WO2006125050A2 - Biobarcode assays for ultra high sensitive detection - Google Patents

Abstract

The invention provides an extremely sensitive, reliable, and adaptable detection method of chemical detection through signal amplification from a reporter reagent capable of selectively binding to the target chemical of interest. The invention utilizes a detection reagent containing a streptavidin-biotin complex or a particle that has been derivatized to include on its surface selective binding compounds that specifically bind to the target chemical species and a large number of copies of a pre-selected reporter moiety.

Description

BIOBARCODE ASSAYS FOR ULTRA HIGH SENSITIVE DETECTION

This application claims priority to U.S. provisional patent applications, Serial Nos. 60/681,74O₅ filed May 17, 2005, and 60/682,160, filed May 18, 2005, the disclosures of each of which are explicitly incorporated by reference herein.

FIELD OF THE INVENTION

The invention resides in the field of chemical detection and specifically in methods and materials useful for the amplification of signals generated by the detection of chemicals including, but not limited to, biological molecules, present at low concentrations.

BACKGROUND OF THE INVENTION

Every biological entity (e.g. viruses, bacteria, human cells) carries with it signature chemicals such as proteins and nucleic acid sequences that can serve as specific targets for detection. Ideally, detection platforms for such molecules should be general so that only minor target-specific changes in the assay need to be made for each analyte.

In the detection of specific nucleic acid molecules, the gold standards in sequence- specific detection are the polymerase chain reaction (PCR) and molecular fluorophore probe technology. PCR is an extraordinarily powerful technique. However, successful amplification of target nucleic acid sequences is hampered by: 1) complexity, 2) cost, 3) major multiplexing challenges and 4) high susceptibility to contamination. For these reasons, point-of-care, general, reliable and cost effective PCR testing (e.g. doctor's offices, battlefield, first-responder sites) may never be realized. While molecular fluorophores are useful for labeling reactions, they exhibit broad absorption and emission bands, require expensive equipment for readout and are quite susceptible to photobleaching.

For protein targets, the enzyme-linked immunosorbent assay (ELISA) is the standard detection technique. The ELISA is an extremely general technique which relies on target-specific antibody labeling and colorimetric readout based either on fluorophores or chromophores. Unfortunately, current assay sensitivities for proteins (pM ranges generally) are nowhere near the sensitivities achieved with PCR for nucleic acids (often fewer than ten copies). Therefore, there is much room to improve upon the sensitivity of protein detection. ELISAs also require fairly expensive colorimetric readout instruments. Additionally, as noted above, ELISA assays based on fluorophore reporters suffer from problems of nonspecific detection, cost and photocatalyzed deterioration.

A successful alternative to these chemical detection assays that has recently been reported is the Bio Bar Code Assay as disclosed in U.S. serial no. 10/877,750, filed June 25, 2004 and U.S. serial no. 11/127,808, filed May 12, 2005, which are incorporated by reference in their entirety. This is a nanoparticle-based approach to the detection of protein and DNA targets (Nam JM, Thaxton CS, Mirkin CA Nanoparticle-based bio-bar codes for the ultrasensitive detection of proteins, Science 301 (5641):1884-1886 Sept. 26, 2003; Nam JM, Stoeva SI, Mirkin CA Bio-bar-code-based DNA detection with PCR-like sensitivity, J. Am. Chem. Soc. 126 (19):5932-5933 May 19, 2004.) The biobarcode assay takes advantage of two target-seeking probes. First, a magnetic probe, surface- functionalized with the appropriate recognition element (monoclonal Ab for proteins and a complementary DNA oligomer for nucleic acid targets) captures the target analyte present in a small detection volume where the recognition elements far outnumber the target analyte. The magnetic particles make washing to remove unbound and non- specifically bound portions in the mixture simple and highly efficient. Next, gold nanoparticles with the appropriate surface-bound recognition element (poly- or monoclonal antibodies for proteins and a non-overlapping complementary DNA oligomer for nucleic acid targets) are added to the magnetic-target analyte hybrid structures. Recognition of the hybrid structures by the gold nanoparticles results in the formation of a "sandwich" structure. Importantly, in addition to the target analyte recognition elements, the gold nanoparticles also carry with them surface-bound DNA oligomers that are hybridized to their anti-parallel complements by DNA base pairing. The complement sequence, referred to as the "biobarcode," has a sequence that has been chosen to serve as a surrogate for the target of detection. As each gold nanoparticle carries with it hundreds to thousands of bio-bar-code strands, there is a huge amplification of the detection signal for each sandwiched target. The bio-bar-code is easily released from the nanoparticle surface in the last step of the assay and further amplified and detected using conventional DNA detection techniques. The biobarcode approach has advantageous sensitivity with regard to detecting protein targets (aM sensitivities versus the typical pM sensitivities of ELISA). Further, the use of biobarcode assays has been demonstrated to be as sensitive for DNA targets as PCR, without the need for enzymatic amplification of the target sequence. The assay allows one to identify protein markers down to the low attomolar (about 20 copies in a 10 ul sample) concentration limit. Because non-specific nanoparticle binding to magnetic microparticle (MMP) probes may affect the level of signal/noise or signal/background, one significant advance would be to separate the recognition moiety from the nanoparticles. Alternatively, another significant advance would be to improve the design of the target-seeking nanoparticle probe to reduce non-specific binding to the MMP probes, thus improving the sensitivity of the biobarcode assay. The subsequently released biobarcodes can then be detected by nanoparticle-based and conventional, e.g., fluorophore, detection methods.

SUMMARY OF THE INVENTION

The invention provides a detection reagent comprising a reporter complex and at least two reporter moieties, wherein the reporter complex comprises a streptavidin-biotin complex and selective binding compounds specific to a target analyte, and wherein the reporter moieties are biotinylated biobarcodes. In one aspect, the biobarcodes are duplex nucleic acid molecules having at least one 3' or 5' end labeled with biotin, and having at least one portion capable of binding to a plurality of other duplexed nucleic acid molecules.

The invention also provides a detection reagent comprising a reporter complex and at least two reporter moieties, wherein the reporter complex comprises selective binding compounds specific to a target analyte and a nanoparticle coated with streptavidin, and wherein the reporter moieties are biotinylated biobarcodes. In one aspect, the biobarcodes are duplex nucleic acid molecules having at least one 3' or 5' end labeled with biotin, and having at least one portion capable of binding to a plurality of other duplexed nucleic acid molecules. In addition, the invention provides a detection reagent comprising a reporter complex and at least two reporter moieties, wherein the reporter complex comprises a plurality of streptavidin-biotin complexes and selective binding compounds specific to a target analyte, and wherein the reporter moieties are double-biotinylated biobarcodes.

The invention further provides a detection reagent comprising a reporter complex and at least two reporter moieties, wherein the reporter complex comprises selective binding compounds specific to a target analyte and a plurality of nanoparticles coated with streptavidin, and wherein the reporter moieties are double-biotinylated biobarcodes.

The invention also provides a detection reagent comprising a reporter complex and at least two reporter moieties, wherein the reporter complex comprises streptavidin, selective binding compounds specific to a target analyte, and a nanoparticle coated with biotinylated biobarcodes, and wherein the reporter moieties are biotinylated biobarcodes.

Furthermore, the invention provides a detection reagent comprising a reporter complex and at least two reporter moieties, wherein the reporter complex comprises a nanoparticle coated with streptavidin, selective binding compounds specific to a target analyte, and a nanoparticle coated with biotinylated biobarcodes, and wherein the reporter moieties are biotinylated biobarcodes.

The invention also provides a detection reagent comprising a reporter complex and at least two reporter moieties, wherein the reporter complex comprises streptavidin, selective binding compounds specific to a target analyte, double biotinylated linkers, and at least one nanoparticle coated with biobarcodes capable of binding to the linkers.

The invention further provides a detection reagent comprising a reporter complex and at least two reporter moieties, wherein the reporter complex comprises a nanoparticle coated with streptavidin, selective binding compounds specific to a target analyte, double biotinylated linkers, and at least one nanoparticle coated with biobarcodes capable of binding to the linkers.

In one aspect, the selective binding compound of a detection reagent of the invention is a protein, an oligonucleotide, or an inorganic compound. For example, when the selective binding compound is a protein, the protein can be an enzyme or an antibody. In another aspect, the reporter moieties can comprise fluorophores, chromophores, oligonucleotides with or without attached fluorophores or chromophores, proteins, porphyrins, lipids, carbohydrates, synthetic polymers, isotopic tags, or radioactive tags.

The invention also provides methods for detecting at least one target analyte in a sample, comprising the steps of: (a) providing at least one capture probe, said capture probe comprising at least one binding complement specific to the target analyte; (b) providing at least one detection reagent according to any of claims 1-10; (c) incubating the capture probe with the target analyte and the detection reagent under conditions effective to allow complex formation between the capture probe, the target analyte, and the detection reagent; (d) separating the complex from any unbound detection reagent; (e) selectively releasing at least a portion of the reporter moieties from the complex; and (f) analyzing the presence or absence of the reporter moieties, wherein the presence or absence of reporter moieties is indicative of the presence or absence of the target analyte.

In one aspect, the capture probe can be immobilized on an insoluble material.

In another aspect, the capture probe can comprise a magnetic particle, and the complex can be separated from any unbound detection reagent by the application of a magnetic field.

In a particular aspect, reporter moieties can be selectively released from the complex by dehybridization. In yet another aspect, biotinylated intermediate oligonucleotides capable of binding to the target analyte are provided in the methods of the invention, and the biotinylated intermediate oligonucleotides can form a complex with the detection reagent.

Specific preferred embodiments of the invention will become evident from the following more detailed description of certain preferred embodiments and the claims.

DESCRIPTION OF THE DRAWINGS

Figure 1. Streptavidin and biotinylated barcode for signal enhancement in biobarcode assay. The detection reagent shown is a streptavidin-biotin complex including streptavidin, a biotinylated antibody, and biotinylated DNA barcodes. In the presence of a target analyte, the capture probe, e.g., a magnetic bead, and the detection reagent form a complex. The complex is then subjected to conditions that result in the release of the barcodes. The barcodes may be detected by any suitable means including a nanoparticle- based detection system on an array plate shown in the figure. Figure 2. Streptavidin coated nanoparticles for signal enhancement in the biobarcode assay. The detection reagent shown is a streptavidin-coated reporter particle including streptavidin-coated nanoparticle, biotinylated antibodies, and biotinylated DNA barcodes. In the presence of a target analyte, the capture probe, e.g., a magnetic bead, and the detection reagent form a complex. The complex is then subjected to conditions that result in the release of the barcodes. The barcodes may be detected by any suitable means including a nanoparticle-based detection system on an array plate shown in the figure.

Figure 3. Streptavidin and biotinylated barcode complex formation for signal enhancement in biobarcode assay. The detection reagent shown is a streptavidin-coated reporter particle including streptavidin-coated nanoparticle, biotinylated antibodies, strepavidin and double biotinylated DNA barcodes. In the presence of a target analyte, the capture probe, e.g., a magnetic bead, and the detection reagent form a complex. The complex is then subjected to conditions that result in the release of the barcodes. The barcodes may be detected by any suitable means including a nanoparticle-based detection system on an array plate shown in the figure.

Figure 4. Streptavidin coated nanoparticles and biotinylated barcode complex formation for signal enhancement in biobarcode assay. The detection reagent shown is a streptavidin-coated reporter particle including a reporter particle complex comprised of streptavidin-coated nanoparticles, biotinylated antibodies, and double biotinylated DNA barcodes. In the presence of a target analyte, the capture probe, e.g., a magnetic bead, and the detection reagent form a complex. The complex is then subjected to conditions that result in the release of the barcodes. The barcodes may be detected by any suitable means including a nanoparticle-based detection system on an array plate shown in the figure.

Figure 5. Streptavidin coated nanoparticles and biotinylated barcode complex formation for signal enhancement in biobarcode assay. The detection reagent shown is a streptavidin-coated reporter particle including a streptavidin-coated nanoparticle, biotinylated antibodies, and biotinylated DNA barcodes. The DNA barcodes shown are complexes of at least one duplex nucleic acid molecules having at least one 3' or 5' end labeled with a biotin to enable binding to the strepavidin. The duplex nucleic acid molecules have at least one, preferably three portions, of the sequence available to bind to a plurality of other duplexed nucleic acid molecules. In the presence of a target analyte, the capture probe, e.g., a magnetic bead, and the detection reagent form a complex. The complex is then subjected to conditions that result in the release of the barcodes. The barcodes may be detected by any suitable means including a nanoparticle-based detection system on an array plate shown in the figure.

Figure 6. Streptavidin and biotinylated barcode complex formation for signal enhancement in biobarcode assay. The detection reagent shown is a streptavidin biotin complex including a biotinylated antibody and biotinylated DNA barcodes. The DNA barcodes shown are complexes of at least one duplex nucleic acid molecules having at least one 3' or 5' end labeled with a biotin to enable binding to the strepavidin. The duplex nucleic acid molecules have at least one, preferably three portions, of the sequence available to bind to a plurality of other duplexed nucleic acid molecues. In the presence of a target analyte, the capture probe, e.g., a magnetic bead, and the detection reagent form a complex. The complex is then subjected to conditions that result in the release of the barcodes. The barcodes may be detected by any suitable means including a nanoparticle- based detection system on an array plate shown in the figure.

Figure 7. Streptavidin and biotinylated barcode nanoparticles for signal enhancement in biobarcode assay. The detection reagent shown is a streptavidin-coated reporter particle including streptavidin-coated nanoparticle, biotinylated antibodies, streptavidin for binding the biotinylated antibodies, and biotinylated DNA barcodes. In the presence of a target analyte, the capture probe, e.g., a magnetic bead, and the detection reagent form a complex. The complex is then subjected to conditions that result in the release of the barcodes. The barcodes may be detected by any suitable means including a nanoparticle-based detection system on an array plate shown in the figure.

Figure 8. Streptavidin coated nanoparticles and biotinylated barcode nanoparticles for signal enhancement in the biobarcode assay. The detection reagent shown includes a first streptavidin-coated nanoparticle bound to biotinylated antibodies and a second streptavidin-coated nanoparticle bound to biotinylated DNA barcodes. In the presence of a target analyte, the capture probe, e.g., a magnetic bead, and the detection reagent form a complex. The complex is then subjected to conditions that result in the release of the barcodes. The barcodes may be detected by any suitable means including a nanoparticle-based detection system on an array plate shown in the figure.

Figure 9. Streptavidin, biotinylated barcode complex, and barcode coated nanoparticles for signal enhancement in the barcode assay. The detection reagent shown includes (i) a streptavidin-biotin complex including streptavidin complexed to biotinylated antibodies, streptavidin, and double biotinylated linkers and (H) nanoparticles having biobarcodes bound thereto either directly or indirectly. In the presence of a target analyte, the capture probe, e.g., a magnetic bead, and the detection reagent form a complex. The complex is then subjected to conditions that result in the release of the barcodes. The barcodes may be detected by any suitable means including a nanoparticle- based detection system on an array plate shown in the figure. Figure 10. Streptavidin coated nanoparticles and barcode coated nanoparticles for signal enhancement in biobarcode assay. The detection reagent shown includes (i) an aggregate of streptavidin-coated nanoparticles held together with double biotinylated linkers, e.g., oligonucleotides, and (ii) nanoparticles having biobarcodes bound thereto either directly or indirectly. In the presence of a target analyte, the capture probe, e.g., a magnetic bead, and the detection reagent form a complex. The complex is then subjected to conditions that result in the release of the barcodes. The barcodes may be detected by any suitable means including a nanoparticle-based detection system on an array plate shown in the figure.

Figure 11. Streptavidin coated nanoparticles for nucleic acid detection in biobarcode assay. The detection reagent includes a streptavidin-coated nanoparticle bound to biotinylated biocodes. In the presence of a target nucleic acid, the capture probe, e.g., a magnetic bead, and the detection reagent form a complex. The complex is then subjected to conditions that result in the release of the barcodes. The barcodes may be detected by any suitable means including a nanoparticle-based detection system on an array plate shown in the figure.

Figure 12. PSA detection with the biobarcode assay. In this example, a magnetic bead having PSA-specific mAb bound thereto was with mixed with a sample having PSA (prostate specific antigen). Thereafter, a biotin-labeled mAb was then added, followed by addition of streptavidin coated 15nm gold-nanoparticles. The resulting complex was washed and followed by addition of biotin-labeled biobarcodes. The resulting complex was washed and subject to conditions resulting in the release of the biobarcodes. The biobarcodes was then detected using silver-enhanced nanoparticle-based detection on an array plate.

DESCRIPTION OF THE INVENTION

Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

As utilized in accordance with the present disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings:

An "insoluble substrate" used in a method of the invention can be any surface capable of having analytes bound thereto. Such surfaces include, but are not limited to, glass, metal, plastic, or materials coated with a functional group designed for binding of analytes. The coating may be thicker than a monomolecular layer; in fact, the coating could involve porous materials of sufficient thickness to generate a porous 3-dimensional structure into which the analytes can diffuse and bind to the internal surfaces.

The term "capture probe" refers to any antibody, oligonucleotide, lectin or similar material that is capable of selectively and specifically binding to the target species of interest. Target analytes such as proteins, polypeptides, fragments, variants, and derivatives may be used to prepare antibodies using methods known in the art. Antibodies may be polyclonal, monospecific polyclonal, monoclonal, recombinant, chimeric, humanized, fully human, single chain and/or bispecific.

Polyclonal antibodies directed toward a target analyte generally are raised in animals (e.g., rabbits or mice) by multiple subcutaneous or intraperitoneal injections of JNK activating phosphatase polypeptide and an adjuvant. It may be useful to conjugate an target analyte protein, polypeptide, or a variant, fragment or derivative thereof to a carrier protein that is immunogenic in the species to be immunized, such as keyhole limpet heocyanin, serum, albumin, bovine thyroglobulin, or soybean trypsin inhibitor. Also, aggregating agents such as alum are used to enhance the immune response. After immunization, the animals are bled and the serum is assayed for anti-target analyte antibody titer.

Monoclonal antibodies directed toward target analytes are produced using any method that provides for the production of antibody molecules by continuous cell lines in culture. Examples of suitable methods for preparing monoclonal antibodies include hybridoma methods of Kohler, et al., Nature 256:495-97 (1975), and the human B-cell hybridoma method, Kozbor, J. Immunol. 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications 51-63 (Marcel Dekker 1987).

The term "oligonucleotide" referred to herein includes naturally occurring, and modified nucleotides linked together by naturally occurring, and/or non-naturally occurring oligonucleotide linkages. Oligonucleotides are a polynucleotide subset comprising members that are generally single-stranded and have a length of 200 bases or fewer. In certain embodiments, oligonucleotides are 10 to 60 bases in length. In certain embodiments, oligonucleotides are 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 to 40 bases in length. Oligonucleotides may be single stranded or double stranded, e.g. for use in the construction of a gene mutant. Oligonucleotides of the invention may be sense or antisense oligonucleotides with reference to a protein-coding sequence.

The term "naturally occurring nucleotides" includes deoxyribonucleotides and ribonucleotides. The term "modified nucleotides" includes nucleotides with modified or substituted sugar groups and the like. The term "oligonucleotide linkages" includes oligonucleotide linkages such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate, phosphoroamidate, and the like. See, e.g., LaPlanche et al, 1986, Nucl. Acids Res., 14:9081; Stec et al, 1984, J. Am. Chem. Soc, 106:6077; Stein et al, 1988, Nucl Acids Res., 16:3209; Zon et al, 1991, Anti-Cancer Drug Design, 6:539; Zon et al, 1991, OLIGONUCLEOTIDES AND ANALOGUES: A PRACTICAL APPROACH, pp. 87- 108 (F. Eckstein, Ed.), Oxford University Press, Oxford England; Stec et al, U.S. Pat. No. 5,151,510; Uhlniann and Peyman, 1990, Chemical Reviews, 90:543, the disclosures of which are hereby incorporated by reference for any purpose. An oligonucleotide can include a detectable label to enable detection of the oligonucleotide or hybridization thereof.

The term "aptamers" refers to nucleic acids (typically DNA, RNA or oligonucleotides) that emerge from in vitro selections or other types of aptamer selection procedures well known in the art (e.g. bead-based selection with flow cytometry or high density aptamer arrays) when the nucleic acid is added to mixtures of molecules. Ligands that bind aptamers include but are not limited to small molecules, peptides, proteins, carbohydrates, hormones, sugar, metabolic byproducts, cofactors, drugs and toxins. Aptamers of the invention are preferably specific for a particular analyte. Aptamers can have diagnostic, target validation and therapeutic applications. The specificity of the binding is defined in terms of the dissociation constant Kd of the aptamer for its ligand. Aptamers can have high affinity with Kd range similar to antibody (pM to nM) and specificity similar/superior to antibody (Tuerk and Gold, 1990, Science, 249:505; Ellington and Szostak, 1990, Nature 346:818). An aptamer will typically be between 10 and 300 nucleotides in length. RNAs and DNAs aptamers can be generated from in vitro selection experiments such as SELEX (Systematic Evolution of Ligands by Exponential Enrichment). Examples of aptamer uses and technology are PhotoSELEX™ and Riboreporters™. Aptamers, their uses, and manufacture are described, for example, in U.S. Patent Nos. 5,840,867, 6,001,648, 6225,058, 6,207,388 and U.S. Patent publication 20020001810, the disclosures of all of which are incorporated by reference in their entireties.

Aptamers configured to bind to specific target analytes can be selected, for example, by synthesizing an initial heterogeneous population of oligonucleotides, and then selecting oligonucleotides within the population that bind tightly to a particular target analyte. Once an aptamer that binds to a particular target molecule has been identified, it can be replicated using a variety of techniques known in biological and other arts, for example, by cloning and polymerase chain reaction (PCR) amplification followed by transcription.

The term "analyte" refers to a substance to be detected or assayed by the method of the invention. Typical analytes may include, but are not limited to proteins, peptides, nucleic acid segments, molecules, cells, microorganisms and fragments and products thereof, or any substance for which attachment sites, binding members or receptors (such as antibodies) can be developed. The analytes have at least one binding site, preferably at least two binding sites, e.g., epitopes, that can be targeted by a capture probe and a detection probe, e.g. antibodies or aptamers or both.

"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, In₂ S₃, In₂ Se₃, Cd₃ P₂, Cd₃ As₂, InAs, and GaAs. The size of the nanoparticles is preferably from about 5 nm to about 150 nm (mean diameter), more preferably from about 5 to about 50 nm, most preferably from about 10 to about 30 nm. The nanoparticles may also be rods. Other nanoparticles useful in the invention include silica and polymer (e.g. latex) nanoparticles.

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 Taransactions 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). Methods of making silica nanoparticles impregnated with fluorophores or phosphors are also well known in the art (see Tan and coworkers, PNAS, 2004, 101, 15027 - 15032). Methods of making ZnS, ZnO, TiO₂, AgI, AgBr, HgI₂, PbS, PbSe, ZnTe, CdTe,

In₂ S₃, In₂ Se₃, Cd₃ P₂, Cd₃ As₂, 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).

As used herein, the terms "label" or "detection label" refers to a detectable marker that may be detected by photonic, electronic, opto-electronic, magnetic, gravity, acoustic, enzymatic, or other physical or chemical means. The term "labeled" refers to incorporation of such a detectable marker (e.g. by incorporation of a radiolabeled nucleotide or attachment to a reporter moiety).

A "sample" as used herein refers to any quantity of a substance that comprises potential target analytes and that can be used in a method of the invention. For example, the sample can be a biological sample or can be extracted from a biological sample derived from humans, animals, plants, fungi, yeast, bacteria, viruses, tissue cultures or viral cultures, or a combination of the above. They may contain or be extracted from solid tissues (e.g. bone marrow, lymph nodes, brain, skin), body fluids (e.g. serum, blood, urine, sputum, seminal or lymph fluids), skeletal tissues, or individual cells. Alternatively, the sample can comprise purified or partially purified nucleic acid molecules or proteins and, for example, buffers and/or reagents that are used to generate appropriate conditions for successfully performing a method of the invention.

The current invention overcomes many of the problems of the prior art while greatly expanding the flexibility, adaptability and usefulness of techniques directed to the amplification of a signal to facilitate detection. In particular, the invention provides detection reagents for accomplishing amplification of a signal.

In certain embodiments, a detection reagent of the invention comprises a reporter complex and at least two reporter moieties. In one embodiment, the invention utilizes novel detection reagents containing a non-nanoparticle reporter (e.g. streptavidin), having some number of selective binding compounds (e.g. biotinylated antibodies), that specifically bind to a pre-selected target chemical species (also referred to herein as "target analytes") and a large number of copies of a pre-selected reporter moiety (e.g. biobarcodes). In one aspect, the biobarcodes may be biotinylated and attached directly to streptavidin. In another aspect, the biobarcodes may be indirectly attached to streptavidin by being hybridized to a complementary biotinylated oligonucleotide, which in turn is attached to the strepavidin. In yet another aspect, the biobarcodes may be optionally labeled (e.g. with fluorophores), to allow detection in solution. In another embodiment, the invention utilizes novel detection reagents containing a reporter complex comprising a streptavidin-coated reporter particle (e.g. a nanoparticle) having some number of selective binding compounds (e.g. biotinylated antibodies), that specifically bind a pre-selected target chemical species and a large number of copies of a pre-selected reporter moiety (e.g. biobarcodes). In one aspect, the biobarcodes may be biotinylated and attached directly to the streptavidin-coated particles. In another aspect, the biobarcodes may be indirectly attached to the streptavidin-coated particles by being hybridized to a complementary biotinylated oligonucleotide, which in turn is attached to the strepavidin coated nanoparticles. The biobarcodes may be optionally labeled (e.g. with fluorophores), to allow detection in solution. In one embodiment of the invention, the detection reagent of the present invention is composed of a reporter complex comprising streptavidin that has been complexed with biotinylated selective binding compounds that specifically bind to the target chemical or target analyte and at least two, and preferably three, of pre-selected reporter moieties. In one aspect, the pre-selected reporter moieties include biotinylated biobarcodes. In another aspect, the pre-selected reporter moieties include biotinylated oligonucleotides that are hybridized to a biobarcode.

In another embodiment of the invention, the detection reagent of the present invention is composed of a reporter complex comprising a particle of a convenient size that has been derivatized to include on its surface selective binding compounds that specifically bind to the target chemical or target analyte and at least two, and preferably a large number, of pre-selected reporter moieties.

The selective binding compound may be any compound capable of selectively recognizing and binding to the target analyte without interfering with the binding between the target analyte and a capture probe to which the target analyte may be attached as described herein. Examples of suitable selective binding compounds include, but are not limited to, antibodies, enzymes, proteins, oligonucleotides and inorganic compounds.

A particle in a reporter complex can be any material that is compatible with the sample containing the target analyte and capable of binding both the selective binding compounds and the reporter moieties. Examples of suitable particles for use in a reporter complex include, but are not limited to, metals, silica, silicon-oxide, and polystyrene. In one aspect, the particle may be a gold nanoparticle coated with streptavidin and the selective binding compounds may be biotinylated antibodies. Suitable, but non-limiting examples of nanoparticles include those described U.S. Patent No. 6,506,564; International Patent Application No. PCT/US02/16382; U.S. Patent Application Serial No. 10/431,341 filed May 7, 2003; and International Patent Application No. PCT/US03/14100; all of which are hereby incorporated by reference in their entirety.

The particles are selected for a functional size typically having a diameter in the nanometer to micrometer range. As the ratio of the numbers of reporter moieties and selective binding moieties initially bound to a reporter particle can be established at greater than one during preparation of the detection reagent of the present invention, release of the reporter moieties from a particle will result in more reporter moieties entering the medium than there are analyte molecules bound to the reporter complex. This ratio establishes the amplification of the signal from the detection of a target analyte molecule. For example, the release of the reporter moieties from one reporter particle bearing 1000 copies of the reporter moiety that is bound to one molecule of immobilized analyte will result in 1000 molecules of reporter moiety appearing in the medium for each molecule of analyte in the original sandwich. This results in the chemical signal represented by the target analyte being amplified by a factor of 1000. This amplification can be adjusted during the synthesis of the detection reagent by manipulating parameters such as the surface area of the reporter particle and the ratio between and the packing densities of the selective binding and reporter moieties on the surface of the reporter particle. Thus, the size of the reporter particle dictates the number of reporter moieties that can be released, and the ultimate amplification factor that is obtained with regard to labeled target molecules. For example, 13 nm strgold particles can carry numerous biotinylated surface oligomers hybridized to biobarcodes serving as reporter moieties thereby achieving a significant amplification signal resulting from a single target molecule bound by a single reporter particle in the sandwich formed in the detection assay. Larger size (e.g. micron-sized) particles will obviously lead to larger amplification factors.

A reporter moiety may be attached to the surface of a particle of a reporter complex by a means sufficiently strong enough to prevent significant non-specific release of the reporter moiety during the steps of the detection method but simultaneously susceptible to separation and release of the reporter moiety immediately prior to the detection step. Thus, the reporter moiety may be attached to the surface of the reporter particle directly through a biotin-strepavidin binding interaction that can be disrupted prior to the detection step. Alternatively, the reporter moiety may be attached to the surface of the reporter particle indirectly through nucleic acid hybridization interaction and release via dehybridization prior to the detection step. Also, biobarcodes in biotinylated form may be directly bound to a strepavidin-coated nanoparticle. Alternatively, biobarcodes may be indirectly bound to the strepavidin-coated nanoparticle by hybridization to a complementary biotinylated oligonucleotide.

If desired, the reporter moieties may optionally include detection labels including, but not limited to, fluorophores, chromophores, oligonucleotides with or without attached fluorophores or chromophores, proteins including enzymes and porphyrins, lipids, carbohydrates, synthetic polymers and tags such as isotopic or radioactive tags.

Several detection reagents of the invention are illustrated, for example, in Figures 1-11 and are described in the Examples below. The invention also provides methods of using the detection reagents of the invention for detecting at least one target analyte in a sample, comprising the steps of: (a) providing at least one capture probe, said capture probe comprising at least one binding complement specific to the target analyte; (b) providing at least one detection reagent according to any of claims 1-10; (c) incubating the capture probe with the target analyte and the detection reagent under conditions effective to allow complex formation between the capture probe, the target analyte, and the detection reagent; (d) separating the complex from any unbound detection reagent; (e) selectively releasing at least a portion of the reporter moieties from the complex; and (f) analyzing the presence or absence of the reporter moieties, wherein the presence or absence of reporter moieties is indicative of the presence or absence of the target analyte.

In certain embodiments, the method is similar to that used in a sandwich immunoassay. In particular, the sample being analyzed is exposed to a capture probe capable of selectively and specifically binding to species of interest, the capture probe can be immobilized on an insoluble material. Any unbound materials are then separated from the immobilized analyte through standard means. Immobilized analyte is then exposed to the detection reagent of this invention. The detection reagent binds to the immobilized analyte through the selective binding moieties incorporated thereon. The "sandwich" structure thus formed (insoluble substrate - analyte - detection reagent) therefore effectively immobilizes the detection reagent on the insoluble substrate. Unbound detection reagent can be separated from this immobilized structure through standard methods. Amplification is performed by exposing the immobilized insoluble substrate - analyte - detection reagent sandwich to some means of separting the reporter moiety (e.g. biobarcode), from the reporter complex, resulting in the release of the reporter moieties into the medium. As the ratio of the numbers of reporter moieties and selective binding compounds initially in the reporter complex can be established at greater than one during preparation of the detection reagent, release of the reporter moieties from a reporter complex results in more reporter moieties entering the medium than there are target analyte molecules bound to the capture probe. Detection, and optionally quantitation, of the released reporter moieties can be performed using any method that is appropriate to the chemical nature of the reporter moiety. The significant amplification of the detected signal of the reporter moiety from the detection of individual target analyte molecules results in an extremely sensitive, reliable and adaptable chemical detection assay.

In the first step of the detection method of the present invention, the sample being analyzed for the presence of the target molecule is exposed to a capture probe (such as an antibody, oligonucleotide, lectin or similar material that is capable of selectively and specifically binding to the target species of interest). The capture probe can be immobilized on an insoluble material that is compatible with the assay chemistry and that it can readily be separated from the reaction medium. The immobilized capture probe can be constructed such that it specifically binds, captures, and immobilizes the analyte of interest, but preferably does not bind any other materials that may be present in the sample. Examples of the insoluble material suitable for use in the methods of the present invention include, but are not limited to, wells of a microtiter plate, a microparticle, fibrous or membrane filters, or other such insoluble materials. The preferred insoluble material is a magnetic particle.

The capture probe is preferably selected such that it binds to a different determinant on the analyte than does the selective binding compound component of the detection reagent. Any unbound materials are then separated from the immobilized target analyte by any suitable means including, for example, decantation, sedimentation, washing, centrifuging or combinations of these processes. The net result of this process is that the analyte of interest is present in a purified and concentrated state on the surface of the insoluble material.

In a subsequent step of the method of the present invention, the immobilized target analyte is exposed to the detection reagent of this invention. The selective binding compound in the detection reagent specifically binds to the target analyte forming a "sandwich" structure including the capture probe bound to the target analyte which is, in turn, bound to the detection reagent. This sandwich structure effectively immobilizes the detection reagent on the insoluble substrate, and any unbound detection reagent can be separated from this immobilized structure by any suitable methods such as decantation, sedimentation, washing, centrifuging or combinations of these processes as noted above.

In another step of the present method the signal from the binding and detection of the target analyte is amplified by exposing the capture probe-target analyte-detection reagent sandwich to conditions that can liberate the reporter moiety from the reporter particle. The liberated reporter moiety then enters the media surrounding the detection reagent bound to the target analyte as described in detail above.

The media containing the released reporter moiety may be analyzed for the presence of the released reporter moieties using any method that is appropriate to the chemical nature of the reporter moiety. For example, a fiuorescently-labeled reporter moiety may be detected and even quantitated by measurement of the fluorescence intensity or fluorescence depolarization of the medium, while the presence of a chemiluminescent-labeled reporter can be determined by measuring the luminescence that occurs upon addition of an appropriate trigger reagent. Oligonucleotide-based reporter moieties can further be amplified by the polymerase chain reaction or captured by complimentary oligonucleotides immobilized on an insoluble substrate and detected and quantitated using methods commonly used in conjunction with nucleic acid-based microarrays. Numerous other options including electrochemical, impedance, enzymatic and radioactivity detection are also available. In yet another embodiment, the invention provides a kit for detecting for one or more analytes in a sample, wherein the kit comprises at least one detection reagent of the invention.

The following examples are offered to illustrate, but not to limit, the invention. Figures 1-12 illustrate these examples.

Examples Example 1

As shown in Figure 1, this Example illustrates a basic core format for antigen detection sandwich assay with limited linear amplification. The formed sandwich MB-Ag- Ab:biotin is reacted with free streptavidin, which, once bound to the sandwich will further capture biotin labeled barcode oligos (with maximum of 3) that can be released for array detection.

Example 2 As shown in Figure 2, this Example illustrates a higher power of linear amplification by using streptavidin coated particles to tag the formed sandwich. The bound streptavidin coated nanoparticles or microparticles bind biotin labeled barcode oligos with much higher magnitude (maximum of 3 multiplied by total number of streptavidin per particle). All bound barcode oligos will then be released for array detection.

Example 3

As shown in Figure 3, this Example illustrates a simple improved format for exponential amplification. The formed sandwich MB-Ag-Ab:biotin are first reacted with free streptavidin. To further amplify signals, barcode oligos labeled with biotin at both ends (B — B) are introduced to the streptavidin bound sandwich and form bridges to capture additional levels of streptavidin. Excess B — B will fill the unoccupied binding sites on streptavidin and later be released for array detection.

Example 4

As shown in Figure 3, this Example illustrates a more complex version of Example 3 using streptavidin coated particles. The formed sandwich MB-Ag-Ab:biotin are first creacted with streptavidin coated particles. Barcode oligos labeled with biotin at both ends (B — B) are introduced to the Streptavidin particles bound sandwich and form bridges to capture additional levels of streptavidin particles. Excess B — B will fill the unoccupied binding sites on streptavidin and later be released for array detection.

Example 5

As shown in Figure 5, this Example shows a more complex version of Example 1 using preformed barcode oligo complexes that are bridged to the sandwich through biotin binding to the bound streptavidin. The formed sandwich MB-Ag-Ab:biotin captures free streptavidin through biotin (Ab-biotin) binding. A pair of barcode oligos (at least one end of one oligo is labeled with biotin) were designed with sequences that can form duplexes with the complementary oligo at each of the three sections as shown in the figure. The duplexes can grow infinitely and form a huge complex. Biotinylated barcode complexes bind to the sandwich through biotin binding to captured streptavidin and later released for detection by array hybridization. T he size of the complex determines the amplification power.

Example 6

As shown in Figure 6, this Example shows a higher power version of example 5 using preformed barcode oligo complexes that are bridged to the sandwich through biotin binding to the bound streptavidin coated particles. The same detection reagent design as shown in Example 5 is used here except that free streptavidin is replaced with streptavidin coated particles. A much higher amplification power is anticipated due to the multiple streptavidin coated on nano/micro particles.

Among the above examples, mix and match between free streptavidin and streptavidin coated particles can be applied to each single format for better assay performance. For Examples 5 and 6, multiple layers of streptavidin or streptavidin particles can be added if the size of the complex is limiting.

Examples 7 and 8 For Examples 7 and 8 as shown in Figures 7 and 8, Examples 1 and 2 can be modified by introducing biotinylated barcode conjugated nanoparticles in place of biotinylated single barcode oligos. Each captured target in sandwich form will be amplified proportionally to the number of arms per barcode nanoparticle as shown in Figures 7 and 8.

Examples 9 and 10

For Examples 9 and 10, Examples 3 and 4 can be further amplified by introducing barcode conjugated nanoparticles with sequences complementary to the B — B oligo bound to streptavidin (free or coated to nanoparticles) as shown in Figures 9 and 10.

Example 11

For Example 11, all above designed formats can be applied to nucleic acid targets detection. Capture oligo coated MB is used in place of the antibody coated MB. Denatured double stranded or single stranded nucleic acids targets (TG) are captured to MB through hybridization to the capture oligos. One or multiple biotinylated intermediate oligos (B-IO) are hybridized to targets (outside the sequences complementary to captures on the MB. The MB-TG-IO sandwich can be detected through the bridges of free streptavidin or streptavidin coated nanoparticles similar to Examples 1-10. In Figure 11, an example was made using streptavidin coated nanoparticles and simple biotinylated barcode see example 2) in signal amplification.

Example 12

In this Example, an experiment was carried out to detect Prostate Specific Antigen (PSA) by using the protein barcode assay as an example of the invention. To capture PSA target, lOμg of PSA antibody (BioDesign, Mab, a-PSA free form, Cat# M86806M, Lot# 21k31504, clone# 8A6) coated magnetic particle was incubated with the recombinant human Kallikrein 3 (rhPSA, R&D System cat# 1344-SE, lot# GDQO14071) in a 50μL volume of binding mixture containing IXPBS (Gibco, Cat# 70013-032, Lot# 1148371), 0.5%BSA (R&D System, Cat# DY995, part# 841380, lot#225340), 0.05% Tween 20 (SigmaUltra, P-7949, Lot# 81K0293), 6.6μglμl tRNA (Sigma cat# R-9001, Lot# 054K0650) for 0.5-2hours at 25⁰C /1200rpm. Then lOOng of the biotinylated anti-human Kallikrein 3 polyclonal goat IgG, (anti-PSA-biotin Ab, R&D System cat# BAF 1344, lot# IRI013071) is added as secondary antibody and incubated for 0.5-2hours at 25°C /1200rpm. After a wash step, 5μL of the streptavidin coated nanoparticles (e.g, streptavidin coated 15nm diameter gold particles, from BBI) is added to the binding mixture and incubated for 0.5hour at 25°C /1200rpm. After a wash step to remove unbound streptavidin coated nanoparticles, the biotin-labeled barcodes, biotin-biotin- (dAdC)i₅-dA ₂5-biotin-biotin, are added to the binding mixture. After a final wash step, the bound barcodes were released from streptavidin by heating in 95% formamide for 5 minutes at 65⁰C (alternatively, the bound barcodes can be released in 95% formamide for 2 minutes at 9O⁰C, or in 0.1% SDS for 5 minutes at 100⁰C ). The eluted barcodes are used for array hybridization. The barcodes are hybridized to a DNA array which contains probe sequence, (dGdT)is in a hybridization mixture containing 3XSSC, 0.02%Tween 20, 0.0125%SDS, 30%formamide. The dT20mer coated gold-nanoparticles are used to hybridize the dA region of the barcode sequence forming the "sandwich". Finally, the hybridized array is stained with silver development solutions and imaged with a light scattering based imaging system (e.g, Verigene ID™, Nanosphere Inc.). Figure 12 showed the PSA detection with barcode assay.

It should be understood that the foregoing disclosure emphasizes certain specific embodiments of the invention and that all modifications or alternatives equivalent thereto are within the spirit and scope of the invention as set forth in the appended claims.

Claims

What is claimed is:

1. A detection reagent comprising a reporter complex and at least two reporter moieties, wherein the reporter complex comprises a streptavidin-biotin complex and selective binding compounds specific to a target analyte, and wherein the reporter moieties are biotinylated biobarcodes.

2. The detection reagent of claim 1, wherein the biobarcodes are duplex nucleic acid molecules having at least one 3' or 5' end labeled with biotin, and having at least one portion capable of binding to a plurality of other duplexed nucleic acid molecules.

3. A detection reagent comprising a reporter complex and at least two reporter moieties, wherein the reporter complex comprises selective binding compounds specific to a target analyte and a nanoparticle coated with streptavidin, and wherein the reporter moieties are biotinylated biobarcodes.

4. The detection reagent of claim 3, wherein the biobarcodes are duplex nucleic acid molecules having at least one 3' or 5' end labeled with biotin, and having at least one portion capable of binding to a plurality of other duplexed nucleic acid molecules.

5. A detection reagent comprising a reporter complex and at least two reporter moieties, wherein the reporter complex comprises a plurality of streptavidin-biotin complexes and selective binding compounds specific to a target analyte, and wherein the reporter moieties are double-biotinylated biobarcodes.

6. A detection reagent comprising a reporter complex and at least two reporter moieties, wherein the reporter complex comprises selective binding compounds specific to a target analyte and a plurality of nanoparticles coated with streptavidin, and wherein the reporter moieties are double-biotinylated biobarcodes.

7. A detection reagent comprising a reporter complex and at least two reporter moieties, wherein the reporter complex comprises streptavidin, selective binding compounds specific to a target analyte, and a nanoparticle coated with biotinylated biobarcodes, and wherein the reporter moieties are biotinylated biobarcodes.

8. A detection reagent comprising a reporter complex and at least two reporter moieties, wherein the reporter complex comprises a nanoparticle coated with streptavidin, selective binding compounds specific to a target analyte, and a nanoparticle coated with biotinylated biobarcodes, and wherein the reporter moieties are biotinylated biobarcodes.

9. A detection reagent comprising a reporter complex and at least two reporter moieties, wherein the reporter complex comprises streptavidin, selective binding compounds specific to a target analyte, double biotinylated linkers, and at least one nanoparticle coated with biobarcodes capable of binding to the linkers.

10. A detection reagent comprising a reporter complex and at least two reporter moieties, wherein the reporter complex comprises a nanoparticle coated with streptavidin, selective binding compounds specific to a target analyte, double biotinylated linkers, and at least one nanoparticle coated with biobarcodes capable of binding to the linkers.

11. The detection reagent of any of claims 1-10, wherein the selective binding compound is a protein, an oligonucleotide, or an inorganic compound.

12. The detection reagent of claim 11, wherein the protein is an enzyme or an antibody.

13. The detection reagent of any of claims 1-10, wherein the at least two reporter moieties comprise fluorophores, chromophores, oligonucleotides with or without attached fluorophores or chromophores, proteins, porphyrins, lipids, carbohydrates, synthetic polymers, isotopic tags, or radioactive tags.

14. A method for detecting at least one target analyte in a sample, comprising steps of: a) providing at least one capture probe, said capture probe comprising at least one binding complement specific to the target analyte; b) providing at least one detection reagent according to any of claims 1-10; c) incubating the capture probe with the target analyte and the detection reagent under conditions effective to allow complex formation between the capture probe, the target analyte, and the detection reagent; d) separating the complex from any unbound detection reagent; e) selectively releasing at least a portion of the reporter moieties from the complex; and f) analyzing the presence or absence of the reporter moieties, wherein the presence or absence of reporter moieties is indicative of the presence or absence of the target analyte.

15. The method of claim 14, wherein the capture probe is immobilized on an insoluble material.

16. The method of claim 14, wherein the capture probe comprises a magnetic particle, and the complex is separated from any unbound detection reagent by the application of a magnetic field.

17. The method of claim 14, wherein the reporter moieties are selectively released from the complex by dehybridization.

18. The method of claim 14, wherein biotinylated intermediate oligonucleotides capable of binding to the target analyte are provided, and wherein the biotinylated intermediate oligonucleotides can form a complex with the detection reagent.