or diagnosis.
  • solid particles which contain reactive groups can be coupled directly to the tether or spacer; lipid-based particles such as oil emulsions, solid lipids, liposomes, and the like, can include lipophilic materials containing reactive groups which may covalently, then, be coupled to linking moieties which bear the dentate portion of the chelating agent, hi one particularly preferred embodiment, the process involves mixing a liquid fluorocarbon compound that forms the core of a nanoparticle and the components of a lipid/surfactant coating for that particle in an aqueous suspension, microfiuidizing, and, if desired, harvesting and sizing the particles.
  • the components to be coupled can be included in the original mixture by virtue of their initial coupling to one or more components of the lipid/surfactant coating, or the coupling to additional moieties can be conducted after the particles are formed.
  • the antibodies of the present invention may be generated by any suitable method known in the art.
  • the antibodies of the present invention can comprise polyclonal antibodies. Methods of preparing polyclonal antibodies are known to the skilled artisan (Harlow, et al., Antibodies: A Laboratory Manual, (Cold spring Harbor Laboratory Press, 2 nd ed. (1988), which is hereby incorporated herein by reference in its entirety).
  • a polypeptide can be administered to various host animals including, but not limited to, rabbits, mice, rats, etc. to induce the production of sera containing polyclonal antibodies specific for the antigen.
  • the administration of the polypeptides may entail one or more injections of an immunizing agent and, if desired, an adjuvant.
  • adjuvants may be used to increase the immunological response, depending on the host species, and include but are not limited to, Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum. Such adjuvants are also well known in the art.
  • immunizing agent may be defined as a polypeptide of the invention, including fragments, variants, and/or derivatives thereof, in addition to fusions with heterologous polypeptides and other forms of the polypeptides as may be described herein.
  • the immunizing agent and/or adjuvant will be injected in the mammal by multiple subcutaneous or intraperitoneal injections, though they may also be given intramuscularly, and/or through IV.
  • the immunizing agent may include polypeptides of the present invention or a fusion protein or variants thereof. Depending upon the nature of the polypeptides (i.e., percent hydrophobicity, percent hydrophilicity, stability, net charge, isoelectric point etc.), it may be useful to conjugate the immunizing agent to a protein known to be immunogenic in the mammal being immunized.
  • Such conjugation includes either chemical conjugation by derivatizing active chemical functional groups to both the polypeptide of the present invention and the immunogenic protein such that a covalent bond is formed, or through fusion-protein based methodology, or other methods known to the skilled artisan.
  • immunogenic proteins include, but are not limited to keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor.
  • adjuvants maybe used to increase the immunological response, depending on the host species, including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum. Additional examples of adjuvants which may be employed includes the MPL-TDM adjuvant (monophosphoryl lipid A, synthetic trehalose dicorynomycolate). The immunization protocol may be selected by one skilled in the art without undue experimentation.
  • the antibodies of the present invention can also comprise monoclonal antibodies.
  • Monoclonal antibodies may be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975) and U.S. Pat. No. 4,376,110, by Harlow, et ah, Antibodies: A Laboratory Manual, (Cold spring Harbor Laboratory Press, 2 nd ed. (1988), by Hammerling, et ah, Monoclonal Antibodies and T-CeIl Hybridomas (Elsevier, N.Y., (1981)), or other methods known to the artisan.
  • a mouse, a humanized mouse, a mouse with a human immune system, hamster, or other appropriate host animal is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent.
  • the lymphocytes may be immunized in vitro.
  • the immunizing agent will typically include antigens form infectious disease organisms, cancer cells, environmental antigens and the like, polypeptides, fragments or a fusion protein thereof.
  • PBLs peripheral blood lymphocytes
  • spleen cells or lymph node cells are used if non-human mammalian sources are desired.
  • the lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986), pp. 59- 103).
  • Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Usually, rat or mouse myeloma cell lines are employed.
  • the hybridoma cells may be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells.
  • a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells.
  • the parental cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT)
  • HGPRT or HPRT hypoxanthine guanine phosphoribosyl transferase
  • the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (“HAT medium”), which substances prevent the growth of HGPRT-deficient cells.
  • Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. More preferred immortalized cell lines are murine myeloma lines, which can be obtained, for instance, from the SaIk Institute Cell Distribution Center, San Diego, Calif, and the American Type Culture Collection, Manassas, Va.
  • human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) pp. 51-63).
  • the culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against the antigens.
  • the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoadsorbant assay (ELISA).
  • RIA radioimmunoassay
  • ELISA enzyme-linked immunoadsorbant assay
  • the binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollart, Anal. Biochem., 107:220 (1980).
  • the clones may be subcloned by limiting dilution procedures and grown by standard methods (Goding, supra). Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI- 1640. Alternatively, the hybridoma cells may be grown in vivo as ascites in a mammal.
  • the monoclonal antibodies secreted by the subclones may be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-sepharose, hydroxyapatite chromatography, gel exclusion chromatography, gel electrophoresis, dialysis, or affinity chromatography.
  • immunoglobulin purification procedures such as, for example, protein A-sepharose, hydroxyapatite chromatography, gel exclusion chromatography, gel electrophoresis, dialysis, or affinity chromatography.
  • the skilled artisan would acknowledge that a variety of methods exist in the art for the production of monoclonal antibodies and thus, the invention is not limited to their sole production in hybridomas.
  • the monoclonal antibodies may be made by recombinant DNA methods, such as those described in U.S. Pat. No. 4,816,567.
  • the term "monoclonal antibody” refers to an antibody derived from a single eukaryotic, phage, or prokaryotic clone.
  • the DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies, or such chains from human, humanized, or other sources).
  • the hybridoma cells of the invention serve as a preferred source of such DNA.
  • the DNA may be placed into expression vectors, which are then transformed into host cells such as Simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells.
  • host cells such as Simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells.
  • mice can be immunized with a polypeptide or a cell expressing such peptide.
  • an immune response e.g., antibodies specific for the antigen are detected in the mouse serum
  • the mouse spleen is harvested and splenocytes isolated.
  • the splenocytes are then fused by well- known techniques to any suitable myeloma cells, for example cells from cell line SP20 available from the ATCC.
  • Hybridomas are selected and cloned by limited dilution.
  • hybridoma clones are then assayed by methods known in the art for cells that secrete antibodies capable of binding a polypeptide of the invention.
  • Ascites fluid which generally contains high levels of antibodies, can be generated by immunizing mice with positive hybridoma clones.
  • the present invention provides methods of generating monoclonal antibodies as well as antibodies produced by the method comprising culturing a hybridoma cell secreting an antibody of the invention wherein, preferably, the hybridoma is generated by fusing splenocytes isolated from a mouse immunized with an antigen of the invention with myeloma cells and then screening the hybridomas resulting from the fusion for hybridoma clones that secrete an antibody able to bind a polypeptide of the invention.
  • Other methods can also be used for the large scale production of antibodies. For example, antibodies can also be generated using various phage display methods known in the art.
  • phage display methods functional antibody domains are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them.
  • phage can be utilized to display antigen binding domains expressed from a repertoire or combinatorial antibody library (e.g., human or murine).
  • Phage expressing an antigen binding domain that binds the antigen of interest can be selected or identified with antigen, e.g., using labeled antigen or antigen bound or captured to a solid surface or bead.
  • Phage used in these methods are typically filamentous phage including ⁇ and Ml 3 binding domains expressed from phage with Fab, Fv or disulfide stabilized Fv antibody domains recombinantly fused to either the phage gene III or gene VIII protein.
  • Examples of phage display methods that can be used to make the antibodies of the present invention include those disclosed in Brinkman et ah, J. Immunol. Methods 182:41-50 (1995); Ames et ah, J. Immunol. Methods 184:177-186 (1995); Kettleborough et ah, Eur. J. Immunol.
  • the antibodies of the present invention have various utilities.
  • such antibodies may be used in diagnostic assays to detect the presence or quantification of the polypeptides of the invention in a sample.
  • Such a diagnostic assay can comprise at least two steps. The first, subjecting a sample with the antibody, wherein the sample is a tissue (e.g., human, animal, etc.), biological fluid (e.g., blood, urine, sputum, semen, amniotic fluid, saliva, etc.), biological extract (e.g., tissue or cellular homogenate, etc.), a protein microchip (e.g., See Arenkov P, et ah, Anal Biochem., 278(2):123-131 (2000)), or a chromatography column, etc.
  • tissue e.g., human, animal, etc.
  • biological fluid e.g., blood, urine, sputum, semen, amniotic fluid, saliva, etc.
  • biological extract e.g., tissue or cellular homogen
  • the method may additionally involve a first step of attaching the antibody, either covalently, electrostatically, or reversibly, to a solid support, and a second step of subjecting the bound antibody to the sample, as defined above and elsewhere herein.
  • the antibodies are labeled with a detectable moiety as described infra.
  • the detectable moiety may be a radioisotope, such as 2 H, 14 C, 32 P, or 125 I, a florescent or chemiluminescent compound, such as fluorescein isothiocyanate, rhodamine, or luciferin, or an enzyme, such as alkaline phosphatase, beta-galactosidase, green fluorescent protein, or horseradish peroxidase. Any method known in the art for conjugating the antibody to the detectable moiety may be employed, including those methods described by Hunter et ah, Nature, 144:945 (1962); David et al, Biochem., 13:1014 (1974); Pain et at, J.
  • antibodies are specific for either prokaryotic or eukaryotic cell antigens. Infectious disease almost invariably results in the acquisition of foreign nucleic acids, which could also be targeted using this technology.
  • Specific targets could be viral, e.g. HIV (virus or provirus) or bacterial, e.g. multi-drug resistant bacteria e.g. TB, fungal or protoazoan. This technology can be especially useful in detecting agents such as microbial or viral agent (e.g. Ebola virus, etc.), or known or novel bio-terrorist agents.
  • Particularly preferred viral organisms causing human diseases include (but not restricted to) Filoviruses, Herpes viruses, Hepatitisviruses, Retroviruses, Orthomyxoviruses, Paramyxoviruses, Togaviruses, Picornaviruses, Papovaviruses and Gastroenteritisviruses.
  • Filoviruses include Herpes viruses, Hepatitisviruses, Retroviruses, Orthomyxoviruses, Paramyxoviruses, Togaviruses, Picornaviruses, Papovaviruses and Gastroenteritisviruses.
  • Other preferred, non-limiting examples of viral agents are listed in Table 1.
  • the antibodies are specific for human or domestic animal bacterial pathogens.
  • Particularly preferred bacteria causing serious human diseases are the Gram positive organisms: Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis and E. faecium, Streptococcus pneumoniae and the Gram negative organisms: Pseudomonas aeruginosa, Burkholdia cepacia, Xanthomonas maltophila, Escherichia coli, Enterobacter spp, Klebsiella pneumoniae and Salmonella spp.
  • the target molecules may include (but are not restricted to) molecules essential to bacterial survival and multiplication in the host organism, virulence gene products, gene products encoding single- or multi-drug resistance.
  • gram negative bacteria are also within the scope of the invention.
  • the antibodies are targeted to toxins produced by a disease agent such as anthrax.
  • anthrax which is one of the agents that can be used in a bioterrorist attack.
  • Anthrax infection is mediated by spores of Bacillus anthracis, which can gain entry to the body through breaks in the skin, through inhalation, or through ingestion.
  • Fatal anthrax is characterized by the establishment of a systemic bacteremia that is accompanied by an overwhelming toxemia. It seems that anthrax is a two- pronged attack with the bacteremia and/or toxemia contributing to the fatal syndrome of shock, hypoperfusion, and multiple organ system failure.
  • the antibodies are specific for protozoa infecting humans and causing human diseases.
  • Particularly preferred protozoan organisms causing human diseases according to the present invention include (but not restricted to) Malaria e.g. Plasmodium falciparum and M. ovale, Trypanosomiasis (sleeping sickness) e.g. Trypanosoma cruzei, Leischmaniasis e.g. Leischmania donovani, Amebiasis e.g. Entamoeba histolytica.
  • the antibodies are specific for fungi causing pathogenic infections in humans.
  • Particularly preferred fungi causing or associated with human diseases according to the present invention include (but not restricted to) Candida albicans, Histoplasma neoformans, Coccidioides immitis and Penicillium marneffei.
  • the antibodies are specific for target oligonucleotides responsible for viral replication; viral infection cycle such as attachment to cellular ligands; viral gene products encoding host immune modulating functions.
  • target oligonucleotides responsible for viral replication
  • viral infection cycle such as attachment to cellular ligands
  • viral gene products encoding host immune modulating functions.
  • examples of viral organisms include, but not restricted to, those listed in table 1. For information about the viral organisms see Fields of Virology, 3. ed., vol 1 and 2, BN Fields et al. (eds.). Non- limiting examples of targets of selected viral organisms are listed in table 2.
  • Table 1 Selected viral organisms causing human diseases.
  • Organism target gene open reading frame gene product
  • HCV hepatitis C virus
  • viral particles are propagated in in vitro culture systems of appropriate mammalian cells. Initial screening is typically performed in transformed cell lines. More thorough screening is typically performed in human diploid cells.
  • WI- 38 is a human diploid fibroblast cell line.
  • MRC-5 is human lung fibroblasts.
  • HeLa is a human aneuploid epithelial cell line.
  • PRMK is primary rhesus monkey kidney cells.
  • PCMK is primary cynomolgus monkey kidney cells.
  • Vero cells green monkey kidney cells
  • Mewo cells will sustain the growth of for example herpesviruses.
  • Bacterial antigens are specific for the human or domestic animal bacterial pathogens listed in (but not restricted to) table 4.
  • the target antigens may include (but are not restricted to) gene products essential to bacterial survival and multiplication in the host organism, virulence gene products encoding single- or multi-drug resistance such as for instance the gene products listed in table 5. Table 4. Selected bacteria causing serious human diseases
  • Table 6 Listed in Table 6 below are examples of genes encoding the protein complexes listed in Table 5 above. The individual genes have homologues in the major human pathogenic bacteria listed in Table 4. Table 6 below depicts an example of a Gram negative (Escherichia coli) and a Gram positive (Staphylococcus aureus) organism, chosen as representatives for the two groups of bacteria. Table 6. Examples of gene products encoding possible gene target proteins.
  • Sequences for the gene products listed in Table 5 and 6 can be found in GenBank (http://www.ncbi.nlm.nih.gov/).
  • GenBank http://www.ncbi.nlm.nih.gov/.
  • the gene sequences maybe genomic, cDNA or mRNA sequences.
  • Preferred sequences are viral gene products containing the complete coding region and 5' untranslated sequences that are involved in viral replication.
  • Protozoan antigens are specific for protozoan organisms infecting humans and causing human diseases.
  • protozoa include, but are not restricted to, the following: 1. Malaria e.g. Plasmodium falciparum and M. ovale, (references: Malaria by M Wahlgren and P Perlman (eds.), Harwood Academic Publishers, 1999. Molecular Immunological Considerations in Malaria Vaccine Development by MF Good and AJ Saul, CRC Press 1993). 2. Trypanosomiasis (sleeping sickness) e.g.
  • the antigens are specific for fungi cause pathogenic infections in humans. These include, but are not restricted to, the following: Candida albicans (references: AH Groll et al., Clinical pharmacology of systemic antifungal agents: a comprehensive review of agents in clinical use, current investigational compounds, and putative targets for antifungal drug development. Adv. Pharmacol. 1998:44:343-501. MDD Backer et al, An antisense-based functional genomics approach for identification of gene products critical for growth of Candida albicans.
  • Host cellular gene products involved in viral diseases are specific for host cellular gene products involved in viral diseases.
  • CD4 chemokine receptors such as CCR3, CCR5 are required for HIV infection.
  • abnormal or cancer cells are detected by the compositions and methods of the invention.
  • many malignancies are associated with the presence of foreign DNA, e.g. Bcr-Abl, Bcl-2, HPV, and these provide unique molecular targets to permit selective malignant cell targeting.
  • the approach can be used to target single base substitutions (e.g. K-ras, p53) or methylation changes.
  • the particles may, along with other reagents, be packaged in a kit useful for conveniently performing the assay methods for the determination of an analyte.
  • reagents can be provided in packaged combination, in the same or separate containers, in liquid or lyophilized form so that the ratio of the reagents provides for substantial optimization of the method and assay.
  • the reagents may each be in separate containers, or various reagents can be combined in one or more containers depending on the cross-reactivity and stability of the reagents.
  • a reagent test kit which may contain, in packaged combination, an antibody specific for a particular analyte, a particle of the present invention containing the same antibody or an analog or derivative thereof, and may optionally also comprise one or more calibrators comprising a known amount of the analyte.
  • a test kit may provide reagents for an assay with enhanced clinical sensitivity for the analyte and structurally related compounds.
  • the self-assembling luminescent protein-metal nanoprobe is a sensor towards particular peptide sequences or organic ligands.
  • the underlying premise of the detection strategy relies on (1) an antibody based fluorescence competition assay and (2) a fluorescence resonance energy transfer (Medintz, I. L. et al. Nature Materials 2, 630-638 (2003); Didenko, V. V. Biotechniques 31, 1106 (2001); Oh, E. et al. Journal of the American Chemical Society 127, 3270-3271 (2005); Perez-Luna, V. H. & Asian, K. Abstracts of Papers of the American Chemical Society 226, U99-U99 (2003); Gueroui, Z. & Libchaber, A.
  • the fluorescence signal may arise from either the labeled antibody or hapten subsequent to analyte detection.
  • Potential arrangements include labeling of the antibody and hapten with a luminescent quantum dot (the donor) and a gold nanoparticle (the quencher), respectively, and the reverse configuration. These assemblies yield either fluorescent antibody ( Figure 2A) or hapten ( Figure 2B) products.
  • nanoprobes specific for anthrax toxin (PA 211 14B7), cancer epitopes, and phosphopeptides can be purchased where available or produced using hybridoma technology.
  • covalent binding of the nanoparticles (or organic donors and quenchers) to the antibody and hapten is accomplished using maleimide activated Qdots® (Quantum Dot Corp.), lysine reactive Nanogold® labeling reagents (Nanoprobes Corp.), or amine-reactive organic donors or quenchers (Molecular Probes).
  • Qdots® Quantum Dot Corp.
  • lysine reactive Nanogold® labeling reagents Nathoprobes Corp.
  • amine-reactive organic donors or quenchers Molecular Probes.
  • the binding sites of the labeled antibody are then loaded with the labeled hapten, thus forming the fluorescently quenched nanosensor.
  • Target detection is then accomplished using a competitive binding assay between the free target analyte and the

Abstract

A luminescent protein-quantum dot biosensor that can be tailor designed towards the specific detection of environmentally or medicinally important compounds. This biosensor utilizes a biomolecule-inorganic or organic nanometer-sized assembly that serves as a scaffold for a fluorescence competition assay. Specific and sensitive detection of chemical poisons, contaminants, and biotoxins can be realized by mediating the structures within the biomolecule-nanoparticle assembly, while maintaining the luminescent properties of the generalized probe. This versatile strategy of biosensing is applicable towards environmental analysis, medicinal diagnostics, and biodefense.

Description

LUMINESCENT NANOSENSORS
FIELD OF THE INVENTION
[0001] The invention relates to sensors and nanoparticles including biosensors and methods for molecular identification.
BACKGROUND OF THE INVENTION
[0002] There is a demand for a rapid, simple, cost-effective technique for screening air, water and blood samples to identify various components therein. Screening can involve detection of harmful chemicals, bacteria and viruses. For example, there is a need for early medical diagnostics, genomics assays, proteomics analyses, drug discovery screening, and detection of biological and chemical warfare agents for homeland security and defense. [0003] Screening can also be used to identify the presence or absence of medical diseases and infectious pathogens. Regarding blood, the use of inexpensive screening analyses can allow the rapid detection and improved treatments of many illnesses. Rapid and effective medical screening tests can also reduce the cost of health care by preventing unnecessary and costly reactive medical treatment.
[0004] A critical factor in many diagnostics is the rapid, selective, and sensitive detection of biochemical substances, such as proteins, metabolites, nucleic acids, biological species or living systems, such as bacteria, virus or related components at ultra-trace levels in samples provided. In the case of medical diagnostic applications, biological samples can include tissues, blood and other bodily fluids.
[0005] There is a need in the art to achieve the required level of sensitivity and specificity in identifying and differentiating between a large number of biochemical constituents in complex samples. Although sensitivities achieved by luminescence techniques are generally adequate for certain applications, alternative techniques with improved spectral selectivities are desirable to overcome the limited spectral specificity generally provided by luminescent labels.
SUMMARY
[0006] A nanoparticle composition comprising a plurality of biomolecules attached thereto, detects analytes or target molecules with specificity and sensitivity. In particular, the nanoparticle composition or nanoprobe utilizes a biomolecule and nanoparticle assembly that serves as a scaffold for a fluorescence competition assay.
[0007] In a preferred embodiment, a nanoprobe for detection of molecular interactions comprises a nanoparticle and a detectably labeled biomolecule attached thereto. Preferably, the nanoparticle is an inorganic or organic nanoparticle.
[0008] In another preferred embodiment, the nanoparticle is a metal. The metal is selected from the group consisting of: gold, silver, copper and platinum.
[0010] In other preferred embodiments, the nanoparticle is selected from the group consisting of : ZnS, ZnO, TiO2, AgI, AgBr, HgI2, PbS, PbSe, ZnTe, CdTe, In2S3, In2S3,
Cd3P2, Cd3As2, InAs, and GaAs.
[0011] hi a preferred embodiment, the nanoparticle is about 5 nm up to 100 nm in diameter., preferably, the nanoparticle is about 5 nm to about 20 nm in diameter.
[0012] Li another preferred embodiment, a plurality of detectably labeled biomolecules are attached to the nanoparticle. The biomolecule can be an antibody, aptamer, hapten, oligonucleotide, protein or peptide.
[0013] In another preferred embodiment, the biomolecule is labeled with a detectable moiety.
[0014] In a preferred embodiment, the detectable moiety is a fluorescent molecule.
Preferred fluorescent molecules comprises at least one of: biotin, fluorescein (5- carboxyfluorescein-N-hydroxysuccinimide ester), rhodamine (5,6-tetramethyl rhodamine),
Cy3, Cy3.5, Cy5, Cy5.5 and Cy7. 6-carboxyfluorescein (6-FAM), 2',4',1,4,- tetrachlorofluorescein (TET), 2l,4',5l,7',l,4-hexachlorofluorescein (HEX), 2',7'-dimethoxy-
4',5'-dichloro-6-carboxyrhodamine (JOE), 2'-chloro-5'-fluoro-7',8'-fused phenyl- 1,4-dichloro-
6-carboxyfluorescein (NED), 2'-chloro-7'-phenyl-l,4-dichloro-6-carboxyfluorescein (VIC), fluorescein isothiocyanate (FITC), 5,6-carboxymethyl fluorescein, Texas red, nitrobenz-2- oxa-l,3-diazol-4-yl (NBD), coumarin, dansyl chloride, rhodamine, amino-methyl coumarin
(AMCA), Eosin, Erythrosin, BODIPY™, Cascade Blue™, Oregon Green™, pyrene, lissamine, xanthenes, acridines, oxazines, phycoerythrin, quantum dye™, fluorescent energy transfer dyes, or thiazole orange-ethidium heterodimer.
[0015] In accordance with the invention, the detectable moiety comprises a donor and acceptor molecule. The donor molecule is a fluorophore and the acceptor molecule is a quencher. [0016] In another preferred embodiment, a quencher molecule comprises at least one of: rhodamine dyes, tetramethyl-6-carboxyrhodamine (TAMRA), tetrapropano-6- carboxyrhodamine (ROX), DABSYL, DABCYL, cyanine dyes, nitrothiazole blue (NTB), anthraquinone, malachite green, nitrothiazole, or nitroimidazole compounds. [0017] In another preferred embodiment, the biomolecules are covalently attached to the nanoparticle. Preferably, the biomolecules are attached to the nanoparticles maleimide linkages, carbodiimide linkages, lysine linkages, amine-reactive organic donors or quenchers. [0018] In another preferred embodiment, the biomolecule comprises a first molecule bound to a second molecule. In accordance with the invention, the first molecule is labeled with a fluorescent moiety and the second molecule is labeled with a quenching moiety. The first molecule and second molecule do not emit a fluorescent signal when the first molecule and second molecule are associated together. However, dissociation of the second molecule from the first molecule results in emission of fluorescence wherein the first molecule remains bound to the nanoparticle. Dissociation of the first and second molecule occurs when an analyte contacts the nanoprobe and the first molecule specifically binds the analyte. [0019] In another preferred embodiment, a method of detecting specific molecular interactions comprises providing a nanoprobe comprising a differentially labeled antibody- hapten complex; contacting the nanoprobe with an analyte, binding of the analyte to the antibody releases the labeled hapten; resulting in a change in fluorescence; and, detecting specific molecular interactions between the nanoprobe and analyte. In a preferred embodiment, the antibody is labeled with a fluorophore and the hapten is labeled with a fluorophore quenching molecule. In one aspect of the invention, the antibody is labeled with a fluorophore quenching molecule and the hapten is labeled with a fluorophore. In accordance with the invention, the nanoprobe is in a quenched fluorescent state as compared to fluorescence emitted when the hapten is released. Binding of the analyte to a detectably labeled antibody is detected by fluorescence as compared to a baseline fluorescence of nanoprobe. This is due to the displacement of the hapten by the binding of an analyte to the detectably labeled antibody and is measured by increase in fluorescence as compared to the baseline fluorescence of nanoprobe. In the alternative, lack of binding of an analyte to the nanoprobe is detected by no increase in fluorescence as compared to binding of a control analyte to the nanoprobe. Thus, the specificity of antibody for an analyte is a measure of fluorescence. The hapten comprises protein, peptide, oligonucleotide or organic molecule. [0020] In accordance with the invention, antibody-analyte binding is determined by affinity of the antibody for the analyte and dissociation constants (KD) between an antibody- hapten complexes and antibody-analyte complexes. The KQ of antibody-hapten is equal or greater than the KD of antibody-analyte complexes.
[0021] In a preferred embodiment, either the antibody or the hapten are fluorescently labeled i.e. one is labeled with a donor molecule and the other is labeled with a quenching molecule. Preferably, the antibody is labeled with a donor molecule and the hapten is labeled with a quenching molecule. Examples of fluorescent molecules include: biotin, fluorescein (5-carboxyfluorescein-N-hydroxysuccinimide ester), rhodamine (5,6-tetramethyl rhodamine), Cy3, Cy3.5, Cy5, Cy5.5 and Cy7. 6-carboxyfluorescein (6-FAM), 2',4',1,4,- tetrachlorofluorescein (TET), 2',4',5',7',l,4-hexachlorofluorescein (HEX), 2',7'-dimethoxy- 4',5'-dichloro-6-carboxyrhodamine (JOE), 2'-chloro-5l-fluoro-7',8'-fused phenyl- 1,4-dichloro- 6-carboxyfluorescein (NED), 2'-chloro-7'-phenyl-l,4-dichloro-6-carboxyfluorescein (VIC), fluorescein isothiocyanate (FITC), 5,6-carboxymethyl fluorescein, Texas red, nitrobenz-2- oxa-l,3-diazol-4-yl (NBD), coumarin, dansyl chloride, rhodamine, amino-methyl coumarin (AMCA), Eosin, Erythrosin, BODIPY™, Cascade Blue™, Oregon Green™, pyrene, lissamine, xanthenes, acridines, oxazines, phycoerythrin, quantum dye™, fluorescent energy transfer dyes, or thiazole orange-ethidium heterodimer.
[0022] Examples of quencher molecules includes: rhodamine dyes, tetramethyl-6- carboxyrhodamine (TAMRA), tetrapropano-6-carboxyrhodamine (ROX), DABSYL, DABCYL, cyanine dyes, nitrothiazole blue (NTB), anthraquinone, malachite green, nitrothiazole, or nitroimidazole compounds.
[0023] In another preferred embodiment, a method or system for simultaneously detecting the presence or absence of one or more different target/analyte molecules in a sample using a plurality of different species of antibodies, aptamers, oligonucleotides, wherein each species of for example, antibody, has a different reporter group, a binding region that binds to a specific non-nucleic acid target molecule, and wherein the binding regions of different antibodies bind to different target molecules, wherein the binding is detected by fluorescent emission as described infra.
[0024] The method can also be carried out with a plurality of antibodies, aptamers etc. For example, each antibody or aptamer can include a reporter such as a molecular beacon that changes fluorescence properties upon target binding, i.e. from quenched state to fluorescence state. Each species of, for example, antibody can be labeled with a different fluorescent dye to allow simultaneous detection of multiple target molecules, e.g., one species might be labeled with fluorescein and another with rhodamine. The fluorescence excitation wavelength (or spectrum) can be varied and/or the emission spectrum can be observed to simultaneously detect the presence of multiple targets.
[0025] The fluorescence measurement can be performed with a number of different instruments, including standard fluorescence spectrophotometers, or in a small volume using a high-intensity source, such a laser, high-efficiency light collection optics, such as a high- numeric aperture microscope objective, and a high-efficiency low-noise detector, such as photo-multiplier tube, a photodiode or a CCD camera.
[0026] The method can further include a computer program that includes instructions for causing the processor to compare the measured fluorescence emission or excitation spectrum with the known spectrum of each of the individual dyes to quantitatively determine the concentration of each of the target molecules in the solution.
[0027] To assist in analyzing the sample, the new detection systems can include pattern recognition software. The software compares the target molecule binding pattern corresponding to the unknown sample with binding patterns corresponding to known compounds. From these comparisons, the software can determine the composition of the sample, or deduce information about the source of the sample. The systems can be used to detect the existence of characteristic compounds, or "molecular fingerprints," associated with certain chemicals or conditions. For example, the systems can be used for human drug testing by detecting the presence of metabolites of particular drugs. The systems can also be used to infer the existence of a disease (e.g., cancer) by detecting the presence of compounds associated with the disease state, or for pollution monitoring by detecting compounds characteristic of the discharge of certain pollutants. Numerous other applications are also possible. [0028] Other aspects of the invention are described infra.
BRIEF DESCRIPTION OF THE DRAWINGS
[0029] There is shown in the drawings embodiments, which are presently preferred, it being understood, however, that the invention can be embodied in other forms without departing from the spirit or essential attributes thereof.
[0030] Figure 1 is a schematic illustration showing the detection strategy utilizing metal nanoparticles as the donor and/or quencher components. [0031] Figures 2A and 2B are a schematic illustration showing that the fluorescence signal may arise from either the labeled antibody or hapten subsequent to analyte detection. Potential arrangements, as shown, include labeling of the antibody and hapten with a luminescent quantum dot (the donor) and a gold nanoparticle (the quencher), respectively, and the reverse configuration. These assemblies yield either fluorescent antibody (Figure 2A) or hapten (Figure 2B) products. [0032] Other aspects of the invention and described infra.
DETAILED DESCRIPTION
[0033] The invention describes a self-assembling fluorescent nanometer-sized probe that structurally and functionally integrates an analyte-specific protein (such as an antibody) with nanoparticles. Detection of the target analyte is afforded through a fluorescence competition assay thereby allowing for a high sensitivity. In addition, the incorporation of metal nanoparticles provides excellent spectral properties, as cadmium nanoparticles are brightly luminescent and gold nanoparticles possess luminescence quenching properties.
Definitions
[0034] Prior to setting forth the invention, it may be helpful to an understanding thereof to set forth definitions of certain terms that will be used hereinafter.
[0035] As used herein, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.
[0036] The term "biomolecule" refers to proteins and peptides, antibodies, aptamers,
DNA, RNA (including mRNA, rRNA, tRNA and tmRNA), nucleotides and nucleosides. The biomolecule is used to detect a complementary biomolecule. Examples include antibodies that detect antigens, oligonucleotides that detect complimentary oligonucleotides, and ligands that detect receptors. Such probes are preferably immobilized on a microelectrode comprising a substrate.
[0037] As used herein, the term "aptamer" or "selected nucleic acid binding species" shall include non-modified or chemically modified RNA or DNA. The method of selection may be by, but is not limited to, affinity chromatography and the method of amplification by reverse transcription (RT) or polymerase chain reaction (PCR).
[0038] As used herein, the term "signaling aptamer" shall include aptamers with reporter molecules, preferably a fluorescent dye, appended to a nanoparticle in such a way that upon conformational changes resulting from the aptamer's interaction with a ligand, the reporter molecules yields a differential signal, preferably a change in fluorescence intensity. [0039] As used herein, the terms "ligand," "target," and "bait" are used interchangeably throughout the specification and includes any molecule that binds to the aptamer. [0040] A "nucleic acid" is a polymeric compound comprised of covalently linked subunits called nucleotides. Nucleic acid includes polyribonucleic acid (RNA) and polydeoxyribonucleic acid (DNA), both of which maybe single-stranded or double-stranded. DNA includes cDNA, genomic DNA, synthetic DNA, and semi-synthetic DNA. The sequence of nucleotides or nucleic acid sequence that encodes a protein is called the sense sequence.
[0041] As used herein, the term "fragment or segment", as applied to a nucleic acid sequence, gene or polypeptide, will ordinarily be at least about 5 contiguous nucleic acid bases (for nucleic acid sequence or gene) or amino acids (for polypeptides), typically at least about 10 contiguous nucleic acid bases or amino acids, more typically at least about 20 contiguous nucleic acid bases or amino acids, usually at least about 30 contiguous nucleic acid bases or amino acids, preferably at least about 40 contiguous nucleic acid bases or amino acids, more preferably at least about 50 contiguous nucleic acid bases or amino acids, and even more preferably at least about 60 to 80 or more contiguous nucleic acid bases or amino acids in length. "Overlapping fragments" as used herein, refer to contiguous nucleic acid or peptide fragments which begin at the amino terminal end of a nucleic acid or protein and end at the carboxy terminal end of the nucleic acid or protein. Each nucleic acid or peptide fragment has at least about one contiguous nucleic acid or amino acid position in common with the next nucleic acid or peptide fragment, more preferably at least about three contiguous nucleic acid bases or amino acid positions in common, most preferably at least about ten contiguous nucleic acid bases amino acid positions in common. [0042] As used herein, the term "oligonucleotide" includes linear or circular oligomers of natural and/or modified monomers or linkages, including deoxyribonucleosides, ribonucleosides, substituted and alpha-anomeric forms thereof, peptide nucleic acids (PNA), locked nucleic acids (LNA), phosphorthiorate, methylphosphonate, and the like. Oligonucleotides are capable of specifically binding to a target polynucleotide by way of a regular pattern of monomer-to-monomer interactions, such as Watson-Crick type of base pairing, Hoδgsteen or reverse Hoδgsteen types of base pairing, or the like. [0043] The oligonucleotide may be "chimeric", that is, composed of different regions.
In the context of this invention "chimeric" compounds are oligonucleotides, which contain two or more chemical regions, for example, DNA region(s), RNA region(s), PNA region(s) etc. Each chemical region is made up of at least one monomer unit, i.e., a nucleotide in the case of an oligonucleotide compound. These oligonucleotides typically comprise at least one region wherein the oligonucleotide is modified in order to exhibit one or more desired properties. The desired properties of the oligonucleotide include, but are not limited, for example, to increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid. Different regions of the oligonucleotide may therefore have different properties. The chimeric oligonucleotides of the present invention can be formed as mixed structures of two or more oligonucleotides, modified oligonucleotides, oligonucleosides and/or oligonucleotide analogs.
[0044] The oligonucleotide can be composed of regions that can be linked in "register", that is, when the monomers are linked consecutively, as in native DNA, or linked via spacers. The spacers are intended to constitute a covalent "bridge" between the regions and have in preferred cases a length not exceeding about 100 carbon atoms. The spacers may carry different functionalities, for example, having positive or negative charge, carry special nucleic acid binding properties (intercalators, groove binders, toxins, fluorophores etc.), being lipophilic, inducing special secondary structures like, for example, alanine containing peptides that induce alpha-helices.
[0045] As used herein, the term "monomers" typically indicates monomers linked by phosphodiester bonds or analogs thereof to form oligonucleotides ranging in size from a few monomeric units, e.g., from about 3-4, to about several hundreds of monomelic units. Analogs of phosphodiester linkages include: phosphorothioate, phosphorodithioate, methylphosphornates, phosphoroselenoate, phosphoramidate, and the like. [0046] In the present context, the terms "nucleobase" covers naturally occurring nucleobases as well as non-naturally occurring nucleobases. It should be clear to the person skilled in the art that various nucleobases which previously have been considered "non- naturally occurring" have subsequently been found in nature. Thus, "nucleobase" includes not only the known purine and pyrimidine heterocycles, but also heterocyclic analogues and tautomers thereof. Illustrative examples of nucleobases are adenine, guanine, thymine, cytosine, uracil, purine, xanthine, diaminopurine, 8-oxo-N6-methyladenine, 7-deazaxanthine, 7-deazaguanine, N^N^ethanocytosin, N6,N6-ethano-2,6-diaminopurine, 5-methylcytosine, 5- (C3-C6)-alkynylcytosine, 5-fluorouracil, 5-bromouracil, pseudoisocytosine, 2-hydroxy-5- methyl-4-triazolopyridin, isocytosine, isoguanin, inosine and the "non-naturally occurring" nucleobases described in Benner et al., U.S. Pat No. 5,432,272. The term "nucleobase" is intended to cover every and all of these examples as well as analogues and tautomers thereof. Especially interesting nucleobases are adenine, guanine, thymine, cytosine, and uracil, which are considered as the naturally occurring nucleobases in relation to therapeutic and diagnostic application in humans.
[0047] As used herein, "nucleoside" includes the natural nucleosides, including 2'-deoxy and 2'-hydroxyl forms, e.g., as described in Kornberg and Baker, DNA Replication, 2nd Ed. (Freeman, San Francisco, 1992).
[0048] "Analogs" in reference to nucleosides includes synthetic nucleosides having modified base moieties and/or modified sugar moieties, e.g., described generally by Scheit, Nucleotide Analogs, John Wiley, New York, 1980; Freier & Altmann, Nucl. Acid. Res., 1997, 25(22), 4429-4443, Toulme, J.J., Nature Biotechnology 19:17-18 (2001); Manoharan M., Biochemica et Biophysica Acta 1489:117-139(1999); Freier S.,M., Nucleic Acid Research, 25:AA29-AAA3 (1997), Uhlman, E., Drug Discovery & Development, 3: 203-213 (2000), Herdewin P., Antisense & Nucleic Acid Drug Dev., 10:297-310 (2000), ); T-O, 3Λ-C-linked [3.2.0] bicycloarabinonucleosides (see e.g. N.K Christiensen., et al, J. Am. Chem. Soc, 120: 5458-5463 (1998). Such analogs include synthetic nucleosides designed to enhance binding properties, e.g., duplex or triplex stability, specificity, or the like. [0049] The term "stability" in reference to duplex or triplex formation generally designates how tightly an antisense oligonucleotide binds to its intended target sequence; more particularly, "stability" designates the free energy of formation of the duplex or triplex under physiological conditions. Melting temperature under a standard set of conditions, e.g., as described below, is a convenient measure of duplex and/or triplex stability. Preferably, oligonucleotides of the invention are selected that have melting temperatures of at least 450C when measured in 100 niM NaCl, 0.1 mM EDTA and 10 mM phosphate buffer aqueous solution, pH 7.0 at a strand concentration of both the oligonucleotide and the target nucleic acid of 1.5 μM. Thus, when used under physiological conditions, duplex or triplex formation will be substantially favored over the state in which the antigen and its target are dissociated. It is understood that a stable duplex or triplex may in some embodiments include mismatches between base pairs and/or among base triplets in the case of triplexes. Preferably, modified oligonucleotides, e.g. comprising LNA units, of the invention form perfectly matched duplexes and/or triplexes with their target nucleic acids.
[0050] "Antibody" refers to a polypeptide ligand substantially encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof, which specifically binds and recognizes an epitope (e.g., an antigen). The recognized immunoglobulin genes include the kappa and lambda light chain constant region genes, the alpha, gamma, delta, epsilon and mu heavy chain constant region genes, and the myriad immunoglobulin variable region genes. Antibodies exist, e.g., as intact immunoglobulins or as a number of well characterized fragments produced by digestion with various peptidases. This includes, e.g., Fab' and F(ab)'2 fragments. The term "antibody," as used herein, also includes antibody fragments either produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA methodologies. It also includes polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies, or single chain antibodies. "Fc" portion of an antibody refers to that portion of an immunoglobulin heavy chain that comprises one or more heavy chain constant region domains, CH2 and CH3, but does not include the CH1 and the heavy chain variable region.
[0051] "Immunoassay" is an assay that uses an antibody to specifically bind an antigen (e.g., a marker). The immunoassay is characterized by the use of specific binding properties of a particular antibody to isolate, target, and/or quantify the antigen. [0052] The phrase "specifically (or selectively) binds" to an antibody or "specifically (or selectively) immunoreactive with," when referring to a protein or peptide, refers to a binding reaction that is determinative of the presence of the protein in a heterogeneous population of proteins and other biologies. Thus, under designated immunoassay conditions, the specified antibodies bind to a particular protein at least two times the background and do not substantially bind in a significant amount to other proteins present in the sample. Specific binding to an antibody under such conditions may require an antibody that is selected for its specificity for a particular protein. For example, antibodies raised to a desired antigen from specific species such as rat, mouse, or human can be selected to obtain only those polyclonal antibodies that are specifically immunoreactive with the desired antigen and not with other proteins, except for polymorphic variants and alleles. This selection may be achieved by subtracting out antibodies that cross-react with the desired antigen molecules from other species. A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein (see, e.g., Harlow & Lane, Antibodies, A Laboratory Manual (1988), for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity). Typically a specific or selective reaction will be at least twice background signal or noise and more typically more than 10 to 100 times background.
[0053] The dissociation constant (KD ) is a useful measure to describe the strength of binding (or affinity) between receptors and their ligands. For example, KD of antibody+antigen is about 10"7 to 10 "π M (i.e. 10'7 M is equivalent to 0.1 mM or 10OnM; TCR+MHC/peptide is about 10"6 M. For example to determine the dissociation constant of antibody (A) and antigen (B), the relationship can be written as an equation: [0054] kl (rate of dissociation) AB → A+B [0055] k2 (rate of association) A+B →AB
[0056] At equilibrium, k2[AB]=kl [A][B], where square brackets mean [concentration]. This equation can be rearranged to: k2 / kl = [A][B] / [AB] = KD. The dissociation constant, Kd, indicates the strength of binding between A and B in terms of how easy it is to separate the complex AB (dissociation or 'off rate').
[0057] "Sample" is used herein in its broadest sense. A sample comprising polypeptides, peptides, antibodies, polynucleotides, organic molecules, and the like, may comprise a bodily fluid; a soluble fraction of a cell preparation, or media in which cells were grown; a chromosome, an organelle, or membrane isolated or extracted from a cell; genomic DNA, RNA, or cDNA, polypeptides, or peptides in solution or bound to a substrate; a cell; a tissue; a tissue print; a fingerprint, skin or hair; environmental samples; and the like. [0058] As used herein, the term "infectious agent" refers to an organism wherein growth/multiplication leads to pathogenic events in humans or animals. Examples of such agents are: bacteria , fungi, protozoa and viruses.
[0059] As used herein, "cancer" refers to all types of cancer or neoplasm or malignant tumors found in mammals, including, but not limited to: leukemias, lymphomas, melanomas, carcinomas and sarcomas. Examples of cancers are cancer of the brain, breast, pancreas, cervix, colon, head & neck, kidney, lung, non-small cell lung, melanoma, mesothelioma, ovary, sarcoma, stomach, uterus and Medulloblastoma. Detection ofAnalytes
[0060] The significance and advantages of the compositions and methods thereof lies both in the biochemical utility of the nanosensor and in its application towards medicinal and environmental diagnostics. To date, most biosensors or probes, which integrate biomolecules and metal nanoparticles, are designed to target nucleic acids (i.e. RNA and DNA) via specific binding events with a concomitant fluorescent detection of the targeted species. Very few of these nanoprobes, however, are designed to target other important molecules such as proteins, biotoxins, and pollutants.
[0061] Detection of the target analyte is afforded through a fluorescence competition assay thus allowing for high sensitivity. The versatility of this probe, however, is fully realized when considering the availability of differing biomolecules such as antibodies (with specificities towards peptides, poisons and other organic molecules) and the ease of, for example, antibody production, m other words, the proposed nanoprobe can be customized towards the application of interest. Judicious selection of the antibody component, therefore, would allow for detection of cancer epitopes, chemical toxins, or environmental contaminants.
[0062] The underlying premise of the detection strategy relies on (1) an antibody based fluorescence competition assay and (2) a fluorescence resonance energy transfer utilizing metal nanoparticles (or organic molecules) as the donor and/or acceptor components. This overall strategy is outlined in Figure 1, where the constructed nanoprobe or sensor contains an antibody-hapten complex containing two differentially labeled spectrally unique nanoparticles or organic donors/acceptors. As a result of the target detection (or analyte binding), the labeled hapten is released from the complex (the nanoprobe) thereby yielding a change in the distances between the donor and acceptor components. This change in distance manifests an increase in overall measured fluorescence of the sample, which therefore serves as a signal confirming the detection of analyte.
[0063] Depending on the identities of the nanoparticles, the fluorescence signal may arise from either the labeled antibody or hapten subsequent to analyte detection. Potential arrangements, as shown in Figure 2, include labeling of the antibody and hapten with a luminescent quantum dot (the donor) and a gold nanoparticle (the quencher), respectively, and the reverse configuration. These assemblies yield either fluorescent antibody (Figure 2A) or hapten (Figure 2B) products. [0064] As an illustrative example, to construct or assemble the nanoprobe, antibodies specific for anthrax toxin (PA 211 14B7), cancer epitopes, and phosphopeptides are purchased or produced using hybridoma technology. The covalent binding of the nanoparticles (or organic donors and quenchers) to the antibody and hapten are accomplished using maleimide activated Qdots® (Quantum Dot Corp.), lysine reactive Nanogold® labeling reagents (Nanoprobes Corp.), or amine-reactive organic donors or quenchers (Molecular Probes). The binding sites of the labeled antibody are then loaded with the labeled hapten, thus forming the fluorescently quenched nanosensor. Target detection is then accomplished using a competitive binding assay between the free target analyte and the quenched hapten- antibody-gold nanoprobe.
[0065] As discussed above, covalent binding of the donor and acceptor components to the antibody and/or hapten will be accomplished using maleimide activated Qdots® (Quantum Dot Corp.), monomaleimido or NHS-Nanogold® labeling reagents (Nanoprobes Corp.), or amine-reactive organic fluorophores or quenchers (Molecular Probes). If necessary, the antibody or hapten will be modified to contain reactive thiol groups using 2- iminothiolane. The self-assembly of the nanoprobe is then afforded by complexation of the labeled hapten to the labeled antibody. The antibody-hapten complex containing differentially labeled donor and acceptor components (the nanoprobe) can then be used for detection of the target analyte (Figure 1).
[0066] This strategy is applied to the detection of particular peptide markers, organic molecules, and nucleic acids used in biodefense, biochemical, medicinal, or environmental diagnostics. Relevant antibodies can be acquired through either commercial or immunological means, whereas fluorescent labeling of the target analog can be synthetically accomplished.
[0067] Incorporation of the donor or acceptor is performed as described. Successful detection of the target analyte, such as a tumor cell, therefore is performed using a rapid fluorometric assay, which can be easily modified into a field — based procedure. [0068] The nanoparticle composition is termed herein as "nanoprobe." The nanoprobe comprises a nanoparticle to which is attached a detectably labeled biomolecule, such as for example an antibody. A second molecule is usually associated with the first molecule, as illustrated in Figure's 1 and 2. For example, the antibody is attached to the nanoparticle and is labeled with a fluorophore. The second molecule is a hapten and is labeled with a quencher. The antibody-hapten complex which is attached to the nanoparticle either by the antibody or, if desired, by the hapten are in a quenched fluorescent state, i.e. little or no fluorescent emissions are detected. If an analyte is provided and the antibody is specific for the analyte, the hapten dissociates from the antibody and is released into the surrounding medium. The fluorophore-quenching moieties, which were in spatial proximity to each other due to the antibody-hapten complex, become dissociated in the presence of analyte for which the antibody is specific for, resulting in the fluorescent emission. [0069] This strategy is applied to the detection of particular nucleic acid sequences, protein sequences or molecules such as those found in cancer epitopes or in environmental analysis. Relevant antibodies can be acquired through either commercial or immunological means, whereas fluorescent labeling of the target analog can be synthetically accomplished. [0070] Incorporation of the donor or acceptor is performed as described. Successful detection of the target analyte, such as a tumor cell, therefore is performed using a rapid fiuorometric assay, which can be easily modified into a field — based procedure. [0071] In a preferred embodiment, a self-assembling luminescent nanoparticle probe that acts as a sensor towards particular peptide sequences, organic ligands, or nucleic acids. Preferably, detection method is the coupling of a fluorescence competition assay with the fluorescence quenching of a gold nanoparticle. Figure 1 is a schematic illustration of the general method and mechanism of the nanoparticle probe.
[0072] In a preferred embodiment, the self-assembling nanoparticle comprises an inert metallic component such as for example, gold particles and gold colloids, CdSe, CdTe, or CdS nanocrsytals, and ceramic particles such as silica. The biomolecule such as an antibody, may be physically adsorbed onto the particle; however, greater stability and longer shelf-life are obtained when the antibody is covalently attached. See for example J. L. Ortega- Vinuesa et al. J. Biomater. Sd. Polymer Edn., 12(4), 379-408 (2001).
[0073] Nanoparticles useful in the practice of the invention include metal (e.g., gold, silver, copper and platinum), semiconductor (e.g., CdSe, CdS, and CdS or CdSe coated with ZnS) and magnetic (e.g., ferromagnetite) colloidal materials. Other nanoparticles useful in the practice of the invention include ZnS, ZnO, TiO2, AgI, AgBr, HgI2, PbS, PbSe, ZnTe, CdTe, In2S3, In2S3, Cd3P2, Cd3As2, InAs, and GaAs. The size of the nanoparticles is preferably from about 5 nm to about 150 run (mean diameter), more preferably from about 5 to about 50 nm, most preferably from about 5 nm to about 20 nm. The nanoparticles may also be rods. [0074] Methods of making metal, semiconductor and magnetic nanoparticles are well- known in the art. See, e.g., Schmid, G. (ed.) Clusters and Colloids (VCH, Weinheim, 1994); Hayat, M. A. (ed.) Colloidal Gold: Principles, Methods, and Applications (Academic Press, San Diego, 1991); Massart, R., IEEE Transactions On Magnetics, 17, 1247 (1981); Ahmadi, T. S. et al, Science, 272, 1924 (1996); Henglein, A. et al., J. Phys. Chem., 99, 14129 (1995); Curtis, A. C, et al., Angew. Chem. Int. Ed. Engl, 27, 1530 (1988).
[0075] Methods of making ZnS, ZnO, TiO2, AgI, AgBr, HgI2, PbS, PbSe, ZnTe, CdTe, In2S3, In2S3, Cd3P2, Cd3As2, InAs, and GaAs nanoparticles are also known in the art. See, e.g., Weller, Angew. Chem. Int. Ed. Engl, 32, 41 (1993); Henglein, Top. Curr. Chem., 143, 113 (1988); Henglein, Chem. Rev., 89, 1861 (1989); Brus, Appl. Phys. A., 53, 465 (1991); Bahncmann, in Photochemical Conversion and Storage of Solar Energy (eds. Pelizetti and Schiavello 1991), page 251; Wang and Herron, J. Phys. Chem., 95, 525 (1991); Olshavsky et al., J. Am. Chem. Soc, 112, 9438 (1990); Ushida et al., J. Phys. Chem., 95, 5382 (1992). [0076] Suitable nanoparticles are also commercially available from, e.g., Ted Pella, Inc. (gold), Amersham Corporation (gold), Nanoprobes, Inc. (gold), and Quantum Dot Corporation (semiconductor). Suitable organic dyes are available from Molecular Probes, Inc.
[0077] Presently preferred for use in detecting biomolecules and nucleic acids are nanoparticle containing probes.
[0078] Gold nanoparticles are also presently preferred for use in nanofabrication for the same reasons given above and because of their stability, ease of imaging by electron microscopy, and well-characterized modification. Also preferred for use in nanofabrication are semiconductor nanoparticles because of their unique electronic and luminescent properties.
[0079] Particles having covalently bound antibodies are typically prepared by activation of the particles, followed by coupling of antibodies to the activated particles. For particles having amine reactive functionalities, such as N-hydroxysuccinimide (NHS), the antibody surface lysine residues will be used to coupling. For thiol reactive functionalities, such as maleimides, native cysteines resulting from mild reduction of the antibody will be used for coupling. In addition, reagents, such as 2-iminothilane, will be used to introduce reactive thiol groups onto the antibody. For particles having carboxylate groups bound to the surface, activation is often achieved by contacting the particles with a solution of a carbodiimide coupling reagent and a succinimide reagent such as N-hydroxysuccinimide (NHS) or N- hydroxysulfosuccinimide (sNHS). The carboxylate groups on the surface are thus converted into NHS-ester or sNHS-ester groups. Carbodiimide couplers include, for example, N-ethyl- N'-(3-dimethyl-aminopropyl)carbodiimide (EDC); dicyclohexylcarbodiimide (DCC); and diisopropylcarbodiimide (DIC). Antibodies, for example IgG, can then be coupled to the particles by mixing the activated particles and the antibodies in an aqueous mixture, thereby forming sensitized particles.
[0080] The nanoprobe of the present invention comprises a labeled biomolecule, such as an antibody, and a labeled hapten. In the presence of the target analyte, the antibody releases the labeled hapten and undergoes a specific interaction with the analyte in the sample. The release of the labeled hapten yields an increase in the fluorescence emission of the nanoprobe, which can then be monitored and correlated with the amount of the analyte in the sample.
[0081] "Analyte" or "target molecule" refers to the substance, or group of substances, whose presence or amount thereof in a liquid medium is to be determined including, but not limited to, any drug or drug derivative, hormone, protein antigen, oligonucleotide, hapten, or hapten-carrier complex. An analyte analog is any substance, or group of substances, which behaves in a similar manner to the analyte, or in a manner conducive to achieving a desired assay result with respect to binding affinity and/or specificity of the antibody for the analyte including, but not limited to, derivatives, metabolites, and isomers thereof. [0082] Antibody means a specific binding partner of the analyte and is meant to include any substance, or group of substances, which has a specific binding affinity for the analyte to the exclusion of other substances. The term includes polyclonal antibodies, monoclonal antibodies and antibody fragments.
[0083] Haptens are substances, typically of low molecular weight, which are not capable of stimulating antibody formation, but which do react with antibodies. The latter are formed by coupling the hapten to a high molecular weight carrier and injecting this coupled product into humans or animals. Examples of haptens include therapeutic drugs such as digoxin and theophylline; drugs of abuse such as morphine, lysergic acid diethylamide (LSD), and Δ9- tetrahydrocannabinol (THC); antibiotics such as aminoglycosides and vancomycin; hormones such as estrogen and progesterone; vitamins such as vitamin B 12 and folic acid; thyroxin; histamine; serotonin; adrenaline and others.
[0084] A carrier refers to an immunogenic substance, commonly a protein that can join with a hapten, thereby enabling the hapten to stimulate an immune response. Carrier substances include proteins, glycoproteins, complex polysaccharides and nucleic acids that are recognized as foreign and thereby elicit an immunologic response from the host. [0085] The terms immunogen and immunogenic refer to substances capable of producing or generating an immune response in an organism.
[0086] A peptide is any compound formed by the linkage of two or more amino acids by amide (peptide) bonds, usually a polymer of α-amino acids in which the α-amino group of each amino acid residue (except the NH2-terminal) is linked to the α-carboxy group of the next residue in a linear chain. The terms peptide, polypeptide and poly(amino acid) are used synonymously herein to refer to this class of compounds without restriction as to size. The largest members of this class are referred to as proteins.
[0087] A covalent bond is a chemical bond between two species, and may involve single bonds or multiple bonds. The term "covalent" does not include hydrophobic/hydrophilic interactions, Hydrogen-bonding, van der Waals interactions, and ionic interactions. [0088] Any sample that is suspected of containing the analyte can be analyzed by the method of the present invention. The sample is typically an aqueous solution such as a body fluid from a host, for example, urine, whole blood, plasma, serum, saliva, semen, stool, sputum, cerebral spinal fluid, tears, mucus or the like, but preferably is urine, plasma or serum. The sample can be pretreated if desired and can be prepared in any convenient medium that does not interfere with the assay. An aqueous medium is preferred. [0089] Calibration material means any standard or reference material containing a known amount of the analyte to be measured. The sample suspected of containing the analyte and the calibration material are assayed under similar conditions. Analyte concentration is then calculated by comparing the results obtained for the unknown specimen with results obtained for the standard.
[0090] Particles, which may be treated according to the present invention, include any type of particle and may be activated with succinimide esters. Such particles include polymer particles including polystyrene and polymethylmethacrylate); gold particles including gold nanoparticles and gold colloids; luminescent semiconductor particles; and ceramic particles including silica, glass, and metal oxide particles. See for example C. R. Martin et al. Analytical Chemistry— News Sc Features, May 1, 1998, 322A-327A. These particles may be activated succinimide esters or maleimides directly, or they may be activated once their surfaces have been modified to contain carboxylate groups. Carboxylate groups can be introduced to surfaces, for example by hydrolysis reactions, by treatment with a carboxylating reagent, or by formation of self-assembled monolayers (SAMs) containing carboxylate groups. See for example J. G. Chapman et al. J. Am. Chem. Soc, 122, 8303- 8304 (2000).
[0091] The amine or thiol compounds are applied to the sensitized particles in an excess amount to provide for maximum reaction of the succinimide ester or maleimide functionalities. Preferably, the particles are contacted with a solution containing at least 50 equivalents of the amine or thiol relative to the amount of reactive groups originally present on the particles before activation. More preferably, the particles are contacted with a solution containing at least 100 equivalents of the amine or thiol. Even more preferably, the particles are contacted with a solution containing at least 200 equivalents of the amine or thiol. The particles are exposed to the contacting solution for a time sufficient to allow an acceptable amount of reaction. This time may be from a few minutes to several hours. After the particles have been treated by exposure to the amine or thiol, the particles are filtered from the reaction solution. These particles thus have a surface, which contains antibody, the reaction products of residual functionalities with the amine or thiol and, optionally, immobilized BSA.
[0092] These particles are then used in the fluorescence competition assay, thus allowing for high sensitivity. The particles can be used in an immunoassay for the corresponding analyte of the antibody using standard immunoassay techniques. Immunoassay mixtures using these particles may also contain a tertiary amine compound to reduce interference due to the presence of tertiary amine groups on the particle surface. Suitable tertiary amine compounds include triethanolamine (TEO).
[0093] Various ancillary materials will frequently be employed in an assay in accordance with the present invention. For example, buffers will normally be present in the assay medium, as well as stabilizers for the assay medium and the assay components. Frequently, in addition to these additives, additional proteins may be included, such as albumin; or surfactants may be included, particularly non-ionic surfactants and the like. [0094] Suitable nanoparticles are also commercially available from, e.g., Ted Pella, Inc. (gold), Amersham Corporation (gold), Nanoprobes, Inc. (gold) and Quantum Dot Corporation.
[0095] In the case of using nucleic acids, the nanoparticles, the oligonucleotides or both are functionalized in order to attach the oligonucleotides to the nanoparticles. Such methods are known in the art. For instance, oligonucleotides functionalized with alkanethiols at their 3'-termini or 5'-termini readily attach to gold nanoparticles. See Whitesides, Proceedings of the Robert A. Welch Foundation 39th Conference On Chemical Research Nanophase Chemistry, Houston, Tex., pages 109-121 (1995). See also, Mucic et al. Chem. Commun. 555-557 (1996) (describes a method of attaching 3' thiol DNA to flat gold surfaces; this method can be used to attach oligonucleotides to nanoparticles). The alkanethiol method can also be used to attach oligonucleotides to other metal, semiconductor and magnetic colloids and to the other nanoparticles listed above. Other functional groups for attaching oligonucleotides to solid surfaces include phosphorothioate groups (see, e.g., U.S. Pat. No. 5,472,881 for the binding of oligonucleotide-phosphorothioates to gold surfaces), substituted alkylsiloxanes (see, e.g. Burwell, Chemical Technology, 4, 370-377 (1974) and Matteucci and Caruthers, J Am. Chem. Soc, 103, 3185-3191 (1981) for binding of oligonucleotides to silica and glass surfaces, and Grabar et al., Anal. Chem., 67, 735-743 for binding of aminoalkylsiloxanes and for similar binding of mercaptoaklylsiloxanes). Oligonucleotides terminated with a 5' thionucleoside or a 3' thionucleoside may also be used for attaching oligonucleotides to solid surfaces. The following references describe other methods which may be employed to attached oligonucleotides to nanoparticles: Nuzzo et al., J. Am. Chem. Soc, 109, 2358 (1987) (disulfides on gold); Allara and Nuzzo, Langmuir, 1, 45 (1985) (carboxylic acids on aluminum); Allara and Tompkins, J. Colloid Interface ScL, 49, 410-421 (1974) (carboxylic acids on copper); Her, The Chemistry Of Silica, Chapter 6, (Wiley 1979) (carboxylic acids on silica); Timmons and Zisman, J Phys. Chem., 69, 984-990 (1965) (carboxylic acids on platinum); Soriaga and Hubbard, J. Am. Chem. Soc, 104, 3937 (1982) (aromatic ring compounds on platinum); Hubbard, Ace. Chem. Res., 13, 177 (1980) (sulfolanes, sulfoxides and other functionalized solvents on platinum); Hickman et al., J Am. Chem. Soc, 111, 7271 (1989) (isonitriles on platinum); Maoz and Sagiv, Langmuir, 3, 1045 (1987) (silanes on silica); Maoz and Sagiv, Langmuir, 3, 1034 (1987) (silanes on silica); Wasserman et al., Langmuir, 5, 1074 (1989) (silanes on silica); Eltekova and Eltekov, Langmuir, 3, 951 (1987) (aromatic carboxylic acids, aldehydes, alcohols and methoxy groups on titanium dioxide and silica); Lee et al., J. Phys. Chem., 92, 2597 (1988) (rigid phosphates on metals).
[0096] Each nanoparticle will have a plurality of oligonucleotides attached to it. As a result, each nanoparticle-oligonucleotide conjugate can bind to a plurality of oligonucleotides or nucleic acids having the complementary sequence. [0097] Oligonucleotides of defined sequences are used for a variety of purposes in the practice of the invention. Methods of making oligonucleotides of a predetermined sequence are well-known. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd ed. 1989) and F. Eckstein (ed.) Oligonucleotides and Analogues, 1st Ed. (Oxford University Press, New York, 1991). Solid-phase synthesis methods are preferred for both oligoribonucleotides and oligodeoxyribonucleotides (the well-known methods of synthesizing DNA are also useful for synthesizing RNA). Oligoribonucleotides and oligodeoxyribonucleotides can also be prepared enzymatically.
[0098] The invention provides methods of detecting nucleic acids. Any type of nucleic acid may be detected, and the methods may be used, e.g., for the diagnosis of disease and in sequencing of nucleic acids. Examples of nucleic acids that can be detected by the methods of the invention include genes (e.g., a gene associated with a particular disease), viral RNA and DNA, bacterial DNA, fungal DNA, cDNA, mRNA, RNA and DNA fragments, oligonucleotides, synthetic oligonucleotides, modified oligonucleotides, single-stranded and double-stranded nucleic acids, natural and synthetic nucleic acids, etc. Thus, examples of the uses of the methods of detecting nucleic acids include: the diagnosis and/or monitoring of viral diseases (e.g., human immunodeficiency virus, hepatitis viruses, herpes viruses, cytomegalovirus, and Epstein-Barr virus), bacterial diseases (e.g., tuberculosis, Lyme disease, H. pylori, Escherichia coli infections, Legionella infections, Mycoplasma infections, Salmonella infections), sexually transmitted diseases (e.g., gonorrhea), inherited disorders (e.g., cystic fibrosis, Duchene muscular dystrophy, phenylketonuria, sickle cell anemia), and cancers (e.g., genes associated with the development of cancer); in forensics; in DNA sequencing; for paternity testing; for cell line authentication; for monitoring gene therapy; and for many other purposes.
[0099] The methods of detecting nucleic acids based on FRET (discussed infra) are cheap, fast, robust (the reagents are stable), do not require specialized or expensive equipment.
[00100] The nucleic acid to be detected may be isolated by known methods, or may be detected directly in cells, tissue samples, biological fluids (e.g., saliva, urine, blood, serum), solutions containing PCR components, solutions containing large excesses of oligonucleotides or high molecular weight DNA, and other samples, as also known in the art. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd ed. 1989) and B. D. Hames and S. J. Higgins, Eds., Gene Probes 1 (IRL Press, New York, 1995). Methods of preparing nucleic acids for detection with hybridizing probes are well known in the art. See, e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual (2nd ed. 1989) and B. D. Hames and S. J. Higgins, Eds., Gene Probes 1 (IRL Press, New York, 1995). [00101] If a nucleic acid is present in small amounts, it may be applied by methods known in the art. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd ed. 1989) and B. D. Hames and S. J. Higgins, Eds., Gene Probes 1 (IRL Press, New York, 1995). Preferred is polymerase chain reaction (PCR) amplification. [00102] One method according to the invention for detecting nucleic acid comprises contacting a nucleic acid with one or more types of nanoparticles having oligonucleotides attached thereto and are fluorescently labeled. The nucleic acid to be detected has at least two portions. The lengths of these portions and the distance(s), if any, between them are chosen so that when the oligonucleotides on the nanoparticles hybridize to the nucleic acid, a detectable change in fluorescence occurs, i.e. quenching. These lengths and distances can be determined empirically and will depend on the type of particle used and its size and the type of electrolyte which will be present in solutions used in the assay (as is known in the art, certain electrolytes affect the conformation of nucleic acids).
[00103] Also, when a nucleic acid is to be detected in the presence of other nucleic acids, the portions of the nucleic acid to which the oligonucleotides on the nanoparticles are to bind must be chosen so that they contain sufficient unique sequence so that detection of the nucleic acid will be specific.
[00104] Methods of labeling oligonucleotides with fluorescent molecules and measuring fluorescence are well known in the art. Suitable fluorescent molecules are also well known in the art and include, but not limited to: the fluoresceins, biotin, rhodamines and Texas Red. The oligonucleotides will be attached to the nanoparticles as described above.
Fluorescence resonance energy transfer (FRET)
[00105] Fluorescence resonance energy transfer (FRET) occurs between the electronic excited states of two fluorophores when they are in sufficient proximity to each other, in which the excited-state energy of the donor fiuorophore is transferred to the acceptor fluorophore. The result is a decrease in the lifetime and a quenching of fluorescence of the donor species and a concomitant increase in the fluorescence intensity of the acceptor species. In one application of this principle, a fluorescent moiety is caused to be in close proximity to a quencher molecule. Donor and acceptor molecules operate in a set wherein one or more acceptor molecules accepts energy from one or more donor molecules, or otherwise quenches signal from the donor molecule, when the donor and acceptor molecules are closely associated. In one embodiment, the donor and acceptor molecules are about 30 to about 200 A apart or about 10 to about 40 nucleotides apart. Transfer of energy may occur through collision of the closely associated molecules of a set, or through a non-radiative process such as fluorescence resonance energy transfer (FRET). For FRET to occur, transfer of energy between donor and acceptor molecules requires that the molecules be close in space and that the emission spectrum of a donor have substantial overlap with the absorption spectrum of the acceptor (Yaron et al. Analytical Biochemistry, 95, 228-235 (1979), the teachings of which are incorporated herein by reference). Alternatively, intramolecular energy transfer may occur between very closely associated donor and acceptor molecules (e.g., within 10 A) whether or not the emission spectrum of a donor molecule has a substantial overlap with the absorption spectrum of the acceptor molecule (Yaron et al.) This process is referred to as intramolecular collision since it is believed that quenching is caused by the direct contact of the donor and acceptor molecule (Yaron et al). [00106] Because the efficiency of both collision and non-radiative transfer of energy between the donor and acceptor molecules is directly dependent on the proximity of the donor and acceptor molecules, formation and dissociation of the complexes of this invention can be monitored by measuring at least one physical property of at least one member of the set which is detectably different when the complex is formed, as compared with when the biomolecules and target/bait exist independently and unassociated. Preferably, the means of detection will involve measuring fluorescence of an acceptor fluorophore of a set or the fluorescence of the donor fluorophore in a set containing a fluorophore and quencher pair (e.g. a donor and acceptor). While not wishing to be bound by theory, the fluorescent molecules may interact with one another via hydrophobic interactions, thereby reducing the adverse effect of distance between the donor and acceptor fluorescent molecules. Thus, fluorescence energy transfer can occur when the donor and acceptor fluorescent molecules are up to about 40 nucleotides away from each other.
[00107] Essentially any fluorophore may be used, including BODIPY, fluoroscein, fluorescein substitutes (Alexa Fluor dye, Oregon green dye), long wavelength dyes, and UV- excited fluorophores. These and additional fiuorophores are listed in Fluorescent and Luminescent Probes for Biological Activity. A Practical Guide to Technology for Quantitative Real-Time Analysis, Second Ed. W. T. Mason, ed. Academic Press (1999) (incorporated herein by reference).
[00108] A quencher is a molecule that absorbs the energy of the excited fluorophore. Close proximity of a fluorophore and a quencher allow for the energy to be transferred from the fluorophore to the quencher. By absorbing this energy, the quencher prevents the fluorophore from releasing the energy in the form of a photon, thereby preventing fluorescence. Fluorescent quenchers tend to be specific to fluorophores that emit at a specific wavelength range.
[00109] Fluorescent quenchers often involve fluorescence resonance energy transfer (FRET). In many instances the fluorescent quencher molecule is also a fluorophore. In such cases, close proximity of the fluorophore and fluorescent quencher is indicated by a decrease in fluorescence of the "fluorophore" and an increase in fluorescence of the fluorescent quencher. Commonly used fluorescent fluorophore pairs (fluorophore/fluorescent quencher) include fluorescein/tetramethyhrhodamine, IAEDANS/fluorescein, fluorescein/fluorescein, and BODIPY FL/BODIPY FL.
[00110] In one embodiment of the present invention, the 3 '-end of the biomolecule is labeled with N, N1, N, N1 -tetramethyl-6-carboxy rhodamine (TAMRA). Donor and acceptor molecules suitable for FRET are well known in the art (see page 46 of R. P. Haugland, Handbook of Fluorescent Probes and Research Chemicals, 6th ed.; Molecular Probes, Oregon, the teachings of which are incorporated herein by reference). Typically, to obtain fluorescence resonance energy transfer, the donor fluorescent molecule has a shorter excitation wavelength than the acceptor fluorescent molecule and the donor emission wavelength overlaps with the acceptor excitation wavelength, to allow transfer of energy from the donor to the acceptor. Preferred fluorescent molecules are: biotin, fluorophores are fluorescein and derivatives thereof, such as 5-(2'-aminoethyl)-aminoapthalene-l -sulfonic acid (EDANS) and rhodamine and derivatives thereof such as Cy3, Cy5 and Texas Red. Suitable donor/acceptor pairs are, for example, fluorescein/tetramethyrhodamine, IAEDANS/fluorescein and ED ANS/D ABCYL. In another embodiment of the present invention, the same fluorescent molecule is used for the donor and acceptor. In this embodiment, the wavelength used to excite the detection complexes is polarized. Unpolarized emission detected is indicative of FRET. In this embodiment, it is preferable to remove unincorporated labeled nucleotides (e.g., by washing) to improve the detection signal. [00111] Those of ordinary skill in the art will recognize that labeled, unlabeled and modified biomolecules such as antibodies, oligonucleotides, aptamers and the like, are readily available for the method of the present invention. They can be synthesized using commercially available instrumentation and reagents or they can be purchased from numerous commercial vendors of custom manufactured oligonucleotides. [00112] In another preferred embodiment, antibodies are labeled with a fluorophore and a quencher to form intra-molecular FRET. Preferably, the folded conformations of the are stabilized by binding to their target molecules and produce a fluorescence signal change of the fluorophore induced by FRET when the antibody binds to its target. Preferably, the target-binding induced FRET cause between about 40% up to 100% fluorescence quenching. [00113] In another preferred embodiment, FRET can be formed within an antibody even if the antibody lacks the necessary conformational changes accompanying the binding to the target molecules.
[00114] Methods and devices for detecting fluorescence are well developed. Essentially any instrument or method for detecting fluorescent emissions may be used. For example, WO 99/27351 (incorporated herein in its entirety) describes a monolithic bioelectrical device comprising a bioreporter and an optical application specific integrated circuit (OASIC). The device allows remote sampling for the presence of substances in solution. Furthermore, the fluorescence may be measured by a number of different modes. Examples include fluorescence intensity, lifetime, and anisotropy in either steady state or kinetic rate change modes (Lakowicz, J. R. In Principles of Fluorescence Spectroscopy; 2nd ed.; Kluwer Academic/Plenum: New York, 1999).
[00115] According to one embodiment of the present invention an oligonucleotide or an assembly of oligonucleotides can also be labeled with at least one pair of resonantly interacting detection moieties. For example, a first detection moiety and a second detection moiety of a pair can each be linked to an oligonucleotide or an assembly of oligonucleotides flanking the cleavage recognition sequence, such that upon cleavage of the recognition sequence by the cleaving agent separation of these moieties occurs. The detection moieties are selected such that at least one of these moieties is capable of producing a detectable signal when separated to a non-interacting distance from the other detection moiety. [00116] Examples of resonantly interacting pairs of detection moieties which can be used, include, but are not limited to, a fluorescer and a quencher and any other type of fluorescent resonant energy transfer (FRET) pairs (for reference, see for example "Fluoroscence resonance energy transfer" by Paul R. Selvin, 1995, Methods in Enzymol. VoI 246, Chap. 13, pp. 300; and "Handbook of fluorescent probes and research chemicals" by Richard P. Haugland, sixth ed. Molecular probes. Specific examples of molecules which can be used in fluorescent resonant energy transfer are listed hereinbelow.
[00117] The optimal distance between a first and a second detection moieties of a pair when linked to a biomolecule such as antibody, aptamer, oligonucleotide, will be that distance wherein the emissions of the first moiety are absorbed by the second moiety. This optimal distance varies with the specific moieties used, and is defined by Forster Radius. Forster Radius (Ro) is the distance between a donor and acceptor that allows quenching of 50% of the excited donor molecules by the quencher. Ro may be defined for any given FRET pair, and may be used as the guideline for designing a FRET-labeled probe. [00118] One of ordinary skill in the art can easily determine, using art-known techniques of spectrophotometry, which fluorophores will make suitable donor-acceptor FRET pairs. For example, FAM (which has an emission maximum of 525 nm) is a suitable donor for TAMRA, ROX, and R6G (all of which have an excitation maximum of 514 nm) in a FRET pair. Additional examples to moieties which can be used include but are not limited to, A- acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid acridine and derivatives such as, acridine, acridine isothiocyanate, 5-(2'-aminoethyl)aminonaphthalene-l-sulfonic acid (EDANS), 4-amino-N-_13-vinylsulfonyl)phenyl!naphthalimide-3,5, disulfonate (Lucifer Yellow VS), N-(4-anilino-l-naphthyl)maleimide, anthranilamide and Brilliant Yellow; coumarin and derivatives such as, coumarin, 7-amino-4-methylcoumarin (AMC, Coumarin 120), 7-amino-4-trifluoromethylcouluarin (Coumaran 151), cyanosine 4',6-diaminidino-2- phenylindole (DAPI), 5', 5"-dibromopyrogallolsulfonephthalein (Bromopyrogallol Red), 7- diethylamino-3-(4'-isothiocyanatophenyl)-4-methylcoumarin, diethylenetriamine pentaacetate, 4,4'-diisothiocyanatodihydro-stilbene-2,2'-disulfonic acid, 4,4'- diisothiocyanatostilbene-2,2'-disulfonic acid, 5-dimethylamino naphthalene- 1-sulfonyl chloride (DNS, dansyl chloride), 4-(4'-dimethylaminophenylazo)benzoic acid (DABCYL) and 4-dimethylaminophenylazophenyl-4'-isothiocyanate (DABITC); eosin and derivatives such as, eosin and eosin isothiocyanate; erythrosin and derivatives such as, erythrosin B and erythrosin isothiocyanate ethidium; fluorescein and derivatives: such as, 5- carboxyfluorescein (FAM), 5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF), 2'7'- dimethoxy-4'5'-dichloro-6-carboxyfluorescein (JOE), fluorescein, fluorescein isothiocyanate, QFITC (XRITC), fluorescamine, IRl 44, IRl 446, Malachite Green isothiocyanate, A- methylumbelliferone, ortho cresolphthalein, nitrotyrosine, pararonsaniline, Phenol Red, B- phycoerythrin and o-phthaldialdehyde; pyrene and derivatives such as, pyrene, pyrene butyrate, succinimidyl 1 -pyrene butyrate and Reactive Red 4 (Cibacron R. T. Brilliant Red 3B-A); rhodamine and derivatives such as, 6-carboxy-X-rhodamine (ROX), 6- carboxyrhodamine (R6G), lissaniine rhodamine B sulfonyl chloride, rhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine X isothiocyanate, sulforhodamine B, sulforhodamine 101, sulforhodamine 101, (Texas Red), N,N,N',N'-tetramethyl-6- carboxyrhodamine (TAMRA), tetramethyl rhodamine, tetramethyl rhodamine isothiocyanate (TRITC), riboflavin, rosolic acid and terbium chelate derivatives.
Detection Labels
[00119] To aid in detection and quantitation of molecules using the disclosed methods detection labels can be directly incorporated into the biomolecules. As used herein, a detection label is any molecule that can be associated with biomolecules, such as antibodies, directly or indirectly, and which results in a measurable, detectable signal, either directly or indirectly. A label may be any moiety covalently attached to a biomolecule. Many such labels for incorporation into antibodies, nucleic acids are known to those of skill in the art. Examples of detection labels suitable for use in the disclosed method are, fluorescent molecules, phosphorescent molecules and radioactive isotopes.
[00120] A preferred class of labels are detection labels, which may provide a signal for detection of the labeled antibodies by fluorescence, chemiluminescence, and electrochemical luminescence. Fluorescent dyes useful for labeling antibodies include fluoresceins, rhodamines, cyanines, and metal porphyrin complexes. Preferred fluorescein dyes include 6- carboxyfluorescein (6-FAM), 2', 4',1,4-tetrachlorofluorescein (TET), 2',4',5',7',1,4- hexachlorofluorescein (HEX), 2',7'-dimethoxy-4',5l-dichloro~6-carboxyrhodamm (JOE), T- chloro-5'-fluoro-7',8'-fused phenyl- l^-dichloro-ό-caroxyflurescein (NED), 2'-chloro-7'- phenyl-l,4-dichloro-6-carboxyfluorescein (VIC), and (JODA). The 5-carboxyl, and other regio-isomers, may also have useful detection properties. Fluorescein, biotin and rhodamine dyes with 1,4-dichloro substituents are especially preferred.
[00121] Another preferred class of labels include quencher moieties. The emission spectra of a quencher moiety overlaps with a proximal intramolecular or intermolecular fluorescent dye such that the fluorescence of the fluorescent dye is substantially diminished, or quenched, by fluorescence resonance energy transfer (FRET). Oligonucleotides which are intramolecularly labeled with both fluorescent dye and quencher moieties are useful in nucleic acid hybridization assays, e.g. the "Taqman™" exonuclease-cleavage PCR assay. [00122] Particularly preferred quenchers include but are not limited to (i) rhodamine dyes selected from the group consisting of tetramethyl-6-carboxyrhodamine (TAMRA), tetrapropano-6-carboxyrhodamine (ROX), and (ii) DABSYL, DABCYL, cyanine dyes including nitrothiazole blue (NTB), anthraquinone, malachite green, nitrothiazole, and nitroimidazole compounds and the like.
[00123] Preferred fluorescent labels are fluorescein (5-carboxyfluorescein-N- hydroxysuccinimide ester), rhodamine (5,6-tetramethyl rhodamine), and the cyanine dyes Cy3, Cy3.5, Cy5, Cy5.5 and Cy7. The absorption and emission maxima, respectively, for these fluors are: FITC (490 nm; 520 nm), Cy3 (554 nm; 568 nm), Cy3.5 (581 run; 588 nm), Cy5 (652 nm: 672 nm), Cy5.5 (682 nm; 703 nm) and Cy7 (755 nm; 778 nm), thus allowing their simultaneous detection. Other examples of fluorescein dyes include 6- carboxyfluorescein (6-FAM), 2l,4',l,4,-tetrachlorofluorescein (TET), 2',4',5',7',1,4- hexachloro fluorescein (HEX), 2',7'-dimethoxy-4',5'-dichloro-6-carboxyrhodamine (JOE), T- chloro-5l-fluoro-7',8'-fused phenyl-l,4-dichloro-6-carboxyfluorescein (NED), and 2'-chloro- 7'-phenyl-l,4-dichloro-6-carboxyfluorescein (VIC). Fluorescent labels can be obtained from a variety of commercial sources, including Amersham Pharmacia Biotech, Piscataway, NJ. ; Molecular Probes, Eugene, Oreg.; and Research Organics, Cleveland, Ohio. [00124] Examples of other suitable fluorescent labels include fluorescein isothiocyanate (FITC), 5,6-carboxymethyl fluorescein, Texas red, nitrobenz-2-oxa-l,3-diazol-4-yl (NBD), coumarin, dansyl chloride, rhodamine, amino-methyl coumarin (AMCA), Eosin, Erythrosin, BODIPY™, Cascade Blue™, Oregon Green™, pyrene, lissamine, xanthenes, acridines, oxazines, phycoerythrin, macrocyclic chelates of lanthanide ions such as quantum dye™, fluorescent energy transfer dyes, such as thiazole orange-ethidium heterodimer, and the cyanine dyes Cy3, Cy3.5, Cy5, Cy5.5 and Cy7. Examples of other specific fluorescent labels include 3-Hydroxypyrene 5,8,10-Tri Sulfonic acid, 5-Hydroxy Tryptamine (5-HT), Acid Fuchsin, Alizarin Complexon, Alizarin Red, Allophycocyanin, Aminocoumarin, Anthroyl Stearate, Astrazon Brilliant Red 4G, Astrazon Orange R, Astrazon Red 6B, Astrazon Yellow 7 GLL, Atabrine, Auramine, Aurophosphine, Aurophosphine G, BAO 9 (Bisaminophenyloxadiazole), BCECF, Berberine Sulphate, Bisbenzamide, Blancophor FFG Solution, Blancophor SV, Bodipy Fl, Brilliant Sulphoflavin FF, Calcien Blue, Calcium Green, Calcofluor RW Solution, Calcofluor White, Calcophor White ABT Solution, Calcophor White Standard Solution, Carbostyryl, Cascade Yellow, Catecholamine, Chinacrine, Coriphosphine O, Coumarin-Phalloidin, CY3.1 8, CY5.1 8, CY7; Dans (1- Dimethyl Amino Naphaline 5 Sulphonic Acid), Dansa (Diamino Naphtyl Sulphonic Acid), Dansyl NH--CH3, Diamino Phenyl Oxydiazole (DAO), Dimethylamino-5-Sulphonic acid, Dipyrrometheneboron Difluoride, Diphenyl Brilliant Flavine 7GFF, Dopamine, Erythrosin ITC, Euchrysin, FIF (Formaldehyde Induced Fluorescence), Flazo Orange, Fluo 3, Fluorescamine, Fura-2, Genacryl Brilliant Red B, Genacryl Brilliant Yellow 10GF, Genacryl Pink 3G, Genacryl Yellow 5GF, Gloxalic Acid, Granular Blue, Haematoporphyrin, Indo-1, Mrawhite Cf Liquid, Leucophor PAF, Leucophor SF, Leucophor WS, Lissamine Rhodamine B200 (RD200), Lucifer Yellow CH, Lucifer Yellow VS, Magdala Red, Marina Blue, Maxilon Brilliant Flavin 10 GFF, Maxilon Brilliant Flavin 8 GFF, MPS (Methyl Green Pyronine Stilbene), Mithramycin, NBD Amine, Nitrobenzoxadidole, Noradrenaline, Nuclear Fast Red, Nuclear Yellow, Nylosan Brilliant Flavin E8G, Oxadiazole, Pacific Blue, Pararosaniline (Feulgen), Phorwite AR Solution, Phorwite BKL, Phorwite Rev, Phorwite RPA, Phosphine 3R, Phthalocyanine, Phycoerythrin R, Polyazaindacene Pontochrome Blue Black, Porphyrin, Primuline, Procion Yellow, Pyronine, Pyronine B, Pyrozal Brilliant Flavin 7GF, Quinacrine Mustard, Rhodamine 123, Rhodamine 5 GLD, Rhodamine 6G, Rhodamine B, Rhodarmine B 200, Rhodamine B Extra, Rhodamine BB, Rhodamine BG, Rhodamine WT, Serotonin, Sevron Brilliant Red 2B, Sevron Brilliant Red 4G, Sevron Brilliant Red B, Sevron Orange, Sevron Yellow L, SITS (Primuline), SITS (Stilbene Isothiosulphonic acid), Stilbene, Snarf 1, sulpho Rhodanine B Can C, Sulpho Rhodamine G Extra, Tetracycline, Thiazine Red R, Thioflavin S, Thioflavin TCN, Thioflavin 5, Thiolyte, Thiozol Orange, Tinopol CBS, True Blue, Ultralite, Uranine B, Uvitex SFC, Xylene Orange, and XRITC. [00125] Additional labels of interest include those that provide for signal only when the antibody with which they are associated is specifically bound to a target molecule, where such labels include: "molecular beacons" as described in Tyagi & Kramer, Nature Biotechnology (1996) 14:303 and EP 0 070 685 Bl. Other labels of interest include those described in U.S. Pat. No. 5,563,037; WO 97/17471 and WO 97/17076. [00126] Labeled nucleotides are also a preferred form of detection label since they can be directly incorporated into the amplification products during synthesis. Examples of detection labels that can be incorporated into amplified nucleic acids include nucleotide analogs such as BrdUrd (5-bromodeoxyuridine, Hoy and Schimke, Mutation Research 290:217-230 (1993)), aminoallyldeoxyuridine (Henegariu et al, Nature Biotechnology 18:345-348 (2000)), 5-methylcytosine (Sano et al., Biochim. Biophys. Acta 951:157-165 (1988)), bromouridine (Wansick et al., J. Cell Biology 122:283-293 (1993)) and nucleotides modified with biotin (Langer et al, Proc. Natl. Acad. Sd. USA 78:6633 (1981)) or with suitable haptens such as digoxygenin (Kerkhof, Anal. Biochem. 205:359-364 (1992)). Suitable fluorescence-labeled nucleotides are Fluorescein-isothiocyanate-dUTP, Cyanine-3-dUTP and Cyanine-5-dUTP (Yu et al, Nucleic Acids Res., 22:3226-3232 (1994)).
Other Nanoparticles
[00127] Generally, nanoparticles of the invention can be purchased commercially, such as Nanogold®, or manufactured according to methods known in the art. Extremely small and well defined structures, for example of inorganic particles, can be formed in association with a surface using self-assembly approaches. Nanoparticles with very uniform sizes are preferred components for forming self-assembled structures, although other functional compositions can be used. The nanoparticles are organized into a well defined structures using fabrication techniques that take advantage of molecular recognition characteristics of self-assembly approaches. Molecular recognition can involve various interactions, such as commingling, key-lock relationships and guest-host interactions. [00128] The deposition techniques are combined with localization techniques that constrain the resulting structures within isolated islands along the substrate surface. The islands can be ordered or disordered arrays. The organized structures or islands are suitable for binding of biomolecules, such as antibodies, peptides, nucleic acid molecules and the like. Preferred nanoparticles can be produced by laser pyrolysis with or without additional processing. Thus, the self-assembly approaches provide an alternative to traditional masking techniques and direct formation approaches for fabricating device structures. [00129] Laser pyrolysis is an excellent approach for efficiently producing a wide range of nanoscale particles with a narrow distribution of average particle diameters. In particular, laser pyrolysis can be used to produce a variety of inorganic particles, such as elemental metal particles, metal/silicon oxide particles, metal/silicon carbide particles, metal/silicon nitride particles and metal/silicon sulfide particles. Alternatively, nanoparticles can be produced using a flame production apparatus such as the apparatus described in U.S. Pat. No. 5,447,708 to Helble et al., entitled "Apparatus for Producing Nanoscale Ceramic Particles," incorporated herein by reference. Furthermore, nanoparticles can be produced with a thermal reaction chamber such as the apparatus described in U.S. Pat. No. 4,842,832 to Inoue et al., "Ultrafme Spherical Particles of Metal Oxide and a Method for the Production Thereof," incorporated herein by reference.
[00130] A basic feature of successful application of laser pyrolysis for the production of desirable inorganic nanoparticles is the generation of a reactant stream containing a metal/silicon precursor compound, a radiation absorber and, generally, a secondary reactant. The secondary reactant can be a source of atoms, such as oxygen, required for the desired product or an oxidizing or reducing agent to drive a desired product formation. A secondary reactant is not needed if the precursor decomposes to the desired product under intense light radiation. The reactant stream is pyrolyzed by an intense light beam, generally a laser beam. As the reactant stream leaves the laser beam, the particles are rapidly quenched. [00131] Nanoparticles produced by laser pyrolysis can be subjected to additional processing to alter the nature of the particles, such as the composition and/or the crystallinity. For example, the nanoparticles can be subjected to heat processing in a gas atmosphere prior to use. Under suitably mild conditions, heat processing is effective to modify the characteristics of the particles without destroying the nanoscale size or the narrow particle size distribution of the initial particles.
[00132] For many applications, the powder is dispersed in a liquid or other fluid for use or for further processing. For the purposes of discussion herein, particle dispersions have concentrations of nanoparticles no more than about 80 weight percent. Appropriate properties of the resulting dispersion may depend on the features of the self-assembly approach, as described below.
[00133] Preferred collections of inorganic nanoparticles have an average diameter less than a 100 nm and a very narrow distribution of primary particle diameters. Preferably, the nanoparticle is between about 1 to 50 nm in diameter, preferably, the nanoparticle is between about 1 to 20 nm in diameter.
[00134] The formation of the structures involves self-assembly approaches that generate well defined organized deposits of nanoparticles. The self-assembly techniques can be used to directly form the deposits of nanoparticles.
[00135] The self-assembled structures can be produced using dispersions of nanoparticles and by manipulating the conditions on the surface of the material and in the solution to lead to the desired structure formation. In some embodiments, a linker is used to chemically bind on one end to the substrate surface and on the other end to the nanoparticle. Selective binding with the linker can be used to direct the self-assembly process. Another alternative approach makes use of natural interactions, such as electrostatic and chemical interactions to help direct the self-assembly process. In other alternative approaches, the nanoparticles are deposited within miniature pores to localize the nanoparticles within the boundaries defined by the porous region. Miniature pores are found within certain materials, such as inorganic oxides or two dimensional organic crystals, or suitable pores can be formed, for example, by ion milling or chemical etching. Further details and additional self-assembly approaches are described below.
[00136] Production ofNanocrystalline/Nanoscale Particles: laser pyrolysis has been discovered to be a valuable tool for the production of nanoscale inorganic particles, including, in particular, carbon particles, elemental metal particles, metal/silicon oxide particles, metal/silicon carbide particles, metal/silicon nitride particles and metal/silicon sulfide particles. In addition, the particles produced by laser pyrolysis are a convenient material for further heat processing under mild conditions to expand the pathways for the production of desirable inorganic nanoparticles, especially particles with high uniformity. T hus, using laser pyrolysis alone or in combination with additional processes, a wide variety of nanoscale particles can be produced.
[00137] The reaction conditions determine the qualities of the particles produced by laser pyrolysis. The reaction conditions for laser pyrolysis can be controlled relatively precisely in order to produce particles with desired properties. The appropriate reaction conditions to produce a certain type of particles generally depend on the design of the particular apparatus. Increasing the laser power results in increased reaction temperatures in the reaction region as well as a faster quenching rate. A rapid quenching rate tends to favor production of high energy phases, which may not be obtained with processes near thermal equilibrium. Similarly, increasing the chamber pressure also tends to favor the production of higher energy structures. Also, increasing the concentration of a reactant, such as a reactant serving as an oxygen source, in the reactant stream favors the production of particles with increased amounts of atoms from the secondary reactant.
[00138] Reactant flow rate and velocity of the reactant gas stream are inversely related to particle size so that increasing the reactant gas flow rate or velocity tends to result in smaller particle sizes. Also, the growth dynamics of the particles have a significant influence on the size of the resulting particles. In other words, different forms of a product compound have a tendency to form different size particles from other phases under relatively similar conditions. Light intensity/laser power also influences particle size with increased light intensity favoring larger particle formation for lower melting materials and smaller particle formation for higher melting materials.
[00139] Laser pyrolysis has been performed generally with gas phase reactants. Many metal/silicon precursor compounds can be delivered into the reaction chamber as a gas. Appropriate metal/silicon precursor compounds for gaseous delivery generally include metal/silicon compounds with reasonable vapor pressures, i.e., vapor pressures sufficient to get desired amounts of precursor vapor in the reactant stream. The vessel holding liquid or solid precursor compounds can be heated to increase the vapor pressure of the metal/silicon precursor, if desired. A carrier gas can be bubbled through a liquid precursor to facilitate delivery of a desired amount of precursor vapor. Similarly, a carrier gas can be passed over the solid precursor to facilitate delivery of the precursor vapor. A suitable container for heating and delivering a solid precursor to a laser pyrolysis apparatus is described below. Solid precursors generally are heated to produce a sufficient vapor pressure. [00140] The use of exclusively gas phase reactants is somewhat limiting with respect to the types of precursor compounds that can be used conveniently. Thus, techniques have been developed to introduce aerosols containing reactant precursors into laser pyrolysis chambers. [00141] Using aerosol delivery apparatuses, solid precursor compounds can be delivered by dissolving the compounds in a solvent. Alternatively, powdered precursor compounds can be dispersed in a liquid/solvent for aerosol delivery. Liquid precursor compounds can be delivered as an aerosol from a neat liquid, a multiple liquid dispersion or a liquid solution. Aerosol reactants can be used to obtain a significant reactant throughput. A solvent/dispersant can be selected to achieve desired properties of the resulting solution/dispersion. Suitable solvents include water, methanol, ethanol, isopropyl alcohol, other organic solvents and mixtures thereof. The solvent should have a desired level of purity such that the resulting particles have a desired purity level. Some solvents, such as isopropyl alcohol, are significant absorbers of infrared light from a CO2 laser such that no additional laser absorbing compound may be needed within the reactant stream if a CO2 laser is used as a light source.
[00142] If aerosol precursors are formed with a solvent present, the solvent preferably is rapidly evaporated by the light beam in the reaction chamber such that a gas phase reaction can take place. Thus, the fundamental features of the laser pyrolysis reaction are unchanged by the presence of an aerosol. Nevertheless, the reaction conditions are affected by the presence of the aerosol. [00143] A number of suitable solid, metal, metal/silicon precursor compounds can be delivered as an aerosol from solution. The compounds are dissolved in a solution generally with a concentration greater than about 0.5 molar. Typically, the greater the concentration of precursor in the solution the greater the throughput of reactant through the reaction chamber. As the concentration increases, however, the solution can become more viscous such that the aerosol may have droplets with larger sizes than desired. Thus, selection of solution concentration can involve a balance of factors in the selection of a preferred solution concentration.
[00144] Preferred secondary reactants serving as oxygen source include, for example, O2, CO, CO2, O3 and mixtures thereof. Oxygen can be supplied as air. T he secondary reactant compound should not react significantly with the metal/silicon precursor prior to entering the reaction zone since this generally would result in the formation of large particles. Alternative secondary reactants can be selected based on the desired product particles and precursors. [00145] Laser pyrolysis can be performed with a variety of optical frequencies. Preferred light sources operate in the infrared portion of the electromagnetic spectrum. CO2 lasers are particularly preferred sources of light. Infrared absorbers for inclusion in the reactant stream include, for example, C2H4, isopropyl alcohol, NH3, SF6, SiH4 and O3. O3 can act as both an infrared absorber and as an oxygen source. The radiation absorber, such as the infrared absorber, absorbs energy from the radiation beam and distributes the energy to the other reactants to drive the pyrolysis.
[00146] Preferably, the energy absorbed from the light beam increases the temperature at a tremendous rate, many times the rate that heat generally would be produced by exothermic reactions under controlled condition. While the process generally involves nonequilibrium conditions, the temperature can be described approximately based on the energy in the absorbing region. The laser pyrolysis process is qualitatively different from the process in a combustion reactor where an energy source initiates a reaction, but the reaction is driven by energy given off by an exothermic reaction. Thus, while this light driven process is referred to as laser pyrolysis, it is not a thermal process even though traditional pyrolysis is a thermal process.
[00147] An inert shielding gas can be used to reduce the amount of reactant and product molecules contacting the reactant chamber components. Inert gases can also be introduced into the reactant stream as a carrier gas and/or as a reaction moderator. Appropriate inert shielding gases include, for example, Ar, He and N2. [00148] An appropriate laser pyrolysis apparatus generally includes a reaction chamber isolated from the ambient environment. A reactant inlet connected to a reactant delivery apparatus produces a reactant stream through the reaction chamber. A laser beam path intersects the reactant stream at a reaction zone. The reactant/product stream continues after the reaction zone to an outlet, where the reactant/product stream exits the reaction chamber and passes into a collection apparatus. Generally, the light source, such as a laser, is located external to the reaction chamber, and the light beam enters the reaction chamber through an appropriate window.
[00149] In another preferred embodiment, the nanoparticles comprise synthetic polymer molecules. The synthetic polymer molecules typically comprise linear or branched polymer chains, where the branched polymer can be a star polymer, a hyperbranched polymer, a graft polymer, a dendritic polymer or a combination thereof. The synthetic polymer molecule can also be a block copolymer, where the crosslinkable groups are typically contained in at least one block of the polymer molecule.
[00150] The polymer molecules will typically have a number average molecular weight in the range of about 500 to 5,000,000, more typically within the range of about 10,000 to 500,000. The molecular weight of the polymer molecules is typically selected so as to provide crosslinked particles that are approximately 2-100 nm in diameter. In one embodiment, the molecular weight is selected to provide crosslinked particles approximately 2-25 nm in diameter and in yet another embodiment, the molecular weight is selected so as to provide approximately 2-10 nm particles.
[00151] In one embodiment of the invention, the synthetic polymer molecule has a backbone comprising monomer units such as ethylenically unsaturated polymerizable monomers, nitrogenous polymers, olefins, condensation monomers, ring-opening monomers including epoxides and norbornenes, esters, sulfones, lactides, lactones, carbonates, imides, arylenes, amides, propylene, ethers, urethanes, vinyl and vinyl derivatives, and organic polysilicas, non-limiting examples of which are described below. [00152] Exemplary ethylenically unsaturated polymerizable monomers include acrylic and methacrylic acids, esters and amides; alkyl acrylates (e.g., methyl acrylate, ethyl acrylate and butyl acrylate); aryl acrylates (e.g., benzyl acrylate); alkyl methacrylates; aryl methacrylates (e.g., methyl acrylate, methyl methacrylate, n-butyl acrylate, n-butyl methacrylate, t-butyl acrylate, t-butyl methacrylate, 2-ethylhexyl methacrylate, benzyl methacrylate and N-phenylacrylamide); alpha-olefins (e.g., ethylene and propylene); and combinations thereof.
[00153] Exemplary nitrogenous polymers include poly(acrylamide); poly(methacrylamide); N,N-dialkyl poly(acrylamide) (particularly wherein the nitrogen- bearing substituents are C1-C12 alkyl); N,N-dialkyl poly(methacrylamide) (particularly wherein the nitrogen-bearing substituents are C1-C12 alkyl); poly(alkoxylated polyamide) (e.g., N-methoxymethylated polyamide and hydroxyethylated polyamide); poly(ε- caprolactam); polypropiolactam; polycapryllactam; polylauryllactam; poly(pyrrolidin-2-one); poly(vinylamine); poly(vinyl pyrrolidone); poly(2-vinylpyridine); poly(3-vinylpyridine); poly(4-vinylpyridine); poly(o-aminostyrene); poly(m-aminostyrene); poly(p-aminostyrene); polyoxazoline; polyethyleneimine; N-alkylated polyethyleneimine (particularly polyethylene imine alkylated with a C1-C12 alkyl substituent); N-acylated polyethylene imine (especially where the nitrogen-bearing substituents are C1-C12 alkyl); poly(p-phenylene terephthalamide); polyetherimides; polyimides; polyurethanes; polyhydrazides; polybenzimidazole; poly(l,2,4- triazole); polyhydantoin; polyimidines; poly(styrene-co-acrylonitrile); poly(butadiene-co- acrylonitrile); and combinations thereof.
[00154] Exemplary olefins are C3-20 (generally C3-15) cyclic olefin monomers such as ethylene, propylene, norbornene and tetracyclododecene.
[00155] Exemplary condensation monomers include dicarboxylic acids, their anhydrides and esters; aminocarboxylic acids and lactams; hydroxycarboxylic acids and lactones; diols, polyether diols and polyester diols; and diisocyanates; and combinations thereof. [00156] Exemplary ring-opening monomers include those monomers that contain a 3- carbon carbocyclic ring or a 5-carbon heterocyclic ring (having up to 2 heteroatoms) and include epoxides and norbornenes.
[00157] Exemplary imides include those polyamides formed by imidization of a poly(amic acid ester) which is formed from a dianhydride selected from the group consisting of pyrometallic dianhydride, benzophenone dianhydride and 9,9-bis-(trifluoromethyl) xanthenetetracarboxylic dianhydride; and a diamine selected from the group consisting of p- phenylene diamine, 4,4'-diamino-diphenyl ether, l,3-bis(p-aminophenoxy) benzene and 2,2- bis[4-aminophenyl]hexa-fluoropropane.
[00158] Exemplary arylenes include phenylenes, phenylquinoxalines, arylene ethers and combinations thereof. [00159] Exemplary vinyl and vinyl derivatives include vinyl acetate, vinyl bromide, vinylidene chloride, butylacrylate unsubstituted styrene and styrene substituted with one or two lower alkyl, halogen or hydroxyl groups (e.g., styrene derivatives such as 4-vinyltoluene, 4-vinylphenol, α-methylstyrene, 2,5-dimethylstyrene, 4-t-butylstyrene and 2-chlorostyrene); and combinations thereof.
[00160] Exemplary organic polysilicas include silsesquioxanes (polymeric silicate materials of the type (RSiO1^)n where R is an organic substituent); alkoxy silanes (particularly, partially condensed alkoxysilanes, e.g., partially condensed by controlled hydrolysis of tetraethoxysilane having an Mn of about 500 to 20,000); organically modified silicates having the composition RSiO3 and R2SiO2 wherein R is an organic substituents; and orthosilicates (particularly, partially condensed orthosilicates having the composition SiOR4); and combinations thereof.
Crosslinkable Groups
[00161] The synthetic polymer molecules used in the methods of the invention can have a plurality of crosslinkable groups that are-inert until-activated, but which when activated undergo a rapid and irreversible intramolecular crosslinking reaction. In order to perform well in the pseudo-high dilution methods of the invention, the crosslinking groups must react at a rapid rate, the crosslinking chemistry must be irreversible and the resulting coupled structure must be unreactive under the conditions required for crosslinking. Accordingly, the crosslinking groups are referred to as "crosslinkable" since they are inert until activated, but which when activated undergo an irreversible intramolecular crosslinking reaction. [00162] There are numerous crosslinkable groups that are suitable for use in the instant invention and they are typically covalently bound to one or more monomer units within a given polymer molecule. They can be directly bound to the monomers or indirectly bound, such as through a linking group. The crosslinkable groups can be thermally activatible; photolytically activatible; activatible with ultraviolet radiation, ionizing radiation, or electron beam radiation; or activatible by a chemical activating agent. The number of crosslinkable groups on the polymer molecules can be is selected to provide a crosslinked particle of suitable size, with the number of crosslinkable groups being inversely related to the particle size since a larger number of groups will provide for more intramolecular crosslinking and thus a smaller particle. For example, the number of crosslinkable groups can be selected so as to provide particles that are approximately 2-100 nm in diameter. In other embodiments, the desired particle diameter may be within the range of 2-25 run, or about 2-10 urn, and the number of crosslinkable groups can be selected accordingly. In a similar manner, the crosslinking density on the polymer molecules can be selected so as to provide the desired particle diameter, for example, within the range of about 2-100 nm, about 2-25 nm or about 2-10 nm.
[00163] Exemplary crosslinkable groups include by way of illustration and not limitation, acryloyl, lower alkyl-substituted acryloyl, vinyl, substituted vinyl, cyclic ether, cyclic ester, activated ester, cycloalkenyl, acid halide, amino, alcohol, phenol, carboxylic acid, diacetylene, unsubstituted and substituted acetylene groups (e.g., optionally substituted with one or more alkyl, aryl, ester; acid or amide groups), eonophiles, dienophiles and substituted and unsubstituted bicyclo[4.2.0]octa-l,3,5-trienyl groups. A particularly suitable crosslinkable group is the benzocyclobutene functionality and its substituted derivatives (especially oxy substituted), a group which has found wide use as a thermally cross-linkable group and in the formulation of thermosetting materials.
[00164] Specific examples of suitable crosslinkable groups include — CH=CH2, — C≡CH, -0(CO)-CH=CH2, — O(CO)— C(alkyl)=CH2 (for example, — O(CO)— C(lower alkyl)=CH2), — (CH2)m— 0(CO)-CH=CH2, — (CH2)m— O(CO)— C(alkyl)=CH2 (for example, — (CH2)m— O(CO)— C(lower alkyl)=CH2), -(CO)-O-CH=CH2, — O— CH=CH2, — C(CH3)=CH2, —C(CF3)=CH2, — C(CH2CH3)=CH2, — C(CH2CF3)=CH2, — C(C6H5)=CH2, -C=CH(C6H5), — C≡C(C6H5), — (CH2)ra— CH=CH2, — (CH2)m— O— CH=CH2, — (CH2)m— (CO)-O-CH=CH2, — (CH2)m— C(CH3)=CH2, — (CH2)m— C(CF3)=CH2, — (CH2)m— C(CH2CHs)=CH2, — (CH2)m— C(CH2CF3)=CH2, — (CH2)m— C(C6H5)=CH2.
Solvents
[00165] Some embodiments of the methods of the invention utilize solvents and there are numerous solvents that are well suited for use in the invention. Preferably, the solvent is inert with respect to the polymer molecules and the produced crosslinked particles.
[00166] High boiling point solvents can be used. These include, by way of illustration and not limitation, benzyl ether; N-cyclohexylpyrrolidinone; N-methylpyrrolidone; dimethylacetamide; dimethylphenyl urea; N,N-dimethyltrimethylene urea; butyl acetate; 2- ethoxyethanol; cyclopentanone; cyclohexanone; γ-butyrolactone; lactate esters such as ethyl lactate; ethoxyethylpropionate; alkylene glycol alkyl ether esters such as propylene glycol methyl ether acetate; alkylene glycol alkyl ethers such as propylene glycol methyl ether and propylene glycol n-propyl ether; alkylene glycol monoalkyl esters such as methyl cellosolve, butyl acetate, 2-ethoxyethanol, and ethyl 3-ethoxypropionate; polyethylene glycols and alkyl and aryl derivatives; diphenyl ether; diphenyl sulfone; ethylene carbonate; and mixtures thereof.
[00167] In addition, there are numerous other common organic solvents that can be utilized. These include, by way of illustration and not limitation, p-xylene, toluene, anisole, mesitylene, 1,3-dimethoxybenzene, trichloroethylene; and mixtures thereof.
Coupling Agents
[00168] It may be desirable to include a coupling agent in the methods of the invention and there are numerous suitable coupling agents that are known in the art. These include, by way of illustration and not limitation, esters, dihaloalkanes such as 1,2-dibromoethane, iodine, bis(bromomethyl)benzene, silicon tetrachloride and tin tetrachloride, di(isopropenyl)benzene and divinyl benzene, alkyltrichlorosilanes and dialkyldichlorosilanes. Selection of the appropriate coupling agent will be determined by the monomers used and/or the nature of the polymer being synthesized as is well known in the art.
Chemical Moieties
[00169] The incorporation of various chemical moieties allows for the preparation of tailored nanoparticles. The chemical moiety can be attached to the preformed polymer molecule or it can be attached to the particle during its formation. Li the latter case, the activation step can be conducted in the presence of a chemical moiety so that the chemical moiety is incorporated into the crosslinked particle. For example, the crosslinked particle can have at least one functional group on its backbone so that the chemical moiety is covalently attached to the crosslinked particle at the functional group.
[00170] Such chemical moieties include, by way of illustration, pharmaceutical agents, catalysts, functional groups, surfactants, sensor groups and photoresponsive units. For example, the polymer molecule can be prepared by first preparing a short carboxy- functionalized polystyrene block, which is then used to initiate the polymerization of a mixture of styrene and vinylbenzocyclobutene. The resulting polymer can then be used as a starting material in the preparation of crosslinked nanoparticles, in which a single carboxy functional group and linear block are now attached. The versatility inherent in the synthesis again allows the length of the linear block, nature of the repeat units and the number of functional groups to be easily varied.
[00171] Exemplary pharmaceutical agents include antibodies, aptamers,peptides, DNA oligomers, lipids, enzymes, carbohydrates and aminoglycosides.
[00172] Exemplary catalysts include metals, acids, bases, oxidizing and reducing agents and chelating groups.
[00173] Exemplary functional groups include acids, esters, alcohols, phenols, amines, thiols, amides, imines, nitriles, ethers, acetylenes, alkenes and heterocyclics.
Surface Coating
[00174] In one embodiment, the nanoparticle surface is coated with a lipophilic material and the tether is anchored into the coating through a hydrophobic moiety such as one or more aliphatic hydrocarbon chains. In one preferred embodiment, the particles themselves can be described generally as nanoparticles having an inert core surrounded by a coating to which any desired materials can be coupled. In the agent of the invention, these materials can include a chelate containing a paramagnetic ion.
[00175] With respect to these nanoparticles, the inert core can be a vegetable, animal or mineral oil, but is preferably a fluorocarbon compound—perfluorinated or otherwise rendered additionally inert. Mineral oils include petroleum derived oils such as paraffin oil and the like. Vegetable oils include, for example, linseed, safflower, soybean, castor, cottonseed, palm and coconut oils. Animal oils include tallow, lard, fish oils, and the like. Many oils are triglycerides.
[00176] Fluorinated liquids are particularly useful as cores. These include straight chain, branched chain, and cyclic hydrocarbons, preferably perfluorinated. Some satisfactorily fluorinated, preferably perfluorinated organic compounds useful in the particles of the invention themselves contain functional groups. However, perfluorinated hydrocarbons are preferred. The nanoparticle core may comprise a mixture of such fluorinated materials.
Typically, at least 50% fluorination is desirable in these inert supports. Preferably, the inert core has a boiling point of above 200C, more preferably above 3O0C, still more preferably above 5O0C, and still more preferably above about 9O0C
[00177] Thus, the perfluoro compounds that are particularly useful in the above-described nanoparticle aspect of the invention include partially or substantially or completely fluorinated compounds. Chlorinated, brominated or iodinated forms may also be used. A detailed list of compounds useful as nanoparticle cores is included below. [00178] With respect to the coating on the nanoparticles in this aspect, the relatively inert core is provided with a lipid/surfactant coating that will serve to anchor the desired moieties to the nanoparticle itself. If an emulsion is to be formed, the coating typically should include a surfactant. Typically, the coating will contain lecithin type compounds which contain both polar and non-polar portions as well as additional agents such as cholesterol. Typical materials for inclusion in the coating include lipid surfactants such as natural or synthetic phospholipids, but also fatty acids, cholesterols, lysolipids, sphingomyelins, tocopherols, glucolipids, stearylamines, cardiolipins, a lipid with ether or ester linked fatty acids, polymerized lipids, and lipid conjugated polyethylene glycol. Other surfactants are commercially available. The foregoing may be mixed with anionic and cationic surfactants. [00179] Fluorochemical surfactants may also be used. These include perfluorinated alcohol phosphate esters and their salts; perfluorinated sulfonamide alcohol phosphate esters and their salts; perfluorinated alkyl sulfonamide alkylene quaternary ammonium salts; N5N- (carboxyl-substituted lower alkyl) perfluorinated alkyl sulfonamides; and mixtures thereof. As used with regard to such surfactants, the term "perfluorinated" means that the surfactant contains at least one perfluorinated alkyl group. A detailed list of surfactants, including fluorinated surfactants that can be used in the coating, is found below. [00180] Typically, the lipids/surfactants are used in a total amount of 0.01-5% by weight of the nanoparticles, preferably 0.1-1% by weight. In one embodiment, lipid/surfactant encapsulated emulsions can be formulated with cationic lipids in the surfactant layer that facilitate the adhesion of nucleic acid material to particle surfaces. Cationic lipids include DOTMA, N-[l-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammoium chloride; DOTAP, 1,2- dioleoyloxy-3-(trimethylammonio)propane; and DOTB, l,2-dioleoyl-3-(4'-trimethyl- ammonio)butanoyl-sn-glycerol may be used. In general the molar ratio of cationic lipid to non-cationic lipid in the lipid/surfactant monolayer may be, for example, 1 : 1000 to 2: 1, preferably, between 2:1 to 1:10, more preferably in the range between 1:1 to 1:2.5 and most preferably 1:1 (ratio of mole amount cationic lipid to mole amount non-cationic lipid, e.g., DPPC). A wide variety of lipids may comprise the non-cationic lipid component of the emulsion surfactant, particularly dipalmitoylphosphatidylcholine, dipahnitoylphosphatidyl- ethanolamine or dioleoylphosphatidylethanolamine in addition to those previously described. In lieu of cationic lipids as described above, lipids bearing cationic polymers such as polyamines, e.g., spermine or polylysine or polyarginine may also be included in the lipid surfactant and afford binding of a negatively charged therapeutic, such as genetic material or analogues there of, to the outside of the emulsion particles.
[00181] In addition to the above-described preferred embodiment, a multiplicity of other particulate supports may be used in carrying out the method of the invention. In other embodiments, for example, the particles may be liposomal particles. The literature describing various types of liposomes is vast and well known to practitioners. As the liposomes themselves are comprised of lipid moieties, the above-described lipids and surfactants are applicable in the description of moieties contained in the liposomes themselves. These lipophilic components can be used to couple to the chelating agent in a manner similar to that described above with respect to the coating on the nanoparticles having an inert core. Micelles are composed of similar materials, and this approach to coupling desired materials, and in particular, the chelating agents applies to them as well. Solid forms of lipids may also be used.
[00182] In another example, proteins or other polymers can be used to form the particulate carrier. These materials can form an inert core to which a lipophilic coating is applied, or the chelating agent can be coupled directly to the polymeric material through techniques employed, for example, in binding affinity reagents to particulate solid supports. Thus, for example, particles formed from proteins can be coupled to tether molecules containing carboxylic acid and/or amino groups through dehydration reactions mediated, for example, by carbodiimides. Sulfur-containing proteins can be coupled through maleimide linkages to other organic molecules which contain tethers to which the chelating agent is bound. Depending on the nature of the particulate carrier, the method of coupling so that an offset is obtained between the dentate portion of the chelating agent and the surface of the particle will be apparent to the ordinarily skilled practitioner.
[00183] The particles can be coupled through the spacer to a chelator in which a transition metal is disposed. Typical chelators include porphyrins, ethylenediaminetetraacetic acid (EDTA), diethylenetriamine-N,N,N',N",N"-ρentaacetate (DTPA), 1 ,4,10,13-tetraoxa-7,16- diazacyclooctadecane-7 (ODDA), 16-diacetate, N-2-(azol-l(2)-yl)ethyliminodiacetic acids, l,4,7,10-tetraazacyclododecane-N,Nl,N",N'"-tetraacetic acid (DOTA), l,7,13-triaza-4,10,16- trioxacyclo-octadecane-N,N',N"-triacetate (TTTA), tetraethylene glycols, 1,5, 9- triazacyclododecane-N,N':,N",-tris(methylenephosphonic acid (DOTRP),N,Nl 5N"- trimethylammonium chloride (DOTMA) and analogues thereof. [00184] Examples of typical core components, referred to above, include, but not limited to: perfluorocarbon compounds which may be employed are perfluorotributylamine (FC47), perfluorodecalin (PP5), perfluoromethyldecalin (PP9), perfluorooctylbromide, perfluorotetrahydrofuran (FC80), perfluroether (PID), [(CF3).sub.2 CFOCF2 (CF2) 2 CF2 OCF(CF3) 2 ]perfluoroether (PIID) [(CF3) 2 CFOCF2 (CF2) 6 CF2 OCF(CF3) 2], perfluoroetheφolymer (Fomblin Y/01), perfluorododecane, perfluorobicyclo[4.3.0.] nonane, perfluorotritrimethylbicyclohexane, perfluorotripropylamine, perfluoroisopropyl cyclohexane, perfluoroendotetrahydrodicyclopentadiene, perfluoroadamantane, perfluoroexotetxahydrodicyclopentadiene, perfluorbicyclo[5.3.0.]decane, perfluorotetramethylcyclohexane, perfluoro- 1 -methyl-4-isopropylcyclohexane, perfluoro-n- butylcyclohexane, perfluorodimethylbicyclo[3.3.1.]nonane, perfluoro- 1 -methyl adamantane, perfluoro- 1 -methyl-4-t butylcyclohexane, perfluorodecahydroacenapthane, perfluorotrimethylbicyclo[3.3.1.]nonane, perfluoro- 1 -methyl adamantane, perfluoro-1- methyl-4-t butylcyclohexane, perfluorodecahydroacenaphthene, perfluorotrimethylbicyclo[3.3.1.]nonane, perfluoro-nundecane, perfluorotetradecahydrophenanthrene, perfluoro-l,3,5,7-tetramethyladamantane, perfluorododecahydrofluorene, perfluoro-l-3-dimethyladamantane, perfluoro-n- octylcyclohexane, perfluoro-7-methyl bicyclo[4.3.0.]nonane, perfluoro-p- diisopropylcyclohexane, perfluoro-m-diisopropylcyclohexane, perfluoro-4- methyloctahydroquinolidizine, perfluoro-N-methyldecahydroquinoline, F-methyl- 1 - oxadecalin, perfluorooctahydroquinolidizine, perfluoro 5,6-dihydro-5-decene, perfluoro-4,5- dihydro-4-octene, perfluorodichlorooctane and perfluorobischlorobutyl ether, perfluorooctane, perfluorodichlorooctane, perfluoro-n-octyl bromide, perfluoroheptane, perfluorodecane, perfluorocyclohexane, perfluoromorpholine, perfluorotripropylamine, perfluortributylamine, perfluorodimethylcyclohexane, perfluorotrimethylcyclohexane, perfluorodicyclohexyl ether, perfluoro-n-butyltetrahydrofuran, and compounds that are structurally similar to these compounds. Chlorinated perfluorocarbons, such as chloroadamantane and chloromethyladamantane as described in U.S. Pat. No. 4,686,024 may be used. Such compounds are described, for example in U.S. Pat. Nos. 3,962,439; 3,493,581, 4,110,474, 4,186,253; 4,187,252; 4,252,824; 4,423,077; 4,443,480; 4,534,978 and 4,542,147. [00185] Commercially available surfactants are Pluronic F-68, Hamposyl™ L30 (W.R. Grace Co., Nashua, N.H.), sodium dodecyl sulfate, Aerosol 413 (American Cyanamid Co., Wayne, NJ.), Aerosol 200 (American Cyanamid Co.), Lipoproteol.TM. LCO (Rhodia Inc., Mammoth, N.J.), Standapol™ SH 135 (Henkel Corp., Teaneck, N.J.), Fizul™ 10-127 (Finetex Inc., Elmwood Park, N.J.), and Cyclopol™ SBFA 30 (Cyclo Chemicals Corp., Miami, FIa.); amphoterics, such as those sold with the trade names: Deriphat™ 170 (Henkel Corp.), Lonzaine™ JS (Lonza, Inc.), Niranol™ C2N-SF (Miranol Chemical Co., Inc., Dayton, N.J.), Amphoterge™ W2 (Lonza, Inc.), and Amphoterge™ 2WAS (Lonza, Inc.); non-ionics, such as those sold with the trade names: Pluronic™ F-68 (BASF Wyandotte, Wyandotte, Mich.), Pluronic™ F-127 (BASF Wyandotte), Brij™ 35 (ICI Americas; Wilmington, Del.), Triton™ X-100 (Rohm and Haas Co., Philadelphia, Pa.), Brij™52 (ICI Americas), Span™ 20 (ICI Americas), Generol™ 122 ES (Henkel Corp.), Triton™ N-42 (Rohm and Haas Co.), Triton™ N-101 (Rohm and Haas Co.), Triton™ X-405 (Rohm and Haas Co.), Tween™ 80 (ICI Americas), Tween™ 85 (ICI Americas), and Brij™ 56 (ICI Americas) and the like.
[00186] Also included may be egg yolk phospholipids, alkylphosphoryl choline or alkylglycerolphosphoryl choline surfactants, and specific examples of these such as 1,2- dioctylglycero-3-phosphoryl choline, l,2-ditetradecylglycero-3-phosphoryl choline, 1,2- dihexadecylglycero-3-phosphoryl choline, l,2-dioctadecylglycero-3-phosphorylcholine, 1- hexadecyl-2-tetradecylglycero-3 -phosphoryl choline, 1 -octadecyl-2-tetradecylglycero-3 - phosphoryl choline, l-tetradecyl-2-octadecylglycero-3-phosphoryl choline, l-hexadecyl-2- octadecylglycero-3-phosphoryl choline, l-2-dioctadecylglycero-3-phosphoryl choline, 1- octadecyl-2-hexadecylglycero-3 -phosphoryl choline, 1 -tetradecyl-2-hexadecylglycero-3 - phosphoryl choline, 2,2-ditetradecyl-l -phosphoryl choline ethane and 1- hexadecyltetradecylglycero-3-phosphoryl choline.
[00187] Suitable perfluorinated alcohol phosphate esters include the free acids of the diethanolamine salts of mono- and bis(lH,lH,2H,2H-perfluoroalkyl)phosphates. The phosphate salts, available under the trade name "Zonyl RP" (E.I. Dupont de Nemours and Co., Wilmington, Del.), are converted to the corresponding free acids by known methods. Suitable perfluorinated sulfonamide alcohol phosphate esters are described in U.S. Pat. No. 3,094,547. Suitable perfluorinated sulfonamide alcohol phosphate esters and salts of these include perfluoro-n-octyl-N-ethylsulfonarnidoethyl phosphate, bis(perfluoro-n-octyl-N- ethylsulfonamidoethyl)phosphate, the ammonium salt of bis(perfluoro-n-octyl-N- ethylsulfonamidoethyl)phosphate,bis(perfluoro-decy l-N-ethylsulfonamidoethyl)-phosphate and bis(perfluorohexyl-N ethylsulfonamidoethyl)-phosphate. The preferred formulations use phosphatidylcholine, derivatized-phosphatidylethanolamine and cholesterol as the aqueous surfactant.
[00188] Suitable paramagnetic metals include a lanthanide element of atomic numbers 58-70 or a transition metal of atomic numbers 21-29, 42 or 44, i.e., for example, scandium, titanium, vanadium, chromium, manganese, iron, cobalt, nickel, copper, molybdenum, ruthenium, cerium, praseodymium, neodymium, promethium, samarium, europium, gadolinium, terbium, dysprosium, holmium, erbium, thulium, and ytterbium, most preferably Gd(III), Mn(II), iron, europium and/or dysprosium.
[00189] The chelating moiety can be coupled to the particle through a spacer or tether which may be an aliphatic chain, a peptide, a polyethylene glycol polymer, or any suitable spacing molecule. One end of the spacer is bound, preferably covalently, to the dentate portion of the chelating agent; the other is anchored to the particle. The coupling to the particle can be covalent or the spacer may be anchored through ionic bonding, hydrogen bonding or van der Waals forces. When the particle surface comprises a lipid surface, particularly preferred anchoring moieties are the hydrocarbon side chains of phosphatides or other di-substituted glycerol derivatives.
[00190] By appropriately coupling the chelating agents, substantial numbers of chelators and paramagnetic ions can be coupled to the particles. Typically, the particles will be coupled to at least 10,000 chelators and/or paramagnetic ions, preferably 20,000 chelators and/or paramagnetic ions, more preferably 50,000 chelators and/or paramagnetic ions, more preferably at least 70,000 chelators and/or paramagnetic ions and more preferably at least 100,000 chelators and/or paramagnetic ions.
[00191] Thus, in addition to the chelated paramagnetic metal ion, the particles may also be coupled to ligands for targeting and/or biologically active molecules. It is possible also to include among the components coupled to the particles bearing the chelated paramagnetic ion, radionuclides for use in treatment or diagnosis.
[00192] As described above, solid particles which contain reactive groups can be coupled directly to the tether or spacer; lipid-based particles such as oil emulsions, solid lipids, liposomes, and the like, can include lipophilic materials containing reactive groups which may covalently, then, be coupled to linking moieties which bear the dentate portion of the chelating agent, hi one particularly preferred embodiment, the process involves mixing a liquid fluorocarbon compound that forms the core of a nanoparticle and the components of a lipid/surfactant coating for that particle in an aqueous suspension, microfiuidizing, and, if desired, harvesting and sizing the particles. The components to be coupled can be included in the original mixture by virtue of their initial coupling to one or more components of the lipid/surfactant coating, or the coupling to additional moieties can be conducted after the particles are formed.
Antibodies
[00193] The antibodies of the present invention may be generated by any suitable method known in the art. The antibodies of the present invention can comprise polyclonal antibodies. Methods of preparing polyclonal antibodies are known to the skilled artisan (Harlow, et al., Antibodies: A Laboratory Manual, (Cold spring Harbor Laboratory Press, 2nd ed. (1988), which is hereby incorporated herein by reference in its entirety). For example, a polypeptide can be administered to various host animals including, but not limited to, rabbits, mice, rats, etc. to induce the production of sera containing polyclonal antibodies specific for the antigen. The administration of the polypeptides may entail one or more injections of an immunizing agent and, if desired, an adjuvant. Various adjuvants may be used to increase the immunological response, depending on the host species, and include but are not limited to, Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum. Such adjuvants are also well known in the art. For the purposes of the invention, "immunizing agent" may be defined as a polypeptide of the invention, including fragments, variants, and/or derivatives thereof, in addition to fusions with heterologous polypeptides and other forms of the polypeptides as may be described herein.
[00194] Typically, the immunizing agent and/or adjuvant will be injected in the mammal by multiple subcutaneous or intraperitoneal injections, though they may also be given intramuscularly, and/or through IV. The immunizing agent may include polypeptides of the present invention or a fusion protein or variants thereof. Depending upon the nature of the polypeptides (i.e., percent hydrophobicity, percent hydrophilicity, stability, net charge, isoelectric point etc.), it may be useful to conjugate the immunizing agent to a protein known to be immunogenic in the mammal being immunized. Such conjugation includes either chemical conjugation by derivatizing active chemical functional groups to both the polypeptide of the present invention and the immunogenic protein such that a covalent bond is formed, or through fusion-protein based methodology, or other methods known to the skilled artisan. Examples of such immunogenic proteins include, but are not limited to keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. Various adjuvants maybe used to increase the immunological response, depending on the host species, including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum. Additional examples of adjuvants which may be employed includes the MPL-TDM adjuvant (monophosphoryl lipid A, synthetic trehalose dicorynomycolate). The immunization protocol may be selected by one skilled in the art without undue experimentation.
[00195] The antibodies of the present invention can also comprise monoclonal antibodies. Monoclonal antibodies may be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975) and U.S. Pat. No. 4,376,110, by Harlow, et ah, Antibodies: A Laboratory Manual, (Cold spring Harbor Laboratory Press, 2nd ed. (1988), by Hammerling, et ah, Monoclonal Antibodies and T-CeIl Hybridomas (Elsevier, N.Y., (1981)), or other methods known to the artisan. Other examples of methods which may be employed for producing monoclonal antibodies includes, but are not limited to, the human B- cell hybridoma technique (Kosbor et ah, 1983, Immunology Today 4:72; Cole et al., 1983, Proc. Natl. Acad. ScL USA 80:2026-2030), and the EBV-hybridoma technique (Cole et al., 1985, Monoclonal Antibodies And Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclass thereof. The hybridoma producing the mAb of this invention may be cultivated in vitro or in vivo. Production of high titers of mAbs in vivo makes this the presently preferred method of production.
[00196] In a hybridoma method, a mouse, a humanized mouse, a mouse with a human immune system, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes may be immunized in vitro.
[00197] The immunizing agent will typically include antigens form infectious disease organisms, cancer cells, environmental antigens and the like, polypeptides, fragments or a fusion protein thereof. Generally, either peripheral blood lymphocytes ("PBLs") are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986), pp. 59- 103). Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Usually, rat or mouse myeloma cell lines are employed. The hybridoma cells may be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells. For example, if the parental cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine ("HAT medium"), which substances prevent the growth of HGPRT-deficient cells.
[00198] Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. More preferred immortalized cell lines are murine myeloma lines, which can be obtained, for instance, from the SaIk Institute Cell Distribution Center, San Diego, Calif, and the American Type Culture Collection, Manassas, Va. As inferred throughout the specification, human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) pp. 51-63). [00199] The culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against the antigens. Preferably, the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoadsorbant assay (ELISA). Such techniques are known in the art and within the skill of the artisan. The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollart, Anal. Biochem., 107:220 (1980).
[00200] After the desired hybridoma cells are identified, the clones may be subcloned by limiting dilution procedures and grown by standard methods (Goding, supra). Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI- 1640. Alternatively, the hybridoma cells may be grown in vivo as ascites in a mammal.
[00201] The monoclonal antibodies secreted by the subclones may be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-sepharose, hydroxyapatite chromatography, gel exclusion chromatography, gel electrophoresis, dialysis, or affinity chromatography. [00202] The skilled artisan would acknowledge that a variety of methods exist in the art for the production of monoclonal antibodies and thus, the invention is not limited to their sole production in hybridomas. For example, the monoclonal antibodies may be made by recombinant DNA methods, such as those described in U.S. Pat. No. 4,816,567. In this context, the term "monoclonal antibody" refers to an antibody derived from a single eukaryotic, phage, or prokaryotic clone. The DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies, or such chains from human, humanized, or other sources). The hybridoma cells of the invention serve as a preferred source of such DNA. Once isolated, the DNA may be placed into expression vectors, which are then transformed into host cells such as Simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells.
[00203] Methods for producing and screening for specific antibodies using hybridoma technology are routine and well known in the art. In a non-limiting example, mice can be immunized with a polypeptide or a cell expressing such peptide. Once an immune response is detected, e.g., antibodies specific for the antigen are detected in the mouse serum, the mouse spleen is harvested and splenocytes isolated. The splenocytes are then fused by well- known techniques to any suitable myeloma cells, for example cells from cell line SP20 available from the ATCC. Hybridomas are selected and cloned by limited dilution. The hybridoma clones are then assayed by methods known in the art for cells that secrete antibodies capable of binding a polypeptide of the invention. Ascites fluid, which generally contains high levels of antibodies, can be generated by immunizing mice with positive hybridoma clones.
[00204] Accordingly, the present invention provides methods of generating monoclonal antibodies as well as antibodies produced by the method comprising culturing a hybridoma cell secreting an antibody of the invention wherein, preferably, the hybridoma is generated by fusing splenocytes isolated from a mouse immunized with an antigen of the invention with myeloma cells and then screening the hybridomas resulting from the fusion for hybridoma clones that secrete an antibody able to bind a polypeptide of the invention. [00205] Other methods can also be used for the large scale production of antibodies. For example, antibodies can also be generated using various phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them. In a particular embodiment, such phage can be utilized to display antigen binding domains expressed from a repertoire or combinatorial antibody library (e.g., human or murine). Phage expressing an antigen binding domain that binds the antigen of interest can be selected or identified with antigen, e.g., using labeled antigen or antigen bound or captured to a solid surface or bead. Phage used in these methods are typically filamentous phage including^ and Ml 3 binding domains expressed from phage with Fab, Fv or disulfide stabilized Fv antibody domains recombinantly fused to either the phage gene III or gene VIII protein. Examples of phage display methods that can be used to make the antibodies of the present invention include those disclosed in Brinkman et ah, J. Immunol. Methods 182:41-50 (1995); Ames et ah, J. Immunol. Methods 184:177-186 (1995); Kettleborough et ah, Eur. J. Immunol. 24:952-958 (1994); Persic et ah, Gene 187 9-18 (1997); Burton et ah, Advances in Immunology 57:191- 280 (1994); PCT application No. PCT/GB91/01134; PCT publications WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108; each of which is incorporated herein by reference in its entirety.
[00206] The antibodies of the present invention have various utilities. For example, such antibodies may be used in diagnostic assays to detect the presence or quantification of the polypeptides of the invention in a sample. Such a diagnostic assay can comprise at least two steps. The first, subjecting a sample with the antibody, wherein the sample is a tissue (e.g., human, animal, etc.), biological fluid (e.g., blood, urine, sputum, semen, amniotic fluid, saliva, etc.), biological extract (e.g., tissue or cellular homogenate, etc.), a protein microchip (e.g., See Arenkov P, et ah, Anal Biochem., 278(2):123-131 (2000)), or a chromatography column, etc. And a second step involving the quantification of antibody bound to the substrate. Alternatively, the method may additionally involve a first step of attaching the antibody, either covalently, electrostatically, or reversibly, to a solid support, and a second step of subjecting the bound antibody to the sample, as defined above and elsewhere herein. [00207] The antibodies are labeled with a detectable moiety as described infra. The detectable moiety may be a radioisotope, such as 2H, 14C, 32P, or 125I, a florescent or chemiluminescent compound, such as fluorescein isothiocyanate, rhodamine, or luciferin, or an enzyme, such as alkaline phosphatase, beta-galactosidase, green fluorescent protein, or horseradish peroxidase. Any method known in the art for conjugating the antibody to the detectable moiety may be employed, including those methods described by Hunter et ah, Nature, 144:945 (1962); David et al, Biochem., 13:1014 (1974); Pain et at, J. Immunol Methods, 40:219(1981); and Nygren, J Histochem. and Cytochem., 30:407 (1982). [00208] In accordance with the invention, antibodies are specific for either prokaryotic or eukaryotic cell antigens. Infectious disease almost invariably results in the acquisition of foreign nucleic acids, which could also be targeted using this technology. Specific targets could be viral, e.g. HIV (virus or provirus) or bacterial, e.g. multi-drug resistant bacteria e.g. TB, fungal or protoazoan. This technology can be especially useful in detecting agents such as microbial or viral agent (e.g. Ebola virus, etc.), or known or novel bio-terrorist agents. [00209] Particularly preferred viral organisms causing human diseases according to the present invention include (but not restricted to) Filoviruses, Herpes viruses, Hepatitisviruses, Retroviruses, Orthomyxoviruses, Paramyxoviruses, Togaviruses, Picornaviruses, Papovaviruses and Gastroenteritisviruses. Other preferred, non-limiting examples of viral agents are listed in Table 1.
[00210] According to another preferred embodiment of the invention, the antibodies are specific for human or domestic animal bacterial pathogens. Particularly preferred bacteria causing serious human diseases are the Gram positive organisms: Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis and E. faecium, Streptococcus pneumoniae and the Gram negative organisms: Pseudomonas aeruginosa, Burkholdia cepacia, Xanthomonas maltophila, Escherichia coli, Enterobacter spp, Klebsiella pneumoniae and Salmonella spp. The target molecules may include (but are not restricted to) molecules essential to bacterial survival and multiplication in the host organism, virulence gene products, gene products encoding single- or multi-drug resistance. However, gram negative bacteria are also within the scope of the invention.
[00211] In another preferred embodiment, the antibodies are targeted to toxins produced by a disease agent such as anthrax. For example, anthrax which is one of the agents that can be used in a bioterrorist attack. Anthrax infection is mediated by spores of Bacillus anthracis, which can gain entry to the body through breaks in the skin, through inhalation, or through ingestion. Fatal anthrax is characterized by the establishment of a systemic bacteremia that is accompanied by an overwhelming toxemia. It seems that anthrax is a two- pronged attack with the bacteremia and/or toxemia contributing to the fatal syndrome of shock, hypoperfusion, and multiple organ system failure. The likelihood of developing systemic disease varies with the portal of organism entry, and is most pronounced for the inhalational route (reviewed in Dixon et al., 1999, New England J. Med. 341: 815-826). [00212] According to one preferred embodiment of the invention, the antibodies are specific for protozoa infecting humans and causing human diseases. Particularly preferred protozoan organisms causing human diseases according to the present invention include (but not restricted to) Malaria e.g. Plasmodium falciparum and M. ovale, Trypanosomiasis (sleeping sickness) e.g. Trypanosoma cruzei, Leischmaniasis e.g. Leischmania donovani, Amebiasis e.g. Entamoeba histolytica.
[00213] According to one preferred embodiment of the invention, the antibodies are specific for fungi causing pathogenic infections in humans. Particularly preferred fungi causing or associated with human diseases according to the present invention include (but not restricted to) Candida albicans, Histoplasma neoformans, Coccidioides immitis and Penicillium marneffei.
[00214] According to one preferred embodiment, the antibodies are specific for target oligonucleotides responsible for viral replication; viral infection cycle such as attachment to cellular ligands; viral gene products encoding host immune modulating functions. Examples of viral organisms include, but not restricted to, those listed in table 1. For information about the viral organisms see Fields of Virology, 3. ed., vol 1 and 2, BN Fields et al. (eds.). Non- limiting examples of targets of selected viral organisms are listed in table 2.
Table 1. Selected viral organisms causing human diseases.
Figure imgf000052_0001
Figure imgf000053_0001
Table 2 Target gene products of viral organisms
Organism target gene open reading frame gene product
HIV gag: MA pl7
CA p24
NC
≠ pol: PR pl5
RT p66
P31 env: gpl20
gp41 tat transcriptional transactivator rev regulator of viral expression vif vpr vpu nef
RSV NSl
NS2
L
2-5A-dependent Rnase L
HPV El helicase
E2 transcription regulator
E3
E4 late NS protein
E5 transforming protein
E6 transforming protein
E7 transforming protein
E8
Ll major capsid protein
L2 minor capsid protein
HCV NS3 protease
NS3 helicase
HCV-IRES
NS5B polymerase
HCMV DNA polymerase
IEl Organism target gene open reading frame gene product
IE2
UL36
UL37
UL44 polymerase asc. protein
UL54 polymerase
UL57 DNA binding protein
UL70 primase
UL102 primase asc. protein
ULl 12
ULl 13
IRSl
VZV 6
16
18
19
28
29
31
39
42
45
47
51
52
55
62
71
HSV IE4 USl
IE5 US12
IEI lO ICPO
IE175 ICP4
UL5 helicase
UL8 helicase
UL13 capsid protein
UL30 polymerase
UL39 ICP6
UL42 DNA binding protein [00215] Information about the above selected gene products, open reading frames and gene products is found in the following references: Field A.K. and Biron, K.K. "The end of innocence" revisited: resistance of herpesviruses to antiviral drugs. Clin. Microbiol. Rev. 199 A; 7: 1-13. Anonymous. Drug resistance in cytomegalovirus: current knowledge and implications for patient management. J. Acquir. Immune Defic. Syndr. Hum. Retrovir. 1996; 12: S1-SS22. Kelley R et ah. Varicella in children with perinatally acquired human immunodeficiency virus infection. JPediatr 1994; 124: 271-273. Hanecak et al. Antisense oligonucleotides inhibition of hepatitis C virus, gene expression in transformed hepatocytes. J Virol 1996; 70: 5203-12. Walker Drug discovery Today 1999; 4: 518-529. Zhang et al. Antisense oligonucleotides inhibition of hepatitis C virus (HCV) gene expression in livers of mice infected with an HCV- Vaccinia virus recombinant. Antim. Agents Chemotherapy 1999; 43, 347- 53. Feigin RD, Cherry JD, eds. Textbook of pediatric infectious diseases. Philadelphia: WB Saunders, 1981. Chen B.et al., Induction of apoptosis of human cervical carcinoma cell line SiHa by antisense oligonucleotide og human papillomavirus type 16 E6 gene. 2000; 21(3): 335-339. The human herpesviruses. New York: Raven Press; 1993. DeClerque E, Walker RT, eds. Antiviral drug development: a multi-disciplinary approach. Plenum; 1987. Antiviral Drug Resistance (Richman, D.D., ed.), Wiley, Chichester, 1995. Flint SJ et al. eds. Principles of virology: Molecular biology, pathogene productsis and control.
[00216] In vitro propagation of virus causing human diseases: To screen for viral antigens, viral particles are propagated in in vitro culture systems of appropriate mammalian cells. Initial screening is typically performed in transformed cell lines. More thorough screening is typically performed in human diploid cells.
Table 3. Examples of in vitro propagation of viruses.
Figure imgf000056_0001
C is cytomegaly, D is cell destruction, F is marked focality, H is hemadsorption and S is formation of syncytium. "-" means that the cell line does not sustain growth of the virus. WI- 38 is a human diploid fibroblast cell line. MRC-5 is human lung fibroblasts. HeLa is a human aneuploid epithelial cell line. PRMK is primary rhesus monkey kidney cells. PCMK is primary cynomolgus monkey kidney cells.
[00217] Likewise Vero cells (green monkey kidney cells) and Mewo cells will sustain the growth of for example herpesviruses. References: DeClerque E, Walker RT, eds. Antiviral drug development: a multi-disciplinary approach. Plenum; 1987. Antiviral Drug Resistance (Richman, D.D., ed.), Wiley, Chichester, 1995. Cytomegalovirus protocols, J. Sinclair (ed.), Humana Press. HIV Protocols, N. Michael and JH Kim (eds.), Humana press. Hepatitis C Protocols, JYN Lau (ed.), Humana Press. Antiviral Methods and Protocols, D Kinchington and RF Schinazi, Humana Press.
[00218] Bacterial antigens: According to another preferred embodiment of the invention, the antibodies are specific for the human or domestic animal bacterial pathogens listed in (but not restricted to) table 4. The target antigens may include (but are not restricted to) gene products essential to bacterial survival and multiplication in the host organism, virulence gene products encoding single- or multi-drug resistance such as for instance the gene products listed in table 5. Table 4. Selected bacteria causing serious human diseases
Figure imgf000057_0001
[00219] References: Cookson B.D., Nosocomial antimicrobial resistance surveillance. J Hosp. Infect. 1999:97-103. Richards MJ. et ah. Nosocomial infections in medical intensive care units in the United States. National Nosocomial Infections Surveillance System. Crit. Care. Med. 1999;5:887-92. House of Lords Select Committee on Science and Technology. Resistance to antibiotics and other antimicrobial agents. London: 1998; Her Majesty's Stationary Office. Johnson A.P.. Intermediate vancomycin resistance in S. aureus: a major threat or a minor inconveniance? J. Antimicrobial. Chemother. 1998;42:289-91. Baquero F.. Pneumococcal resistance to beta-lactam antibiotics: a global overview. Microb. Drug Resist. 1995;1:115-20. Hsueh P.R. et al.. Persistence of a multidrug resistant Pseudomonas aeruginosa clone in an intensive care burn unit. J. Clin. Microbiol. 1998;36:1347-51. Livermore D.. Multiresistance and Superbugs. Commun. Dis. Public Health 1998;l:74-76.
Table 5. Examples of potential antigens in bacteria.
Figure imgf000058_0001
[00220] References: Escherichia coli and Salmonella in Cellular and Molecular Biology, vol 1 & 2. C Neidhardt and R Curtiss (eds.), American Society for Microbiology Press. Gram-Positive Pathogens. VA Fischetti et al. (eds.), American Society for Microbiology Press. Bacterial Pathogene productsis: A Molecular Approach. AA Salyers and DD Whitt (eds.), American Society for Microbiology Press. Organization of the Procaryotic Genome. RL Charlebois (ed.), American Society for Microbiology Press.
[00221] Listed in Table 6 below are examples of genes encoding the protein complexes listed in Table 5 above. The individual genes have homologues in the major human pathogenic bacteria listed in Table 4. Table 6 below depicts an example of a Gram negative (Escherichia coli) and a Gram positive (Staphylococcus aureus) organism, chosen as representatives for the two groups of bacteria. Table 6. Examples of gene products encoding possible gene target proteins.
Figure imgf000059_0001
[00222] References: Escherichia coli and Salmonella in Cellular and Molecular Biology, vol 1 & 2. C Neidhardt and R Curtiss (eds.), American Society for Microbiology Press. Gram-Positive Pathogens. VA Fischetti et al. {eds.), American Society for Microbiology Press. Bacterial Pathogenesis: A Molecular Approach. AA Salyers and DD Whitt (eds.), American Society for Microbiology Press. Organization of the Prokaryotic Genome. RL Charlebois (ed.), American Society for Microbiology Press.
[00223] Sequences for the gene products listed in Table 5 and 6 can be found in GenBank (http://www.ncbi.nlm.nih.gov/). The gene sequences maybe genomic, cDNA or mRNA sequences. Preferred sequences are viral gene products containing the complete coding region and 5' untranslated sequences that are involved in viral replication.
[00224] Protozoan antigens: According to one preferred embodiment of the invention, the antibodies are specific for protozoan organisms infecting humans and causing human diseases. Such protozoa include, but are not restricted to, the following: 1. Malaria e.g. Plasmodium falciparum and M. ovale, (references: Malaria by M Wahlgren and P Perlman (eds.), Harwood Academic Publishers, 1999. Molecular Immunological Considerations in Malaria Vaccine Development by MF Good and AJ Saul, CRC Press 1993). 2. Trypanosomiasis (sleeping sickness) e.g. Trypanosoma cruzei (reference: Progress in Human African Trypanosomiasis, Sleeping Sickness by M Dumas et al. {eds.), Springer Verlag 1998). 3. Leischmaniasis e.g. Leischmania donovani (reference: AL Banuals et ah, Molecular Epidemiology and Evolutionary Genetics of Leischmania Parasites. Int JParasitol 1999;29:1137-47). 4. Amebiasis e.g. Entamoeba histolytica (RP Stock et al, Inhibition of Gene Expression in Entamoeba histolytica with Antisense Peptide Nucleic Acid Oligomers. Nature Biotechnology 2001;19:231-34).
[00225] Fungal infections: According to one preferred embodiment of the invention, the antigens are specific for fungi cause pathogenic infections in humans. These include, but are not restricted to, the following: Candida albicans (references: AH Groll et al., Clinical pharmacology of systemic antifungal agents: a comprehensive review of agents in clinical use, current investigational compounds, and putative targets for antifungal drug development. Adv. Pharmacol. 1998:44:343-501. MDD Backer et al, An antisense-based functional genomics approach for identification of gene products critical for growth of Candida albicans. Nature Biotechnology 2001;19:235-241) and others, e.g., Histoplasma neoformans, Coccidioides immitis and Penicillium marneffei (reference: SA Marques et al, Mycoses associated with AIDS in the Third World. Med Mycol 2000; 38 Suppl 1 :269-79). [00226] Host cellular gene products involved in viral diseases: According to one preferred embodiment of the invention, the antibodies are specific for host cellular gene products involved in viral diseases. For example CD4, chemokine receptors such as CCR3, CCR5 are required for HIV infection.
[00227] hi another preferred embodiment, abnormal or cancer cells are detected by the compositions and methods of the invention. For example, many malignancies are associated with the presence of foreign DNA, e.g. Bcr-Abl, Bcl-2, HPV, and these provide unique molecular targets to permit selective malignant cell targeting. The approach can be used to target single base substitutions (e.g. K-ras, p53) or methylation changes.
Kits
[00228] The particles may, along with other reagents, be packaged in a kit useful for conveniently performing the assay methods for the determination of an analyte. To enhance the versatility of the subject invention, reagents can be provided in packaged combination, in the same or separate containers, in liquid or lyophilized form so that the ratio of the reagents provides for substantial optimization of the method and assay. The reagents may each be in separate containers, or various reagents can be combined in one or more containers depending on the cross-reactivity and stability of the reagents.
[00229] For example, a reagent test kit which may contain, in packaged combination, an antibody specific for a particular analyte, a particle of the present invention containing the same antibody or an analog or derivative thereof, and may optionally also comprise one or more calibrators comprising a known amount of the analyte. Such a test kit may provide reagents for an assay with enhanced clinical sensitivity for the analyte and structurally related compounds.
[00230] The following examples are offered by way of illustration, not by way of limitation. While specific examples have been provided, the above description is illustrative and not restrictive. Any one or more of the features of the previously described embodiments can be combined in any manner with one or more features of any other embodiments in the present invention. Furthermore, many variations of the invention will become apparent to those skilled in the art upon review of the specification. The scope of the invention should, therefore, be determined not with reference to the above description, but instead should be determined with reference to the appended claims along with their full scope of equivalents. [00231] AU publications and patent documents cited in this application are incorporated by reference for all purposes to the same extent as if each individual publication or patent document were so individually denoted. By their citation of various references in this document, Applicants do not admit any particular reference is "prior art" to their invention.
EXAMPLES Example 1
[00232] The self-assembling luminescent protein-metal nanoprobe is a sensor towards particular peptide sequences or organic ligands. The underlying premise of the detection strategy relies on (1) an antibody based fluorescence competition assay and (2) a fluorescence resonance energy transfer (Medintz, I. L. et al. Nature Materials 2, 630-638 (2003); Didenko, V. V. Biotechniques 31, 1106 (2001); Oh, E. et al. Journal of the American Chemical Society 127, 3270-3271 (2005); Perez-Luna, V. H. & Asian, K. Abstracts of Papers of the American Chemical Society 226, U99-U99 (2003); Gueroui, Z. & Libchaber, A. Single-molecule measurements of gold-quenched quantum dots. Physical Review Letters 93, - (2004)) utilizing metal nanoparticles as the donor and/or quencher components. This overall strategy is schematically illustrated in Figure 1, where the constructed nanoprobe or sensor contains a differentially labeled antibody-hapten complex containing two spectrally unique nanoparticles. As a result of the target detection (or analyte binding), the labeled hapten is released from the complex (the nanoprobe) thereby yielding a change in the distances between the two differing nanoparticles. This change in distance manifests an increase in measured overall fluorescence of the sample, which serves as a signal confirming the detection of analyte.
[00233] Depending on the identities of the nanoparticles, the fluorescence signal may arise from either the labeled antibody or hapten subsequent to analyte detection. Potential arrangements, as shown in Figure 2, include labeling of the antibody and hapten with a luminescent quantum dot (the donor) and a gold nanoparticle (the quencher), respectively, and the reverse configuration. These assemblies yield either fluorescent antibody (Figure 2A) or hapten (Figure 2B) products.
[00234] To construct or assemble the nanoprobe, antibodies specific for anthrax toxin (PA 211 14B7), cancer epitopes, and phosphopeptides can be purchased where available or produced using hybridoma technology. Experimentally, covalent binding of the nanoparticles (or organic donors and quenchers) to the antibody and hapten is accomplished using maleimide activated Qdots® (Quantum Dot Corp.), lysine reactive Nanogold® labeling reagents (Nanoprobes Corp.), or amine-reactive organic donors or quenchers (Molecular Probes). The binding sites of the labeled antibody are then loaded with the labeled hapten, thus forming the fluorescently quenched nanosensor. Target detection is then accomplished using a competitive binding assay between the free target analyte and the quenched hapten- antibody-gold nanoprobe.
Other Embodiments
[00235] While the above specification contains many specifics, these should not be construed as limitations on the scope of the invention, but rather as examples of preferred embodiments thereof. Many other variations are possible. Accordingly, the scope of the invention should be determined not by the embodiments illustrated, but by the appended claims and their legal equivalents.

Claims

What is claimed is:
1. A nanoprobe for detection of molecular interactions comprising: a nanoparticle and a detectably labeled biomolecule attached thereto.
2. The nanoprobe of claim 1, wherein the nanoparticle is an inorganic or organic nanoparticle.
3. The nanoprobe of claim 1, wherein the nanoparticle is a metal.
4. The nanoprobe of claim 3, wherein the metal is selected from the group consisting of: gold, silver, copper and platinum.
5. The nanoprobe of claim 2, wherein the nanoparticle is selected from the group consisting of : ZnS, ZnO, TiO2, AgI, AgBr, HgI2, PbS, PbSe, ZnTe, CdTe, In2S3, In2S3, Cd3P2, Cd3As2, InAs, and GaAs.
6. The nanoprobe of claim 1, wherein the nanoparticle is about 5 nm up to 100 nm in diameter.
7. The nanoprobe of claim 1, wherein the nanoparticle is about 5 nm to about 20 nm in diameter.
8. The nanoprobe of claim 1, wherein a plurality of detectably labeled biomolecules are attached to the nanoparticle.
9. The nanoprobe of claim 1, wherein the biomolecule is an antibody, aptamer, hapten, oligonucleotide, protein or peptide.
10. The nanoprobe of claim 1 , wherein the biomolecule is labeled with a detectable moiety.
11. The nanoprobe of claim 10, wherein the detectable moiety is a fluorescent molecule.
12. The nanoprobe of claim 10, wherein the detectable moiety comprises at least one of: biotin, luorescein (5-carboxyfluorescein-N-hydroxysuccinimide ester), rhodamine (5,6-tetramethyl rhodamine), Cy3, Cy3.5, Cy5, Cy5.5 and Cy7. 6-carboxyfluorescein (6- FAM), 2',4',1 A-tetrachlorofluorescein (TET), 2',4I,5I,7',l,4-hexachlorofluorescein (HEX), 2',7t-dimethoxy-4',5l-dichloro-6-carboxyrhodamine (JOE), 2'-chloro-5'-fluoro-7',8'-fused phenyl- 1 ^-dichloro-ό-carboxyfluorescein (NED), 2'-chloro-7'-phenyl- 1 ,4-dichloro-6- carboxyfluorescein (VIC), fluorescein isothiocyanate (FITC), 5,6-carboxymethyl fluorescein, Texas red, nitrobenz-2-oxa-l,3-diazol-4-yl (NBD), coumarin, dansyl chloride, rhodamine, amino-methyl coumarin (AMCA), Eosin, Erythrosin, BODIPY™, Cascade Blue™, Oregon Green™, pyrene, lissamine, xanthenes, acridines, oxazines, phycoerythrin, quantum dye™, fluorescent energy transfer dyes, or thiazole orange-ethidium heterodimer.
13. The nanoprobe of claim 1 , wherein the biomolecules are covalently attached to the nanoparticle.
14. The nanoprobe of claim 1, wherein the biomolecules are attached to the nanoparticles maleimide linkages, carbodiimide linkages, lysine linkages, amine-reactive organic donors or quenchers.
15. The nanoprobe of claim 1 , wherein the biomolecule comprises first molecule bound to a second molecule.
16. The nanoprobe of claim 1, wherein the first molecule is labeled with a fluorescent moiety and the second molecule is labeled with a quenching moiety.
17. The nanoprobe of claim 16, wherein the first molecule and second molecule do not emit a fluorescent signal when the first molecule and second molecule are associated together.
18. The nanoprobe of claim 16, wherein dissociation of the second molecule from the first molecule results in emission of fluorescence.
19. The nanoprobe of claim 18, wherein the first molecule remains bound to the nanoparticle.
20. The nanoprobe of claim 16, wherein dissociation of the first and second molecule occurs when an analyte contacts the nanoprobe.
21. The nanoprobe of claim 20, wherein the first molecule specifically binds the analyte.
22. A method of detecting specific molecular interactions comprising: providing a nanoprobe comprising a differentially labeled antibody-hapten complex; contacting the nanoprobe with an analyte, binding of the analyte to the antibody releases the labeled hapten; resulting in a change in fluorescence; and, detecting specific molecular interactions between the nanoprobe and analyte.
23. The method of claim 22, wherein the antibody is labeled with a fluorophore and the hapten is labeled with a fluorophore quenching molecule.
24. The method of claim 22, wherein the antibody is labeled with a fluorophore quenching molecule and the hapten is labeled with a fluorophore.
25. The method of claim 22, wherein the nanoprobe is in a quenched fluorescent state as compared to fluorescence emitted when the hapten is released.
26. The method of claim 22, wherein binding of the analyte to a detectably labeled antibody is detected by fluorescence as compared to a baseline fluorescence of nanoprobe.
27. The method of claim 26, wherein binding of an analyte to the detectably labeled antibody is detected by displacement of the hapten as measured by increase in fluorescence as compared to the baseline fluorescence of nanoprobe.
28. The method of claim 22, wherein absence of binding between the analyte and the nanoprobe is detected by no increase in fluorescence as compared to binding of a control analyte to the nanoprobe.
29. The method of claim 22, wherein specificity of antibody for an analyte is a measure of fluorescence.
30. The method of claim 22, wherein antibody-analyte binding is determined by affinity of the antibody for the analyte and dissociation constants (KD) between an antibody- hapten complexes and antibody-analyte complexes.
31. The method of claim 23, wherein the KD of antibody-hapten is less than the KD of antibody-analyte complexes.
32. The method of claim 22, wherein the hapten comprises protein, peptide, oligonucleotide or organic molecule.
33. The method of claim 22, wherein a fluorescent molecule comprises at least one of: biotin, luorescein (5-carboxyfluorescein-N-hydroxysuccinimide ester), rhodamine (5,6-tetramethyl rhodamine), Cy3, Cy3.5, Cy5, Cy5.5 and Cy7. 6-carboxyfluorescein (6- FAM), 2',4',1,4,-tetrachlorofluorescein (TET), 2',4l,5I,7',l,4-hexachlorofluorescein (HEX), 2',7'-dimethoxy-4',5'-dichloro-6-carboxyrhodamine (JOE), 2'-chloro-5'-fluoro-7',8'-fused phenyl- 1 ^-dichloro-ό-carboxyfiuorescein (NED), 2'-chloro-7'-phenyl- 1 ,4-dichloro-6- carboxyfluorescein (VIC), fluorescein isothiocyanate (FITC), 5,6-carboxymethyl fluorescein, Texas red, nitrobenz-2-oxa-l,3-diazol-4-yl (NBD), coumarin, dansyl chloride, rhodamine, amino-methyl coumarin (AMCA), Eosin, Erythrosin, BODIPY™, Cascade Blue™, Oregon Green™, pyrene, lissamine, xanthenes, acridines, oxazines, phycoerythrin, quantum dye™, fluorescent energy transfer dyes, or thiazole orange-ethidium heterodimer.
34. The method of claim 22, wherein a quencher molecule comprises at least one of: rhodamine dyes, tetramethyl-6-carboxyrhodamine (TAMRA), tetrapropano-6- carboxyrhodamine (ROX), DABSYL, DABCYL, cyanine dyes, nitrothiazole blue (NTB), anthraquinone, malachite green, nitrothiazole, or nitroimidazole compounds.
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