HK1127093B - Multiplexed analyses of test samples - Google Patents

Description

Multiplex analysis of test samples

Technical Field

The present invention relates to methods, devices, reagents and kits for detecting target molecules in a sample, and more particularly to detecting and/or quantifying one or more target molecules contained in a test sample.

Background

The following description provides a summary of information relevant to the present invention and is not an admission that any of the information provided herein or publications cited are prior art to the present invention.

Assays for the detection and quantification of physiologically important molecules in biological and other samples are important tools in the fields of scientific research and medicine. One class of such assays involves the use of microarrays comprising one or more aptamers immobilized on a solid support. These aptamers are each capable of binding to a target molecule with high specificity and high affinity. See U.S. Pat. No. 5,475,096 entitled "Nucleic Acid Ligands" and also U.S. Pat. Nos. 6,242,246, 6,458,543 and 6,503,715 entitled "Nucleic Acid Ligand Diagnostic Biochip". Once the microarray and sample are contacted, the aptamers bind to their respective target molecules present in the sample, so that the presence, amount and/or concentration of the target molecules in the sample can be determined.

One variation of this assay uses aptamers that include photoreactive functional groups such that the aptamers can be covalently bound or "photocrosslinked" to their target molecules. See U.S. Pat. No. 6,544,776 entitled "Nucleic Acid Ligand Diagnostic Biochip". These photoreactive aptamers are also referred to as "photoaptamers". See the title "Systematic Evolution of nucleic Acid Ligands by exponentiall entity: U.S. Pat. Nos. 5,763,177, 6,001,577 and 6,291,184 to Photoelection of nucleic Acid Ligands and Solution SELEX ". See also U.S. Pat. No. 6,458,539 entitled "Photoelection of Nucleic acid ligands". Upon contacting the microarray with the sample, the photoaptamers have an opportunity to bind their target molecules, the photoaptamers are activated by light, and the solid support is washed to remove any non-specifically bound molecules. Harsh washing conditions can be used because target molecules bound to the photoaptamer are not typically removed due to covalent bonding of the photoactivating functional groups on the photoaptamer. In this manner, the assay can determine the presence, amount and/or concentration of the target molecule in the test sample.

In both assay formats, the aptamer is immobilized on a solid support prior to contacting the sample. However, in certain circumstances, immobilization of the aptamer prior to contact with the sample may not provide an optimal assay. For example, pre-immobilization of the aptamer may result in insufficient mixing of the aptamer and target molecule on the surface of the solid support, which may result in an excessively long reaction time, and thus an extended incubation time to allow adequate binding of the aptamer to its target molecule. Furthermore, when photoaptamers are used in assays and depending on the material used as the solid support, the solid support may disperse or absorb the light used to effect the formation of covalent bonds between the photoaptamer and its target molecule. Furthermore, depending on the method used, detection of the target molecule bound to its aptamer may be inaccurate, as the solid support surface may likewise be exposed to and affected by any labeling substance used. Finally, immobilization of aptamers to a solid support typically involves an aptamer preparation step (i.e., immobilization) prior to exposing the aptamer to a sample, which preparation step may affect the activity or functionality of the aptamer.

Thus, there is a need for methods, apparatus, time and kits that provide highly sensitive assays for detecting and/or quantifying target molecules in a test sample by optimizing conditions that affect: (1) the activity of the aptamer, (2) the efficiency with which the equilibrium of the binding of the aptamer-target complex is achieved, (3) the formation of covalent bonds between the aptamer and its target, and (4) the detection of the aptamer-target complex.

Brief description of the invention

The present invention includes methods, devices, reagents and kits for detecting and/or quantifying one or more target molecules that may be present in a test sample. In one embodiment, the test sample is contacted with an aptamer that includes a label and has a specific affinity for the target molecule. An aptamer affinity complex is formed that includes an aptamer that binds to its target molecule. If the test sample contains the target molecule, an aptamer affinity complex will typically form in the test sample. Such aptamer affinity complexes are optionally converted to aptamer covalent complexes that include an aptamer covalently bound to its target molecule. Subsequently, such aptamer affinity complexes (or optionally aptamer covalent complexes) can be detected and/or quantified by any of a variety of methods known to those skilled in the art including, but not limited to, the use of solid supports, the use of mass spectrometry, and the use of quantitative polymerase chain reaction (Q-PCR).

In one embodiment, the aptamer affinity complex (or optionally aptamer covalent complex) is detected and/or quantified by using a solid support. In this embodiment, the aptamer affinity complex (or optionally the aptamer covalent complex) is attached to a solid support. Attachment is achieved by contacting the solid support with an aptamer affinity complex (or optionally an aptamer covalent complex) and allowing the tag comprised in the aptamer to associate, directly or indirectly, with the probe attached to the solid support. Aptamer affinity complexes (or optionally aptamer covalent complexes) associated with the probes on the solid support are then detected and optionally quantified. The complex is contacted with a labeling substance at any time prior to detection and optionally quantification, i.e. before or after the aptamer affinity complex (or optionally the aptamer covalent complex) is attached to the solid support, such that the bound target molecule can be detected.

In another embodiment, the aptamer affinity complex (or optionally aptamer covalent complex) is detected and/or quantified using mass spectrometry. In this embodiment, the aptamer affinity complex (or optionally the aptamer covalent complex) is attached to the solid support by contacting the solid support with the aptamer affinity complex (or optionally the aptamer covalent complex) and allowing the tag comprised in the aptamer to associate, directly or indirectly, with the probe on the solid support. This facilitates separation of the aptamer affinity complex (or optionally the aptamer covalent complex) from the remainder of the test sample, thereby concentrating the target molecule prior to mass spectrometry analysis and improving the detection and quantification of analytes in the complex mixture using such analytical tools. The aptamer affinity complex (or optionally aptamer covalent complex) associated with the probe on the solid support is then eluted and analyzed by mass spectrometry, which generates a peak spectrum that can be used to identify and thereby detect the target molecule. Once the target molecule is detected, it can optionally also be quantified using standard techniques known to those skilled in the art. In one embodiment, where the target molecule is a protein, prior to analyzing the aptamer affinity complex (or optionally the aptamer covalent complex) using mass spectrometry, the aptamer affinity complex (or optionally the aptamer covalent complex) can be digested with a protease, such as proteinase K or trypsin, to produce a fragment of the bound target molecule that can be used to identify the target molecule, so that the target molecule can be detected and optionally quantified.

In further embodiments, the aptamer affinity complex (or optionally aptamer covalent complex) is detected and/or quantified using Q-PCR. In this embodiment, the free aptamer in the test sample is separated from the aptamer affinity complex (or optionally the aptamer covalent complex) prior to detection and/or quantification. Quantification of the aptamer affinity complex (or optionally the aptamer covalent complex) is performed by first performing PCR and then directly or indirectly determining the amount or concentration of aptamer bound to the target molecule in the test sample. The amount or concentration of the target molecule in the test sample is generally directly proportional to the amount and concentration of aptamer quantified using Q-PCR. A typical method that can be used in this way to quantify the aptamer affinity complex (or optionally the aptamer covalent complex) is TaqManAssays (PE Biosystems, Foster City, Calif.; see also U.S. Pat. No. 5,210,015).

Brief Description of Drawings

FIGS. 1A and 1B illustrate a method of detecting and/or quantifying one or more target molecules that may be present in a test sample.

FIGS. 2A, 2B, and 2C illustrate a method of detecting and/or quantifying one or more target molecules that may be present in a test sample.

FIG. 3 illustrates a method of detecting and/or quantifying one or more target molecules that may be present in a test sample.

Figure 4 shows dose response curves for VEGF in serial dilutions of buffer (figure 4A) and plasma (figure 4B) determined using the assays depicted in figures 2A, 2B and 2C. The non-protein buffer response was subtracted from the midpoint in each data of both groups. The least squares line fitting the log transformed data is shown.

Fig. 5A-5J show dose response curves for compounding 41 photoaptamers with 10 target proteins in serial dilutions of buffer determined using the assays depicted in fig. 2A, 2B, and 2C. The non-protein buffer response for each aptamer was subtracted in each data point of the set. The least squares line fitted to the Log transform data is also plotted. Only the data points used in the fit line are shown.

Fig. 6A and 6B show repeated measurements in RFU of the response of 57 photoaptamers in two individual serum samples obtained from the analysis depicted in fig. 2A, 2B and 2C. Both sets of repeated measurements showed very good repeatability for 57 targets of measurement, with Pearson correlation coefficient greater than 0.99.

Fig. 7 shows dose response curves for tPA in buffer (●) and plasma (a-solidup) using an optional kinetic challenge (kinetic challenge) UPS hybrid capture assay. The non-protein buffer responses were averaged and subtracted from both curves. In plasma samples without the target protein, the diluted plasma response without kinetic challenge in tPA at 0.1pM is indicated by (□) and with kinetic challenge by (. DELTA.). The response measured in plasma was reduced by almost 1 log due to kinetic challenge, whereas no change in target-aptamer response was demonstrated from the results for buffer (. smallcircle.) and plasma (. DELTA.) with 10nM tPA.

Figure 8 shows the dose response curve of tPA in plasma measured with an optional kinetic-stimulated assay with competitor (■) and without competitor (●). Non-protein plasma values of 1pM [ tPA ] are depicted and reduced by 70% due to the addition of competitor, whereas the response in 30nM tPA demonstrates no change in target-aptamer response, which is essentially the same with and without competitor.

FIG. 9 shows dose response curves for 3 target proteins (tPA (FIG. 9A), PAI-1 (FIG. 9B) and IL-6 (FIG. 9C)) in buffer (●) and plasma (a). RFU values were corrected by subtracting the non-protein buffer RFU values for each aptamer. The least squares line of the buffer data fit to the log transformed data is depicted. The aptamer (Δ at 1pM) corrected non-protein plasma RFU values were 66, 26 and 49RFU, respectively.

FIGS. 10A-10D show dose response curves for 4 target proteins added to plasma crosslinked in buffer and prior to optional removal of free aptamer by K +/SDS precipitation (●), compared to the curve generated without removal of free aptamer (■). The signal is enhanced after removal of free aptamer, and the kinetic range of the measurement is also generally increased.

FIG. 11 shows the effect of photoinduced chemical cross-linking of target proteins (bFGF) and their photoaptamers when detergents and high salt concentrations are used in hybridization. In the absence of light, and thus no target covalently attached to its photoaptamer, the assay signal in the buffer decreased by more than 2 orders of magnitude. The endogenous bFGF concentration was rather low, as reflected by a weak signal relative to the no light control and general background response.

FIG. 12 shows the dose response of the target protein (C4b) in buffer, using a modified 5-benzyl-dT library instead of dT to develop the photoaptamer. A linear response of 3 log values of target concentration indicates the activity of the modified nucleotide aptamer in the assay.

FIG. 13 shows the dose response curves generated by direct labeling of the target protein (. tangle-solidup.) or fluorescently labeled streptavidin (■) after biotinylation on either the Schott Nexterion surface (FIG. 13A) or the methacrylate copolymer surface (FIG. 13B). Both surfaces perform well and both marking strategies are comparable.

FIG. 14 illustrates hybridization of aptamer-target complexes in buffer (FIG. 14A) or 10% serum (FIG. 14B) and in Affymetrix GeneChipTest3 Array with NHS-PEO₄-a biotin label. Staining with Phoroyrythronin-R was performed in Affymetrix GeneChipApplication is performed on a fluidics workstation. In buffer (FIG. 14A), VEGF aptamer hybridized to probe 201(1) at an intensity of 3500RFU, bFGF aptamer hybridized to probe 1121(2) at an intensity of 23000 RFU. In serum (fig. 14B), VEGF and bFGF aptamers corresponded to relative intensities of 5000(1) and 18000(2), respectively.

FIG. 15 illustrates blocking of AffymetrixGeneChip prior to aptamer-target complex hybridization in plasma samplesTest3 Array. Biotinylated probes were hybridized to surfaces blocked with skim milk (fig. 15B), "starter block" (fig. 15C) and unlabeled plasma (fig. 15D) in buffer (fig. 15A) and in plasma samples. The background values for these four surfaces were 49, 300, 400 and 500RFU, while the hybridization signals for these three probes were 16000, 33000 and 18000 in (fig. 15A) and (fig. 15B), 17000, 35000 and 18000 in (fig. 15C), and 20000, 36000 and 18000 in (fig. 15D).

FIG. 16 illustrates addition to plasma, cross-linking with photoaptamers, and use in Affymetrix GeneChipQuantitative detection of the hybridized target protein on Test3 Array. The hybridization response of the aptamer complex of the target proteins IL1-R4 (. tangle-solidup.) and bFGF (■) formed in plasma was subtracted by the non-protein responseThe RFU value answered. After blocking the array surface to reduce adsorption of molecules in the sample matrix, a quantitative range of two-log was observed from the plasma samples.

Figure 17 shows the target protein dose response curves for 3 target proteins multiplexed with photoaptamers, serially diluted in buffer. Photoaptamer-cross-linked target proteins by Luminex Seromap conjugated with specific oligonucleotides^TMMicrosphere hybridization is captured. The Luminex 100 IS equipment system IS used for signal quantification. MFI (intermediate fluorescence intensity) values were corrected by subtracting non-protein control MFI values from each aptamer.

Detailed Description

The practice of the invention disclosed herein employs, unless otherwise indicated, conventional chemical, microbiological, molecular biological and recombinant DNA techniques within the skill of the art. These techniques are explained in detail in the following documents: see, e.g., Sambrook et al, molecular cloning: a Laboratory Manual (Current Edition); DNA Cloning: APractcal Approach, vol.I & II (D.Glover, ed.); oligonucleotide Synthesis (n.gait, ed., Current Edition); nucleic Acid Hybridization (b.hames & s.higgins, eds., Current Edition); transformation and transformation (b.hames & s.higgins, eds., Current Edition).

All publications, published patent documents and patent applications cited in this specification are indicative of the level of skill in the art to which this invention pertains. Accordingly, all publications, published patent documents and patent applications cited herein are incorporated by reference to the same extent as if each individual publication, published patent document or patent application were specifically and individually indicated to be incorporated by reference.

As used in this specification, including the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise, and are to be construed interchangeably with "at least one" and "one or more". Thus, "an aptamer" includes mixtures of aptamers, "a probe" includes mixtures of probes, and the like.

As used herein, the terms "comprises," "comprising," "includes," "including," "contains," "containing," and variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article of manufacture, or composition of matter, which comprises, includes, or contains an element or a series of elements, does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article of manufacture, or composition of matter.

The present invention encompasses methods, devices, reagents and kits for detecting and/or quantifying one or more target molecules that may be present in a test sample. The disclosed methods, apparatus, reagents and kits provide highly sensitive assays for detecting and/or quantifying target molecules in a test sample by optimizing conditions that affect: (1) the activity of the aptamer, (2) the efficiency with which the equilibrium of aptamer-target complex binding is achieved, (3) the formation of covalent bonds between the aptamer and its target and (4) the detection of the aptamer-target complex.

Referring to fig. 1A and 1B, a test sample is first contacted with an aptamer having a specific affinity for a target molecule to detect and/or quantify the presence of the target molecule in the test sample. Aptamer affinity complexes are formed that include an aptamer that binds to its target molecule. If the test sample contains the target molecule, aptamer affinity complexes will typically form in the test sample. Aptamer affinity complexes are optionally converted to aptamer covalent complexes, including aptamers covalently bound to their target molecules, using methods appropriate to the aptamer used. Subsequently, the aptamer affinity complex (or optionally the aptamer covalent complex) is detected and/or quantified.

In one embodiment, the aptamer affinity complex (or optionally aptamer covalent complex) is detected and/or quantified by attaching the aptamer affinity complex (or optionally aptamer covalent complex) to a solid support. Referring to fig. 2A, 2B and 2C, in an exemplary method of detecting and/or quantifying a target molecule that may be present in a test sample, the test sample is contacted with an aptamer that includes a label and has a specific affinity for the target molecule. Forming an aptamer affinity complex comprising an aptamer that binds to its target molecule. If the test sample contains the target molecule, aptamer affinity complexes will typically form in the test sample. The aptamer affinity complex is optionally converted to an aptamer covalent complex comprising an aptamer covalently bound to its target molecule using methods appropriate for the aptamer used. The aptamer affinity complex (or optionally the aptamer covalent complex) is attached to a solid support. Attachment is achieved by contacting the solid support with an aptamer affinity complex (or optionally an aptamer covalent complex) and allowing the tag included on the aptamer to associate, directly or indirectly, with the probe attached to the solid support. Aptamer affinity complexes (or optionally aptamer covalent complexes) associated with the probes on the solid support are then detected and optionally quantified. The complex is contacted with a labeling substance at any time prior to detection and optionally quantification, i.e. before or after the aptamer affinity complex (or optionally the aptamer covalent complex) is attached to the solid support, such that the bound target molecule can be detected.

As used herein, "nucleic acid," "oligonucleotide," and "polynucleotide" are used interchangeably to refer to a polymer of nucleotides of any length, such nucleotides can include deoxyribonucleotides, ribonucleotides and/or analogs, or chemically modified deoxyribonucleotides or ribonucleotides. The terms "polynucleotide", "oligonucleotide" and "nucleic acid" encompass double-or single-stranded molecules, as well as triple-helical molecules.

Chemical modifications of nucleotides, if present, may include single modifications or any combination of the following: 2-sugar modifications, 5-pyrimidine modifications (e.g., 5- (N-benzylcarboxamide) -2 '-deoxyuridine, 5- (N-isobutylcarboxamide) -2' -deoxyuridine, 5- (N- [2- (1H-indol-3 yl) ethyl ] carboxamide) -2 '-deoxyuridine, 5- (N- [1- (3-trimethylammonium) propylcarboxamide) -2' -deoxyuridine chloride, 5- (N-naphthylcarboxamide) -2 '-deoxyuridine and 5- (N- [1- (2, 3-dihydroxypropyl) ] carboxamide) -2' -deoxyuridine), 8-purine modifications, exocyclic amine modifications, 4-thiouridine substitutions, 5-bromo or 5-iodouracil substitutions, backbone modifications, methylation, aberrant base-pairing combinations such as isobasesiocytidine and isoguanidine (isoglunidine), and the like. Modifications may also include 3 'and 5' modifications such as capping. Other modifications may include substitution of one or more of the natural nucleotides with an analog, internucleotide modifications such as those without electrical linkages (methyl phosphates, phosphotriesters, phosphoramides, carbamates, etc.) and charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), intercalator modifications (e.g., acridine, psoralen), chelator-containing modifications (e.g., metals, radioactive metals, boron, and oxidative metals, etc.), alkylator-containing modifications, and modifications with modified linkages (e.g., alpha anomeric nucleic acids, etc.). Furthermore, any hydroxyl groups typically present in sugars may be replaced with phosphate or phosphate groups, protected with standard protecting groups, or activated to make additional linkages to additional nucleotides or solid supports. The 5 'and 3' terminal OH groups may be phosphorylated or substituted with an amine, an organic capping group moiety of about 1 to about 20 carbon atoms, or an organic capping group moiety of about 1 to about 20 polyethylene glycol (PEG) polymers or other hydrophilic or hydrophobic biological or synthetic polymers. Modifications to the nucleotide structure, if present, may be made before or after assembly of the polymer. The nucleotide sequence may be interrupted by non-nucleotide components. The polynucleotide may be further modified after polymerization, such as by conjugation with a labeling element.

Polynucleotides may also contain analogs of ribose or deoxyribose commonly known in the art, including 2 '-O-methyl-, 2' -O-allyl, 2 '-fluoro-or 2' -azido-ribose, carbocyclic sugar analogs, alpha-anomeric sugars, epimeric sugars such as arabinose, xylose, or lyxose, pyranose, furanose, sedoheptulose, acyclic analogs, and abasic nucleoside (abasic nucleoside) analogs such as methyl nucleosides. As mentioned above, one or more phosphodiester bonds may be replaced by other linking groups. These other linking groups include embodiments in which the phosphate is replaced with: p (O) S ("thioate"), p (S) S ("dithioate"), (O) NR2 ("amide"), p (O) R, p (O) OR ', CO OR CH2 ("formacetal"), wherein R OR R' are each independently H OR substituted OR unsubstituted alkyl (1-20C), optionally containing an ether (-O-) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl OR araldyl. The linkage in not all polynucleotides must be identical. Substitution of analogues of sugars, purines and pyrimidines may be advantageous for designing the final product, for example the backbone structure may be altered like that of polyamides.

As used herein, "aptamer" and "nucleic acid ligand" are used interchangeably to refer to a nucleic acid having specific binding affinity for a target molecule. Affinity interactions are known to be a matter of degree, however, in this context, "specific binding affinity" of an aptamer to its target means that the aptamer binds its target generally with much higher affinity than to other components in the test sample. An "aptamer" is a set of copies of one type or more nucleic acid molecules having a particular nucleotide sequence. Aptamers can include any suitable number of nucleotides. "aptamer" refers to more than one group of such molecules. Different aptamers may have the same or different number of nucleotides. Any of the methods disclosed herein can include the use of one or more aptamers. Any of the methods disclosed herein may also include the use of two or more aptamers that specifically bind to the same target molecule. In further description below, aptamers may include labels. If the aptamer includes a tag, all aptamer copies need not have the same tag. Moreover, if different aptamers each include a label, these different aptamers may have the same or different labels.

Aptamers can be identified by any known method, including the SELEX method. See, for example, U.S. Pat. No. 5,475,096 entitled "Nucleic Acid Ligands". Once identified, aptamers may be prepared or synthesized by any method known, including chemical and enzymatic synthesis methods.

The terms "SELEX" and "SELEX method" are used interchangeably herein and generally refer to a combination of the following methods: (1) selecting a nucleic acid that can interact with the target molecule in a desired manner, e.g., a high affinity binding protein, (2) amplifying such selected nucleic acid. See, for example, U.S. Pat. No. 5,475,096 entitled "nucleic acids Ligands". The SELEX method can be used to produce aptamers that bind covalently to a target as well as aptamers that bind non-covalently to a target. See, for example, the title "systematic evolution of Nucleic Acid Ligands by Exponential entity: U.S. Pat. No. 5,705,337 to Chemi-SELEX.

As indicated herein, an aptamer may further comprise a "label," which refers to a component that provides a means to attach or immobilize the aptamer (and any target molecule bound to it) on a solid support. A "tag" is a set of copies of one or more species of a component that can associate with a probe. "multiple indicia" refers to more than one such grouping of ingredients. Labels may be attached to or included on the aptamer by any method known in the art. Typically, the label is such that the aptamer can be associated directly or indirectly with a probe attached to a solid support. The label can localize the aptamer covalent complex to a spatially specific address on the solid support. Thus, different labels may localize different aptamer covalent complexes to different spatially specific addresses on the solid support. The label may be a polynucleotide, a polypeptide, a peptide nucleic acid, a locked nucleic acid, an oligosaccharide, a polysaccharide, an antibody, an affybody, an antibody mimetic, a cellular receptor, a ligand, a lipid, any fragment or derivative of these structures, any combination of the above, or any other structure to which a probe (or linker molecule, as described below) can be designed to specifically bind or associate. Typically, the label is designed not to interact intramolecularly with the aptamer itself or to which it is attached or part of. If SELEX is used to identify aptamers, a marker may be added to the aptamers before or after SELEX. In one embodiment, the tag is included at the 5' end of the aptamer after SELEX. In another embodiment, the label is included at the 3' end of the aptamer after SELEX.

In one embodiment, the tag comprises a polynucleotide designed to associate with a probe comprising a complementary nucleotide sequence by direct hybridization to the probe sequence. In this embodiment, the label is generally designed and the hybridization reaction is carried out under conditions such that the label does not hybridize to probes other than the probe to which the label includes a sequence that is fully complementary.

In some embodiments, the tag comprises a nucleotide that is part of the aptamer itself. For example, if SELEX is used to identify aptamers, aptamers typically include a5 'fixed end that is separated from a 3' fixed end by a variable nucleotide sequence, i.e., a variable region, that depends on the aptamer. In one embodiment, the tag may comprise any suitable number of nucleotides included in the immobilized end of the aptamer therein, e.g., all or part of the immobilized end, including nucleotides internal to the immobilized end. In another embodiment, the tag may comprise any suitable number of nucleotides included in the variable region of the aptamer, e.g., all or part of the variable region. In further embodiments, the tag may comprise any suitable number of nucleotides that overlap the variable region and one fixed end, i.e., the tag may comprise a nucleotide sequence that includes any portion (including all) of the variable region and any portion (including all) of the fixed end.

In another embodiment, the label is directly associated with the probe and covalently binds to the probe, thereby covalently linking the aptamer to the surface of the solid support. In this embodiment, the label and probe may include suitable reactive groups that, upon association of the label and probe, are sufficiently close to one another to undergo a chemical reaction that forms a covalent bond. This reaction may occur spontaneously or may require activation, for example photo-or chemical activation. In an exemplary embodiment, the label comprises a diene moiety and the probe comprises a diene affibody, the diene and diene affibodies forming a covalent bond by a spontaneous Diels-Alder conjugation reaction. Any suitable complementary chemistry may be used, for example, N-Mannich reactions, disulfide bond formation, Curtius reactions, aldol condensations, Schiff base formation, and Michael additions.

In another embodiment, the label is indirectly associated with the probe, for example, via a linker molecule, as described below. In this embodiment, the label comprises a polynucleotide sequence complementary to a particular region or component of the linker molecule. The label is typically designed and the hybridization reaction is performed such that the label does not hybridize to other polynucleotide sequences than the polynucleotide sequence comprised by the adapter molecule.

If the tag comprises a polynucleotide, this polynucleotide may comprise any suitable number of nucleotides. In one embodiment, the label comprises at least about 10 nucleotides. In another embodiment, the label comprises from about 10 to about 45 nucleotides. In another embodiment, the label comprises at least about 30 nucleotides. Different labels comprising polynucleotides may comprise the same or different numbers of nucleotides.

The term "about" as used herein denotes an insignificant modification or variation of the numerical value which does not alter the basic function to which it is related.

As used herein, "associate" and any variation thereof refers to an interaction or coordination between the label and probe and creates a digger-stable complex such that other "unassociated" or unbound substances, such as components of an unbound test sample, can be separated from the label-probe complex, e.g., under a given coordination or reaction condition. Labels and probes can be directly associated with each other by specifically interacting and binding to each other. Labels and probes may also be associated with each other indirectly, for example, at a time via coordination mediated by a linker molecule.

As used herein, a "probe" refers to a molecule that is designed to associate, either directly or indirectly, with a label. A "probe" is a set of copies of a molecule or a multi-molecular structure of a type that can immobilize an aptamer on a solid support by direct or indirect association with a label. "multiple probes" refers to more than one such molecular group. Probes may be polynucleotides, polypeptides, peptide nucleic acids, locked nucleic acids, oligosaccharides, polysaccharides, antibodies, affybods, antibody mimetics, cell receptors, ligands, lipids, any fragment or derivative of these structures, any combination of the above, or any other structure to which a label (or linker molecule) may be designed to specifically bind or associate. Probes may be covalently or non-covalently attached to a solid support using any method known in the art.

In one embodiment, the probe comprises a polynucleotide having a sequence complementary to a polynucleotide tag sequence. In this embodiment, the probe is typically designed to perform a hybridization reaction under conditions such that the probe does not hybridize to a nucleotide sequence other than a label that includes a sequence complementary to the probe (i.e., the probe is typically configured such that the hybridization reaction is performed under conditions such that the probe does not hybridize to a different label or aptamer).

In another embodiment, the probe is indirectly associated with the label, e.g., via a linker molecule. In this embodiment, the probe may comprise a polynucleotide sequence complementary to a particular region or component of the linker molecule. The probe is generally designed to undergo a hybridization reaction so that the probe does not hybridize to a nucleotide sequence other than the polynucleotide sequence included in the linker moiety.

If the probe comprises a polynucleotide, this polynucleotide may comprise any suitable number of nucleotides. In one embodiment, the probe comprises at least about 10 nucleotides. In another embodiment, the probe comprises about 10-45 nucleotides. In another embodiment, the probe comprises at least about 30 nucleotides. Different probes comprising a polynucleotide may comprise the same or different number of nucleotides.

As used herein, a "linker molecule" refers to one or more molecules designed to mediate the association of a label with a probe. Typically, the linker molecule is bifunctional, comprising a functional group attached to the label and a functional group attached to the probe. A "linker molecule" is a set of copies of one or more types of molecules or multi-molecular structures that can associate labels and probes. "multiple linker molecules" refers to a set or more of such molecules or multimolecular structures. The linker molecule may have any suitable configuration and may comprise any suitable components, including polynucleotides, polypeptides, peptide nucleic acids, locked nucleic acids, oligosaccharides, polysaccharides, antibodies, affybods, antibody mimetics, polyethylene glycol (PEG) molecules, cellular receptors, ligands, lipids, any fragment or derivative of these structures, any combination of the above, or any other structure or chemical component that may be designed to mediate the specific association of a label and a probe. The linker molecule may be aliphatic or aromatic.

The composition of the linker molecule is not important to any of the methods disclosed herein. It is generally preferred that the linker molecule is hydrophilic. In general, the length of a particular linker molecule can be selected for ease of synthesis and ease of mediating association of labels and probes. The linker molecule should not contain functionality or length that would interfere with the reactions required for the methods of the invention.

Referring to FIGS. 2A, 2B and 2C, when the linker molecule is used in any of the methods disclosed herein, the linker molecule can be introduced at any suitable time for performing the assay and can be contacted with the label or probe first. For example, a label included on the aptamer may be contacted with the linker molecule at any time prior to contacting the aptamer covalent complex with the probe on the solid support. In another embodiment, the probe attached to the solid support may be contacted with the linker molecule at any time prior to exposure of the probe to the label on the aptamer covalent complex. In further embodiments, the probe may be contacted with the label on both the linker molecule and the aptamer covalent complex, e.g., depending on the complexity of the particular assay being performed and the reaction conditions.

Linker molecules typically comprise a tag association component and a probe association component. The label association component and probe association component are independently selected based on the particular label and probe used in a particular assay. In one embodiment, the tag associates into a polynucleotide that is complementary to a polynucleotide sequence included in the tag. In another embodiment, the probe associates as a polynucleotide that is complementary to a polynucleotide sequence included in the probe. In a further embodiment, the tag association component is a polynucleotide and the probe association component is a polynucleotide.

In a further embodiment, the linker molecule comprises a tag association component separated from a probe association component by a third component. In this embodiment, this third component may include one or more molecules or subcomponents, including polynucleotides, polypeptides, peptide nucleic acids, locked nucleic acids, oligosaccharides, polysaccharides, antibodies, affybods, antibody mimetics, aliphatic carbon molecules, polyethylene glycol (PEG) molecules, cellular receptors, ligands, lipids, any fragments or derivatives of these structures, any combinations of the above, or any other chemical structure or component that may aid in label and probe association, such as increasing the elasticity between the label association component and the probe association component.

The polynucleotide component of the linker molecule may comprise any suitable number of nucleotides. In one embodiment, the polynucleotide component of the linker molecule comprises at least about 10 nucleotides. In another embodiment, the polynucleotide component of the linker molecule comprises about 10-45 nucleotides. In another embodiment, the polynucleotide component of the linker molecule comprises at least about 30 nucleotides. Linker molecules used in any of the methods disclosed herein can include polynucleotide components having the same or different number of nucleotides.

As used herein, "photoaptamer," "photoreactive nucleic acid ligand," and "photoreactive aptamer" are used interchangeably to refer to aptamers that contain one or more photoreactive functional groups that can be covalently bound or "crosslinked" to a target molecule. For example, a native nucleic acid residue can be modified to include a chemical functionality that confers photoreactivity to the nucleic acid residue upon exposure to a radiation source of suitable wavelength. Photoaptamers can be identified and/or prepared by any known method. In some embodiments, the photoreactive aptamer is identified using the light SELEX method. See, for example, the headings "Systematic Evolution of nucleic Acid Ligands by Exponential entity: photoselect of nucleic Acid Ligands and Solution SELEX U.S. Pat. Nos. 5,763,177, 6,001,577, and 6,291,184. See also, for example, U.S. patent 6,458,539 entitled "Photoselection of nucleic acids Ligands". In other embodiments, aptamers are prepared and subsequently modified to incorporate one or more photoreactive functional groups, thereby generating photoaptamers. In these embodiments, one or more photoreactive nucleic acid residues may be incorporated into the aptamer by replacing one or more other nucleotides, such as one or more thymine and/or cytosine nucleotides in the aptamer, with a photoreactive nucleic acid residue or by modifying one or more nucleic acid residues to include a photoreactive functional group.

Exemplary photoreactive functional groups that can be used for incorporation into the photoaptamer include: 5-bromouracil, 5-iodouracil, 5-bromovinyluracil, 5-iodovinyluracil, 5-azidouracil, 4-thiouracil, 5-thiouracil, 4-thiocytosine, 5-bromocytosine, 5-iodocytosine, 5-bromovinylcytosine, 5-iodovinylcytosine, 5-azidocytosine, 8-azidoadenine, 8-bromoadenine, 8-iodoadenine, 8-azidoguanine, 8-bromoguanine, 8-iodoguanine, 8-azidohypoxanthine, 8-bromohypoxanthine, 8-iodohypoxanthine, 8-azidoxanthine, 8-bromoxanthine, 8-iodoxanthine, 5- [ (4-azidophenacyl) thio ] cytosine, 5- [ (4-azidophenacyl) thio ] uracil, 7-deaza-7-iodoadenine, 7-deaza-7-iodoguanine, 7-deaza-7-bromoadenine and 7-deaza-7-bromoguanine.

In addition to these exemplary nucleoside-based photoreactive functional groups, other photoreactive functional groups that can be added to the aptamer ends with suitable linker molecules can also be used. These photoreactive functional groups include: benzophenone, anthraquinone, 4-azido-2-nitroaniline, psoralen, any derivative of these, and the like.

The photoreactive functional group incorporated into the photoaptamer can be activated by any suitable method. In one embodiment, the photoaptamer containing the photoreactive functional group is cross-linked to its target by exposing the photoaptamer affinity complex to a source of electromagnetic radiation. Suitable types of electromagnetic radiation include ultraviolet, visible, X-ray, and gamma ray. Suitable radiation sources include the use of monochromatic or filtered polychromatic light sources.

In one embodiment, a photoreactive nucleotide such as 4-azido-2-nitroaniline may be incorporated into a photoaptamer, and light in the wavelength range of about 325nm to about 470nm may be used to irradiate the photoaptamer affinity complex comprising this photoaptamer. Excitation in these wavelength ranges can be achieved by inexpensive Light Emitting Diodes (LEDs) using a single LED or an array of LEDs because the energy required is small. Monochromatic light in the almost wavelength range of 465 to 475nm, 100 degree viewing angle and providing 38 lumens can be provided by one or more high energy LEDs. In some cases when the desired photoreactive group cannot be excited with the wavelength generated by the LED, substitution of a suitable electron-donating or electron-donating group is often used to shift the excitation wavelength of the photoreactive functional group moderately so that the wavelength generated by the LED can be used to excite the photoreactive functional group.

In one embodiment, where a photoreactive nucleotide is incorporated into a photoaptamer, light having a wavelength in the range of about 300nm to about 350nm may be used to illuminate a photoaptamer affinity complex comprising the photoaptamer to convert the photoaptamer affinity complex into a photoaptamer covalent complex.

In one embodiment, a photoreactive nucleotide such as 5-iodouracil or 5-iodocytosine may be incorporated into a photoaptamer, and light having a wavelength in the range of about 320nm to about 325nm may be used to illuminate a photoaptamer affinity complex comprising the photoaptamer. This combination allows for selective cross-linking of the aptamer containing the chromophore with its target molecule without inducing other non-specific photoreactions. For example, in the case of a target protein, any tryptophan residues that may be included in the target protein and any thymine and uracil bases that may be included in the photoaptamer may also be photoreactive. Since 5-iodouracil or 5-iodocytosine absorbs light at a wavelength of about 325nm and tryptophan and the natural nucleobases do not, the use of light of this wavelength allows for selective photoreaction at 5-iodouracil or 5-iodocytosine in the photoreactive affinity complex. For example, monochromatic light in the wavelength range of about 320nm to 325nm may be provided by a frequency-multiplied tunable dye laser emitting light at a wavelength of about 320nm or a helium-cadmium laser emitting light at a wavelength of about 325 nm.

In a further embodiment, the photoaptamer affinity complex may be exposed to a xenon chloride (XeCl) excimer laser with an emission wavelength set at about 308 nm. In this embodiment, the photoaptamer may include a photoreactive functional group (e.g., 5-bromouracil or 5-bromocytosine), and the photoaptamer affinity complex is treated with a light source to photoactivate the photoreactive functional group, whereby the photoaptamer is cross-linked with its target molecule to form a photoaptamer covalent complex.

In another embodiment, the photoaptamer may be cross-linked to its target by exposing the photoaptamer affinity complex to a high pressure mercury lamp with emission wavelength set to about 313 nm. In further embodiments, a wavelength filter may be used to limit the emitted light to greater than about 300nm to minimize the activation of chromophores other than those included in the photoaptamer affinity complex.

In a further embodiment, the photoaptamer may be cross-linked to its target by exposing the photoaptamer affinity complex to a low pressure mercury lamp with an emission wavelength set to about 254nm, which is subsequently absorbed by the phosphorous and re-emitted at a wavelength of about 300nm to about 325 nm. In this embodiment, the re-emitted light passes through a filter to remove light of about 254nm that is not absorbed by the phosphor and any light having a wavelength of about 290nm to about 305nm, which may be harmful to the target protein.

In another embodiment, a halogen photoreactive functional group such as iodouracil or bromocytosine may be incorporated into a photoaptamer, and a photoaptamer affinity complex comprising such a photoaptamer may be treated with light having a wavelength of about 350nm to about 400 nm. For example, monochromatic light set at about 355nm by the third harmonic of a YAG neodymium laser or monochromatic light set at about 351nm by the first harmonic of a xenon fluoride (XeF) excimer laser may be used.

Reference herein to "target molecule" and "target" are used interchangeably to refer to any molecule of interest that may be present in a test sample to which an aptamer may bind with high affinity and specificity. "molecule of interest" includes any minor variant of a particular molecule, for example, for proteins, in amino acid sequence, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation and modification, for example, conjugation to a labeling element that does not substantially alter the properties of the molecule. "target molecules" and "targets" are copies of a set of one or more types of molecules or multi-molecular structures that can bind to an aptamer. "multiple target molecules" and "multiple targets" refer to more than one group of such molecules. Exemplary target molecules include proteins, polypeptides, nucleic acids, carbohydrates, lipids, polysaccharides, glycoproteins, hormones, receptors, antigens, antibodies, affybodes, antibody mimetics, viruses, pathogens, toxic substances, substrates, metabolites, transition state analogs, cofactors, inhibitors, drugs, dyes, nutrients, growth factors, cells, tissues, and any fragment or portion of any of these. Aptamers can be chemical or biological molecules of virtually any size, and thus virtually any size chemical or biological molecule can be a suitable target. The target may also be modified to enhance the likelihood and strength of interaction between the target and the aptamer. In an exemplary embodiment, the target molecule is a protein. See U.S. Pat. No. 6,376,190 entitled Modified SELEX Process with Purified Protein, wherein the SELEX target is a peptide.

"polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acids of any length. The polymer may be linear or branched, it may comprise modified amino acids, it may be interrupted by non-amino acids. The term also encompasses amino acid polymers that are naturally or artificially modified, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation and modification, such as conjugation to a labeling element. Also included in the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art. The polypeptide may be single chain or associated chain.

As used herein, the terms "non-target molecule" and "non-target" are used interchangeably and refer to a molecule contained in a test sample that can form a non-specific complex with an aptamer. "non-target molecules" and "non-target" are a set of one or more types of molecular or multi-molecular structural copies that can bind to an aptamer. "multiple non-target molecules" or "multiple non-target" refers to more than one group of such molecules. It is known that one molecule is not a target for a first aptamer, but may be a target for a second aptamer. Likewise, one molecule may be the target of a first aptamer, but not the target of a second aptamer.

The term "aptamer affinity complex" as referred to herein refers to a non-covalent complex formed by the interaction of an aptamer with its target molecule. An "aptamer affinity complex" is a set of copies of one type or more species of complex formed by an aptamer that binds to its corresponding target molecule. "multiple aptamer affinity complexes" refers to more than one group of such complexes. Aptamer affinity complexes can typically be inverted or dissociated under ambient conditions, such as increased temperature, increased salt concentration, or the addition of denaturants.

The term "aptamer covalent complex" as referred to herein refers to an aptamer affinity complex in which an aptamer is induced or otherwise forms a covalent bond with its target molecule. An "aptamer covalent complex" is a set of copies of one type or more species of complex formed by an aptamer covalently bound to its corresponding target molecule. "multiple aptamer covalent complexes" refers to more than one group of such complexes. Covalent bonds or linkages between an aptamer and its target molecule can be induced by photoactivation of chemical moieties on the aptamer, including those described above for photoaptamers. Covalent bonds or linkages between an aptamer and its target molecule can also be chemically induced. Chemical groups that may be included on the aptamer for inducing covalent attachment to the target include, but are not limited to, aldehydes, maleimides, acryloyl derivatives, diazo derivatives, thiols, and the like. In some embodiments, chemical crosslinking groups such as maleimides or diazonium salts may convert aptamer affinity complexes into aptamer covalent complexes simply by providing the appropriate environment and arrangement of reactive groups necessary for the specific and sufficiently robust chemical reaction to occur. In other embodiments, chemical cross-linkers such as aldehyde groups may require the addition of other components, such as sodium cyanoborohydride, to convert the aptamer affinity complex into a stable, irreversible aptamer covalent complex. In other embodiments, such chemical cross-linkers are not included in the aptamer, but rather a third agent is used to transform the aptamer affinity complex into an aptamer covalent complex by facilitating covalent attachment of the aptamer to its target. For example, a homo-or heterobifunctional reagent containing an amine reactive moiety (e.g., an N-hydroxysuccinimide ester, aldehyde, or amide) and a nucleoside reactive group (e.g., iodoacetamide or an activated aldehyde) can induce covalent complexation of an aptamer affinity complex, such as an affinity complex formed by an aptamer and a target protein.

The term "test sample" as used herein refers to any material, solution or mixture containing a plurality of molecules and including at least one target molecule. The term test sample includes biological samples as defined below and samples that may be used for environmental or toxicity tests, such as contaminated or potentially contaminated water and industrial sewage. The test sample may also be an end product, an intermediate product or a by-product of a manufacturing process, such as a manufacturing process. The test sample may include any suitable assay medium, buffer or diluent added to a material, solution or mixture obtained from an organism or other source (e.g., environmental or industrial source).

The term "biological sample" refers to any material, solution or mixture obtained from an organism. This includes blood (including whole blood, leukocytes, peripheral blood mononuclear cells, plasma and serum), sputum, breath, urine, semen, saliva, meningeal fluid, amniotic fluid, glandular fluid, lymph fluid, nipple aspirate, bronchial aspirate, synovial fluid, joint aspirate, cells, cell extracts and cerebrospinal fluid. This also includes all experimental separation fractions previously described. The term "biological sample" also includes materials, solutions or mixtures containing homogeneous solid materials, e.g., obtained from a stool sample, a tissue sample or a tissue biopsy. The term "biological sample" also includes materials, solutions or mixtures derived from tissue culture, cell culture, bacterial culture and viral culture.

"solid support" refers to any substrate having a surface to which molecules can be attached, either directly or indirectly, by covalent or non-covalent bonds. The solid support may comprise any matrix material that provides physical support for the probes attached to the surface. Such materials are generally capable of withstanding the conditions required to attach the probe to its surface or any subsequent processing or handling in the assay. Such materials may be natural, synthetic or modified natural materials. Suitable solid support materials may include silicon, graphite, mirrors, flakes, ceramics, plastics (including polymers such as polyvinyl chloride, cyclic olefin copolymers, polyacrylamides, polyacrylates, polyethylene, polypropylene, poly (4-methylbutene), polystyrene, polymethacrylates, poly (ethylene terephthalate), polytetrafluoroethylene (PTEE or Teflon @)) Nylon, poly (vinyl butyrate)), germanium, gallium arsenide, gold, silver, and the like, either alone or in combination with other materials. Other hard materials are also contemplated, such as glass, which includes silicon, and further includes, for example, available Bioglass glass. Other materials that may be used include porous materials such as controlled pore glass beads. Any material known in the art that is capable of incorporating one or more functional groups such as any amino, carboxyl, thiol, or hydroxyl functional group on the surface may also be used.

The materials used for the solid support may have any of a wide variety of configurations ranging from simple to complex. The solid support may have any of a variety of shapes including a strip, a plate, a disk, a particle (including beads), a tube, a pore, and the like. Typically, the material is relatively flat, such as a glass slide, however it may also be spherical, such as beads or cylindrical (e.g., pillars). In many embodiments, the material is generally molded into a rectangular solid. A number of predetermined arrays, such as probe arrays, can be synthesized on the sheet and then cut along the graduation marks into individual array matrices. Exemplary solid supports that may be used include microtiter wells, microscope slides, membranes, paramagnetic beads, charged paper, Langmuir-Bodgett membranes, silicon wafer chips, flow through chips, and microbeads.

The surface of the solid support is typically an outer portion of the matrix material forming the solid support. The surface of the solid support to which the probes are bound may be smooth or substantially flat, or irregular, such as depressions, grooves, protrusions, or other textures. The surface may be modified by one or more layers of different compounds for modifying the surface properties into a desired pattern. In many embodiments, such surface modifying layers, when present, can generally have a thickness of from a single molecule thickness to about 1mm, or from a single molecule thickness to about 0.1mm, or from a single molecule thickness to about 0.001 mm.

Surface modification layers of interest include inorganic and organic layers, such as metals, metal oxides, polymers, small organic molecules, and the like. Polymer layers of interest include methacrylate copolymers, polyacrylamides, polysaccharides, phospholipids, polyurethanes, polyesters, polycarbonates, polyureas, polyamides, polyvinylamines, polyarylene sulfides, polysiloxanes, polyimides, polyacetates (polyacetates) and the like, wherein the polymer may be heteropolymers or homopolymers with or without separate functional moieties attached thereto (e.g., conjugated moieties). Other surface modifications of interest include three-dimensional networks, such as hydrogels. Any suitable hydrogel known in the art may be used. See, for example, U.S. patent application publication No.2003/0218130 entitled "Biochips with Surfaces Coated with polysaccharides-Based Hydrogels", U.S. patent application publication No.20050147994 entitled "Method for enhancing a biological in a Polyurethane-Hydrogel Composition, a Composition Prepared from the Method, and Biomedical Applications", U.S. patent application publication No. 2005005908 entitled "PhotosalsslinkedHydrogel Surface Coatings", and references cited in these publications.

In one embodiment, the solid support surface comprises a hydrogel. The hydrogel may comprise, for example, a polymer matrix. The hydrogel may be chemically attached to the surface of the solid support and may include binding functionalities that are capable of directly or indirectly attaching the probes to the hydrogel. Exemplary binding functionalities include hydrophobic groups, hydrophilic groups, reactive groups such as aldehydes, epoxies, carbonates, and the like, carboxyl groups, thiols, sulfonates, sulfates, amino groups, substituted amino groups, phosphates, metal chelators, thioethers, biotin, borates, and the like.

Any surface suitable for gene expression or SNP analysis may also be used, including substrates or surfaces provided by the following companies: affymetrix, General Electric (e.g., CodeLink), Agilent, and Schott Nexterion, either in the form of a substrate and surface, or in the form of a product ingredient further comprising other ingredients.

The probes may be bound to the surface of the solid support by any suitable means, as long as the probes do not fall off during subsequent incubation and processing according to the disclosed methods, for example, washing the surface to remove non-specific complexes. The probes may be bound by non-covalent attachment, such as adhesion, adsorption, or covalent attachment to the surface of a solid support. When the probe is covalently attached to the solid support, the surface of the solid support will contain the functional group to which the probe is attached. The nature of the functional group used depends on the nature of the probe. There have been a number of reports of methods for covalently attaching molecules to surfaces. Typically, these reactions are carried out by reacting reactive functional groups on the molecule with activated functional groups on the surface. For example, amine-containing compounds can be attached to carboxylic acid-containing surfaces by forming activated esters of the carboxylic acid, such as N-hydroxysuccinimide derivatives. This amine readily reacts with the activated ester to form a stable amide bond. This reaction is useful where the desired amine reacts significantly faster than the other nucleophiles in the system.

Examples of methods previously described in the art include activating a surface with: cyanogen bromide, N-hydroxysuccinimide esters, carbonyldiimidazole, carbodiimides, azlactones, cyanuric chloride, organic sulfonyl chlorides, divinyl sulfone, nitrophenyl esters, iodoacetyl groups, maleimides, epoxies, hydrazides, reductive amination, diazonium salts and Mannich condensation. Molecules that react with the activated surface include amines, alcohols, carboxylic acids, thiols, carbonyls and active hydrogen containing compounds.

In one embodiment, the probes are bound to the surface of the solid support in a predetermined spatial arrangement or pattern, which refers to any arrangement where the characteristics (features) of the specific locations on the surface where the probes are located are known. In one embodiment, the predetermined arrangement is an array. An array generally comprises any one-, two-, or three-dimensional arrangement of addressable regions having a particular probe associated with that region. An array is addressable by having a plurality of distinct probe regions, such that a particular aptamer covalent complex can be detected at a particular predetermined location or address on the array, thereby allowing the detection of a particular target molecule by specific association with a tag on the aptamer covalent complex.

A collection of arrays on a solid support surface refers to one or more arrays disposed on an individual solid support surface separated by inter-array regions. Typically, the surface of the solid support opposite the surface having the array (the opposite surface) does not contain any array. The array may be designed to detect any type of test sample. The solid support surface may carry at least 1, 2, 4, 20, 100 or at least 500 arrays. Any or all of the arrays may be the same or different from one another, and each may contain a number of spots or features of probes distributed thereon, as desired. A typical array may be less than about 20cm²Or less than about 10cm²Has an area containing more than 10, more than 100, more than 1000 or 1 ten thousand, or even more than 10 ten thousand features. For example, a feature may have a width (i.e., the diameter of a dot) of about 10 μm to about 1.0 cm. In other embodiments, each featureFeatures may have a width of about 10 μm to about 1.0mm, or a width of about 5.0 μm to about 500 μm, or a width of about 10 μm to about 200 μm. Non-circular features may have a range of widths (diameters) equivalent to the above circular features.

Any of a variety of geometric arrays on a solid support may be used. As mentioned above, a solid support may contain 1 or more arrays. The features on the array may be arranged in straight rows and columns. This is particularly attractive for single arrays on solid supports. If multiple arrays are present, such arrays may be arranged, for example, in a series of curvilinear rows (e.g., a series of concentric circles or half-dots) across the surface of the solid support, or the like. Similarly, the pattern of features may differ from a pattern of straight rows and columns of dots by including, for example, a series of curved rows (e.g., a series of concentric circles or half-dots) across the surface of the solid support, or the like. The configuration of the array and its characteristics may be selected based on production, handling and use factors.

Each feature or element in the array is defined as a small, regularly shaped region of the surface of the solid support. These features are arranged in a predetermined manner. Each feature of the array typically carries a predetermined probe or mixture of features. Each feature within a molecular array may contain different probes, and probes in a given feature may differ from probes in other features in the array. Some or all of the features may be different compositions. Each array contains a plurality of dots or features, and each array may be separated from the other arrays by spaces or areas. It is also known that no space is required to separate the arrays from each other. Inter-array regions and inter-feature regions are typically present but not necessary. These inter-array and inter-feature areas do not carry any probes if any edge areas are present. It is known that if inter-array and inter-feature regions are present, these regions can be of various sizes and configurations.

In some embodiments, an array may be formed by attaching probes to a first solid support, such as a bead, and then arranging this first solid support in an array format on a second solid support, such as a microtiter plate. In other embodiments, arrays may be formed by attaching probes to addressable beads. "addressable beads" include dyes, barcodes, and transponders.

In some embodiments, depending on the solid support surface selected, the surface may be "blocked" or "passivated" to reduce or inhibit non-specific binding of molecules to the solid support surface. Blocking or inactivating agents include milk powder, casein, pooled serum, pooled plasma, BSA, PEG-PLL, PEG-silane, SuperBlock or StarterBlock (Pierce Biotechnology, Rockford, Ill) and any combination of these.

The term "labeling substance" as referred to herein refers to one or more reagents that can be used to detect the target molecule that binds to the aptamer on the aptamer covalent complex.

In one embodiment for detecting a target molecule, the aptamer covalent complex is contacted with a labeling substance that includes a binding partner specific to the target molecule to which the target molecule binds the aptamer. The specific binding partner may be any suitable moiety, including an antibody, antibody fragment, synthetic antibody mimetic, biomimetic, aptamer, molecularly imprinted ligand, and the like. The specific binding partner is typically conjugated or linked to a detectable moiety or label, to another labeling substance component. The binding of the specific binding partner to the label may be carried out by any of the methods described above for attaching the probe to the surface of a solid support. It is known that in the detection of multiple target molecules, multiple aptamer covalent complexes can be contacted with a mixture of specific binding partners, each specific for the target molecule suspected of being present. The label used may be a label known in the art for multiplexed detection of multiple target molecules.

The detectable moiety or label can be detected directly or indirectly. In general, any detectable reporter molecule can serve as a label. Labels include, for example, (i) a reporter molecule that is directly detected by virtue of generating a signal, (ii) a specific binding partner member that can be indirectly detected by subsequent binding to a homologue (cognate) containing the reporter molecule, (iii) a mass label that can be detected by mass spectrometry, (iv) an oligonucleotide primer that can provide an amplification or ligation template, and (v) a specific polynucleotide sequence or recognition sequence capable of acting as a ligand, for example a repressor protein, which in the latter two instances will have or can have a reporter molecule, and the like. The reporter molecule can be a catalyst, e.g., an enzyme, a polynucleotide encoding a catalyst, a promoter, a dye, a fluorescent molecule, a quantum dot, a chemiluminescent molecule, a coenzyme, an enzyme substrate, a radioactive group, a small organic molecule, an amplifiable polynucleotide sequence, a particle such as a latex or carbon particle, a metal sol, a crystallite, a liposome, a cell, and the like, which may or may not be labeled with a dye, a catalyst or other detectable group, a mass label that alters the weight of the molecule to which it is conjugated for mass spectrometric detection, and the like. The label may be selected from electromagnetic or electrochemical materials. In one embodiment, the detectable label is a fluorescent dye. Other labels and labeling schemes will also be apparent to those skilled in the art based on the present invention.

In another embodiment for detecting a target molecule, the target molecule is a protein and the aptamer covalent complex is contacted with a labeling agent comprising a universal protein stain. As used herein, "universal protein stain" and "UPS" are used interchangeably to refer to any labeling substance that labels most, if not all, of the proteins present in a test sample with a detectable moiety, but does not label or minimally label nucleic acids or other components of an assay, such as a solid support. Any chemically reactive group found on proteins other than nucleic acids or other substrate surfaces can serve as a site for covalent attachment. Exemplary chemically reactive groups include primary amines (e.g., on lysine residues), thiols (e.g., on cysteines that may be formed as a result of reducing disulfide bonds), sugar moieties on alcohols (e.g., serine, threonine, tyrosine, and glycoproteins (including products that oxidize cis-diols on these sugars), and carboxylates (e.g., on glutamic acid and aspartic acid).

The detectable moiety may comprise any of the reporter molecules described above and any other chemical or composition that may be used in any manner to generate a detectable signal. The detectable moiety may be detected by a fluorescent signal, a chemiluminescent signal, or any other detectable signal based on the characteristics of the moiety. When the detectable moiety is an enzyme (e.g., alkaline phosphatase), the signal can be generated in the presence of the enzyme substrate and any additional factors required for enzymatic activity. When the detectable moiety is an enzyme substrate, the signal may be generated in the presence of the enzyme and any additional factors required for enzyme activity. Suitable reagent methods for attaching a detectable moiety to a target protein include covalently attaching the detectable moiety to the target protein, non-covalently associating the detectable moiety with another labeling substance component covalently attached to the target protein, and covalently attaching the detectable moiety to another labeling substance component non-covalently associated with the target protein. General protein staining is described in detail in U.S. patent application Ser. No. 10/504,696 entitled "Methods and Reagents for Detecting Targetbinding by Nucleic Acid Ligands", filed on 12.8.2004.

In some embodiments, the UPS is a single chemical reagent comprising a detectable moiety that covalently reacts with a functional group specific to the protein, but not the aptamer, and in such a reaction, covalently attaches the detectable moiety to the target protein. The UPS of this embodiment includes a dye having a group capable of covalently reacting with a functional group specific to a protein. These groups may be added to the dye by derivatization, or may be present in the unmodified dye. In one embodiment, the UPS comprises an N-hydroxysuccinimide-activated dye that reacts with an amine group, such as an N-hydroxysuccinimide-activated fluorophore, including an NHS-Alexa fluorophore (e.g., NHS-Alexa 647). Another UPS that may be used is CBQCA (3- (4-carboxybenzoyl) quinoline-2-carbaldehyde), which also reacts with amines in the presence of cyanides or thiols to form highly fluorescent isoindoles. Additional amine reactive groups suitable for use in the UPS reagent include isocyanates, isothiocyanates, acyl azides, sulfonyl chlorides, aldehydes, 4-thio-2, 3, 5, 6-tetrafluorophenol (STP) ester, TFP-Alexa 647 and arylating reagents such as NBD (7-nitrobenzo-2-oxa-1, 3-diazole) chloride, NBD fluoride and dichlorotriazine.

In other embodiments, the UPS comprises a plurality of reagents. For example, the UPS may comprise a first agent that covalently reacts with the target protein, one or more additional agents that attach a detectable moiety to the target protein directly or indirectly covalently or non-covalently through a chemical group or other functional group introduced by the first agent. When the UPS contains multiple reagents, it is understood that in some cases the reagents are added sequentially, and in other cases the reagents are added simultaneously.

In one embodiment, a suitable UPS comprises (a) a biotin derivative that reacts with a target protein and (b) a streptavidin detectable moiety conjugate, such as a fluorescent streptavidin derivative or streptavidin-enzyme conjugate. The biotin derivative and the amine group react to covalently attach biotin to the target protein, and the streptavidin detectable moiety conjugate binds to the immobilized biotin group, thereby localizing the detectable moiety on the protein-bound solid support site. In this embodiment, suitable reagents include PFP-biotin, NHS-PEO₄Biotin (spacer 29)) thio-NHS-LC-Biotin (spacer 22.4)) And TFP-PEO₃Biotin (spacer 32.6))。

In another embodiment, a suitable UPS comprises (a) biotin or a biotin derivative conjugated to a reactive group capable of covalently attaching biotin or a biotin derivative to a bound target protein; (b) avidin and/or streptavidin, and (c) a biotin detectable moiety conjugate, such as a fluorescent biotin derivative. The biotin derivative in (a) mayIs an amine-reactive biotin derivative, such as NHS-biotin, wherein the biotin is optionally separated from NHS by a spacer atom (Calbiochem, Inc.). The reaction of the NHS group with the primary amine on the bound target protein results in the covalent attachment of biotin to the target protein bound to its corresponding aptamer. The target protein complexed with its corresponding aptamer can then be treated with streptavidin or avidin. Since streptavidin and avidin are each capable of binding 4 biotins, the addition of these proteins can provide 3 biotin binding sites for each biotin originally coupled to the bound target protein via NHS-biotin. The biotin detectable moiety derivative of (c) is then added so that it binds tightly to the unoccupied biotin sites on the streptavidin or avidin. In this embodiment, suitable reagents include PFP-biotin, NHS-PEO₄Small biotin (spacer arm 29)) thio-NHS-LC-Biotin (spacer 22.4)) And TFP-PEO₃Biotin (spacer 32.6))。

The concentration of the labeling substance, including the concentration of each particular agent that may be used, is generally determined in view of, for example, the nature of the labeling substance, the nature of the target molecule and the predetermined cut-off level, the biological significance of the specific target level, and the like. The final concentration of each reagent is typically determined empirically to optimize the sensitivity of the method. In one embodiment, the concentration of the labeling substance is generally sufficient to detect at least about 1% of the target molecules. In another embodiment, the concentration of the labeling substance is generally sufficient to detect at least about 10% of the target molecules. In a further embodiment, the concentration of the labeling substance is generally sufficient to detect at least about 90% of the target molecules.

The activation of the labeling substance depends on the nature of the reagent used. For example, for those agents that are activated by light, the agent is irradiated with light of an appropriate wavelength. Other methods of activation will be apparent to those skilled in the art in light of this disclosure. For some labeling substances, e.g., those involving radioactive labels, enzymes, etc., no activator is required. For enzyme systems, it may be necessary to add substrates and/or cofactors.

Detecting the presence and/or amount of the signal generated by the labeling substance on the solid support comprises detecting the signal, which is typically only a step of reading the signal. The signal is typically read with a device, the nature of which depends on the nature of the signal. The device may be a spectrophotometer, fluorometer, absorption spectrophotometer, photometer, chemo-photometer, exposure meter, photographic device, or the like. The presence and/or amount of the detected signal correlates with the presence and/or amount of any target molecules present in the test sample above the predetermined cut-off level. The temperature during measurement typically ranges from about 10 ° to about 70 ℃, or from about 20 ° to about 45 ℃, or from about 20 ° to about 25 ℃. In one method, a standard curve is plotted using measurements of known concentrations of target molecules. Calibrators and other controls may also be used.

In one embodiment, the solid support comprising the array is, for example, moved into a detection apparatus, thereby detecting the presence of any bound target molecules on the surface of the solid support. The detection device may be a scanning device comprising an optical system. The array may be examined or read by illuminating the array and reading the location and intensity of the signal (e.g., fluorescence) produced at each array feature. The scanner may be similar to, for example, a Tecan LS300 scanner available from Tecan Systems, San Jose, California. However, the array may be inspected or read by methods or instruments other than those previously described, including other optical techniques (e.g., detection of chemiluminescent or electroluminescent labels) and electronic techniques to inspect the array.

The results from the detection array may be raw data (e.g., fluorescence intensity readings for each feature read in one or more color channels) or processed results, such as by removing feature readings below a predetermined threshold and/or based on the manner in which the array is read (e.g., whether a particular target molecule is present in the test sample). If desired, the detection results (processed or unprocessed) may be communicated (e.g., by communication) to a remote location and received there for further use (e.g., further processing).

In another embodiment, the method is performed by being under computer control, i.e. with the aid of a computer. For example, IBM may be usedA compatible personal computer. The computer is driven by software specific to the method of the invention. Computer hardware capable of facilitating the implementation of the method of the invention may include the following systems: pentium (Pentium)A processor or higher having a dominant frequency of at least 100MHz, at least 32M memory (RAM), at least 80M virtual memory, in Microsoft Windows95 or WindowsNT 4.0 operating system (or a subsequent system).

Software which can be used for carrying out the method can be, for example, Microsoft Excel or Microsoft AccessIt can be extended appropriately according to the function and template written by the user and connected with the independent program according to the need and the need. Examples of software or computer programs useful in facilitating the practice of the methods of the present invention may be written in the following programming languages: visual BASICFORTRAN, C + +, Java, Python, and any other suitable programming language currently or available in the future. It should be understood that the above-described computer information and software used herein is by way of example only, and not by way of limitation. Any of the methods disclosed herein may be applied to other computers, computer systems, and software. Other possible languages include, for example, PASCAL, PERL, or assembly language.

In another embodiment, the aptamer affinity complex (or optionally the aptamer covalent complex) is detected and/or quantified with a mass spectrometer. Referring to fig. 1B, in an exemplary method of detecting and/or quantifying a target molecule that may be present in a test sample, the test sample is contacted with an aptamer that includes a label and has a specific affinity for the target molecule. Aptamer affinity complexes including aptamers that bind to their target molecules can be formed. If the test sample contains the target molecule, aptamer affinity complexes will typically form in the test sample. Aptamer affinity complexes are optionally converted to aptamer covalent complexes comprising an aptamer covalently bound to its target molecule using methods appropriate for the aptamer used. The aptamer affinity complex (or optionally the aptamer covalent complex) is attached to a solid support. Attachment is achieved by contacting the solid support with an aptamer affinity complex (or optionally an aptamer covalent complex) and allowing the label on the aptamer to associate, directly or indirectly, with a probe attached to the solid support. Aptamer affinity complexes (or optionally aptamer covalent complexes) associated with the probes on the solid support are then prepared for mass spectrometer detection (or optionally quantitation).

The aptamer affinity complex (or optionally aptamer covalent complex) may be prepared by any method for detection and optionally quantification by a mass spectrometer. For example, in one embodiment, when the target molecule is a protein, the aptamer affinity complex (or optionally the aptamer covalent complex) is prepared by digestion with a protease either before or after the complex is removed from the solid support. In another embodiment, the aptamer affinity complex (or optionally the aptamer covalent complex) is released from the solid support and prepared for mass spectrometer analysis by any method known in the art, including matrix-assisted laser desorption ionization (MALDI), surface-enhanced laser desorption ionization (SELDI), electrospray ionization or electron impact ionization. In one embodiment, the aptamer affinity complex (or optionally the aptamer covalent complex) may be eluted directly into an electrospray ionization mass spectrometer. In another embodiment, the eluted test sample may be subjected to further processing, such as enzymatic digestion or chemical modification, prior to mass spectrometer analysis. Mass spectra can be obtained by electrospray ionization, matrix assisted laser desorption ionization or electron impact ionization.

Typically, quantification of target molecules with a mass spectrometer requires an internal standard, i.e. the introduction of a compound of known concentration in the test sample to be analyzed. The ideal internal standard would have similar elution and ionization characteristics to those of the target molecule, but would produce ions with different mass-to-charge ratios. A common internal standard is a stable isotopically labeled target molecule. In one embodiment, a stable isotopically labeled target molecule is added to the test sample as an intrinsic standard. The spectral peaks corresponding to the various components in the test sample are then compared to the peak heights or areas of the internal standards to quantify the target molecule.

In another embodiment, quantification of the target molecule is achieved by comparing the height or area of a spectral peak corresponding to the target molecule to a set of spectral peaks produced by a sample containing a known concentration of the target molecule. The height and area of the spectral peaks obtained for samples containing known concentrations of target molecule constitute a standard curve from which the unknown concentration of target molecule in the test sample can be calculated.

In another embodiment, the aptamer affinity complex (or optionally aptamer covalent complex) is detected and/or quantified using Q-PCR. As used herein, "Q-PCR" refers to a PCR reaction that is conducted under conditions that are controlled and in a manner such that the results of the assay are quantitative, i.e., the assay is capable of quantifying the amount or concentration of aptamer present in a test sample. Referring to fig. 1B, in an exemplary method of detecting and/or quantifying a target molecule that may be present in a test sample, the test sample is contacted with an aptamer that includes a label and has a specific affinity for the target molecule. Aptamer affinity complexes including aptamers that bind to their target molecules can be formed. If the test sample contains the target molecule, aptamer affinity complexes will typically form in the test sample. Aptamer affinity complexes are optionally converted to aptamer covalent complexes comprising an aptamer covalently bound to its target molecule by using methods appropriate to the aptamer used. As further described herein, any free aptamer present in the test sample is subsequently separated from the aptamer affinity complex (or optional aptamer covalent complex) following conversion of the aptamer affinity complex and any optional aptamer covalent complex to. The aptamer affinity complex (or optionally aptamer covalent complex) is then quantified using known methods for quantifying the replication of polynucleotides.

In one embodiment, the amount or concentration of aptamer affinity complex (or optionally aptamer covalent complex) in the test sample is measured using TaqManAnd (3) PCR. This technique relies primarily on the 5 '-3' exonuclease activity of oligonucleotide replicase to generate signals from the targeted sequence. TaqMan probes are selected based on the sequence of the aptamer to be quantified and typically include a5 '-terminal fluorescein, such as 6-carboxyfluorescein, and a 3' -terminal quenching group, such as 6-carboxytetramethylfluorescein, to generate a signal when the aptamer is amplified by Polymerase Chain Reaction (PCR). When the polymerase replicates the aptamer sequence, exonuclease activity releases the fluorescein annealed on the probe downstream of the PCR primer, thereby generating a signal. The signal increases with the generation of replication products. The amount of PCR product depends on the number of replication cycles performed and the initial concentration of aptamer.

In another embodiment, the amount or concentration of aptamer affinity complex (or optionally aptamer covalent complex) is determined using an intercalating fluorescent dye during replication. Intercalating dyes, e.g. SYBRgreen, which produces a greater amount of fluorescent signal in the presence of double-stranded DNA compared to the fluorescent signal produced in the presence of single-stranded DNA. The signal generated by the dye increases when a double stranded DNA product is formed during PCR. The amount of signal generated depends on the number of PCR cycles and the initial concentration of aptamer.

In another embodiment, the amount or concentration of aptamer affinity complex (or optionally aptamer covalent complex) is determined using a "molecular beacon" during replication (see Tyagi et al, nat. Biotech.16: 4953, 1998; U.S. Pat. No. 5,925,517). A molecular beacon is a specific nucleic acid probe that folds to form a hairpin loop and contains a fluorescein at one end and a quencher at the other end of the hairpin structure, such that the fluorescein produces no or little signal when the hairpin structure is formed. The loop sequence is specific to the target polynucleotide sequence and when hybridized to the aptamer sequence, the hairpin loop opens, thereby generating a fluorescent signal.

One or more steps of any of the methods disclosed herein may be performed using a computer program. Another aspect of the invention is a computer program product comprising a computer readable storage medium having a computer program stored thereon which, when loaded into a computer, performs or facilitates the performance of any of the methods disclosed herein.

One aspect of the present invention is that the product of any of the methods disclosed herein, i.e., the results of the analysis, may be evaluated at the testing site, or transferred to another site for evaluation and communication to the interested party at a remote location, as desired. Reference herein to a "remote location" refers to a location that is physically distinct from the location at which the results are obtained. Thus, the results may be sent to another room, another building, or another part of a city, another city, and so on. Data may be transmitted by standard means such as facsimile, mail, courier, email, ftp, voicemail, and the like.

"communicating" information refers to the transmission of data representing the information by way of electronic signals over a suitable communication channel, such as a private or public network. "shipping" an item refers to any method of taking the item from one location to another, by physically transporting the item or other methods (if possible), including, at least for data, physically transporting a medium containing the data or transmitting the data.

Referring to fig. 3, in another exemplary method of detecting and/or quantifying one or more target molecules that may be present in a test sample, a test sample that may comprise a target molecule and at least one non-target molecule may optionally be contacted with a competitor molecule (as shown in fig. 3, option a). One or more non-specific complexes may form. The test sample is then contacted with an aptamer comprising a label and having a specific affinity for the target molecule. Aptamer affinity complexes including aptamers that bind to their target molecules can be formed. If the test sample contains the target molecule, aptamer affinity complexes will typically form in the test sample. Depending on the nature of the test sample, one or more non-specific complexes may also form between the aptamer and one or more non-target molecules. If the test sample is contacted with a competitor molecule, a variety of non-specific complexes containing competitor can also form and be present in the test sample.

The test sample may then optionally be exposed to conditions that kinetically excite the constituents of the test sample (as shown in fig. 3, option B). In further description below, kinetic excitation may comprise diluting the test sample, introducing the competitor molecule into the test sample, or capturing the aptamer affinity complex on a solid support, followed by washing with or without the competitor molecule in a wash solution. If a kinetic challenge is introduced, non-specific complexes between the aptamer and any non-target molecules are less likely to reform upon dissociation. Because nonspecific complexes generally dissociate more rapidly than aptamer affinity complexes, kinetic excitation reduces the probability of aptamer involvement with non-target nonspecific complexes. An efficient kinetic challenge may provide the assay with additional specificity beyond the initial aptamer binding event and subsequent covalent interactions.

Whether or not kinetic excitation is used, the formed aptamer affinity complex is then converted to an aptamer covalent complex comprising an aptamer covalently bound to its target molecule by using methods appropriate to the aptamer used. After aptamer covalent complex formation, any free or uncomplexed aptamers that may be present in the test sample can optionally be isolated from the test sample (as shown in FIG. 3, option C). Optionally, free or uncomplexed non-target and target molecules that may be present in any test sample can be isolated from the test sample (as shown in figure 3, option D). Optionally, free aptamer and free non-target and target molecules may be removed in any order after formation of the aptamer covalent complex.

If dilution is used to introduce a kinetic challenge, subsequent test samples containing aptamer covalent complexes are preferably concentrated. If applicable, concentration may be achieved using the methods described below with respect to optionally separating any free aptamer from the test sample and/or optionally removing other components of the test sample that may react with the labeling substance.

The aptamer covalent complex in the test sample is then detected and/or quantified using any of the methods described herein or any other suitable method known to those of skill in the art. For example, the aptamer covalent complex can be attached to the solid support surface by contacting the solid support with the test sample such that the label on the aptamer associates, directly or indirectly, with a probe immobilized on the solid support surface. The aptamer covalent complexes associated with the probes on the solid support are then detected and optionally quantified. The aptamer covalent complex is contacted with a labeling agent at any time prior to detection and optionally quantification, i.e., before or after attachment of the aptamer covalent complex to the solid support, to detect the bound target molecule. Those skilled in the art will appreciate that aptamer covalent complexes can also be detected and/or quantified using mass spectrometry, Q-PCR, or any other suitable method known in the art.

As used herein, "competitor" and "competitor" are used interchangeably to refer to any molecule that can form a non-specific complex with a non-target molecule. "competitor" and "competitor" are copies of a set of one or more species of molecules. "multiple competitor molecules" and "multiple competitors" refer to more than one group of such molecules. Competitor molecules include polynucleotides, polyanions (e.g., heparin, single-stranded salmon sperm DNA, and polyglucans (e.g., dextran sulfate), abasic phosphodiester polymers (abasic phosphodiester polymers), dNTPs, and pyrophosphates.

Reference herein to a "non-specific complex" refers to a non-covalent association between 2 or more molecules in addition to an aptamer and its target molecule. Because the non-specific complex is not selected based on affinity interactions between its constituent molecules, but rather represents an interaction between classes of molecules, the molecules associated in the non-specific complex will, on average, exhibit much lower affinity for each other and will have a correspondingly higher off-rate than the aptamer and its target molecule. Non-specific complexes include complexes formed between aptamers and non-target molecules, competitors and target molecules, and target molecules and non-target molecules.

As used herein, "kinetic excitation" refers to the process of enriching aptamer affinity complexes from a group of complexes including aptamer affinity complexes and non-specific complexes by providing kinetic pressure and using different affinity properties, including off-rates, of the compositions of these classes of complexes. Kinetic excitation usually results in an increase in specificity, since aptamer-non-target complexes are significantly reduced compared to aptamer-target complexes. As used herein, "kinetic pressure" refers to a means of providing an opportunity for the complex to naturally dissociate and/or inhibit the re-association of molecules that naturally dissociate from the complex. Kinetic pressure may be applied by addition of competitor molecules or sample dilution or thorough washing after binding of the complex to the solid support or any other method known to those skilled in the art. As is known to those skilled in the art, because kinetic excitation generally depends on the different off-rates of aptamer affinity complexes and aptamer non-target complexes, the duration of kinetic excitation is selected to maintain a high proportion of aptamer affinity complexes while substantially reducing the number of aptamer non-target complexes. For efficient kinetic excitation, the dissociation rate of the aptamer affinity complex is preferably much lower than that of the aptamer non-target complex. Because aptamers can be selected to include specific properties, the composition of aptamer affinity complexes can be designed to have a relatively off-rate.

The term "isolating" herein refers to separating or removing one or more molecular species from a test sample. Separation may be used to increase sensitivity and/or reduce background. Separation after aptamer covalent complex formation is most efficient when the aptamer affinity complex becomes irreversible due to covalent bonds.

For example, removing free aptamer from the test sample may increase the sensitivity of the assay, as the free aptamer may compete with the aptamer covalent complex when it attaches to the probe on the surface of the solid support. When QPCR is used for detection or optional quantification, removal of free aptamer facilitates detection and quantification of the target molecule. In one embodiment, the target molecule is a protein and the free aptamer is separated from the aptamer covalent complex (and other parts of the test sample) by using a reagent that precipitates the protein in the sample and complexes including the protein, such as aptamer covalent complexes and non-precipitating free nucleic acids. Such reagents may include K +/SDS, acetone, (NH4)₂SO₄ProCipitate and other charged polymers known in the art.

In one embodiment, aptamers, such as labeled photoaptamers having specific affinity for a target molecule, in this case a target protein, are introduced into a test sample. As further described herein, after formation of the aptamer affinity complex and conversion to the aptamer covalent complex, the aptamer-protein covalent complex and uncomplexed protein are precipitated from the test sample with a suitable reagent, such as any of the reagents listed above or any other suitable reagent. The precipitated components of the test sample were pelleted by centrifugation and the supernatant containing free aptamer was decanted. The pellet containing the free protein and the aptamer-protein covalent complex is then suspended in a suitable solution, such as an assay binding diluent, and the aptamer covalent complex can then be contacted with a labeling substance either before or after the aptamer covalent complex is attached to the solid support. The target molecule, if present in the test sample, can then be detected and/or quantified by detecting the labeling substance on the aptamer covalent complex.

To reduce the background of the assay, molecules that are reactive with the label and those that are not covalently linked to the aptamer will be removed from the test sample. In one embodiment, this is achieved by precipitating free and complexed aptamer in the test sample while leaving other molecules that can react with the marker substance in the supernatant to be decanted. Such nucleic acid precipitation may be achieved with reagents comprising cetyltrimethylammonium bromide (CTAB), dodecyltrimethylammonium bromide (DTAB) and organic solvents such as ethanol. In another embodiment, free and complexed aptamers are isolated from the sample by hybridizing the aptamers to a solid support. The solid support in this example can include microbeads (e.g., paramagnetic beads), any other suitable solid support described herein, and the like. After hybridization of the aptamer to a suitable solid support surface, the solution containing the molecule capable of reacting with the label is easily removed, thereby concentrating the aptamer covalent complex. One skilled in the art will appreciate that isolating aptamers in this manner may use labels included on the aptamers or some other nucleic acid sequence on the aptamers to hybridize the aptamers to a suitable complementary nucleotide sequence attached to a solid support.

In one embodiment, a labeled aptamer, such as a labeled photoaptamer with specific affinity for a target protein, is introduced into the test sample. As further described herein, after aptamer affinity complex formation and conversion to an aptamer covalent complex, the aptamer-protein covalent complex and free aptamer are isolated from the test sample using a suitable reagent, such as any of the reagents listed above or any other suitable reagent. The precipitated components of the test sample are precipitated by centrifugation and the supernatant containing uncomplexed target and the remainder of the test sample is decanted. The pellet containing the free aptamer and aptamer covalent complex is then suspended in a suitable solution, such as an assay binding dilution, and the aptamer covalent complex and free aptamer may then be contacted with the labeling substance either before or after the aptamer covalent complex is attached to the solid support. If the target molecule is present in the test sample, it will be detected and/or quantified by detecting the labeling substance on the aptamer covalent complex.

In another embodiment, a labeled aptamer, such as a labeled photoaptamer with specific affinity for the target protein, is introduced into the test sample. As further described herein, after aptamer affinity complex formation and conversion to an aptamer covalent complex, the aptamer-protein covalent complex and free aptamer are captured on a solid support using beads containing a probe complementary to the aptamer label. The beads are pelleted by magnetic force (for paramagnetic beads) or centrifugation (for non-paramagnetic beads) and the supernatant containing uncomplexed target and test sample is decanted. The pellet is then suspended in a suitable solution, such as an assay binding diluent, and the aptamer covalent complex and free aptamer are eluted using any suitable method that disrupts the hybridization interaction, including, for example, heat, high pH, distilled water, combinations of these, or any other known method. The beads are then pelleted and the supernatant containing the free aptamer and the aptamer covalent complex can be contacted with the labeling substance either before or after the aptamer covalent complex is attached to the solid support. If the target molecule is present in the test sample, it will be detected and/or quantified by detecting the labeling substance on the aptamer covalent complex.

In another embodiment, the assay is performed as described above up to and including the step of suspending the beads after the supernatant containing uncomplexed target and test sample is decanted. The aptamer covalent complex can then be contacted with a labeling substance prior to eluting the free aptamer and aptamer covalent complex from the bead, followed by repeated precipitation and washing to remove unreacted labeling substance prior to contacting the solid support and aptamer covalent complex for detection and/or quantification of the target molecule.

In any of the methods disclosed herein, the test sample can be prepared as a 2-fold or multiple dilution of the test sample, which can increase the kinetic range of the concentration of the target molecule present in the test sample for detection by the methods disclosed herein. The diluted test samples are analyzed separately until and including aptamer covalent complex formation, and then the diluted test samples may be combined for the remainder of the analysis and simultaneously detected on a solid support. In one embodiment, each diluted test sample comprises a unique aptamer, and thus the corresponding target can be measured individually. In another embodiment, aptamers may be added to 2 or more dilutions, each dilution contacting a unique and labeled aptamer to a specific target, such that specific aptamer signals can be detected in each different diluted sample on one solid support. Linking diluted samples in this manner can extend the kinetic range of a target molecule by many orders of magnitude, increasing accuracy when the overlapping regions of quantitation result in multiplexed detection of the concentration of a target.

In one embodiment, a series of dilutions of a test sample are prepared into which a labeled aptamer, such as a labeled photoaptamer having a specific affinity for the target molecule, is introduced. The same aptamer with a different label can be added to each test sample dilution. As further described herein, after formation of the aptamer affinity complex and conversion to an aptamer covalent complex, each test sample can be combined and contacted with a labeling substance either before or after the aptamer covalent complex is attached to the solid support. If the target molecule is present in the test sample, it will be detected and/or quantified by detecting the labeling substance on the aptamer covalent complex. The detected signal for each aptamer with a different label can be combined to accurately quantify the amount or concentration of the target molecule in the original test sample. For example, a first dilution may yield the greatest signal to the target, yielding only semi-quantitative information, while a second dilution may yield a signal less than saturation, allowing accurate quantification of the target in the original test sample.

In another embodiment, serial dilutions of test samples are prepared, into which labeled aptamers, such as labeled photoaptamers having specific affinity for the target molecule, are introduced. Different aptamers with unique labels can be added to each sample dilution. As further described herein, after formation of the aptamer affinity complex and conversion to an aptamer covalent complex, each test sample can be combined and contacted with a labeling substance either before or after the aptamer covalent complex is attached to the solid support. The presence of the target molecule in the test sample will be detected and/or quantified by detecting the labeling substance on the aptamer covalent complex. Depending on the different serial dilutions of the original sample, the resulting signal can be used to quantify targets over a range of several orders of magnitude.

In any of the methods disclosed herein, the test sample can be compared to a reference sample. As used herein, a "reference sample" refers to any material, solution or mixture that contains a plurality of molecules and is known to include at least one target molecule. The precise amount or concentration of any target molecule present in the reference sample is also known. The term reference sample includes biological samples as defined herein and samples that may be used for environmental or toxicological tests, such as contaminated or potentially contaminated water and industrial wastewater. The reference sample may also be an end product, an intermediate product or a by-product of a manufacturing process, such as a processing process. The reference sample may comprise any suitable analysis medium, buffer or diluent added to a material, solution or mixture obtained from an organism or other source (e.g., environmental or industrial source).

In one embodiment, the reference sample is treated in the same manner as the test sample separately until, but not including, exposure to the labeling substance, but only in this embodiment, this step must occur prior to the aptamer covalent complex being attached to the solid support. Two different labeling substances are used to distinguish between target levels in the reference sample and the test sample. After the sample and the labeling substance are contacted, they may be mixed together and simultaneously contacted with the solid support. Thus, by measuring the signal of each marker substance separately, any differential behaviour (i.e. different amounts or concentrations of target in the sample) between the reference sample and the test sample can be directly compared. The two labeling substances may include pairs of Cy3 and Cy5, and pairs of Alexa555 and Alexa 647. In one embodiment, the reference sample may be a mixed biological sample representing a control group. In another embodiment, the reference sample may be a biological sample collected from an individual for the first time and the test sample may be a sample collected from the same individual but for a second time, thereby facilitating longitudinal studies of individuals by measuring and assessing any changes in the amount or concentration of one or more target molecules in a plurality of biological samples provided by the individual over a period of time.

In one embodiment, labeled aptamers, such as photoaptamers labeled and having specific affinity for a target molecule, are introduced into the reference sample and the test sample. As further described herein, after formation of the aptamer affinity complex and conversion to an aptamer covalent complex, the two samples can be contacted with two different labeling substances containing two different detection molecules. Two separately labeled samples containing aptamer covalent complexes can then be attached to the solid support. If the target molecule is present in one or both of the samples, it will be detected and/or quantified by detecting the two labeled species on the aptamer covalent complex, typically by performing multiple scans at different excitation and emission wavelengths, for example when the detection label is a fluorescent molecule. Such quantification may result in a more accurate assessment of the amount or concentration of different targets in the reference sample and the test sample, and may facilitate comparison of different test samples when the same reference sample is used.

In any of the methods disclosed herein, a plurality of marker substances can be used to analyze a test sample. In one embodiment, 2 or more labeling substances, each having a unique chemical composition to label different target molecule moieties and optionally different detection groups, may be used in a test sample. Different chemical moieties will label different functional groups on the target molecule, from which more information can be obtained. For example, in the case of a target protein, one tag may be attached to the target using a chemical moiety that reacts with a primary amine (e.g., lysine), while a second tag is attached to the target via a chemical moiety that reacts with, for example, a carbohydrate group typically associated with a glycosylated protein. The relative quantification of these two moieties on the same target molecule recognized by a common aptamer can provide useful information about the degree of glycosylation of the target in the test sample.

In one embodiment, a labeled aptamer, such as a photoaptamer that is labeled and has a specific affinity for a target molecule, is introduced into a test sample. As further described herein, after formation of the aptamer affinity complex and conversion to an aptamer covalent complex, the test sample can be contacted with two different labeling substances containing two different detection molecules before or after contacting the aptamer complex with the solid support. In one embodiment, the detection label and the chemical component are unique such that the same test sample containing the aptamer covalent complex can be labeled consecutively. In another embodiment, the chemical components may be carried out simultaneously in one reaction. The multi-labeled aptamer covalent complex can then be detected and/or quantified by detecting the two labeled species on the aptamer covalent complex, typically by performing multiple scans at different excitation and emission wavelengths, for example, when the detection label is a fluorescent molecule.

In another embodiment, two or more unique labeling substances use the same detection label. The assay is performed as described above until and including conversion to the aptamer covalent complex, after which the test sample is divided into a number of aliquots based on different labeling substances, each test sample being contacted with the labeling substance before or after the aptamer covalent complex is attached to the solid support. Separately labeled aliquots of the test sample containing the aptamer covalent complexes can be separately detected and/or quantified.

Any of the methods described herein can be used to perform multiplexed analysis of a test sample. Any such multiplexed analysis may involve the use of at least 2, at least tens, at least hundreds, or at least tens of thousands of aptamers to simultaneously analyze an equivalent number of target molecules in a test sample, e.g., a biological sample. In these embodiments, a plurality of labeled aptamers, e.g., labeled photoaptamers having a specific affinity for a target molecule, are introduced into a test sample. As further described herein, after formation of the aptamer affinity complex and conversion to an aptamer covalent complex, the aptamer covalent complex is attached to a solid support via a plurality of corresponding probes immobilized on the solid support. The aptamer covalent complex can be contacted with the labeling substance before or after the aptamer covalent complex is attached to the solid support. The presence of the target molecule in the test sample can be detected and/or quantified by detecting the labeling substance on the aptamer covalent complex.

Another aspect of the invention relates to kits that are convenient for performing the methods of the invention to analyze a test sample. To enhance the versatility of the present invention, the reagents may be provided in combination packages, either in the same container or in different containers, so that the ratio of the various reagents provides a substantially optimized method and assay. The reagents may be in separate containers or multiple reagents combined in 1 or more containers depending on the cross-reactivity and stability of the reagents.

The kit package combination comprises at least one labeled aptamer, a solid support comprising at least one probe and a suitable labeling substance. The kit may also include wash solutions such as buffers for sample dilution and washing of the array, sample preparation reagents, and the like. The relative amounts of the various reagents in the kit can vary widely to provide reagent concentrations that optimize the reaction to be performed in the assay and further optimize the sensitivity of the assay. Under appropriate circumstances, one or more of the reagents in the kit may be provided in dry powder form, typically in lyophilized form, including excipients, which upon dissolution will provide a reagent solution of appropriate concentration for performing the methods and assays of the invention. The kit may further comprise instructions for the methods of the invention described herein.

In an exemplary embodiment, a kit for detecting and/or quantifying one or more target molecules that may be present in a test sample comprises at least one aptamer having a specific affinity for the target molecule and comprises a label, a labeling substance, and a solid support, wherein the solid support comprises at least one probe distributed thereon, wherein the probe is capable of associating with the label on the aptamer.

Examples

The following examples are provided for illustrative purposes only and are not intended to limit the scope of the invention as defined in the appended claims.

Example 1 measurement of buffer and plasma serially diluted VEGF for detection and quantification by surface immobilized probes Using hybrid Capture Photoaptamer assay

This example illustrates the steps of analysis for a single photoaptamer and its target protein as shown in figures 2A, 2B and 2C. The analysis was performed with two different test samples, buffer and plasma.

A. Preparing oligonucleotides: here, a DNA oligonucleotide label included at the 3' end of the photoaptamer is used. In this example, the marker is a 3' immobilization region used in the SELEX method. VEGF photoaptamer 509-80 was synthesized using standard methods for synthesizing solid phase phosphoramidite DNA synthesis on a conventional synthesizer. The 5-BrdU containing the photoaptamer was cleaved and deprotected under milder conditions than those for DNA synthesis (tert-butylamine: methanol: water 1: 2, 70 ℃,5 h), filtered and evaporated to dryness. Aptamers were precipitated with ethanol and purified by reverse phase HPLC. DNA probes complementary to the label on the photoaptamer and comprising an amine reactive group were synthesized as described above and cleaved and deprotected using standard DNA methods. The probe was purified by ethanol precipitation only.

B. Immobilization of capture probes on amine reaction surfaces: reverse complement Capture tags were synthesized as described above, with a 5' amine and coupled to an amine reverseA surface of a glass slide comprising a Cyclic Olefin Copolymer (COC) substrate coated with a methacrylate copolymer (for further description see international application No. PCT/US2006/008877(WO 2006/101798), filed 3/14/2006, entitled "Polymer Compound for biological Use and biochemical substrate Using Such a Polymer Compound"), referred to herein as a "methacrylate copolymer surface". A microarray of capture probes is printed onto the surface. Briefly, the amine containing capture probe reacts with the surface-embedded active ester group. Capture probes in buffer (300mM sodium phosphate, 25mM sodium borate (pH9.5, 21 ℃), 0.01% Tween20 and 1mM 4-Dimethylaminopyridine (DMAP)) to 20. mu.M. Capture probes were deposited in triplicate at 250pL spots on the slide surface of the array using a GeneMachines OmniGrid access microarray (microarray). The arrayed slides were incubated at room temperature and 65% humidity for 1 hour, followed by 1 hour at 65 ℃ and finally overnight at room temperature. The remaining amine reactive groups were hydrolyzed with 20mM NaOH for 5 minutes, followed by 10H₂O, then in N₂Drying under air flow. Slides were stored at room temperature in a dry room in a dark room prior to use.

VEGF aptamer 509-80 was mixed at 1nM concentration with 100. mu.L assay diluent (1xSB17(40mM HEPES, 102mM NaCl, 5mM KCl, 5mM MgCl)₂，1mMEDTA)，0.1％Tween 20, 0.05% BSA, 100. mu.g/mL herring sperm DNA) or 10% plasma (10% plasma in 1xSB18(40mM HEPES, 102mM NaCl, 5mM KCl, 5mM MgCl)₂) 200 ug/mL herring sperm DNA, 0.1% Tween20) Serial dilutions of the target protein VEGF from 5nM to 1.6pM were incubated. The sample was gently mixed and incubated at 37 ℃ for 30 minutes. Excimer Laser (Tui Laser Excisstar S200, 10ns pulse width) for sampleDegree, 200Hz, 0.8 mJ/pulse) emitted 1 joule of 308nm light. Staining was performed by adding 1. mu.L of succinimidyl-Alexa647 at 10mg/mL in DMSO to each sample. The solution was mixed and reacted at 4 ℃ overnight. The staining reaction was quenched by the addition of 5. mu.L of 10% BSA and incubation for 2 hours. Finally, 18. mu.L of 5M NaCl and 4.5. mu.L of 10% Triton were added to 90. mu.L of 10% plasma sample, and 18. mu.L of 5M NaCl and 0.9. mu.L of 10% Triton were added to 90. mu.L of buffer sample. Grace Plate pads were attached to the slide containing the immobilized probes to form 16 wells. Each well was incubated with 80. mu.L of a solution containing 600. mu.L of 5M NaCl, 30. mu.L of 10% Triton and 3mL of assay diluent at 42 ℃ for 15 minutes. The solution was then removed and replaced with 80. mu.L of sample. The wells were sealed with Microseal 'F' membrane and the slides were mixed for 3 hours at 600rpm at 37 ℃ in Eppendorf Thermomixer R. The hybridization solution was removed and the wells were washed 3 times with a solution containing SB17, 0.5% Triton, 100. mu.g/mL herring sperm DNA and 1M NaCl. The pad was then removed and the entire slide placed in a 30mL solution containing SB17, 0.5% Triton and 1M NaCl in a pap jar for 30 minutes, followed by 1xSB17, 0.05% TweenThe rinse was performed for 2 minutes and 0.5xSB17 for 30 seconds. Slide glass on N₂Dried in air stream and scanned with a TECAN LS300 fluorescence scanner (excitation 633 nm/emission 670 nm). The resulting TIF images were analyzed using Imaging Research, inc. ArrayVision software, using standard techniques for processing microarray images to isolate features and calculate average intensities.

The results are shown in FIG. 4, where the results of buffer (FIG. 4A) and plasma (FIG. 4B) were used to quantify VEGF aptamer capture probes as described above. Linear reactions serially diluted in two media, obtained by subtracting the no protein buffer control in each reaction, contained VEGF concentrations ranging from 2pM to 5 nM.

Example 2 detection and quantification of multiple target proteins in buffer by photoaptamer assay with hybrid Capture Using surface immobilized probes

This example illustrates the steps of multiplex format analysis with 10 photoaptamers and their target proteins in buffer as shown in FIGS. 2A, 2B and 2C.

A. Preparation of labeled photoaptamers: unique oligonucleotide tags assigned to Affymetrix GeneChipTest3 Array obtained a panel of gene expression probes reverse complementary sequence of each of the aptamers. Labeled photoaptamers were prepared as described in example 1, except that an amine group was added to the 5' end of the photoaptamer. Prior to use, the aptamer solution was heated at 95 ℃ for 3 minutes, then cooled to 37 ℃ under control at a rate of 0.1 ℃ per second.

B. Immobilization of capture probes on amine reaction surfaces: reverse complement capture tags were immobilized as described in example 1, except that here 3' amine was used for the probe and DMAP was removed in printing (print) buffer and 0.0025% Tween was used20. The printed slides were incubated at 65 ℃ for 2 hours and then stored in a desiccator overnight. The slides were then treated with methoxyethylamine (pH 9.537 ℃) to remove unbound probes and consume excess active ester groups on the surface.

The analysis was performed as the protocol shown in FIGS. 2A, 2B and 2C. The basic method described in example 1 was used. However, 41 photoaptamers were complexed and 10 target proteins of these aptamer subgroups were serially diluted in the sample. In this example, the protein quantified: the photoaptamer pair includes: bFGF: 6-7, ERBB 4: 1797-38, IL-1R 4: 1472-3, MCP-3: 851-90, PAI-1: 1921-52, TIMP-1: 90536. tPA: 987-51, uPA: 1162-70, VEGF: 509-80, VEGF sR 2: 1546-23.

41 photoaptamers, 6 serial dilutions of each 2nM and 10 target proteins and protein-free control in 100. mu.L assay buffer (SB17, 0.1% Tween)20) Incubate at 37 ℃ for 30 minutes. After the sample was irradiated, it was stained as described in example 1 except that 2.1. mu.L of succinimidyl-Alexa647 was used and the reaction was incubated for 2 hours at room temperature in a dark room. The staining reaction was quenched by the addition of 25. mu.L of 5M NaCl, 5. mu.L of Triton X-100, 13.5. mu.L of 100mM glycine and 1.5. mu.L of 10mg/mL herring sperm DNA at room temperature for 40 minutes in the dark, followed by 2 minutes at 70 ℃. Before hybridization, a glass slide with a pad arrayed with hybridization probes was prepared as described in example 1. The prehybridization solution was removed, the hybridization sample was added to the well and incubated in a humidified chamber at 45 ℃ for 2 hours. The hybridization solution was removed and the wells were washed 3 times with 45 ℃ wash (1xSB17, 0.33% Triton X-100 and 1M guanidine hydrochloride). The pad was then removed and each whole slide was placed in a pap jar with wash buffer for 20 minutes at 45 ℃ followed by 1xSB17, 0.1% TweenRinse for 2 minutes at 20 and finally rinse at 0.25xSB 17. Slides were dried, scanned and quantified as described in example 1. The results are shown in FIG. 5, which shows the signals generated by multiplex analysis of 10 target proteins in buffer at protein concentrations ranging from 10pM to 1 nM.

Example 3 detection and quantification of multiple target proteins in serum by photoaptamer assay with hybrid Capture Using surface immobilized probes

This example demonstrates the analysis of multiplexed versions of serum samples measured with 57 photoaptamers as shown in figures 2A, 2B and 2C.

A. Preparation of labeled photoaptamers: labeled photoaptamers were prepared as described in example 2.

B. Immobilization of capture probes on amine reaction surfaces: the reverse complement of capture labels was immobilized as described in example 2.

57 photoaptamers were divided into 2 groupsDiversification was performed with 1 group of 27 photoaptamers for low abundance targets in human serum or plasma and a group of 30 photoaptamers for high abundance targets. 2 dilutions of 14 serum samples were prepared and mixed with 2 aptamer groups such that the final concentration of each aptamer was 1nM and 10% serum, 0.9xSB1, 0.1% Tween was used for the 27 aptamer groups20，50mg/mL lghsDNA，10mg/mL(BrdU)₃₀For 30 aptamer groups 1% serum, 0.99xSB1, 0.1% Tween was used20，5mg/mL lghsDNA，1mg/mL(BrdU)₃₀. SB1 contains 40mM HEPES, 102mM NaCl, 5mM KCl, 1mM MgCl₂And 1mM CaCl₂. The samples were equilibrated, crosslinked and stained as described in example 2. The staining reaction was quenched by addition of 10 μ L of 10% low Fatty Acid (FA) BSA, 25 μ L of 5nM NaCl, 5 μ L of 10% Triton X100 and 7 μ L of 100mM glycine and incubation at room temperature for 40 min. After quenching, 1.5. mu.L of 10% SDS was added and the sample was heated at 42 ℃ for 2 minutes.

Slides were prepared as described in example 1, and loaded with 1xSB1, 0.1% Tween20, 0.66% Low FA BSA, 830mM NaCl, 0.33% Triton X100, 0.1% SDS, 50mg/mL Large herring sperm DNA and 10mg/mL (BrdU)₃₀The temperature was maintained for 15 minutes. The prehybridization buffer was removed, stained samples were added and incubated in a humidified incubator at 60 ℃ for 2 hours. After hybridization, the slides were washed, dried, scanned and quantified as described in example 2.

Fig. 6 shows signals quantified by scanning pictures repeatedly measuring 57 photoaptamers in two individual serum samples. The measurements proved to be highly reproducible, with Pearson correlation values measured for aptamers in duplicate samples better than 0.99.

Example 4 kinetic excitation by dilution in photoaptamer assays with hybrid Capture with surface immobilized probes

This example illustrates the use of two optional steps in the analysis depicted in FIG. 3, kinetic excitation followed by removal of free protein. Kinetic excitation by dilution elucidates the removal of aptamer-protein non-specific complexes and the retention of aptamer-target affinity complexes in plasma. This example also illustrates the use of bead capture after dilution to concentrate the sample and allow free protein to be removed prior to staining.

A. Preparation of labeled photoaptamer 987-51: labeled photoaptamers were prepared as described in example 2.

B. Immobilization of capture probes on amine reaction surfaces: the reverse complement of capture labels was immobilized as described in example 2.

Light aptamer 987-51 at a concentration of 20nM and 2 protein-free controls and 10 serial dilutions of the target protein tPA in assay buffer (SB17, 0.1% Tween)20) Or plasma (10% plasma in SB18, 0.1% Tween)20) are mixed. To evaluate the response without an optical challenge, 2 additional control samples were prepared in buffer and plasma, 1 without protein and the other with the highest protein concentration. The 28 sample volumes were 10 μ L, 14 in buffer, 14 in plasma, and incubated at 37 ℃ for 30 minutes. The first 12 samples in buffer and plasma were prepared by adding SB17, 0.1% Tween20 diluted 50-fold and incubated for 5 minutes, followed by irradiation of all 28 samples with 1J UV (OAI Hg lamp filtered light source). After irradiation, the remaining 2 samples in buffer and plasma were also diluted 50-fold as above.

Each sample was adjusted to 1M NaCl and incubated with 25. mu.g Dynal paramagnetic beads coupled to an oligonucleotide complementary to the 3' end of aptamer 987-51 for 90 minutes at 45 ℃. The sample was centrifuged at 1000g for 2 minutes and the supernatant removed. The beads were incubated with 100. mu.L of SB17, 0.1% Tween20 washes 3 times. The beads were suspended in 100mM sodium bicarbonate, 1mM EDTA, 0.02% Tween-20, 0.2mg/mL Alexa647-NHS dye and shaking at room temperature for 1 hour. The staining solution was removed and replaced with 100. mu.L of SB17, 0.1% Tween-20, 10mM glycine to quench the staining reaction. The beads were then washed 3 times with 100. mu.L of guanidine wash buffer (SB17, 0.1% Tween-20, 0.33% Triton X-100, 1M guanidine hydrochloride), suspended in 70. mu.L of 0.33% Triton X-100, 1mg/mL dextran sulfate, and heated at 95 ℃ for 5 minutes to elute the aptamer from the magnetic beads. The lined slides were prepared and pre-hybridized as described in example 2. Each 65. mu.L of the supernatant containing the eluted aptamer was transferred to wells containing 25. mu.L of 3.6M NaCl, 144mM HEPES, 0.33% Triton X-100. Slides were incubated overnight at 45 ℃ in a humid incubator, then washed, dried, scanned and quantified as described in example 2.

Fig. 7 shows a schematic of the results after 50-fold dilution in buffer (●) and plasma (a). The signal obtained from the high protein control undiluted sample (o-buffer,. DELTA.plasma) is in good agreement with the dilution values, indicating that there is little loss of target signal during the kinetic challenge. The protein-free plasma RFU without dilution and with dilution was 838 RFU (□ 0.1.1 pM) and 131 RFU (Δ), resulting in an 84% reduction in signal, presumably due to removal of aptamer non-specific complexes during kinetic challenge.

Example 5 kinetic excitation of competitors in photoaptamer assays with hybrid Capture Using surface immobilized probes

This example illustrates the optional step of introducing kinetic excitation in the analysis depicted in FIG. 3. The kinetic excitation by addition of competitor molecules in this example illustrates the removal of aptamer-protein non-specific complexes and the retention of aptamer-target affinity complexes in plasma.

A. Preparation of labeled photoaptamer 987-51: labeled photoaptamers were prepared as described in example 2.

B. Immobilization of capture probes on amine reaction surfaces: the reverse complement of capture labels was immobilized as described in example 2.

Light aptamer 987-51 at a concentration of 20nM and protein-free controls and 10% plasma in SB18, 0.1% TweenThe target protein tPA was mixed at 6 serial dilution concentrations diluted in 20. Groups 2 were prepared to facilitate comparison of samples with or without competitor addition. Each solution was incubated at 37 ℃ for 30 minutes. After 30 minutes equilibration, half 1 sample of one set was added to an equal volume of 550. mu. MdN₁₅Competitors (at SB17, 0.1% Tween)20) at 37 ℃ for 9 minutes, followed by irradiation as described in example 4. After irradiation, SB17, 0.1% Tween-20 was added to the competitor-free set of samples to equalize the competitor addition volume. The samples were stained as described in example 4, except that the incubation time was 2 hours. Staining reactions were quenched by the addition of 45. mu.L of 0.83M NaCl, 0.33% Triton X-100, 0.1mg/mL big herring sperm DNA and 9mM glycine. The samples were mixed and incubated at room temperature for 40 minutes in a dark room followed by heating at 70 ℃ for 2 minutes.

Slides were prepared with pads and in solution (SB18, 0.1% Tween) as described in example 220, 0.33% Triton X-100 and 100. mu.g/mL big herring sperm DNA) in a containerAnd (4) warming. The samples were added to the slide wells and incubated overnight at 45 ℃ in a humidified incubator, then rinsed, dried, scanned and quantified as described in example 2.

The results are shown in fig. 8, where the dose response curve for tPA in plasma with or without added competitor is shown. The plasma values of the non-added protein decreased by 70% due to the addition of competitor, while the response to the highest target concentration was not altered in the presence of competitor.

Example 6 kinetic excitation by bead Capture and washing of aptamer affinity complexes in Photoaptamer assays with surface immobilized probes for hybrid Capture

This example illustrates the optional step of introducing kinetic excitation in the analysis depicted in FIG. 3. Kinetic excitation was achieved in this example by capturing aptamer affinity complexes on beads and washing the immobilized complexes to remove unassociated target protein prior to cross-linking.

A. Preparation of labeled photoaptamers 987-51, 1152-46 and 1920-1: labeled photoaptamers were prepared as described in example 2.

B. Immobilization of capture probes on amine reaction surfaces: the reverse complement of capture labels was immobilized as described in example 2.

The photoaptamers 987-51, 1152-46 and 1920-1 at a concentration of 4nM were mixed with biotinylated probes complementary to the unique tag sequence of each photoaptamer at a concentration of 8nM and incubated at 95 ℃ for 15 seconds, followed by slow cooling to 37 ℃ at 0.1 ℃ per second. Photoaptamer at concentration 0.2 nM: photoaptamer with 0.4nM probe: biotinylated Probe complexes and detection in assay buffer (SB17, 0.1% Tween)20) Or plasma (10% plasma in SB18, 0.1% Tween)20) and 6 serial dilution concentrations of the target proteins tPA, PAI-1 and IL-6. These 14 samples were 100 μ L in volume, 7 in buffer, 7 in plasma, all incubated at 37 ℃ for 30 minutes, after which 50 μ g of Dynal MyOne streptavidin beads were added to each sample and incubated at 37 ℃ for 2 minutes, mixed to capture the non-covalent aptamer: biotinylated probe: a protein complex. The beads were incubated with 100. mu.L of SB17, 0.1% Tween20, 0.1mg/mL herring sperm DNA was washed 3 times for 30 seconds, resuspended in 100. mu.L of the same solution, irradiated with 4J UV light (OAI Hg lamp filtered light source) and mixed.

The beads were treated with 100mM sodium bicarbonate, 1mM EDTA, 0.02% Tween20 washes 1, resuspension in 100mM sodium bicarbonate, 1mM EDTA, 0.02% Tween-20, 0.2mg/ml of LAlexa647-NHS dye and shaking at room temperature for 1 hour. The beads were washed 3 times with 100. mu.L of SB17, 0.1% Tween-20, 0.33% Triton X-100, 1M guanidine hydrochloride, 25mM glycine, 1 time with 100. mu.L of SB17, 0.1% Tween-20, 0.33% Triton X-100, resuspended in 95. mu.L of 40mM HEPES, pH7.5, 0.33% Triton X-100, and heated at 70 ℃ for 5 minutes to elute the aptamer from the magnetic beads. The lined slides were prepared and pre-hybridized as described in example 2. mu.L each of the supernatant containing the eluted aptamer and 30. mu.L of 40mM HEPES, pH7.5, 3M NaCl, 0.33% Triton X-100 were combined, incubated at 70 ℃ for 2 minutes, and 110. mu.L each of the solutions was transferred to a slide well. Slides were incubated overnight at 45 ℃ in a humidified incubator. Samples were removed and washed 3 times with 150. mu.L of SB17, 0.1% Tween-20, 0.33% Triton X-100, 1M guanidine hydrochloride at 45 ℃. The padded slides were disassembled and placed in a pap jar containing 30mL SB17, 0.1% Tween-20, 0.33% Triton X-100, 1M guanidine hydrochloride and vortexed at 45 ℃ for 20 minutes. The slide is thenTransferred to a pap jar containing 30mL SB17, 0.1% TWEEN-20 and rotary mixing at 20 ℃ for 2 minutes, transferred to a pap jar containing 30mL 0.2X SB17, 0.02% TWEEN-20 for 15 seconds without mixing, dried, scanned and quantified as described in example 2.

FIG. 9 shows a graphical representation of the results for tPA aptamer 987-51 (FIG. 9A), PAI-1 aptamer 1152-46 (FIG. 9B) and IL-6 aptamer 1920-1 (FIG. 9C) in buffer (●) and plasma (. tangle-solidup.). RFU values have been corrected by subtracting protein free buffer RFU values for each aptamer. These aptamer (Δ, 1pM) corrected protein-free plasma RFU values were 66, 26, and 49RFU, respectively.

Example 7 removal of free aptamer in Photoaptamer assay with hybrid Capture with surface-immobilized probes

This example illustrates the optional step depicted in FIG. 3 of removing free aptamer prior to hybrid capture on a surface. Free aptamers were obtained by using K⁺the/SDS precipitates the protein and aptamer-protein covalent complexes while leaving free aptamer in solution to remove free aptamer. The supernatant containing free aptamer was decanted and the pellet was suspended to complete the procedure.

A. Preparation of labeled photoaptamers: labeled photoaptamers were prepared as described in example 2.

B. Immobilization of capture probes on amine-reacted surfaces: the reverse complement of capture labels was immobilized as described in example 2.

Binding reactions were prepared as described in example 1 in SB18, with a final concentration of 2nM photoaptamer and serial dilutions of 2nM to 0.64pM target protein and no protein control. The reaction was incubated at 37 ℃ for 30 minutes and irradiated as described in example 1. The samples were then transferred to 1.5-mleppendorf tubes and 10 μ L of pooled plasma was added. To each sample was added 300. mu.L of SDS solution as follows: 1.33% SDS, 1.33. mu.g/ml tRNA and 10mM HEPES (pH7.5) (final volume 400. mu.L). The sample was vortexed vigorously for 10 seconds and then incubated at 37 ℃ for 10 minutes. 6 is added into each reactantμ L2.5M KCl, the sample vortexed vigorously for an additional 10 seconds and then placed on ice for 10 minutes. The pellet was centrifuged at 8,000g for 5 minutes at 4 ℃ in a small centrifuge. The resulting supernatant was removed. The pellet was washed with cold 200mM KCl, 10mM HEPES (pH7.5) followed by gentle vortexing for 5 seconds. The precipitate was again centrifuged at 8,000g at 4 ℃ for 5 minutes. The wash supernatant was removed. Each pellet was suspended in 200. mu.L of warm (> 37 ℃)1mM EDTA, 10mM HEPES, pH 7.5. Paramagnetic beads as described in example 4 were added to each sample and the bead suspension was mixed vigorously for 30 minutes at 50 ℃. Using a microwell plate magnet, beads were incubated with 100. mu.L of SB17, 0.1% Tween20 buffer washes 3 times. The beads were suspended in NHS-Alexa647 carbonate buffer (pH8.5) containing 0.2mg/ml amine reaction and incubated for 60 min at 25 ℃ with constant mixing. The reaction was quenched, the beads washed, and the aptamers were eluted and hybridized as described in example 4. Slides were scanned and quantified as described in example 1.

FIG. 10 shows the results obtained with and without treatment for free aptamer removal. The signal is higher when uncomplexed aptamer is removed from the sample before the sample is introduced to the probe.

Example 8 Generation of UPS assay signals under conditions that affect aptamer affinity complex signals without affecting aptamer covalent complex signals

This example demonstrates the effect of using detergents and high salt concentrations on the covalent attachment of a target molecule to its photoaptamer upon hybridization of the photoaptamer complex to its probe on a solid support. The results of the analysis performed with or without covalent cross-linking (mediated here by photoactivation) of the plasma samples and samples containing bFGF in the buffer were compared.

A. Preparation of labeled photoaptamer 6-7: the oligonucleotide marker used here is the 3' immobilization region used in the SELEX method. The bFGF photoaptamers were synthesized as described in example 1.

B. Immobilization of capture probes on amine-reacted surfaces: the reverse complement capture tags were immobilized as described in example 1.

1nM 6-7 was prepared in buffer and plasma binding solution as described in example 1. Only 10nM bFGF protein was added to the buffer samples. The samples were mixed gently and incubated at 37 ℃ for 30 minutes. One set of samples was irradiated as described in example 1 and another set of replicate samples were not irradiated. The samples were then stained as described in example 1, except that the reaction time was 3 hours at room temperature, followed by addition of 10. mu.L BSA, 25. mu.L 5M NaCl and 5. mu.L 10% Triton X-100 and incubation for 2 hours. Slides were prepared as described in example 1, and 80. mu.L of prehybridization solution (500. mu.L of 5M NaCl and 100. mu.L of 10% Triton in 2mL of sample buffer) was added to each well for 15 minutes at 42 ℃. The prehybridization solution was removed and replaced with 80. mu.L of sample. The wells were sealed with Microseal 'F' membrane and the slides were mixed in Eppendorf Thermomixer R at 600rpm for 2 hours at 40 ℃ followed by 20 minutes at 35 ℃. The wells were washed 2 times with 1xSB17, 0.5% Triton, 1M NaCl and then incubated for 15 minutes in the second wash. After hybridization, the slides were treated as described in example 1, except that the 3 wash times were 15 minutes, 2 minutes, and 30 seconds. Slides were dried, scanned and quantified as described in example 1.

The results of bFGF aptamer 6-7 are shown in FIG. 11. It appears that the signal obtained with 10nM bGFG in buffer and 10% plasma is dependent on light. The signal of a non-illuminated sample in which covalent attachment of target and photoaptamer cannot be formed is comparable to the signal observed outside the feature, referred to herein as "general background". The concentration of endogenous bFGF in 10% plasma is rather low, as reflected by a very weak plasma signal relative to no light and general background response.

Example 9 detection of C4b crosslinked to DNA photoaptamer comprising 5-benzyl-dT and 5-bromodC nucleotides in buffer

This example illustrates the activity of photoaptamers containing modified nucleotides in the assay format shown in fig. 2A, 2B and 2C. DNA photoaptamers of C4b protein (1987-74) consisted of 5-benzyl-dT and 5-bromo-dC nucleotides instead of standard dT and dC.

A. Preparation of labeled photoaptamer 1987-74: vector inserts containing aptamer sequences were amplified from E.coli (E.coli) cells by PCR with primers specific for the aptamer immobilization region. The 3' primer is biotinylated and can capture the PCR product on MyOne-streptavidin beads. After washing, the non-biotinylated strand of the captured duplex was removed by washing with 20mM NaOH and aptamers were formed by extension of oligonucleotide primers with unique capture tags conjugated to the 5' primer sequence. Primer extension with template DNA still coupled to streptavidin beads was performed using KOD DNA polymerase in a mixture containing dATP, 5-Br-dCTP, dGTP and 5-benzyl-dUTP to incorporate the modified nucleotides. Aptamers were collected from the beads by washing with 20mM NaOH followed by neutralization with HCl.

B. Immobilization of capture probes on amine-reacted surfaces: the reverse complement capture tags were immobilized as described in example 1.

1987-74 and a series of C4b proteins at a final concentration of 2nM, SB17, 0.1% Tween20 and a protein-free control, wherein the concentration of the diluent is as follows: 25nM, 5nM, 1nM, 0.2nM, 0.04 nM. Samples were equilibrated and irradiated as described in example 1, after which 2. mu.L of herring sperm DNA (10mg/ml) was added. Add 7.5. mu.L of NHS-Alexa-647(1.33mg/mL in DMSO) to each sample and incubate for 2 hours at room temperature to fluorescently label C4b protein. Addition of 25. mu.L of 5M NaCl, 5. mu.L of 10% Triton-100 and 1. mu.L of 100mM glycine quenched unreacted label, disrupting non-covalent interactions to facilitate subsequent hybridization.

Slides were prepared as described in example 1, and mounted with SB17, 0.1% TweenA solution of 20, 0.8M NaCl, 20. mu.g/mL herring sperm DNA and 0.33% Triton X-100 at 42 deg.CThe temperature was maintained for 15 minutes. After removing the solution, 80. mu.L of labeled sample, 1 per subarray (subarray), was added to the slide and incubated at 42 ℃ for 30 minutes on a rotating platform. The samples were removed and the substrate washed 2 times with prehybridization solution and with herring sperm DNA or Tween20 prehybridization solution was washed once with 1xSB17, 0.1% Tween20 washes 1 time, 1 time with 0.25xSB 17. The slides were then dried, scanned and quantified as described in example 1.

FIG. 12 shows the results of dose response curves from 1987-74 obtained as a function of target concentration, illustrating the activity of the modified nucleotide aptamers in the assay.

Example 10 UPS hybrid Capture assay on two different surfaces Using two independent staining methods

This example demonstrates the functionality analyzed on the methacrylate copolymer surface of the previous example and on the Schott Nexterion amine-reacted surface on a glass substrate on the surface of two different compositions. In addition, the target protein staining reaction was performed as in the previous examples, attaching Alexa-647 directly to the target protein or labeling the target protein with biotin first followed by streptavidin-Alexa-647. This assay allows quantitative detection of VEGF protein in buffer using surface or staining methods.

A. Preparation of labeled photoaptamer 509-80: labeled photoaptamers were prepared as described in example 1.

B. Immobilization of capture probes on amine-reacted surfaces: capture probes with 5' amines were synthesized as described in example 1 and immobilized on both surfaces. Immobilization on Schott Nexterion slides was performed by dissolving the probes at 40 or 20. mu.M in 300mM sodium phosphate (pH8.5),. 005% Tween20 and 0.001% sarkosyl. The capture probes were deposited as described in example 1. After probe deposition, slides were incubated overnight in a dry box and then incubated with 100mM sodium bicarbonate (pH8.5) and 0.1% Tween20 at room temperature for 8 hours. The slides were then rinsed 10 times with water at N₂Drying under air flow. The capture probes were immobilized on the methacrylate copolymer surface as described in example 1.

VEGF aptamer 590-80 at a concentration of 1nM in a volume of 100. mu.L, was diluted in 6 serial dilutions from 50nM to 16pM of VEGF and no protein control in assay dilutions (SB17, 0.1% Tween)20, 0.05% BSA, 100. mu.g/mL herring sperm DNA). The samples were gently mixed, incubated at 37 ℃ for 15 minutes, and then irradiated as described in example 1. The samples were separated, stained directly with NHS-Alexa-647, or with NHS-PEO₄Biotin reaction and subsequent staining with streptavidin Alexa-647. For staining with NHS-Alexa-647, the procedure of example 1 was used except that the reaction was at room temperature for 4 hours. For with NHS-PEO₄Biotin reaction, adding 1. mu.L of 20mM NHS-PEO dissolved in DMSO to 100. mu.L of sample₄-biotin. The solution was mixed and incubated at room temperature for 4 hours. Both staining reactions were quenched by the addition of 23. mu.L of 5M NaCl, 11.4. mu.L of 10% BSA and 1.1. mu.L of 10% Triton X-100 and incubation for 2 hours.

Two different slides were padded as described in example 1 and prehybridized with 1xSB17, 0.1% Triton X-100, 0.5% BSA and 100. mu.g/mL herring sperm DNA for 15 min at 42 ℃. The solution was then removed and replaced with 80 μ L of sample. The wells were sealed with Microseal 'F' membrane and the slides were mixed with Eppendorf Thermomixer R at 600rpm, 42 ℃ for 3 hours followed by 34 ℃ for 1 hour. For with NHS-Alexa-647 direct labeled samples, wells were washed 3 times with 1xSB17, 0.5% Triton and 1M NaCl. The pad was removed and the entire slide was placed in 30mL of SB17, 0.5% Triton, 1M NaCl in a pap jar for 30 minutes, followed by 1xSB17, 0.1% TweenRinsing for 2 minutes at 20 and then for 20 seconds at 0.25xSB 17. For with NHS-PEO₄Biotin-reacted samples, wells with 1xSB17, 0.1% Tween20, 0.5M NaCl, 100. mu.g/mL herring sperm DNA, 0.5% BSA (streptavidin staining buffer) 3 times. A4. mu.g/mL solution of streptavidin Alexa-647 was prepared in streptavidin staining buffer and 80. mu.L of this solution was added to each well for 15 min at 37 ℃. The wells were then washed with 1xSB17, 0.1% Tween20 washes 3 times. The pad was removed and the entire slide was 1xSB17, 0.1% Tween in a 30mL pap jar20 for 30 minutes followed by a rinse of 0.25xSB17 for 20 seconds. Slides were dried, scanned and quantified as described in example 1.

Figure 13 shows the results of dose response of Schott Nexterion (figure 13A) and methacrylate copolymer surface (figure 13B) as a function of protein concentration. Both surfaces gave similar quantitative results, indicating that the analysis was surface independent. It can also be seen that both staining strategies are equally efficient.

Example 11 Affymetrix GeneChip on coated Quartz surfacesTest3 array by hybridizationDetection buffer for capture photocrosslinked protein and target proteins VEGF and bFGF in serum

This example demonstrates another surface Affymetrix GeneChipUse of the Test3 array (quartz glass surface) in the hybridization capture step of the assay. The analysis was performed in buffer and serum.

A. Synthesis of labeled photoaptamers 509-80 and 6-7: reverse complementary AffymetrixGeneChipTwo probes named 201 and 1121 of the Test3 array were assigned to aptamers 509-80 and 6-7 prepared as described in example 1.

B. Immobilization of capture probes on amine-reacted surfaces: GeneChip with in situ synthesized probesTest3 array was purchased from Affymetrix.

VEGF aptamers 509-80 and bFGF aptamers 6-7, each at a concentration of 2nM, in a volume of 100. mu.L, were assayed in dilutions (1xSB17, 0.1% Tween)20, 0.02% B SA, 100. mu.g/mL herring sperm DNA) with 20nM VEGF and bFGF protein. The samples were gently mixed and incubated at 37 ℃ for 60 minutes, then irradiated and treated with NHS-PEO as described in example 9₄Biotin staining, except that the reaction time was 1 hour. The reaction was quenched by the addition of 10. mu.L 10% BSA, 1. mu.L 10mg/mL herring sperm DNA, 5. mu.L 10% Triton X-100 and 25. mu.L 5M NaCl. 10 μ L of serum was added to one sample to achieve approximately 10% serum sample.

GeneChip from AffymetrixTest3The arrays were incubated with 100. mu.L of solution (100. mu.L of assay diluent, 10. mu.L of 10% BSA, 1. mu.L of 10mg/mL herring sperm DNA, 25. mu.L of 5M NaCl, 5. mu.L of 10% Triton X-100 and 7. mu.L DMSO) at 45 ℃ for 1 hour, then 100. mu.L of the sample was added to Test3 array chamber, incubated at 45 ℃ for 60 minutes, and then washed with buffer (1xSB1, 0.5% Triton X-100 and 1M NaCl). The array was then placed on an Affymetrix GeneChipApplied to a fluidics bench used to perform standard Affymetrix washing and staining procedures. The array was then read and quantified on an Affymetrix scanner. The results are shown in fig. 14. In the buffer (FIG. 14A), VEGF aptamer hybridized to probe 201 (shown as 1 in the figure) at an intensity of 3500RFU, bFGF aptamer hybridized to probe 1121 (shown as 2 in the figure) at an intensity of 23000 RFU. In serum (fig. 14B), the relative strengths of VEGF and bFGF were 5000(1) and 18000 (2).

Example 12 Affymetrix GeneChip Pre-blocked on coated Quartz surfaces with skim milk, Superblock or unstained plasmaSurface passivation of Test3 array this example illustrates blocking of Affymetrix GeneChip with skim milk, Superblock or unstained plasmaThe surface of the serum and plasma adsorption on the Test3 array was passivated.

A. Preparation of biotinylated oligonucleotides: and immobilization on GeneChip3 different probe-annealed biotinylated oligonucleotides, denoted 201, 1121, and 108 on the Test3 array, were synthesized as described in example 1.

B. Plasma biotinylation: 160 μ L of 21mM NHS-PEO₄Biotin addition to 100mL of 1xSB18, 0.1% Tween20 for 2 hours, and then quenched by the addition of 1mL of 100mM glycine (pH 7.5). Biotinylated plasma was stored at-20 ℃.

Control buffer contained 0.75xSB17, 0.1% BSA, 100. mu.g/mL herring sperm DNA, 0.1% Tween20 and 0.8M NaCl. 10% biotinylated plasma was prepared with control buffer and biotinylated plasma. To 1mL of 10% biotinylated plasma was added 50. mu.L of 10% TritonX-100, 10. mu.L of 10% SDS and 10. mu.L of 10mg/mL herring sperm DNA to pretreat the plasma, which was heated to 95 ℃ for 10 minutes, followed by addition of 200. mu.L of 5M NaCl. mu.L of 1000 Xbiotinylated probe mixture was added to 1mL of control buffer or 10% plasma.

4 Affymetrix Test3 arrays were incubated in 1xSB17, 1% BSA, 0.4% Triton X-100, 0.1% SDS, 1M NaCl and 100. mu.g/mL herring sperm DNA. Array B was blocked with a solution of 2% non-fat DRY MILK (Nestle Carnation, INSTANT NONFAT DRY MILK) suspended in PBS, 0.1% SDS and 0.4%, and array C was blocked with StarterBlock (PIERCE), 0.1% SDS and 0.4% solution. Array D was blocked with 10% pooled plasma, 0.1% SDS and 0.4% Triton X-100 solution. Each blocking solution was heated to 95 ℃ for 10 minutes and cooled to room temperature before being added to the array. Each Test3 array was blocked with 100. mu.L of the respective blocking solution at 45 ℃ for 1.5 hours. Then 100. mu.L of sample was added to each array. Array A was spiked with control buffer with probes and arrays B, C and D were spiked with 10% plasma with probes. The array was incubated at 45 ℃ for 45 minutes.

The array was then washed with 1xSB17, 0.4% Triton X-100, 0.1% SDS, 1M NaCl, 1% BSA by flowing 1mL of wash solution through the chamber, incubating the array for 5 minutes, then flowing another 1mL of wash solution through the chamber, and finally replacing the last 100. mu.L of wash solution with 100. mu.L of fresh wash solution. This is achieved byEach procedure was performed 3 times. The array was placed on an Affymetrix GeneChipThe incubation was performed for approximately 1 hour prior to application of the fluidics bench and then read on an Affymetrix scanner.

The results are shown in FIGS. 15A-D. The general background for 4 arrays was 40, 300, 400 and 500RFU, and the measured values for 3 biotinylated probes on the array were-17,000, -34000 and 18000.

Example 13 GeneChip Pre-blocked with skim milkDetection of target proteins by hybrid Capture of Photocrosslinked proteins in Test3 array

This example illustrates Affymetrix GeneChip on a coated glass surface blocked with skim milkHybridization capture of the target proteins IL-1R 4 and bFGF on the Test3 array.

A. Synthesis of labeled photoaptamers 1472-3 and 6-7: affymetrix GeneChipThe reverse complement of the two probes represented as 1364 and 1121 in the Test3 array were assigned to aptamers 1472-3 and 6-7 and synthesized as described in example 1.

4 solutions each containing aptamers 1472-3 and 6-7 at a concentration of 2nM each were prepared in 10% plasma to achieve the following final concentrations of protein pair (IL-1R 4, bFGF): (0, 0), (1nM, 30pM), (100pM, 1nM) and (10pM, 100 pM). Plasma dilutions contained 0.9xSB18, 100. mu.g/mL herring sperm DNA, 5. mu.g/mL (BrdU)₃₀，0.1％Tween 20 and 10% plasma. The samples were incubated at 37 ℃ for 30 minutes and irradiated as described in example 1.5 mg/mL NHS-PEO in 1. mu.L DMSO₄Biotin is added to each sample, incubated at room temperature for 2 hours, followed by the addition of 2. mu.L of 100mM glycine (pH7.5), 1. mu.L of 10% SDS and 5. mu.L of 10% Triton X-100. The sample was then heated to 95 ℃ for 10 minutes, cooled to room temperature, and quenched by the addition of 10. mu.L of 10% BSA and 25. mu.L of 5M NaCl. Then 1. mu.L of 100 XProbe-108 was added as a control sequence.

The 4 Test3 arrays were blocked with skim milk powder solution for 3 hours at 45 ℃ as described in example 11 for array B. The array was then washed with PBS, 0.1% SDS and 0.4% Triton X-100 and incubated with 1xSB17, 1% BSA, 0.5% Triton X-100, 0.1% SDS, 1M NaCl and 0.1mg/mL herring sperm DNA for 20 minutes at 45 ℃. Then 100. mu.L of each sample was added to a separate array. The array was incubated at 45 ℃ for 45 minutes. The array was completed as described in example 12. The results are shown in fig. 16, where a linear dose response can be observed for both targets in plasma.

Example 14 in Luminex SeroMap^TMDetection of target protein C5b, 6 Complex, Neurotropan-3 and troponin I by hybrid Capture of photocrosslinked proteins in microspheres

This example demonstrates a Luminex SeroMap^TMUse of microspheres as solid supports for the detection and optionally quantification of target molecules that may be present in a test sample. These assays were performed with the target protein added to the buffer. The detection equipment IS a Luminex 100 IS equipment system.

Amine-terminated probes assigned to Photoaptamers 2184-64(C5b, 6 complex aptamer), 2273-34(neuroptropin-3 aptamer) and 2338-12 (troponin I aptamer) were probed with EDC (1-ethyl-3- [ 3-dimethylaminopropyl ] amine]Carbodiimide hydrochloride) chemical component to-COOH functionalized SeroMap^TMOn the microspheres.

Photoaptamers 2184-64, 2273-34 and 23338-12 and aptamer 3 'at final concentrations of 2nM each'The end-complementary biotinylated oligonucleotide was preannealed and then combined in buffer SB17, 0.05% Tween20 at a concentration in the range of 169fM to 3.33nM of a mixture of protein C5b, 6 complex, neotropin-3 and troponin I. Duplicate no-protein control assay samples were also prepared. A volume of 100. mu.L of these analytical samples was incubated at 37 ℃ for 15 minutes and then photocrosslinked. Dynal MyOne streptavidin beads (400. mu.g) were added to each assay sample, incubated for 10 minutes at 25 ℃ and mixed to capture the photoaptamer: biotinylated oligonucleotide and protein-photoaptamer: biotinylated oligonucleotide hybrids. The beads were treated with 100. mu.L of 100mM sodium bicarbonate, 1mM EDTA, 0.02% Tween20 and 10. mu. M D-Biotin (pH8.5) for 30 seconds, 2 times. The purpose of the D-biotin component in the buffer is to saturate the free streptavidin binding sites. The washed beads were suspended in 100. mu.L of 100mM sodium bicarbonate (pH8.5), 1mM EDTA, 0.02% Tween20 and 150 μ M thio-NHS-LC-Biotin (Pierce Biotechnology) to facilitate conjugation of the target protein with biotinylated photoaptamers. This biotinylation reaction was incubated at 25 ℃ for 1 hour with constant mixing. The beads were then incubated with 100. mu.L of SB17, 3.14M guanidine hydrochloride, 0.05% Tween20 washes 3 times followed by 2 washes with 100. mu.L SB17, 0.33% TritonX-100. The washed beads were suspended in 100. mu.L of 10. mu. M D-biotin, 0.05% Tween20, 10mM HEPES, pH7.5, and then heated to 70 ℃ for 5 minutes to release the photoaptamer from the bead-bound complementary biotinylated oligonucleotide. For each sample analyzed, 75 μ Ι _ of bead-eluting bulk volume was combined with 25 μ Ι _ of the following high salt buffer: 4M NaCl，0.4％Tween 20, 160mM Tris-Cl, pH 8.0. Add 11.25. mu.L of 20% SDS and transfer 30. mu.L of the appropriate probe-conjugated SeroMap to each assay sample^TMMicrospheres (1500 color coded microspheres/probes, 0.1% Tween)20, 1M NaCl, 1.25% BSA, 40mM Tris-Cl, pH 8.0). To facilitate hybridization of the photoaptamer and the microsphere conjugated probe, the assay samples were incubated at 65 ℃ for 2 hours with constant mixing. At 65 ℃, assay samples were transferred to 96-well microtiter vacuum filter plates using 200mM NaCl, 0.1% Tween20, 40mM Tris-Cl (pH8.0) was washed 4 times at 65 ℃. The microspheres were then suspended in 80. mu.L of 200mM NaCl, 0.1% Tween20, 40mM Tris-Cl (pH8.0), and transferred to a 96-well microtiter plate. mu.L of 10. mu.g/ml streptavidin-R-phycoerythrin (Molecular Probes # S866) was added to detect the crosslinking of the aptamer to the biotinylated target protein. After incubation at 37 ℃ for 15 minutes, the samples were analyzed for quantification of the standard Luminex apparatus signal (R-phycoerythrin).

FIG. 17 graphically depicts the results for C5B, 6 complex photoaptamer 2184-64 (FIG. 17A), neurotropin-3 photoaptamer 2273-34 (FIG. 17B), and troponin I photoaptamer 2338-12 (FIG. 17C). For each aptamer, MFI (median fluorescence intensity) values were corrected by subtracting the no protein control MFI values.

The invention described above relates to various embodiments and examples. No single particular embodiment, example, or element of a particular embodiment or example is to be construed as a critical, required, or essential element or feature of any or all the claims. Further, no element described in connection with the recitation of "essential" or "critical" is essential to the practice of the invention unless explicitly described as such.

It will be understood that various modifications and substitutions may be made to the disclosed embodiments without departing from the scope of the invention as defined in the following claims. The specification, including the drawings and examples, is to be regarded in an illustrative rather than a restrictive sense, and all such modifications and alterations are intended to be included within the scope of the present invention. The scope of the invention is, therefore, indicated by the appended claims and their legal equivalents, rather than by the examples given above. For example, the steps recited in any method claims may be executed in any order practicable and are not limited to the order presented in any embodiment, example, or claim.

Claims (17)

1. A method for detecting a target molecule that may be present in a test sample, the method comprising:

(a) contacting a test sample with an aptamer having a specific affinity for a target molecule, wherein if the target molecule is present in the test sample, an aptamer affinity complex is formed by contact of the aptamer with its target molecule;

(b) exposing the test sample to conditions that kinetically stimulate components of the test sample after equilibration of aptamer affinity complex formation;

(c) at any time prior to (d), contacting the aptamer affinity complex with a labeling agent; and

(d) detecting and/or quantifying said aptamer affinity complex separated from the remainder of the test sample,

wherein the kinetic challenge comprises (i) introducing a competitor molecule into the test sample, or (ii) diluting the test sample, or (iii) capturing the aptamer affinity complex on a solid support followed by washing in the presence of the competitor molecule in a wash solution.

2. The method of claim 1, wherein the at least one competitor molecule is independently selected from an oligonucleotide, a polyanion, an abasic phosphodiester polymer, a dNTP, or a pyrophosphate.

3. The method of claim 2, wherein the competitor molecule is a polyanion.

4. The method of claim 3, wherein said polyanion is dextran sulfate.

5. The method of claim 1, wherein said conditions that kinetically provoke said aptamer comprise dilution of said test sample.

6. The method of claim 1, wherein the aptamer is a single-stranded nucleic acid or a double-stranded nucleic acid.

7. The method of claim 1, wherein the target molecule is selected from the group consisting of a protein, a carbohydrate, a hormone, an antigen, a growth factor, and a tissue.

8. The method of claim 1, wherein the test sample is a biological sample, wherein the biological sample is selected from the group consisting of whole blood, plasma, serum, sputum, breath, urine, semen, saliva, meningeal fluid, amniotic fluid, glandular fluid, lymph fluid, nipple aspirate, bronchial aspirate, synovial fluid, joint aspirate, cells, cell extract, stool, tissue extract, tissue biopsy, and cerebrospinal fluid.

9. The method of claim 3, wherein said polyanion is heparin or polyglucan.

10. The method of claim 6, wherein the aptamer is DNA or RNA.

11. The method of claim 7, wherein the target molecule is a protein.

12. The method of claim 11, wherein the protein is a glycoprotein.

13. The method of claim 11, wherein the protein is a receptor or an antibody.

14. The method of claim 7, wherein the target molecule is a carbohydrate.

15. The method of claim 14, wherein the carbohydrate is a polysaccharide.

16. The method of claim 8, wherein the test sample is a biological sample of cells.

17. The method of claim 16, wherein the cell is a leukocyte or a peripheral blood mononuclear cell.