WO2009097470A1

WO2009097470A1 - High specifity immuno-amplification

Info

Publication number: WO2009097470A1
Application number: PCT/US2009/032505
Authority: WO
Inventors: Victor Manneh
Original assignee: Victor Manneh
Priority date: 2008-01-29
Filing date: 2009-01-29
Publication date: 2009-08-06
Also published as: EP2245461A1

Abstract

A method is described for detecting analytes which specifically bind to a specific binding molecule using proximity modulated signal generation. The method uses a first construct which includes a specific binding moiety, such as an antibody, attached to a photosensitizer moiety, and a second construct which includes a second binding molecule which recognizes the bound analyte and which also contains a photocleavable linker linked to an amplifiable nucleic acid oligomer. Upon exposure to activating light, the photosensitizer generates singlet oxygen causing cleavage of the photocleavable linker in analyte-bound second constructs and releasing the corresponding amplifiable oligomer. Following amplification, the amplified oligomer can be detected and/or quantitated in conventional ways, thereby identifying the corresponding analyte.

Description

HIGH SPECIFICITY IMMUNO-AMPLIFICATION

FIELD OF THE INVENTION

[0001] The present invention relates to assays for the detection of specific analytes.

BACKGROUND OF THE INVENTION

[0002] The following discussion is provided solely to assist the understanding of the reader, and does not constitute an admission that any of the information discussed or references cited constitute prior art to the present invention.

[0003] Important characteristics of an effective commercial assay include specificity, precision, the ability to perform the assay rapidly and most importantly the requirement of high sensitivity. For nucleic acid assays, many strategies have been employed to improve sensitivity through amplification of the target template sequence, e. g. polymerase chain reaction (PCR), or isothermal amplification.

[0004] In contrast, for non-nucleic acid analytes such as proteins, small molecules and microorganisms, the option of target amplification is largely not available; thus, increases in sensitivity are brought about by signal amplification, e.g. enzyme-linked immunoabsorbent assays (ELISAs).

[0005] In spite of the tremendous progress that has been made in recent years toward developing highly sensitive and robust assay systems for non-nucleic acid samples, few if any of the current technologies has high sensitivity at the sensitivity level of nucleic acid assays. As a result, we are excluded from measuring many useful markers, including many proteins and microorganisms, that have tremendous medical application for monitoring and prevention of disease because of the high sensitivity requirement. The requirements for extreme high sensitivity using the existing technology exclude many useful markers. Additionally, one typically has to use large amount of sample to compensate for lack of sensitivity, while often the available sample volume is rather scarce. [0006] As described above, even though ultrasensitive detection has become routine for nucleic acids, it remains challenging for proteins. A primary reason for this is the lack of a direct amplification method, analogous to the PCR for nucleic acids. Although proteins cannot be directly copied, they can be indirectly amplified by a process termed immuno-PCR (IPCR). In IPCR, a sandwich immunoassay is performed on a solid support, similar to traditional ELISA, except that the secondary antibody is covalently coupled to a DNA oligonucleotide rather than an enzyme (1 ). This DNA is then PCR-amplified and ean be detected afterward by, e.g., gel electrophoresis, or in real time by specially designed fluorescent probes, e.g., TaqMan (12). IPCR provides substantial increases in sensitivity as compared with ELISA but requires conjugation of the secondary antibodies to DNA strands as well as the thermocycling and enzymatic amplification common to PCR. Variations have been reported in which improved reagents or other forms of enzymatic amplification provide improvements in sensitivity, quantification, or ease of use (e.g., by avoiding thermocycling) (3-10). For example, IPCR using oligovalent streptavidin-DNA assemblies has been reported to provide 1 , 000-fold increases in sensitivity as compared with traditional ELISA (6, 7).

1. Sano, T., Smith, C. L & Cantor, C. R. (1992) Science 258, 120-122.

2. Brackmann, S. (2004) Angew. Chem. Int. Ed. 43, 5730-5734.

3. Schweitzer, B., Wiltshire, S., Lambert, J., O'Malley, S., Kukanskis, K., Zhu,

Z., Kingsmore, S. F., Lizardi, P. M. & Ward, D. C. (2000) Proc. Natl. Acad. Sci. USA 92, 10113-101 19.

4. Zhang, H. -T., Kacharmina, J. E., Miyashiro, K., Greene, M. I. & Eberwine,

J. (2001 ) Proc. Natl. Acad. Sci. USA 98, 5497-5502.

5. Niemeyer, C. M., Adler, M., Pignataro, B., Lenhert, S., Gao, S., Chi, L.,

Fuchs, H. & Blohm, D. (1999) Nucleic Acids Res. 27, 4553-4561.

6. Adler, M., Langer, M., Witthohn, K., Eck, J., Blohm, D. & Niemeyer, C. M.

(2003) Biochem. Biophys. Res. Commun. 300, 757-763.

7. Adler, M., Wacker, R. & Niemeyer, C. M. (2003) Biochem. Biophys. Res.

Commun. 308, 240-250.

8. Niemeyer, C. M., Wacker, R. & Adler, M. (2003) Nucleic Acids Res. 31 , e90. 9. Fredriksson, S., Gulberg, M., Jarius, J., Olsson, C, Pietras, K.,

Gustafsdottir, S. M., Ostman, A. & Landegren, U. (2002) Nat. Biotechnol. 20, 473-477.

10. Gullberg, M., Gustafsdottir, S. M., Schallmeiner, E., Jarvius, J., Bjarnegard, M., Betsholtz, C, Landegren, U. & Fredriksson, S. (2004) Proc. Natl. Acad. USA 101 , 8420-8424.

[0007] Immuno-PCR is described, for example, in Sano et al., US Pat 5,665,539 which is incorporated herein by reference in its entirety. Additional Immuno-PCR and other Immuno-Amplification variants are described, for example, in Baez et al., 6,51 1 ,809; Collier et al., 5,985,548; Dodge et al., 6,927,024; Greene et al., 7,341 ,831 ; Lawton, 7,341 ,837; Barletta et al., 2005/0239108; Grossman et al., 2005/0214805; Grossman et al., 2006/0199194; Karlsen, 2004/0076983; McCready et al., 2007/0166709; Nadeau et al., 2005/0009050; and Wu, 2005/0079520, each of which is incorporated herein by reference in its entirety.

[0008] A different type of assay signal amplification, termed the Invader assay, is described in U.S. patent 5,846,717.

SUMMARY OF THE INVENTION

[0009] The present invention provides an advantageous assay method and materials for detecting and/or quantifying the presence of one or more analytes in a sample by utilizing proximity mediated cleavage to release a signal molecule. In particularly advantageous configurations, the assay uses amplifiable nucleic acid oligomers as signal molecules, which allow signal amplification from even a very small number of bound analyte molecules. In addition, when used in conjunction with a separation moiety, the proximity mediated cleavage allows the assay to be configured such that a high degree of both specificity and sensitivity is provided. Advantageously, the proximity mediated cleavage is provided by the combination of a photosensitizer, which generates singlet oxygen upon exposure to an appropriate wavelength of light, and a corresponding photocleavable linker, which undergoes cleavage upon reaction with singlet oxygen. That cleavage will then release a desired moiety, e.g., a signal moiety such as an amplifiable nucleic acid oligomer.

[0010] Thus, a first aspect of the invention concerns a method for detecting at least one analyte, often a non-nucleic acid analyte, in a sample solution by detecting the presence and/or amount of a pre-selected amplified nucleic acid molecule, where the nucleic acid molecule is amplified from an oligomer released from an analyte-linked oligomer-containing construct by cleavage of a photocleavable linker. In advantageous embodiments, the cleavage is proximity- mediated.

[0011] In many embodiments, the method further involves binding the analyte to a first analyte-specific binding construct which includes a photosensitizer moiety and a first analyte-specific binding moiety, and to a second analyte-specific binding construct which includes a second analyte-specific binding moiety linked with an amplifiable nucleic acid oligomer through a photocleavable linker. In advantageous embodiments, the cleavage is proximity-mediated and will substantially only occur when first and second analyte-specific binding constructs are bound to the same analyte entity. [0012] In advantageous embodiments, the second analyte-specific binding construct also includes a separation moiety, e.g., a magnetic moiety such as a magnetic bead; the amplifiable nucleic acid oligomer is linked with such magnetic bead through the photocleavable linker.

[0013] In certain embodiments, the amplifiable nucleic acid oligomer is dsDNA, ssDNA, or RNA; the amplification is performed using a DNA polymerase, a replicase, or an RNA polymerase; the amplified nucleic acid is DNA or RNA.

[0014] In particular embodiments, the bound analyte is separated from the sample solution, e.g., before release and/or detection of the signal moiety; uncleaved second analyte-specific binding construct is separated from released amplifiable nucleic acid oligomer, e.g., using a magnetic field to move magnetic moieties; amplifiable nucleic acid oligomer is cleaved from the second analyte- specific binding moiety, e.g., by singlet oxygen induced cleavage of a photocleavable linker.

[0015] In certain advantageous embodiments, the method includes cleaving the amplifiable nucleic acid oligomer from the second analyte-specific binding moiety thereby releasing amplifiable nucleic acid oligomer, separating unbound second analyte specific binding construct and the remainder of the bound second analyte specific binding constructs from the amplifiable nucleic acid oligomer; and amplifying the released amplifiable nucleic acid oligomer, e.g., using isothermal amplification or PCR or other nucleic acid amplification technique.

[0016] In certain embodiments, the method includes distinguishably detecting a plurality of different pre-selected amplified nucleic acid molecules (amplified from a corresponding plurality of different amplifiable nucleic acid oligomers) corresponding to a plurality of different analytes, e.g., at least 2, 3, 4, 5, 7, 10, 15, 20, or more, or 2-5, 3-7, 3-10, 5-10, 5-15, 7-15, 7-20, 10-15, or 10-20 different analytes (with corresponding different amplified nucleic acid molecules.

[0017] The first and/or second binding moieties may be any of a variety different molecules or moieties which provide specific or at least highly preferential binding to target analyte, such as antibodies (which may be antibody fragments such as Fab fragments) which may be polyclonal but are advantageously monoclonal, a receptor or fragment thereof, an aptamer, or a ligand or ligand analog.

[0018] The analyte may be essentially any entity, e.g., molecule, which can be recognized and bound by a specific binding molecule or moiety, e.g., a peptide, a protein, a cell surface receptor or other cell surface protein, a soluble protein, a polysaccharide, a molecular complex (e.g., a nucleoprotein or glycoprotein), a nucleic acid, a small molecule, a drug, a pesticide, a toxin, a cell, a microorganism, a bacterium, a virus. A variety of analytes which may be used in the present invention are described in Ullman et al., US Pat 5,340,716, which is incorporated herein by reference in its entirety.

[0019] Also in particular embodiments, the photosensitizer absorbs light within a defined waveband and generates singlet oxygen in response to absorbing that light; the photosensitizer absorbs light above 450 nm, above 550 nm, above 600 nm, above 700 nm, above 800 nm, above 900 nm, in the range of 450-1000 nm, in the range of 550-900 nm, in the range of 550-700 nm, in the range of 650-800 nm, or in the range of 700-900 nm, and generates singlet oxygen in response to absorbing that light; the photosensitizer is selected from the group consisting of pthalocyanines, naphthalocyanines, 7,8-dihydro-5, 10, 15, 20- tetrakis (3- hydroxyphenyl)-21 -23-[H]porphyrin (THPC), PEG-m-THPC, temoporfin, meta-tetra (hydroxyphenyl)chlorine, and photofrin.

[0020] Also in certain embodiments, the photocleavable linker is hydrolyzed upon interaction with singlet oxygen; the photocleavable linker is selected from the group consisting of oxazoles, thiazoles, olefins, thioethers, selenoethers, and imidazoles.

[0021] In certain embodiments, singlet oxygen is generated in "close proximity" to a photocleavable linker which is linked through binding to an analyte species. In most cases, "close proximity" will be within the diffusion distance for the lifetime of the singlet oxygen in the particular actual or intended assay environment, e.g., in an aqueous solution such as, for example, an aqueous buffer. In many cases,

"close proximity" will be 100 nm or less, and often 50 nm or less, 40 nm or less, 30 nm or less, 20 nm or less, 10 nm or less, 5 nm or less, 2 nm or less, 1 nm or less, 500 angstroms or less, 200 angstroms or less, 100 angstroms or less, or 50 angstroms or less.

[0022] In particular embodiments, the first analyte-binding construct includes a photosensitizer and is shielded or otherwise protected from exposure to light or from exposure to light of wavelengths effective to cause the photosensitizer to generate substantial amounts of singlet oxygen until such time in the performance of an assay as cleavage of photocleavable linker is desired.

[0023] Also in particular embodiments, at least one first analyte-specific binding construct is immobilized on a solid phase surface (e.g., a bead, plate, well, sheet, film, or chip) and includes a first analyte-specific binding moiety. The first analyte- specific binding construct includes or is immobilized in close proximity to at least one photosensitizer; the immobilized first analyte-specific binding construct is contacted with a sample which contains or potentially contains analyte which binds to the first analyte-specific binding construct forming a two-part complex; sample may be displaced or washed away from the first analyte-specific binding construct; immobilized first analyte-specific binding construct with bound analyte is contacted with at least one second analyte-specific binding construct which includes a second analyte-specific binding moiety linked (directly or indirectly) with an amplifiable nucleic acid oligomer through a photocleavable linker, and optionally a separation moiety (e.g., a magnetic moiety such as a magnetic bead) and which binds to the two-part complex forming a three-part complex; unbound second analyte-specific binding construct is removed and/or immobilized (e.g., by washing and/or using the separation moiety; the photosensitizer is exposed to light effective to induce substantial production of singlet oxygen resulting in cleavage of the photocleavable linker and release of amplifiable oligomer; amplifiable oligomer is amplified (preferably after separation from any non- specifically bound second analyte-specific binding construct, such as by solution displacement or washing); amplified oligomer is detected and/or quantitated thereby identifying and/or quantitating corresponding analyte. In advantageous cases, a plurality of different analyte-specific binding constructs are used having different analyte-specific binding moieties recognizing and binding different analytes and also respectively having distinguishably different amplifiable oligomers (e.g., differing in length and/or nucleotide sequence).

[0024] In some embodiments, at least one first analyte-specific binding construct is used which includes a first analyte-specific binding moiety, a photosensitizer, and a separation moiety (e.g., a magnetic moiety); at least one second analyte-specific binding construct is used which includes a second analyte-specific binding moiety linked with an amplifiable oligomer through a photocleavable linker; the first and second analyte-specific binding constructs are contacted (simultaneously or sequentially in either order) with sample which contains or may contain analyte which binds to the first and second analyte- specific binding moieties forming three-part complexes, or first or second analyte specific binding construct is contacted with sample then the sample and analyte- specific binding construct are separated and the sample-contacted analyte- specific binding construct is contacted with the other analyte-specific binding construct forming three part complexes; three part complexes are preferably separated from unbound and/or non-specifically bound second analyte-specific binding constructs; three part complexes are exposed to light effective to induce substantial production of singlet oxygen from the photosensitizers causing cleavage of photocleavable linkers and release of amplifiable oligomers; preferably released amplifiable oligomers are separated from first analyte-specific binding constructs; released amplifiable oligomers are amplified and the amplification products are detected and/or quantitated, where the presence and/or amount of amplification product is indicative of the presence and/or amount of corresponding analyte. In advantageous cases, a plurality of different analyte- specific binding constructs are used having different analyte-specific binding moieties recognizing and binding different analytes and also respectively having distinguishably different amplifiable oligomers (e.g., differing in length and/or nucleotide sequence).

[0025] In certain advantageous embodiments, the method uses a first analyte- specific binding construct which includes a first analyte-specific binding moiety linked with a photosensitizer, and a second analyte-specific binding construct which includes a second analyte-specific binding moiety linked with an amplifiable oligomer through a photocleavable linker and also includes a separation moiety (e.g., a magnetic moiety such as a magnetic bead); the first and second analyte- specific binding constructs are contacted (simultaneously or sequentially in either order) with sample which contains or may contain analyte which binds to the first and second analyte-specific binding moieties forming three-part complexes, or first or second analyte specific binding construct is contacted with sample then the sample and analyte-specific binding construct are separated and the sample- contacted analyte-specific binding construct is contacted with the other analyte- specific binding construct forming three part complexes; three part complexes are optionally separated from unbound and/or non-specifically bound second analyte- specific binding constructs; three part complexes are exposed to light effective to induce substantial production of singlet oxygen from the photosensitizers causing cleavage of photocleavable linkers and release of amplifiable oligomers; preferably released amplifiable oligomers are separated from second analyte- specific binding constructs having unreleased oligomers; released amplifiable oligomers are amplified and the amplification products are detected and/or quantitated, where the presence and/or amount of amplification product is indicative of the presence and/or amount of corresponding analyte. In advantageous cases, a plurality of different analyte-specific binding constructs are used having different analyte-specific binding moieties recognizing and binding different analytes and also respectively having distinguishably different amplifiable oligomers (e.g., differing in length and/or nucleotide sequence).

[0026] For some constructs, additional specific binding pairs are utilized to assemble components. For example, biotin is commonly used with avidin or streptavidin. Thus, for example, a second analyte-specific binding construct may be assembled from sub-constructs, one which is biotinylated and one which includes avidin or streptavidin. Thus, for example, a magnetic bead bearing second analyte-specific binding moiety can also be biotinylated on its surface. Either before or after binding of the second analyte-specific binding construct with analyte, the bead can be contacted with avidin or streptavidin linked with amplifiable oligomer. Alternatively the positions of biotin and avidin/streptavidin can be reversed. [0027] A related aspect concerns a method for amplifying a specific binding event signal. The method includes releasing an amplifiable nucleic acid oligomer from an analyte-bound analyte specific binding moiety by cleaving a photocleavable linker located between the analyte specific binding moiety and the amplifiable nucleic acid oligomer, generally by reaction with singlet oxygen, thereby providing a released oligomer, and amplifying the released oligomer.

[0028] In certain embodiments, the method is carried out as described for the first aspect above.

[0029] In particular embodiments, the amplifying is performed using isothermal amplification or PCR; a plurality of distinguishable oligomers respectively bound to different analytes are released, and in most cases amplified.

[0030] Also in particular embodiments, the analyte specific binding moiety includes a magnetic bead, and the amplifiable oligomer(s) is linked to the bead through a photocleavable linker; the magnetic bead is separated from the released amplifiable oligomer prior to amplifying; bound and unbound magnetic beads are separated from released amplifiable oligomer prior to amplifying.

[0031] Another related aspect concerns a kit for detecting an analyte in a solution, where the kit contains a first analyte-specific binding construct, which includes a photosensitizer moiety and a first analyte-specific binding moiety. Together with or separate from the first analyte-specific binding construct, the kit contains a second analyte-specific binding construct which includes a second analyte-specific binding moiety linked with an amplifiable nucleic acid oligomer through a photocleavable linker. Preferably the second analyte specific binding construct also includes a separation moiety such as a magnetic moiety, e.g., a magnetic bead.

[0032] In certain embodiments the first and/or second construct is as described for an aspect above or otherwise described herein.

[0033] In some cases, the kit includes directions for use of the kit to detect and/or quantitate the amount of an analyte which binds to the first and second analyte specific binding moieties; the first and/or second analyte specific binding constructs are mixed together (e.g., in solution or in a dry preparation which can be dissolved or suspended, such as in aqueous solution). The kit may also include amplification reagents (e.g., primers) and/or detection reagents (e.g., labeled probes).

[0034] Yet another related aspect concerns a signal amplification construct, which includes an analyte-specific binding molecule linked with a photocleavable linker, which is also linked with an amplifiable nucleic acid molecule.

[0035] In certain embodiments, the construct is as described for an aspect above or otherwise described herein for a second analyte specific binding construct.

[0036] In particularly advantageous embodiments, the construct also includes a magnetic bead; usually the construct is configured such that the amplifiable nucleic acid molecule is linked with the magnetic bead through the photocleavable linker.

[0037] In particular embodiments, the analyte-specific binding molecule is bound with an analyte, which may also be bound with a photosensitizer construct, where the photosensitizer construct includes another analyte specific binding molecule and a photosensitizer moiety.

[0038] In the constructs described for the aspects above or otherwise described herein, highly preferably, for the constructs containing a photosensitizer moiety and a photocleavable linker respectively, when both are bound to an analyte species the photosensitizer moiety and the photocleavable linker are in close proximity (e.g., the distance between the photosensitizer moiety and the photocleavable linker is no greater than the diffusion distance for singlet oxygen in the solution in which the combined construct is present or is intended to be present when exposed to singlet oxygen inducing light during an assay). [0039] For the present conjugates, the term "photosensitizer" refers to a molecule or moiety of the conjugate which generates singlet oxygen when exposed to light of a particular wavelength.

[0040] Also in reference to the present conjugate molecules, the term "photocleavable linker" refers to a moiety which reacts with singlet oxygen resulting in the cleavage of the moiety. For the present constructs, in most cases a photocleavable linker links an amplifiable nucleic acid oligomer and a separation moiety.

[0041] In connection to the distances between photosensitizer and photocleavable linker in the present invention, "close proximity" to a photocleavable linker which is linked through binding to an analyte species, means that the singlet oxygen is generated sufficiently close to the photocleavable linker that the concentration of singlet oxygen is sufficient at the cleavable linker such that the cleavable linker will more likely than not react with a singlet oxygen within one minute.

[0042] In the context of the present invention, the term "small molecule" refers to a molecule or a moiety of a conjugate that has a MW of 1000 daltons or less, usually 600, 500, 400, 300, 200, 100 daltons or less.

[0043] The term "specific binding agent" is used to refer to a molecule or moiety which binds to a particular other molecule or complex with a significant level of specificity. That is, the specific binding agent binds to the particular other molecule or complex to a substantially greater degree than to other molecules or complexes that are normally present in the particular environment. In many case the specific binding agent binds with another particular molecule, and the two binding molecules constitute a specific binding pair. Examples include antibody/antigen pairs (including specific binding antibody fragments), antibody/hapten pairs (including specific binding antibody fragments), ligand/receptor pairs (including ligand analogs), aptamer/target molecule pairs (including nucleic acid and peptide aptamers), and enzyme/substrate pairs (including substrate analogs). [0044] The term "analyte-specific binding moiety" refers to a specific binding agent which is a portion of a construct and which has binding specificity for a corresponding particular analyte.

[0045] In the context of this invention, the term "analyte" refers to an entity which is to be detected using an embodiment of the present assays. Such entity will, in many cases be a molecule, e.g., a small molecule such as a drug, toxin, or pesticide compound, a single molecule protein, single molecule RNA, or single molecule polysaccharide, but may also be a more complex structure such as a multi-polypeptide protein complex, a complex of protein and nucleic acid (often RNA), a virus, or a bacterium or other cell. Of course, analyte-specific binding moieties may recognize and bind to only a portion of the analyte, e.g., an antibody binding to an epitope of a protein on or in the surface of a cell.

[0046] Indication that a molecule (or molecule/complex or complex/complex) "specifically binds" with another means that the molecule binds with the other to a substantially greater extent than to other similar molecules in the binding environment. Highly preferably, the molecule does not bind to a significant extent to any other molecules in the relevant binding environment.

[0047] As used in the context of the present invention, the term "oligomer" refers to a nucleic acid oligomer, i.e., a chain of nucleotides and/or nucleotide analogs which is no more than about 500 nucleotides in length, and often shorter, e.g., no more than 400, 300, 200, 150, or 100 nucleotides in length, more often in a range of 5 to 300, 5 to 200, 5 to 150, 5 to 100, 10 to 300, 10 to 200, 10 to 150, 10 to 100, 20 to 300, 20 to 200, 20 to 150, 20 to 100, 50 to 300, 50 to 200, 50 to 150, or 50 to 100 nucleotides in length.

[0048] The term "amplifiable nucleic acid oligomer" refers to a nucleic acid oligomer which has a length and nucleotide sequence such that it can readily amplified using conventional nucleic acid amplification techniques, e.g., PCR, RT- PCR, isothermal amplification, or ligase chain reaction.

[0049] The term "antibody" is used to refer to an immunoglobulin molecule or specifically binding fragment thereof, e.g., an Fab fragment. [0050] A "hapten" is a molecule which can be recognized and bound by an antibody but does not elicit production of antibodies unless combined with a carrier.

[0051] As an example of a molecule or structure having specific binding properties, the term "receptor" refers to a molecule normally produced by a cell and normally found within a cell or on the surface characterized by selective binding of a specific substance and a specific physiologic effect that accompanies the binding. Unless clearly indicated to the contrary, the term also includes derivatives and fragments of native receptors. For use in the present invention, such a receptor is highly preferably a single molecule.

[0052] As uses in the context of the present invention, the term "separation moiety" refers to a portion of a first or second analyte specific construct which allows the construct containing the moiety to be held in and/or moved to a particular spatial volume, space, or location. A particularly advantageous example is a magnetic moiety, e.g., a magnetic bead, which allows the corresponding construct to be moved or held by a magnetic field.

[0053] As used in connection with the present constructs, the term "linked" means that the references items are physically associated together, either directly or indirectly. Such association will typically be through a series or set of covalent and/or non-covalent bonds. For example, a binding moiety may be associated with an oligomer through a photocleavable linker with covalent bonding directly between the binding moiety and photocleavable linker and covalent bonding directly between the photocleavable linker and oligomer, or, in an alternative example, the binding moiety may be directly attached to a bead (covalently or non-covalently) and attached to the same bead is a photocleavable linker which attached to an oligomer. In either case, the binding moiety is regarded as "linked" with the oligomer.

[0054] Additional embodiments will be apparent from the Detailed Description and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Fig. 1 is a schematic illustration of the conduct of an exemplary assay using magnetic beads bearing releasable nucleic acid oligomers.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0056] The present invention provides a highly advantageous approach for analyte assays which involves nucleic acid amplification to amplify a signal corresponding to an analyte binding event, in most cases using a proximity- mediated signal generator. Within this approach, it is highly beneficial to configure the constructs to be used to distinguish specifically bound signal generator from non-specifically bound and unbound signal generator. That is, it is highly beneficial for the generated and detected signal to be specific for analyte binding events, without (or at least with a large reduction in) the background associated with non-specific binding and residual unbound assay components. The result is an assay format which provides both high sensitivity and high specificity for specific analytes, e.g., protein analytes. In conjunction with a suitable nucleic acid amplification technique, this further can allow the assay to be quantitative for the analyte.

[0057] The present method has some similarities to recently developed ImmunoPCR methods, in that both the present methods and ImmunoPCR involve binding of an analyte to an antibody or other analyte binding molecule, binding a nucleic acid oligomer to the bound analyte, amplifying the nucleic acid oligomer, and detecting the amplified nucleic acid. A significant limitation of conventional ImmunoPCR is that the amplification step will amplify all of the particular nucleic acid oligomer present, including any non-specifically bound oligomer (e.g., associated with a solid phase surface) and any oligomer which has not otherwise been washed away. Amplified oligomer resulting from oligomer which is not specifically bound with the particular analyte results in background signal. Such background can be quite variable, and can cause significant reduction in assay sensitivity, precision, and/or accuracy.

[0058] The present invention can significantly reduce or even substantially eliminate that difficulty by introducing a second level of specificity. That is, the binding of the nucleic acid oligomer to the bound analyte is preferably followed by a proximity sensitive initial signal generation, usually involving cleavage of a photocleavable linker resulting from exposure to singlet oxygen produced by a photosensitizer linked or otherwise associated with a binding moiety which resulted in initial binding of the analyte. For example, the singlet oxygen generator (photosensitizer) and the photocleavable linker (with its associated oligomer) are on different analyte binding constructs. As a result, the photosensitizer and photocleavable linker will only be in close proximity with each other when both are bound to the same analyte molecule. Due to the short lifetime of singlet oxygen in most aqueous solutions, the result is the specific release of oligomer which was bound to analyte and no or essentially no release of oligomer which is non-specifically bound, e.g., to a solid phase surface or other system component. If the oligomers corresponding to specific binding events are separated from the unbound and non-specifically bound oligomers prior to amplification, only the specifically released oligomer corresponding to binding events will be available for amplification and detection. Advantageously, the assay can be configured and carried out in an essentially homogeneous format.

[0059] As indicated above, the benefit of the second level of specificity is a substantial reduction of background and variability. This allows the present system to provide improved assay results, with significant improvement over the current Immuno-PCR and Tadpoles approaches due to specific amplification of the nucleic acid without requiring extensive wash steps or having interference in amplification.

[0060] In order to more fully explain the invention, the various assay components and their use is discussed in greater detail below.

First Analyte-Specific Binding Construct

[0061] The first analyte-specific binding construct includes an analyte specific binding moiety (e.g., an antibody, hapten, or receptor) and a component needed for the proximity sensitive signal generation (e.g., proximity sensitive oligomer release). Advantageously, the first construct is used in solution or suspension, but assays can also be configured such that a first construct will be immobilized on a solid phase surface, e.g., a surface of a plate, well, chip, film, sheet, or filter. [0062] In highly advantageous assays, the first analyte specific binding construct includes both an analyte specific binding moiety and a photosensitizer moiety. If desired, other moieties may also be included, such as a spacer between the binding moiety and the photosensitizer moiety and/or a separation moiety such as a magnetic bead or other magnetic particle. Constructs can also be provided which include a plurality of analyte specific binding moieties, which may be of the same or different type, and which may bind the same or different analytes.

[0063] Likewise, a plurality of different first analyte-specific binding constructs may be utilized in an assay, having different analyte specificities. When used in conjunction with a corresponding set of different second analyte-specific binding constructs having specificity for the same set of different analytes, assay multiplexing can be provided.

[0064] For some constructs to be used for photocleavage, the photosensitizer is not included in the first analyte-specific binding construct but is still in close proximity, e.g., attached in close proximity on a solid phase surface or embedded within the solid phase material.

Second Analyte-Specific Binding Construct

[0065] The second analyte-specific binding construct includes a second analyte specific binding moiety, which may, for example, recognize the analyte or the analyte-first analyte specific binding moiety complex. As with the first analyte specific binding moiety, the second analyte specific binding moiety may, for example, be an antibody, hapten, receptor, aptamer, or the like. The second analyte-specific binding construct also includes an amplifiable nucleic acid oligomer, and often a plurality of such oligomers. In most cases, such a plurality of oligomers will be multiple copies of the same oligomer, but in some cases the construct will include a plurality of different oligomers, e.g., oligomers which have different nucleotide sequence and/or length. The second construct will also usually include a cleavage moiety, advantageously a photocleavable linker. Highly advantageous photocleavable linkers react with singlet oxygen resulting in cleavage of the linker. Such a photocleable linker can advantageously be positioned such that cleavage of the linker releases the amplifiable oligomer.

[0066] In advantageous embodiments, in addition to the analyte specific binding moiety(ies) and one or more oligomers, the construct includes a separation moiety, e.g., a magnetic particle such as a magnetic bead. The bead or other particle can be linked with one or more amplifiable nucleic acid oligomers through cleavable linkers. Such a separation moiety allows constructs which include the separation moiety to be moved to, moved from, and/or held in a desired space. For example, a magnetic bead allows a construct including such bead to be moved and/or held using a magnetic field, thus allowing separation of such constructs from other components in a solution or suspension.

[0067] For use with relatively large analyte entities, e.g., cells, it can advantageous if both the first and second analyte-specific binding constructs bind to the analyte in close proximity, e.g., to the same cell surface protein. In this way, proximity mediated release of amplifable nucleic acid oligomer can be maintained.

Multiplexing

[0068] The selection of different nucleic acid oligomers linked with different second analyte-specific binding moieties also provides effective multiplexing capability, that is, the ability to detect and distinguish a number of different analytes in the same assay. In such assays, the different analyte specific binding moieties, with their corresponding different oligonucleotides, will each specifically bind to the respective analytes. Release and separation of the respective oligomers will create an oligomer mixture in which the different oligomer species represent the different analytes. Amplification and detection of the respective oligomers will then identify and/or quantify the respective analytes bound from the sample.

[0069] The oligomers may be distinguished based on conventional distinguishable characteristics. For example, the oligomers can be distinguished based on nucleotide sequence and/or length and/or different detectable labels. Techniques for distinguishing oligomers based on such parameters are well- known.

[0070] Usually, the second analyte-specific binding constructs having specificity for different analytes (and/or for different portions of an analyte) are used in conjunction with a corresponding set of first analyte-specific binding constructs. In this way, a "sandwich" complex will be created with the first and second analyte- specific binding constructs both recognizing and binding a single analyte entity.

Photosensitizer Moieties

[0071] As indicated, in certain particularly advantageous embodiments, the present invention utilizes at least one photosensitizer moiety.

[0072] Photosensitizers are compounds (attached as moieties in the present conjugates) that photochemically generate a reactive form of oxygen called singlet oxygen (Haugland, R. P. et al. Handbook of Fluorescent Probes and Research Products. Molecular Probes, 9^th Ed. 2002). A number of these photosensitizers, such as phthalocyanine, have found utility as anticancer drugs when used as singlet oxygen generators in Photodynamic Therapy (PDT). Those photosensitizers identified for PDT can be used in the present invention also with corresponding suitable cleavable compounds. In some cases, a single molecule of the photosensitizer can generate half a million molecules of singlet oxygen per second (Youngjae, Y., et al. Water Soluble, Core-Modified Porphyrins. 3. Synthesis, Photophysical Properties, and in Vitro Studies of Photosensitization, Uptake, and Localization with Carboxylic Acid-Substituted Derivatives J. Med. Chem., 2003, Vol. 46: 3734-3747).

[0073] Upon exposure of the photosensitizer to light of appropriate wavelength, singlet oxygen is generated. As a result, in many applications it is desirable to prevent or at least limit the exposure of the photosensitizer to light until the desired time and/or photosensitizer position. At the desired time and/or location, the photosensitizer is exposed to light (e.g., from a laser) causing single oxygen generation. For the present invention, the higher wavelengths (650 nm and above) of activating light energy (e.g., from lasers) are preferred because they can penetrate deeper into solution.

[0074] The limited diffusion distance of singlet oxygen in aqueous solutions provides a proximity requirement for the present assay system. That is, when free in solution, it is unlikely that during exposure to activating light a photosensitizer moiety will be found sufficiently close to a construct containing a corresponding photocleavable linker that the linker will be cleaved. It is only when the construct containing the photosensitizer and the construct containing the photocleavable linker are brought into proximity (preferably close proximity) by being bound to the same analyte structure (e.g., analyte molecule) that photocleavage will occur.

[0075] A non-limiting list of examples of useful photosensitizers include the pthalocyanines, naphthalocyanines, 7,8-dihydro-5, 10, 15, 20- tetrakis (3- hydroxyphenyl)-21 -23-[H]porphyrin (THPC), PEG-m-THPC, temoporfin, meta-tetra (hydroxyphenyl)chlorine, photofrin. Other photosensitizers are also known and can be used in this invention.

C. Photocleavable Linkers

[0076] A variety of photocleavable linkers are known, which will react with singlet oxygen in solution, resulting in cleavage of the linker.

[0077] Such a photocleavable linkage includes an oxidation-labile linkage that is cleaved by singlet oxygen. Examples of such linkers are heterocyclic compounds, such as diheterocyclopentadienes, as exemplified by substituted imidazoles, thiazoles, oxazoles, etc., where the rings will usually be substituted with at least one aromatic which contains carbon-carbon double bonds. Upon reaction with singlet oxygen, these compounds form an oxo group which then hydrolyzes into two separate molecules or moieties (Ando et al., Singlet Oxygen Reaction--ll Alkylthiosubstituted Ethylene, Tetrahedron, Pergamon Press 1973, Vol. 29: 1507-1513).

[0078] The linker may, for example, be a thioether or its selenium analog; or an olefin, which contains carbon-carbon double bonds. Cleavage of a double bond to an oxo group releases the active moiety, e.g., an amplifiable nucleic acid oligomer or an anticancer drug. Additional examples of olefins which may be used include vinyl sulfides, vinyl ethers, enamines, imines substituted at the carbon atoms with an a-methine (CH, a carbon atom having at least one hydrogen atom), where the vinyl group may be in a ring, the heteroatom may be in a ring, or substituted on the cyclicolefinic carbon atom, and there will be at least one and up to four heteroatoms bonded to the olefinic carbon atoms. The resulting dioxetane may decompose spontaneously or, highly preferably, by reaction with singlet oxygen from a photosensitizer. Such reactions are described in the following exemplary references: Adam and Liu, J. Amer. Chem. Soc. 94,1206-1209,1972; Ando, et al., J. C. S. Chem. Comm. 1972,477-8; Ando, et al., J. Amer. Chem. Soc. 96,6766- 8,1974; Wasserman and Terao, Tetra. Lett. 21 ,1735-38, 1975; and U. S. Patent No. 5,756,726 (which is incorporated herein by reference in its entirety).

[0079] The dioxetane occurs upon reaction of singlet oxygen with an activated olefin substituted with a drug moiety or other active moiety at one carbon atom and the second binding agent at the other carbon atom of the olefin. See, for example, U. S. Patent No. 5,807,675 (incorporated herein by reference in its entirety).

[0080] Exemplary cleavable linkages include S-3-thiolacrylic acid, -N, N-methyl 4-amino-4butenoicacid,-0, 3-hydroxyacrolein, N- (4-carboxyphenyl) 2-imidazole, oxazole, and thiazole. Other useful cleavable linkers include N-alkyl acridinyl derivatives, substituted at the 9 position with a divalent group of the formula: - (CO) X' (A)wherein: X' is a heteroatom selected from the group consisting of O, S, N, and Se, usually one of the first three; and A is a chain of at least 2 carbon atoms and usually not more than 6 carbon atoms substituted with anticancer drug, where preferably the other valences of A are satisfied by hydrogen, although the chain may be substituted with other groups, such as alkyl, aryl, heterocyclic groups, etc., A generally being not more than 10 carbon atoms.

[0081] Other cleavable linkers are heterocyclic compounds, such as diheterocyclopentadienes, as exemplified by substituted imidazoles, thiazoles, oxazoles, etc., where the rings will, in some cases, be substituted with at least one aromatic group and in some instances hydrolysis will be necessary to release the drug. The oxazole cleavable linkage, "-CH2-oxazole- (CH2) n-C (=O)-NH-.

[0082] Still other cleavable linkers are tellurium (Te) derivatives, where the Te is bonded to an ethylene group having a hydrogen atom beta to the Te atom. The ethylene group is part of an alicyclic or heterocyclic ring that may have an oxo group, preferably fused to an aromatic ring and the other valence of the Te is bonded to the drug. The rings may be, for example, coumarin, benzoxazine, tetralin, etc.

[0083] Photocleavable linkers suitable for use in this invention which are currently regarded as preferable include, for example, oxazoles, thiazoles, olefins, thioethers, selenoethers, and imidazoles.

Analytes

[0084] The present materials and method are applicable to an extremely broad range of analytes. Analytes appropriate for use in the invention will specifically bind with a specific binding agent under suitable binding conditions, i.e., are part of a specific binding pair. In most cases, the binding will be performed in solution. Useful binding agents include but are not limited to antibodies (including antibody fragments); receptors (including receptor fragments), e.g., for binding to ligands or ligand analogs), enzymes and derivatives thereof (e.g., for binding to corresponding substrates and substrate analogs), ligands and ligand analogs (e.g., for binding to corresponding receptors), and aptamers. Suitable binding agents can be selected and/or constructed by methods well-known in the field.

[0085] Analytes for which the present invention is well suited include, for example, proteins (including both soluble and membrane bound, e.g., cell surface, proteins, drug entities (including both legal and illegal drugs), toxins (e.g., compounds which are damaging to humans, dogs, cats, bovines, porcines,

[0086] Samples may be used, for example, from a large variety of animals, including but not limited to mammals such as humans, dogs, cats, ruminants, porcines, equines, ovines, animals of Family Bovidae, e.g., bovines and caprines, as well as others. Samples from animals can be of various types, e.g., blood, serum, urine, stool, tissue, saliva, sweat, and exhalate. Samples may also be obtained from many other sources, such as from tissue culture, cell culture, microorganism culture (e.g., bacterial, fungal, viral), environmental samples (e.g., soil, water, or air), plants, and the like. In most case, samples will be in liquid form, or will be treated to be in liquid form.

[0087] Some exemplary analytes and types of analytes are pointed out in references cited in the following section.

Representative Application of the Invention

[0088] The present assays may be configured in various ways. An exemplary set of constructs suitable for a homogeneous assay is illustrated schematically in Fig. 1. As shown, the assay uses two constructs, one of which includes a first analyte-specific antibody linked with a photosensitizer. The other construct includes a magnetic bead to which second analyte specific antibodies are linked (should recognize epitopes on the analyte which are distinct from those recognized by the first analyte specific antibodies). The bead is also linked with nucleic acid oligomers through photocleavable linkers (i.e., moieties which are cleaved by reaction with singlet oxygen generated by the photosensitizer.

[0089] As illustrated in Panel A of Fig. 1 , when the first and second constructs are contacted with the specific analyte, a sandwich structure is formed in which both first and second constructs are bound to the analyte. Exposure of the photosentizer to light (hv), e.g., at 680 nm, causes the generation of singlet oxygen (step 1 ) which diffuses the short distance to the photocleavable linker. The singlet oxygen can react with the linker, causing cleavable of the linker and release of the associated oligomer (step 2). At this stage, the result is free oligomer in solution, which corresponds to analyte binding events. At this point, the magnetic particles can be separated from the released oligomers by applying a magnetic field. This is done in such a manner that the magnetic particles are isolated from the solution containing the released oligomers. This separation thus removes all oligomers which were not associated with an analyte binding event, as well as magnetic particles which did not bind, or which bound non-specifically to other components in the sample solution. The result is that the only oligomers left in the solution are oligomers which correspond to a specific analyte binding event. Those oligomers are then amplified (step 3) and detected. The amplified oligomers may be detected using techniques for detection of nucleic acid oligomers, e.g., using electrophoresis, binding with labeled probes, or direct labeling of oligomers during amplification and detection of the labeled oligomers.

[0090] In contrast, Panel B of Fig. 1 shows the process when specific analyte is not bound. In this case, the sandwich structure in which both first and second constructs are bound to the analyte is not formed. Therefore, first and second analyte-specific binding constructs are not held in close proximity. As a result, when light (hv) is applied (step 1 ) single oxygen is generated but is rapidly quenched in solution such that the cleavable linkers in the second analyte-specific binding constructs are not cleaved and the attached oligomers are not released (step 2). If the magnetic beads with oligomers still attached are removed from the solution to be amplied, upon application of amplification conditions no oligomers corresponding to specific analyte binding events will be present in solution for amplification and no amplification of such oligomers will occur and be detected. Of course, in many cases it will be desirable to have positive amplification control oligomers in solution to demonstrate that that amplification process was properly performed.

[0091] The present assay approach is applicable to one or multiple analytes. For multiple analytes (e.g., multiplexing), it is advantageous to distinguishably detect oligomers corresponding to binding events for the respective distinct analytes.

[0092] Analytes, analyte specific binding moieties, binding conditions, methods and materials for forming conjugates (e.g., conjugates including oligomers), amplification processes and components, and detection methods useful in the present invention are described, for example, in Sano et al., US Pat 5,665,539; Baez et al., 6,511 ,809; Collier et al., 5,985,548; Dodge et al., 6,927,024; Greene et al., 7,341 ,831 ; Lawton, 7,341 ,837; Barletta et al., 2005/0239108; Grossman et al., 2005/0214805; Grossman et al., 2006/0199194; Karlsen, 2004/0076983; McCready et al., 2007/0166709; Nadeau et al., 2005/0009050; and Wu, 2005/0079520, each of which is incorporated herein by reference in its entirety.

Kits

[0093] Advantageously, assay components for the present system can be provided in the form of a kit, e.g., for carrying out the assays a plurality of times. Thus, for example, a kit can contain a quantity of a first analyte specific binding construct and/or a quantity of corresponding second analyte specific binding construct. In certain advantageous embodiments, the second construct includes a separation moiety such as a magnetic bead.

[0094] The kit can be packaged, e.g., in single use or multiple use form. One or both of the constructs can be provided in solution (or suspension) or in dry form (e.g., lyophilized). The constructs may be contained in containers such as bottles or vials, or may be contained in assay devices, e.g., microfluidic assay devices.

[0095] The kit can also include written and/or pictorial directions for use of the kit, typically for performing an assay for a particular analyte or set of analytes.

[0096] The kit can also include other components, e.g., components for carrying out steps in an assay. For example, the kit may include amplification components (e.g., amplification enzymes, primers and/or buffers) and/or detection components (e.g., probes, labeled probes, and/or labels for incorporation in amplified oligomers, or electrophoretic gels for separation of amplification products).

EXAMPLES

[0097] The following example illustrates a basic application of the assay's technical approach.

EXAMPLE 1 : Demonstration of Release of a DNA Surrogate Preliminary Studies:

[0098] The following example serves as a basic demonstration of the present immuno-PCR technical approach using a photosensitizer nanoconjugate, a cleavable linker, and a DNA surrogate (a fluorophore in order to follow the release of the surrogate, Molecular Weight = 1079 g/mol).

[0099] Materials/Methods - A photosensitizer (Phthalocyanine) was incorporated into white, carboxy modified, polystyrene nanoparticles. These particles were then aminated by coating them with amino-dextran polymer in the presence of EDC. After extensive washing, the amine-modified nanoconjugate was resuspended, characterized for the presence of amines and monodispersity, and stored in double-deionized water for later use (see Structure 1 ).

Structure 1 - Amine-modified nanoconjugate.

[00100] The photocleavabe linker 2-Amino-4-thiazoleacetic acid (Alpha-Aesar) was modified with amine reactive, N-hydroxysuccinimide activated Alexa-546 dye (NHS-Alexa-546, Invitrogen) as a DNA surrogate to create the following subcomponent (see Structure 2). The use of this DNA surrogate enables tracking its presence throughout the experiment.

DNA Surrogate

Structure 2 - Photocleavable Linker with conjugated DNA Surrogate.

[00101] This subcomponent was then reacted with an excess of amine-modified, photosensitizer nanoconjugate from above in the presence of EDC. After extensive washing, the final nanoconjugate (see Structure 3) was resuspended and stored in double-deionized water for later use.

Surrogate

Structure 3 - Final nanoconjugate. [00102] Experimental - Ten microliters of the final nanoconjugate were activated for ten seconds using a 680 nm, 300 mW laser diode (Perkin-Elmer). The resulting nanoconjugate suspension and its fluorescence characteristics were compared to the non-Alexa-dyed amine-modified nanoconjugate and the non- activated, final nanoconjugate using a fluorescence microscope equipped with a scientific grade, CCD camera (Leica). Briefly, each nanoconjugate suspension was diluted 1 :10 in double-deionized water and applied to a glass slide for analysis. Each suspension was then centrifuged at 13,000 x g for five minutes and the resulting supernatants applied to glass slides for analysis. Fluorometric measurements from the phthalocyanine photosensitizer dye (Excitation 680 nm / Emission 700 nm) and the Alexa-546, DNA surrogate dye (Excitation 556 nm / Emission 570 nm) were performed. The nanoconjugate was then resuspended and washed again by centrifugation. The resulting nanoconjugate suspensions were applied to glass slides and measured fluorometrically as well.

[00103] Results - Each of the nanoconjugate suspension samples were analyzed microscopically to confirm the presence and release of the Alexa-546 fluorophore. The analysis included imaging of the photosensitizer nanoconjugate using the excitation/emission wavelengths of the phthalocyanine dye, as well as imaging of the location of signal from the DNA surrogate using its excitation/emission wavelengths. Comparison of the images indicated that:

(a) amine-modified photosensitizer nanoconjugate (1 ) (No Alexa-546 DNA surrogate) has pthalocyanine dye fluorescence but no Alexa-546 fluorescence,

(b) final nanoconjugate pre-laser activation (2) (with attached DNA surrogate) has Alexa-546 fluorescence associated with the nanoconjugate as well as the pthalocyanine fluorescence, and

(c) the final nanoconjugate post-laser activation (3) (released DNA surrogate) has pthalocyanine fluorescence but has little Alexa-546 fluorescence remaining associated with the nanoconjugate. [00104] In accordance with the description above, the amine-modified nanoconjugate (1 ) has only phthalocyanine dye present, and thus fluoresces only in the phthalocyanine channel. The final nanoconjugate pre-laser activation (2) has both phthalocyanine and Alexa-546 dye present, and fluoresces in both channels. The final nanoconjugate post-laser activation (3), following 2 cycles of washing, has little Alexa-546 fluorescence remaining, indicative of the release of the Alexa-546 from the nanoconjugate surface due to photoactivation of the phthalocyanine. These results indicate that upon activation of the photosensitizer the bond between the polymer and DNA surrogate is cleaved, releasing the DNA surrogate as a free compound.

[00105] To confirm the release of Alexa-546 fluorophore following phthalocyanine activation, fluorescence images were obtained and intensities (in Relative Fluorescence Units, RFU) from the Alexa-546 fluorescence channel for each of the nanoconjugate suspensions' supernatant were analyzed and the intensities are shown below in Table 1 below.

Table 1

[00106] These results indicate that: The amine-modified nanoconjugate in (1 ) and the final nanoconjugate pre-laser activation in (2) show no fluorescence in their supernatants in the Alexa-546 channel, indicating no DNA surrogate in the supernatant. The final nanoconjugate post-laser activation in (3) shows high levels of Alexa-546 fluorophore in its supernatant, confirming the release of the fluorophore from the nanoconjugate surface into solution following activation of the phthalocyanine.

[00107] The fluorescence intensity measured in the final nanoconjugate post- laser activation's supernatant exhibits the successful cleavage of the photocleavable linker holding the Alexa-546 fluorophore to the nanoconjugate. It is clear from the lack of fluorescence measured in the pre-laser activated and amino-modified nanoconjugate supernatants that it is the activation of the photosensitizer dye that has caused this cleavage.

[00108] Conclusion - The preparation of a photosensitizer-cleavable linker-DNA surrogate complex conjugated to a nanopolymer has been achieved. Activation of the photosensitizer with a long wavelength laser cleaves the linker between the DNA surrogate and polymer and initiates the rapid release of the DNA surrogate from the final nanoconjugate.

EXAMPLE 2: Photocleavage and Amplification of Released Oligomer Exemplary Experimental Protocol:

[00109] Materials - 6 urn, carboxy-modified magnetic particles (Bangs Laboratories) are functionalized with amino-PEG-biotin (Molecular Biosciences) and nucleic acid template sequence (Integrated DNA Technologies) through a photocleavable linker (Alfa-Aesar).

[00110] White, carboxy modified, polystyrene nanoparticles are dyed with photosensitizer dye (Phthalocyanine) using a procedure similar to that described in Ullman, et al., 1996, Clin Chem 42:1518. A variety of other methods known in the art for loading dyes on particles can also be used. These particles are then functionalized with streptavidin via EDC. After extensive washing, the particles are resuspended, sonicated to ensure monodispersity, characterized and stored in double-deionized water for later use.

[00111] Experimental - Four reactions containing Mag-biotin-DNA particles and streptavidin photosensitizer particles are incubated in buffer to form Mag-biotin- DNA: streptavidin photosensitizer complexes. As a set of binding specificity controls, four reactions containing Mag-biotin-DNA particles and streptavidin photosensitizer particles plus excess free biotin are also incubated.

[00112] Two reactions from each set are excited with 680 nm light to activate the photosensitizer, while two reactions from each set are left untreated. Following separation of the magnetic particles from the solution, the resulting supernatants are extracted and used as PCR template material for the known synthetic template on the Mag-biotin-DNA particles. After sufficient cycles (e.g., 20-40 cycles) of PCR, the reactions are loaded onto a gel for analysis and the levels of amplicon from each condition compared.

[00113] All patents and other references cited in the specification are indicative of the level of skill of those skilled in the art to which the invention pertains, and are incorporated by reference in their entireties, including any tables and figures, to the same extent as if each reference had been incorporated by reference in its entirety individually.

[00114] One skilled in the art would readily appreciate that the present invention is well adapted to obtain the ends and advantages mentioned, as well as those inherent therein. The methods, variances, and compositions described herein as presently representative of preferred embodiments are exemplary and are not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art, which are encompassed within the spirit of the invention, are defined by the scope of the claims.

[00115] It will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. For example, variations can be made to selection of photosensitizer and/or photocleavable linker, as well as to the type and binding specificity of the analyte specific binding moieties. Thus, such additional embodiments are within the scope of the present invention and the following claims. [00116] The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein. Thus, for example, in each instance herein any of the terms "comprising", "consisting essentially of" and "consisting of" may be replaced with either of the other two terms. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims.

[00117] In addition, where features or aspects of the invention are described in terms of Markush groups or other grouping of alternatives, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group or other group.

[00118] Also, unless indicated to the contrary, where various numerical values or value range endpoints are provided for embodiments, additional embodiments are described by taking any 2 different values as the endpoints of a range or by taking two different range endpoints from specified ranges as the endpoints of an additional range. Such ranges are also within the scope of the described invention. Further, specification of a numerical range including values greater than one includes specific description of each integer value within that range.

[00119] Thus, additional embodiments are within the scope of the invention and within the following claims.

Claims

WHAT IS CLAIMED IS:

1. A method for detecting at least one non-nucleic acid analyte in a sample solution, comprising detecting the presence of a pre-selected amplified nucleic acid molecule, wherein said nucleic acid molecule is amplified from an oligomer released from an analyte-linked oligomer-containing construct by cleavage of a photocleavable linker.

2. The method of claim 1 , wherein said method further comprises binding said analyte to a first analyte-specific binding construct comprising a photosensitizer moiety and a first analyte-specific binding moiety, and to a second analyte-specific binding construct comprising a second analyte-specific binding moiety, linked with an amplifiable nucleic acid oligomer through a photocleavable linker, wherein said cleavage substantially only occurs when said first and second analyte specific binding constructs are bound to the same analyte entity.

3. The method of claim 2, wherein said second analyte-specific binding construct further comprises a separation moiety.

4. The method of claim 3, wherein said separation moiety comprises a magnetic bead.

5. The method of claim 4, wherein said amplifiable nucleic acid oligomer is linked with said magnetic bead through said photocleavable linker.

6. The method of claim 2, further comprising separating bound analyte from said sample solution.

7. The method of claim 2, further comprising separating uncleaved second analyte-specific binding construct from released amplifiable nucleic acid oligomer.

8. The method of claim 2, further comprising cleaving said amplifiable nucleic acid oligomer from said second analyte-specific binding moiety.

9. The method of claim 2, further comprising cleaving said amplifiable nucleic acid oligomer from said second analyte- specific binding moiety thereby providing released amplifiable nucleic acid oligomer; separating the remainder of said second analyte specific binding moiety from said released amplifiable nucleic acid oligomer; and amplifying said released amplifiable nucleic acid oligomer.

10. The method of claim 1 , wherein said amplification is performed using isothermal amplification.

1 1. The method of claim 1 , wherein said amplification is performed using PCR.

12. The method of claim 1 , wherein said detecting comprises distinguishably detecting a plurality of different pre-selected amplified nucleic acid molecules, wherein each of said plurality of different pre-selected amplified nucleic molecules corresponds to a different analyte.

13. The method of claim 12, wherein said plurality of different pre-selected amplified nucleic acid molecules is at least 3 different pre-selected amplified nucleic acid molecules.

14. The method of claim 1 , wherein said first analyte-specific binding moiety consists essentially of an antibody.

15. The method of claim 1 , wherein said first analyte-specific binding moiety consists essentially of a receptor or fragment thereof.

16. The method of claim 1 , wherein said first analyte-specific binding moiety consists essentially of an aptamer.

17. The method of claim 1 , wherein said first analyte-specific binding moiety consists essentially of a ligand or ligand analog.

18. The method of claim 1 , wherein said second analyte-specific binding moiety consists essentially of an antibody.

19. The method of claim 1 , wherein said second analyte-specific binding moiety consists essentially of a receptor or fragment thereof.

20. The method of claim 1 , wherein said second analyte-specific binding moiety consists essentially of an aptamer.

21. The method of claim 1 , wherein said second analyte-specific binding moiety consists essentially of a ligand or ligand analog.

22. The method of claim 1 , wherein said analyte is a protein.

23. The method of claim 1 , wherein said analyte is a nucleic acid molecule.

24. The method of claim 1 , wherein said analyte is a small molecule.

25. The method of claim 1 , wherein said analyte is a drug.

26. The method of claim 1 , wherein said analyte is a toxin.

27. A method for amplifying a specific binding event signal, comprising releasing an amplifiable nucleic acid oligomer from an analyte-bound analyte specific binding moiety by cleaving a photocleavable linker located between said analyte-bound analyte specific binding moiety and said amplifiable nucleic acid oligomer, thereby providing a released oligomer; and amplifying said released oligomer.

28. The method of claim 27, wherein said amplifying is performed using isothermal amplification.

29. The method of claim 27, wherein said amplifying is performed using PCR.

30. The method of claim 27, wherein a plurality of distinguishable oligomers respectively bound to different analytes is released.

31. The method of claim 27, wherein said analyte specific binding moiety comprises a magnetic bead, and said amplifiable oligomer is linked to said bead through a photocleavable linker.

32. The method of claim 31 , wherein said magnetic bead is separated from said amplifiable oligomer prior to said amplifying.

33. A kit for detecting an analyte in a solution, comprising a first analyte-specific binding construct, comprising a photosensitizer moiety and a first analyte-specific binding moiety; and separate from said first analyte-specific binding construct, a second analyte-specific binding construct comprising a second analyte-specific binding moiety linked with an amplifiable nucleic acid oligomer through a photocleavable linker.

34. A signal amplification construct, comprising an analyte-specific binding molecule linked with a photocleavable linker, which is also linked with an amplifiable nucleic acid molecule.

35. The construct of claim 34, further comprising a magnetic bead.

36. The construct of claim 35, wherein said amplifiable nucleic acid molecule is linked with said magnetic bead through said photocleavable linker.

37. The construct of claim 34, wherein said analyte-specific binding molecule is bound with an analyte.

38. The construct of claim 37, wherein said analyte is also bound with a photosensitizer construct, wherein said photosensitizer construct comprises a second analyte specific binding molecule and a photosensitizer moiety.