analyte detection by selective labeling of biological samples

Abstract

To detect biological analytes in a sample by selective labeling of the sample.SOLUTION: A method includes: contacting a biological sample having a first target analyte with a first agent, where the first agent includes a first binding species that specifically binds to the first target analyte, and a first oligonucleotide conjugated to the binding species; contacting the biological sample with a second agent, where the second agent includes a first reactive species and a second oligonucleotide conjugated to the first reactive species, so as to hybridize at least a portion of the second oligonucleotide to at least a portion of the first oligonucleotide; and contacting the biological sample with a first labeling species, where the first labeling species reacts with the first reactive species to deposit the first labeling species or a derivative thereof in the biological sample.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 62 / 801,011 and U.S. Provisional Patent Application No. 62 / 801,009, filed on February 4, 2019, the entire contents of each of these U.S. Provisional Patent Applications are incorporated herein by reference.

[0002] This disclosure relates to the detection of biological analytes in a sample by selective labeling of the sample. [Background technology]

[0003] Antibodies are used to selectively bind to a wide variety of proteins in biological samples. Immunohistochemical methods typically involve conjugating a dye-conjugated antibody to a complementary marker, thereby labeling the marker with a dye. Detection of fluorescence emission from the dye label reveals the presence of the marker in the sample. [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] U.S. Patent No. 10,126,242 [Patent Document 2] U.S. Patent No. 7,555,155 [Patent Document 3] PCT Patent Publication Number WO2005 / 040769 [Non-patent literature]

[0005] [Non-Patent Document 1] Faget et al., Methods Mol. Biol. 1318: pp. 161-1672 (2015) [Non-Patent Document 2] L. Jimenez and D. Landgrebe, "Hyperspectral Data Analysis and Feature Reduction Via Projection Pursuit," IEEE Transactions on Geoscience and Remote Sensing, Vol. 37, No. 6, pp. 2653-2667, November 1999. [Overview of the Initiative] [Means for solving the problem]

[0006] This disclosure features a method for selectively applying dyes and other labeling species to a sample to identify and quantify specific target analytes in the sample. After identification and / or quantification, additional dyes and labeling substances can be added to the sample to identify and quantify additional specific target analytes. In this manner, a series of sequential labeling and detection cycles can be performed to selectively identify and quantify specific target analytes in a sample.

[0007] The method can be performed by a step of binding a plurality of first substances to a sample, wherein each first substance comprises a binding species that specifically binds to a different target analyte in the sample, and a different first oligonucleotide conjugated to the binding species. Thereafter, a second substance is introduced, which comprises a second oligonucleotide conjugated to a reactive species. The second oligonucleotide hybridizes with the first oligonucleotide of one of the first substances, and localizes the second substance at a position corresponding to the target analyte associated with the first substance in the sample. The reaction between the reactive species and an introduced labeling substance causes the labeling substance to deposit in the immediate vicinity of the target analyte. Thereafter, the second substance can be removed by dehybridization under relatively mild conditions, ensuring that each of the first substances remains bound to the sample. Subsequently, additional labeling cycles can be performed in which different second substances are introduced, each of the different second substances comprising a different second oligonucleotide conjugated to a reactive species. By selecting a second substance having a second oligonucleotide that is complementary to a specific first oligonucleotide, a specific target analyte can be selectively labeled with a different labeling substance. The relatively mild conditions under which the second substance is removed from the sample ensure that the first substance remains bound to the sample and sample integrity is maintained.

[0008] In one aspect, the present disclosure features a method comprising the following steps: (i) contacting a biological sample comprising a first target analyte with a first substance, wherein the first substance comprises a first binding species that specifically binds to the first target analyte and a first oligonucleotide conjugated to the binding species; (ii) contacting the biological sample with a second substance comprising a first reactive species and a second oligonucleotide conjugated to the first reactive species, and hybridizing at least a portion of the second oligonucleotide to at least a portion of the first oligonucleotide; (iii) contacting the biological sample with a first labeling species, wherein the first labeling species reacts with the first reactive species to deposit the first labeling species or a derivative thereof in the biological sample; (iv) removing the second substance from the biological sample after deposition of the first labeling species or a derivative thereof; (v) contacting the biological sample with a third substance, wherein the third substance comprises a second binding species that specifically binds to a second target analyte in the biological sample, and a third oligonucleotide conjugated to the second binding species; (vi) contacting the biological sample with a fourth substance comprising a second reactive species and a fourth oligonucleotide conjugated to the second reactive species, and hybridizing at least a portion of the fourth oligonucleotide to at least a portion of the third oligonucleotide; (vii) contacting the biological sample with a second labeling species, wherein the second labeling species reacts with the second reactive species to deposit the second labeling species or a derivative thereof in the biological sample.

[0009] Embodiments of the method may include any one or more of the following features.

[0010] The first reactive species may comprise a catalytic substance, for example, an enzyme such as horseradish peroxidase. The first labeling species may comprise a dye. The first labeling species may comprise a conjugate of an inactive tyramide or a derivative thereof and a dye.

[0011] The step of contacting a biological sample with a first labeled species includes converting the first labeled species into a conjugate of an active tyramide or a derivative thereof and a dye, wherein the active tyramide or a derivative thereof binds to the biological sample in close proximity to the second substance.

[0012] The first conjugated species may include an antibody or an antibody fragment.

[0013] The first oligonucleotide and / or the second oligonucleotide may contain at least 10 nucleotides. The nucleotide sequences of the first and second oligonucleotides may be at least 70% complementary. The second oligonucleotide may contain more nucleotides than the first oligonucleotide.

[0014] The second oligonucleotide may contain multiple adjacent, discontinuous nucleotide sequences that are complementary to different parts of the sequence of the first oligonucleotide.

[0015] The first and second reactants may be the same. The first and second reactants may each include an enzyme, such as horseradish peroxidase.

[0016] The first and third oligonucleotides may differ. The second and fourth oligonucleotides may differ.

[0017] The first labeled species may contain a first pigment, and the second labeled species may contain a second pigment different from the first pigment.

[0018] The first conjugate may comprise a first antibody or a first antibody fragment, and the second conjugate may comprise a second antibody or a second antibody fragment. The first and second conjugates can selectively bind to different first and second target analytes in a biological sample.

[0019] The first oligonucleotide may comprise a nucleotide sequence of RNA bases and / or a nucleotide sequence of DNA bases. The first oligonucleotide may comprise at least one synthetic nucleotide. The first oligonucleotide may be entirely single-stranded, or alternatively, partially double-stranded.

[0020] The pigments may include chromogenic or fluorescent species.

[0021] Embodiments of this method may also include any combination of other features described herein, even if described in relation to a different embodiment, unless otherwise explicitly stated.

[0022] In another embodiment, the present disclosure features a reagent kit comprising: a first substance comprising a first conjugate species that specifically binds to a first target analyte of a biological sample and a first oligonucleotide conjugated to the first conjugate species; a second substance comprising a second conjugate species that specifically binds to a second target analyte of a biological sample and a second oligonucleotide conjugated to the second conjugate species; a third substance comprising a reactant species and a third oligonucleotide conjugated to the reactant species; a fourth substance comprising a reactant species and a fourth oligonucleotide conjugated to the reactant species; a first labeled species; and a second labeled species, wherein each of the first and second labeled species reacts with the reactant species to deposit the first and second labeled species or derivatives thereof, respectively, in the biological sample.

[0023] Embodiments of the reagent kit may include any combination of features described herein, even if described in relation to a different embodiment, unless otherwise explicitly stated.

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which this disclosure belongs. Similar or equivalent methods and materials may be used in carrying out or testing the subject matter herein, but suitable methods and materials are listed below. All publications, patent applications, patents, and other references referenced herein are incorporated herein by reference as a whole. In case of any conflict, this specification, including definitions, shall prevail. Furthermore, materials, methods, and examples are illustrative only and not intended to limit.

[0025] Details of one or more embodiments are shown in the accompanying drawings and the description below. Other features and advantages will be apparent from the description, drawings, and claims. [Brief explanation of the drawing]

[0026] [Figure 1] This is a schematic diagram illustrating an example of a series of steps for the analysis of biological samples. [Figure 2A] This is a schematic diagram showing the steps of the method for sample analysis. [Figure 2B] This is a schematic diagram showing the steps of the method for sample analysis. [Figure 2C] This is a schematic diagram showing the steps of the method for sample analysis. [Figure 2D] This is a schematic diagram showing the steps of the method for sample analysis. [Figure 3A] This flowchart shows an example of a series of steps for the analysis of biological samples. [Figure 3B] This is a schematic diagram showing the steps of the method for sample analysis. [Figure 3C] This is a schematic diagram showing the steps of the method for sample analysis. [Figure 3D] This is a schematic diagram showing the steps of the method for sample analysis. [Figure 3E] This is a schematic diagram showing the steps of the method for sample analysis. [Figure 4A]This is a schematic diagram showing the steps of the method for sample analysis. [Figure 4B] This is a schematic diagram showing the steps of the method for sample analysis. [Figure 4C] This is a schematic diagram showing the steps of the method for sample analysis. [Figure 4D] This is a schematic diagram showing the steps of the method for sample analysis. [Figure 4E] This is a schematic diagram showing the steps of the method for sample analysis. [Figure 5A] This is a schematic diagram showing an example of an oligonucleotide. [Figure 5B] This is a schematic diagram showing an example of an oligonucleotide. [Figure 5C] This is a schematic diagram showing an example of an oligonucleotide. [Figure 5D] This is a schematic diagram showing an example of an oligonucleotide. [Figure 5E] This is a schematic diagram showing an example of the first substance. [Figure 5F] This is a schematic diagram showing an example of the second substance. [Figure 6] This is a schematic diagram illustrating an example of a multispectral imaging system. [Figure 7] This is a schematic diagram showing an example of a control device. [Figure 8A] These are images of tissue sections labeled with different labeling regions. [Figure 8B] These are images of tissue sections labeled with different labeling regions. [Figure 8C] These are images of tissue sections labeled with different labeling regions. [Figure 8D] These are images of tissue sections labeled with different labeling regions. [Figure 9A] These are images of tissue sections labeled with different reporting substances and labeling regions. [Figure 9B] These are images of tissue sections labeled with different reporting substances and labeling regions. [Figure 9C] These are images of tissue sections labeled with different reporting substances and labeling regions. [Figure 10A]These are images of tissue sections labeled with different labeling regions. [Figure 10B] These are images of tissue sections labeled with different labeling regions. [Figure 10C] These are images of tissue sections labeled with different labeling regions. [Figure 10D] These are images of tissue sections labeled with different labeling regions. [Figure 10E] These are images of tissue sections labeled with different labeling regions. [Figure 10F] These are images of tissue sections labeled with different labeling regions. [Modes for carrying out the invention]

[0027] Similar reference symbols in various drawings represent the same elements.

[0028] Introduction The analysis of multiple target analytes in biological samples is a crucial aspect of modern research methods. For example, identifying and quantifying multiple disease markers can elucidate complex regulatory and co-expression relationships in cells, which can confirm models of disease progression and aid in the development of targeted therapies that can intervene in critical stages of that progression.

[0029] Various techniques are used to identify and quantify multiple target analytes in biological samples. For example, a technique for targeting multiple expressed biomarkers (e.g., antigens, peptides) involves exposing the sample to a set of probes, each of which contains an antibody that specifically binds to one of the biomarkers and is conjugated to a different dye species. To target N different biomarkers in a sample, N different types of probes are introduced, each of which stains one type of biomarker with one of the N different dyes. The emission (e.g., fluorescence) from each of the different probe dyes is then measured to identify and quantify the N different biomarkers in the sample.

[0030] In such a method, the emission from each of the different probe dyes is identified, and each of the different biomarkers targeted by the probe is identified and quantified separately. In practice, this can impose an effective limit on the extent to which multiple biomarkers can be analyzed, as separating and quantifying the radiometric measurements corresponding to different probe dyes becomes increasingly difficult as the number of such dyes in the sample increases. For example, even with strategic selection of probe dyes and relatively sophisticated methods for decomposing fluorescence radiometric measurements into contributions from individual component dye emission spectra, the aforementioned method may be limited to simultaneous multiplexed investigations of about 10 different biomarkers in a sample.

[0031] The aforementioned immunohistochemical methods for labeling target analytes may also be limited by the amount of probe dye that can be delivered to specifically label each of the target analytes in the sample. Since the probe dye is conjugated to a specific antibody, the amount of probe dye that can be applied to a particular location in the sample directly depends on the number of probe dye portions directly attached to the antibody that binds to the analyte at that location. Therefore, certain immunohistochemical methods are somewhat limited in their ability to amplify the signal emanating from a particular target analyte by selectively depositing a larger amount of probe dye at the locations corresponding to those target analytes.

[0032] To further increase the number of target analytes in a sample that can be identified and quantified, conventional histochemical labeling methods can be modified to include an antibody removal step. For example, in the first round of sample analysis, the sample can be labeled with a set of N different dye-conjugate antibodies, and N different biomarkers corresponding to N different probes can be identified and quantified in the sample by measuring the emission from the N different dyes. Subsequently, an antibody removal step may be performed in which the N different probes are removed from the sample. Subsequently, the sample is labeled with a new set of M different dye-conjugate antibodies, and the M antibodies label target analytes different from the previous N probes. Measuring the emission from the M different dyes of this second set of probes results in the identification and quantification of a second set of M different biomarkers in the sample. This methodology can be expanded with multiple cycles of antibody removal and multiplex labeling.

[0033] However, the preparation conditions used to remove conjugated probe antibodies from a sample can be relatively time-consuming and aggressive, potentially detrimental to the integrity of certain types of samples. Therefore, the number of such labeling and antibody removal cycles that can be performed before the sample is damaged may be limited. Furthermore, depending on the nature of binding between a particular antibody and the corresponding biomarker in the sample, it may be difficult to completely remove each probe from the sample after each analytical cycle. If a probe is not completely removed from the sample, a certain amount of its corresponding dye label remains in the sample. Emission from the remaining dye label in subsequent analytical cycles can interfere with emission from dyes conjugated to other probes, leading to inaccurate quantification of certain biomarkers in the sample. Therefore, the number of multiplexed labeling and detections that can be effectively performed may be limited by the extent to which the dye-conjugated antibody-based probes can be removed from the sample.

[0034] In certain standard analytical procedures, dye quenching methods are used to eliminate signals generated from dyes conjugated to residual antibodies in a sample. However, these methods can also be difficult and time-consuming to implement. For example, applying a dye quenching reagent that completely eliminates the contribution from dyes conjugated to residual antibodies, without interfering with signals generated by dyes conjugated to subsequent antibodies, and without excessively affecting the biochemical, structural, and spectral properties of the sample can be extremely challenging.

[0035] This disclosure features a method for performing multiplex labeling, identification, signal amplification, and quantification of target analytes in biological samples. The method can be used to perform multiple cycles of target analyte labeling, detection, and removal of certain active substances involved in the labeling process without disrupting antibody-biomarker binding in the sample. Rather, the removal of active substances involved in the labeling process is performed by dehybridizing the active substances under relatively mild conditions, preserving sample integrity and ensuring that the removal of active substances during each labeling and detection cycle is nearly complete. As a result, cross-labeling of species is minimal or nonexistent. Moreover, each target analyte in the sample can be selectively labeled with a different labeling species, and the deposition of each labeling species is highly constrained to a region of the sample that specifically corresponds to the location of a particular target analyte.

[0036] Analysis of target analytes This disclosure describes various different analytical methodologies for identifying and quantifying multiple target analytes in a biological sample. Figure 1 is a flowchart 100 illustrating an example of a series of steps for performing one method of sample analysis. In the first step 102, the biological sample containing the target analyte comes into contact with a first substance that specifically binds to the target analyte. This first step is schematically illustrated in Figure 2A. In Figure 2A, the biological sample 202 contains the target analyte 210. The sample 202 comes into contact with the first substance 204. The first substance 204 contains a binding species 206 that specifically binds to the target analyte 210, and a first oligonucleotide 208 conjugated to the binding species 206. In this way, the first substance 204 is specifically localized to the location in the sample corresponding to the target analyte 210.

[0037] As used herein, the terms “to bring into contact” and “to bring into contact” mean to associate a substance, species, part, or other element with a sample, or another substance, species, part, or element, such that the two interact with each other. For example, when sample 202 “comes into contact” with the first and second substances, labeled species, and reporting substance, these substances and species may associate with the sample at close enough that they interact with the sample, and may bind with the sample, or with other substances, species, parts, and elements that are already in contact with, bound to, hybridized with, and / or deposited in the sample.

[0038] Returning to Figure 1, in the next step 104, the sample comes into contact with a second substance that associates with the first substance. This step is schematically illustrated in Figure 2B. In Figure 2B, the second substance 216 comes into contact with the sample 202. The second substance 216 contains a second oligonucleotide 212 conjugated to the reactant 214. The second oligonucleotide 212 is at least partially complementary to the first oligonucleotide 208, and as a result, the first and second oligonucleotides hybridize. In this way, the second substance 216 is localized in the sample at the same position as the first substance 204, and therefore at the position corresponding to the target analyte 210.

[0039] Returning to Figure 1, in step 106, the sample comes into contact with the labeled species. The labeled species reacts with the reactant of the second substance from step 104, depositing the labeled species in the sample in proximity to the second substance. This step is schematically illustrated in Figure 2C, in which the labeled species 218 comes into contact with the sample. As shown in Figure 2C, the labeled species 218 reacts with the reactant 214 in the reaction indicated by arrow 220. This reaction deposits the labeled species 218 or its derivative in the sample in proximity to the second substance 216 222, and therefore in proximity to the target analyte 210. In this way, the deposited labeled species 218 (or its derivative) spatially colocalizes with the target analyte 210.

[0040] Referring again to Figure 1, after the deposition of the labeled species or its derivative in step 106, the labeled species is detected in step 108 to identify and / or quantify the target analyte 210 in the sample 202. After the detection of the labeled species or its derivative, the procedure shown in flowchart 100 is completed.

[0041] The procedures described above and other methods described herein can be used to identify and quantify a wide variety of different analytes 210 in a biological sample 202. Examples of analytes 210 include, but are not limited to, antigens, peptides, proteins, and other amino acid-containing moieties. Additional examples of analytes 210 include, but are not limited to, oligonucleotides, nucleic acid fragments, including oligonucleotides containing DNA bases, RNA bases, both DNA and RNA bases, and synthetic bases, as well as lipids.

[0042] The methods described herein are suitable for the identification and quantification of many different clinically relevant biomarkers in biological samples, particularly those expressed in tumor tissue, the tumor microenvironment, and tissues representing other disease states. Examples of such biomarkers corresponding to analytes 210 include, but are not limited to, tumor markers such as Sox10, S100, pancytokeratin, PAX5, and PAX8; immune cell identifiers such as CD3, CD4, CD8, CD20, FoxP3, CD45RA, CD45LCA, CD68, CD163, CD11c, CD33, and HLADR; activation markers such as Ki67 and granzyme B; and checkpoint-related markers such as TIM3, LAG3, PD1, PDL1, CTLA4, CD80, CD86, IDO-1, VISTA, CD47, and CD26.

[0043] The methods described herein can be used to analyze various different types of biological samples 202. In some embodiments, the biological sample 202 may be fresh, frozen, or fixed. The biological sample may be of animal origin, for example, from humans, mice, rats, cattle, pigs, sheep, monkeys, rabbits, fruit flies, frogs, nematodes, or marmots. The biological sample may include formalin-fixed paraffin-embedded (FFPE) tissue sections, frozen tissue sections, fresh tissue, cells obtained from the subject (e.g., by fine-needle aspiration or other techniques), cultured cells, biological tissues, biological fluids, homogenates, or unknown biological samples.

[0044] In certain embodiments, the biological sample 202 can be immobilized on a surface. For example, the surface may be a slide, plate, well, tube, membrane, or film. In some embodiments, the biological sample 202 can be mounted on a slide. In certain embodiments, the biological sample 202 can be fixed using a fixative such as an aldehyde, alcohol, oxidizing agent, mercury, picrate, HOPE fixative, or another fixative. The biological sample may be fixed by thermal fixation, either as an alternative or additional method. Fixation can also be achieved by immersion or perfusion.

[0045] In some embodiments, the biological sample 202 can be frozen. For example, the biological sample can be frozen at temperatures below 0°C, below -10°C, below -20°C, below -30°C, below -40°C, below -50°C, below -60°C, below -70°C, or below -80°C.

[0046] In certain embodiments, the biological sample 202 can be immobilized in a three-dimensional form. Examples of three-dimensional forms include a frozen block, a paraffin block, or a frozen liquid. For example, the biological sample 202 may be a frozen block of animal tissue in an optimal cutting temperature compound. The tissue block can be frozen or immobilized. In some embodiments, the tissue block can be cut to expose a surface that may be in contact with the first substance, as discussed above.

[0047] In some embodiments, if the biological sample 202 corresponds to a block, the block can be sliced ​​to produce serial sections of the block, each of which can be analyzed by the method described herein. By doing so, three-dimensional information (e.g., information as a function of depth in the sample) about the identity and / or quantity of one or more target analytes in the sample can be obtained.

[0048] Generally, the conjugate species 206 is selected to target a specific analyte in the sample 202. The methods described herein can be carried out using a wide variety of different types of conjugate species. For example, to target a specific antigen, peptide, protein, or other amino acid-containing species in the sample 202, the conjugate species 206 may include an antibody or antibody fragment. The antibody or antibody fragment may include any one of different types of antibody species, including, but not limited to, immunoglobulin G (IgG), immunoglobulin M (IgM), polyclonal antibodies, monoclonal antibodies, single-strand fragment variable (scFv) antibodies, nanobodies, antigen-binding fragments (Fab), and diabodies. The antibodies and antibody fragments may be of mouse, rat, rabbit, human, camel, or goat origin. In some embodiments, the antibodies or antibody fragments may be produced against antigens of human, mouse, rat, cattle, pig, sheep, monkey, rabbit, fruit fly, frog, nematode, or marmot. In certain embodiments, antibodies or antibody fragments may be produced in response to antigens of animals, plants, bacteria, fungi, or protozoa.

[0049] Various different binding mechanisms can occur between the binding species 206 and the target analyte 210 in sample 202. In some embodiments, for example, the binding species 206 (e.g., an antibody or antibody fragment) reversibly binds to the target analyte 210. In certain embodiments, the binding species 206 irreversibly binds to the target analyte 210. Binding between the binding species 206 and the target analyte 210 can occur in some embodiments via the formation of one or more covalent bonds. Alternatively or additionally, the binding species 206 and the target analyte 210 bind via one or more non-covalent bonds. One or more fixatives can be used to facilitate the formation of covalent and / or non-covalent bonds.

[0050] The specific binding mechanism between the binding species 206 and the target analyte 210 in sample 202 depends on the properties of the binding species 206 and the target analyte 210. For example, if the binding species 206 is an antibody or antibody fragment and the target analyte 210 is an antigen, binding occurs between the antigen epitope and the paratope of the antibody or antibody fragment. As another example, if the binding species 206 is an antibody or antibody fragment and the target analyte 210 is a lipid, binding may occur between the recognition site on the antibody or antibody fragment and the lipid head group (e.g., a phospholipid head group).

[0051] Generally, the binding species 206 binds to the target analyte 210 in the sample 202 with a specific sensitivity, where sensitivity refers to the statistical proportion of the target analyte 210 entities in the sample that are correctly recognized and bound by the binding species 206. In some embodiments, the sensitivity of the binding species 206 to the target analyte 210 is 60% or higher (e.g., 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, 99% or higher).

[0052] The binding species 206 also generally binds to the target analyte 210 with a specific degree of specificity, where specificity refers to the statistical ratio or efficiency by which the binding species selectively binds to a particular target analyte 210 in preference to other target analytes in the biological sample. In some embodiments, the binding species 206 has a specificity of at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%) to the target analyte 210.

[0053] The affinity of the binding species 206 to the target analyte 210 generally refers to the strength of the binding between the binding species 206 and the target analyte 210, and the dissociation constant K d It can be characterized by the following. In some embodiments, the affinity of the binding species 206 to the target analyte 210 is 10 -4 M or less (for example, 10 -5 M or less, 10 -6 M or less, 10 -7 M or less, 10 -8 M or less, 10-9 10 M or less -10 10 M or less -11 10 M or less -12 10 M or less -13 10 M or less -14 M or less).

[0054] In some embodiments, binding species 206 binds to at least 20% (e.g., at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%) of target analyte 210 in sample 202. As discussed above, in general, binding species 206 selectively binds to target analyte 210 in sample 202. As used herein, "selective binding" means that at least 70% or more (e.g., 80% or more, 90% or more, 95% or more) of binding species 206 in sample 202 bind to target analyte 210 rather than to other species in sample 202.

[0055] Oligonucleotides and Hybridization In general, first oligonucleotide 208 comprises a plurality of nucleotides. The nucleotides may include, for example, DNA bases (e.g., A, C, G, T), RNA bases (e.g., A, C, G, U), and any combination of DNA and / or RNA bases. First oligonucleotide 208 may also include non-naturally occurring (e.g., synthetic) nucleotides including DNA analogs and / or RNA analogs. Examples of such synthetic analogs include, but are not limited to, peptide nucleic acids, morpholino and locked nucleic acids, glycol nucleic acids, and threose nucleic acids.

[0056] In general, the base sequence in first oligonucleotide 208 may be any sequence. Furthermore, in general, nucleotides and other moieties in first oligonucleotide 208 may be conjugated via natural and / or non-natural (e.g., synthetic) linkages.

[0057] In some embodiments, the first oligonucleotide 208 comprises one or more nucleotides capable of base-pairing with a complementary nucleotide with high reliability. Examples of such nucleotides include, but are not limited to, 7-deaza-adenine, 7-deaza-guanine, adenine, guanine, cytosine, thymine, uracil, 2-deaza-2-thio-guanosine, 2-thio-7-deaza-guanosine, 2-thio-adenine, 2-thio-7-deaza-adenine, isoguanine, 7-deaza-guanine, 5,6-dihydrouridine, 5,6-dihydrothymine, xanthine, 7-deaza-xanthine, hypoxanthine, 7-deaza-xanthine, 2,6-diamino-7-deazapurine, 5-methyl-cytosine, 5-propynyluridine, 5-propynylcytidine, 2-thio-thymine, and 2-thiouridine.

[0058] In certain embodiments, the first oligonucleotide 208 may correspond to or contain one or more fragments of specific nucleic acid species. For example, the first oligonucleotide 208 may correspond to or contain one or more fragments of locked nucleic acid (LNA), peptide nucleic acid (PNA), unlocked nucleic acid (UNA), and / or morpholino oligomers.

[0059] The length of the first oligonucleotide 208 (e.g., the number of nucleotides in the first oligonucleotide 208) can generally be selected as required to ensure efficient and selective hybridization with the second oligonucleotide 212. In some embodiments, the first oligonucleotide 208 may contain at least 5 (e.g., at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100) nucleotides.

[0060] In some embodiments, the first oligonucleotide 208 may have nucleotides between 5 and 30, between 5 and 25, between 5 and 20, between 10 and 20, between 10 and 30, between 10 and 50, between 10 and 70, between 10 and 100, between 20 and 50, between 20 and 70, between 20 and 100, between 30 and 50, between 30 and 70, between 30 and 100, between 40 and 70, between 40 and 100, between 50 and 70, between 50 and 100, between 60 and 70, between 60 and 80, between 60 and 90, or between 60 and 100.

[0061] In a particular embodiment, the first oligonucleotide 208 may have 5 or fewer nucleotides (for example, 10 or fewer, 15 or fewer, 20 or fewer, 25 or fewer, 30 or fewer, 35 or fewer, 40 or fewer, 45 or fewer, 50 or fewer, 55 or fewer, 60 or fewer, 65 or fewer, 70 or fewer, 75 or fewer, 80 or fewer, 85 or fewer, 90 or fewer, 95 or fewer, or 100 or fewer).

[0062] In some embodiments, the first oligonucleotide 208 may be entirely single-stranded. Alternatively, in certain embodiments, the first oligonucleotide 208 may be at least partially double-stranded. The partially double-stranded region of the first oligonucleotide 208 may be at the 3' end of the oligonucleotide, at the 5' end of the oligonucleotide, or between the 5' and 3' ends of the oligonucleotide.

[0063] Figure 5A is a schematic diagram of the first oligonucleotide 208, which includes two single-stranded regions 504 and a double-stranded region 502. As discussed above, the double-stranded region 502 may be located at the 3' end of the first oligonucleotide 208, the 5' end of the first oligonucleotide 208, or an intermediate position between the 3' and 5' ends. In certain embodiments, the first oligonucleotide 208 may contain more than one double-stranded region (e.g., two or more, three or more, four or more, five or more, or even more double-stranded regions).

[0064] The double-stranded region may be formed by a secondary oligonucleotide chain 506 that is bound (e.g., hybridized) to the main oligonucleotide chain 508 of the first oligonucleotide 208, as shown in Figure 5A. Alternatively or additionally, the first oligonucleotide 208 may include a secondary structure that enables the folding of the single-stranded first oligonucleotide 208. At least partial complementarity between different parts of the single strand allows those parts to hybridize to form one or more double-stranded regions from the single strand.

[0065] One or more double-stranded regions 502 of the first oligonucleotide 208 may extend individually, or collectively, over a certain percentage of the total length (e.g., the total number of nucleotides) of the first oligonucleotide 208. In some embodiments, for example, one or more double-stranded regions may extend individually, or all of the double-stranded regions may extend over 1% or more of the total length of the first oligonucleotide 208 (e.g., 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%).

[0066] Generally, the second oligonucleotide 212 may contain any of the features described above for the first oligonucleotide 208. In some embodiments, the second oligonucleotide 212 may contain the same number of nucleotides as the first oligonucleotide 208. Alternatively, in certain embodiments, the second oligonucleotide 212 may contain a different number of nucleotides.

[0067] The second oligonucleotide 212 may have the same or a different chain structure as the first oligonucleotide 208. That is, the second oligonucleotide 212 may be single-stranded, double-stranded, or partially double-stranded, independently of the structure of the first oligonucleotide 208. The second oligonucleotide 212 may generally contain any number of double-stranded regions extending over a portion of the entire length of the second oligonucleotide 212, as described above for the first oligonucleotide 208.

[0068] As discussed above, the second oligonucleotide 212 hybridizes with the first oligonucleotide 208 by base pairing, resulting in the colocalization of the first substance 204 and the second substance 216 at the position of the target analyte 210 in the sample. The efficiency of hybridization is in part related to the degree of complementarity between the sequences of the first oligonucleotide and the second oligonucleotide. As used herein, the percentage of complementary sequences of two sequences refers to the percentage of nucleotides in the shorter of the two sequences that have complementary counterparts at complementary positions in the other sequence, such that the two counterparts pair up during hybridization. In some embodiments, for example, the sequences of two oligonucleotides are at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%) complementary.

[0069] As used herein, the term “at least partially complementary” means that two nucleotide sequences are complementary enough to hybridize. Generally, two nucleotide sequences are at least partially complementary if their sequences are at least 50% complementary.

[0070] Generally, the second oligonucleotide 212 includes at least one binding region that hybridizes with the corresponding binding region of the first oligonucleotide 208. The binding region can be located at the 3' end, the 5' end, or midway between the two ends of the second oligonucleotide. If the second oligonucleotide 212 includes multiple binding regions, any of the binding regions may be located as described above.

[0071] In some embodiments, the binding region of the second oligonucleotide 212 is at least partially complementary to and hybridizes with the 3' end of the first oligonucleotide 208. In certain embodiments, the binding region of the second oligonucleotide 212 is at least partially complementary to and hybridizes with the 5' end of the first oligonucleotide 208. Figure 5B shows schematic diagrams of the first and second oligonucleotides 208 and 212, each having binding regions 510a and 510b. In Figure 5B, the binding region 510b of the second oligonucleotide is at least partially complementary to the 3' or 5' end of the first oligonucleotide 208.

[0072] In a particular embodiment, the binding region 510b of the second oligonucleotide 212 is at least partially complementary to and hybridizes with the intermediate region of the first oligonucleotide 208. Figure 5C shows a schematic diagram of the binding region 510b of the second oligonucleotide 212 binding to the intermediate binding region 510 of the first oligonucleotide 208.

[0073] In some embodiments, the binding region 510b of the second oligonucleotide 212 is at least partially complementary to and hybridizes with the entire first oligonucleotide 208. In certain embodiments, the binding region 510a of the first oligonucleotide 208 is at least partially complementary to and hybridizes with the entire second oligonucleotide.

[0074] In a particular embodiment, one or both of the first and second oligonucleotides 208 and 212 include multiple binding regions separated by one or more non-binding regions. Figure 5D is a schematic diagram showing the first and second oligonucleotides 208 and 212, each of which includes multiple binding regions 510a and 510b, each separated by non-binding regions 512a and 512b, respectively. Generally, each binding region may have any of the properties discussed above with respect to the first and second oligonucleotides 208 and 212 and their respective binding regions.

[0075] The unbinding regions 512a and 512b may be formed by various different linkage species, including non-complementary nucleotide sequences and nucleotide-free spacer portions. The unbinding regions 512a-b may have the same or different geometric lengths, and the binding regions 510a-b may have the same or different lengths (e.g., the same or different number of nucleotides). Within each oligonucleotide (e.g., 208 and / or 212), the binding and unbinding regions may have the same or different lengths.

[0076] In some embodiments, the conjugate species 206 may be conjugated to a plurality of first oligonucleotides 208 in the first substance. Figure 5E is a schematic diagram showing the first substance 204 in which the conjugate species 206 is conjugated to three first oligonucleotides 208. Generally, each of the first oligonucleotides 208 has the same nucleotide sequence, and as a result, the second oligonucleotide 212 can hybridize with any of the first oligonucleotides. In Figure 5E, three first oligonucleotides are conjugated to the conjugate species 206, but more generally, two or more (e.g., three or more, four or more, five or more, six or more, seven or more, eight or more, or even more) first oligonucleotides may be conjugated to the conjugate species 206. By conjugating more than one first oligonucleotide to the binding species 206, the proportion and amount of additional reactive species that can selectively deposit at the location of the target analyte 210 in the sample and deposit in the immediate vicinity of that location in the sample are increased.

[0077] In some embodiments, the second oligonucleotide 212 of the second substance 216 is conjugated to a plurality of reactants 214. Figure 5F is a schematic diagram showing the second substance 216 with the second oligonucleotide 212 conjugated to three reactants 214. The three reactants may all be the same, or one or more may differ from the others. In Figure 5F, three reactants 214 are conjugated to the second oligonucleotide 212, but more generally, two or more (e.g., three or more, four or more, five or more, six or more, seven or more, eight or more, or even more) reactants may be conjugated to the second oligonucleotide 212. By conjugating more than one reactant to the second oligonucleotide 212, the additional reactants can selectively deposit at the location of the target analyte 210 in the sample, increasing the proportion and amount of labeled species that can be deposited in close proximity to that location in the sample.

[0078] Reactive species and label species As discussed above, in the second substance 216, the second oligonucleotide 212 is conjugated to a reactant 214 that reacts with the labeled species 218. The reactant 214 can correspond to one or more of various different chemical or biochemical species and moieties. In some embodiments, for example, the reactant 214 corresponds to a catalytic substance that catalyzes the reaction of the labeled species 218. Examples of catalytic substances that may correspond to the reactant 214 include, but are not limited to, enzymes, organometallic moieties based on transition metals, moieties containing peroxidases, and photoactivatable species. Examples of suitable enzymes include, but are not limited to, horseradish peroxidase (HRP) and soybean peroxidase. In some embodiments, the reactant 214 may include a hemin-containing complex that can mimic HRP, such as hematin.

[0079] Generally, a labeled species 218 includes at least one labeled portion. Various different labeled portions can be used depending on the nature of the methodology used to identify and quantify the target 210 in the sample 202. In some embodiments, for example, the labeled species 218 includes a dye. As used herein, “dye” is a portion that interacts with incident light, and the light emitted therefrom is measured and can be used to detect the presence of the dye in the sample. Generally, the dye may be a fluorescent portion, an absorbent portion (e.g., a color-producing portion), or another type of portion that emits light, or, if a dye is present, the presence of the dye can be determined by altering the incident light passing through or reflected from the sample and measuring the resulting change in the transmitted or reflected light from the sample.

[0080] In certain embodiments, the labeled portion may include a hapten. The hapten can then (or simultaneously) bind to the dye portion to provide a labeled portion that can be detected by measuring the emitted, transmitted, or reflected light from the sample.

[0081] If the label portion of label species 218 contains a dye, a wide variety of different dyes can be used. For example, the dye may be a xanthene-based dye such as fluorescein and / or rhodamine. Suitable examples of fluorescein and rhodamine dyes include, but are not limited to, fluorescein isothiocyanate (FITC), 6-carboxyfluorescein (commonly known by the abbreviations FAM and F), 6-carboxy-2',4',7',4,7-hexachlorofluorescein (HEX), 6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein (JOE or J), N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA or T), 6-carboxy-X-rhodamine (ROX or R), 5-carboxyrhodamine-6G (R6G5 or G5), 6-carboxyrhodamine-6G (R6G6 or G6), and rhodamine 110.

[0082] The pigments may also be cyanine-based pigments. Suitable examples of such pigments include, but are not limited to, the pigments Cy3, Cy5, and Cy7. The pigments may also be coumarin pigments (e.g., umbelliferone), benzimide pigments (e.g., any of the Hoechst pigments such as Hoechst 33258), phenanthridine pigments (e.g., Texas Red), ethidium pigments, acridine pigments, carbazole pigments, phenoxazine pigments, porphyrin pigments, polymethine pigments (e.g., any of the BODIPY pigments), and quinoline pigments.

[0083] If the dye is a fluorescent moiety, the dye may be a moiety corresponding to any of the following non-limiting examples and / or derivatives: pyrene, coumarin, diethylaminocoumarin, FAM, fluorescein chlorotriazinyl, fluorescein, R10, JOE, R6G, tetramethylrhodamine, TAMRA, lisamin, naphthofluorescein, Texas Red, Cy3, and Cy5.

[0084] In certain embodiments, the dye may comprise one or more quantum dot-based species. Quantum dot-based fluorophores having fluorescence emission spectra in many different spectral bands are available, and suitable quantum dot-based dyes can be used as labeling species in the methods described herein.

[0085] Tyramide signal amplification In some embodiments, the reactant 214 is an enzyme involved in an enzyme-mediated reaction that can deposit a labeled species 218 (or a derivative thereof) with a second substance 216 and, therefore, at a location in the sample adjacent to the target analyte 210. As an example of enzyme-mediated deposition of a labeled species, the reactant 214 may be horseradish peroxidase (HRP) or another species that mimics the activity of HRP. HRP can be used in the methods described herein as a catalyst for tyramide signal amplification (TSA).

[0086] To perform TSA, the labeled species 218 contains a labeled moiety (e.g., a dye as described above) conjugated to a tyramide species. When sample 202 first comes into contact with the labeled species 218, the tyramide species is in an inactive form. However, HRP catalyzes the conversion of the tyramide species to an active form that is capable of binding to sample 202. After the conversion of the tyramide species to its active form, its labeled moiety binds to sample 202 at a location close to where it is produced (e.g., at the location of the second substance 216 and the target analyte 210). Figure 2C schematically illustrates the deposition of the labeled species 218 (which may contain an active tyramide species conjugated to the labeled moiety) at a location 222 close to the target analyte 210.

[0087] The amount of tyramide-containing labeled species 218 deposited in sample 202 can be controlled by adjusting the amount of tyramide-containing labeled species 218 introduced into sample 202 and the duration of the enzyme-mediated activation process. As a result, the detected signal corresponding to the labeled species 218 (and therefore, the target analyte 210) can be "amplified." In the context of this disclosure, amplification refers to the conjugation of more than one labeled species 218 to each target analyte 210. With respect to immunohistochemical labeling methods in which each conjugated antibody is conjugated to a single labeled species (e.g., a single fluorophore moiety), TSA techniques can be used to deposit multiple labeled species 218 (or derivatives thereof) in the sample to generate a measurable signal corresponding to a single target analyte 210, thereby increasing the amplitude or intensity of the measurable signal corresponding to the single target analyte with respect to a signal otherwise measured from the single labeled species.

[0088] Generally, the ratio of the number of labeled species 218 that can be deposited in close proximity to a single target analyte 210 in a sample is increased beyond 1:1 by performing the TSA methodology described above. In some embodiments, for example, the ratio is 2:1 or higher (e.g., 3:1 or higher, 4:1 or higher, 5:1 or higher, 6:1 or higher, 8:1 or higher, 10:1 or higher, 20:1 or higher, 30:1 or higher, or even higher).

[0089] Amplification offers several important advantages. Firstly, the measurable signal corresponding to the target analyte has a higher amplitude or intensity than in the absence of amplification, which can reduce exposure and measurement times. Secondly, the increased amplitude or intensity of the measured signal allows for the more reliable detection of target analytes that are present in the sample at relatively low concentrations and whose corresponding measured signal would be relatively weak without amplification by other methods. Thirdly, the increased amplitude or intensity of the measured signal makes it easier to compensate for confounding effects of tissue autofluorescence, as well as the detection of the measured signal against background autofluorescence signals, which would otherwise obscure some or all of the measured signal.

[0090] Amplification can also be used to adjust the measurement signals corresponding to different target analytes. For example, in a sample where a particular analyte is present at a significantly lower concentration than other analytes, the amplitude or intensity of the measurement signal corresponding to the low-concentration analyte can be amplified so that it more closely matches the amplitude or intensity of the signal corresponding to the other target analytes present at higher concentrations in the sample. In this way, the range amplitude or intensity of the measurement signal may be reduced, and as a result, the dynamic range of the measurement system used to detect the measurement signal may also be smaller than the dynamic range that would otherwise be used for the measurement signal in the absence of amplification.

[0091] Furthermore, the presence of low-concentration analytes (e.g., very weakly expressed biomarkers) in a sample can be visualized together with higher-concentration analytes for co-expression analysis, protein regulation evaluation, and other comparative analyses that would be more difficult if both low-concentration and higher-concentration analytes were not detected and visualized simultaneously.

[0092] If the reactant species 214 corresponds to an enzyme or other catalyst, that enzyme or catalyst can mediate the deposition of the labeled species 218 in the sample by any variety of different types of reactions. In some embodiments, for example (TSA for HRP-mediated deposition of tyramide conjugate-type labeled moieties), the reaction mediated by the enzyme or catalyst is a redox reaction. Other examples of suitable enzyme or catalyst-mediated reactions include, but are not limited to, deprotonation, elimination, radical generation, deprotection, and rearrangement.

[0093] For redox reactions (such as TSA for HRP-mediated deposition of labeled species 218), various different oxidizing and / or reducing agents can be used. In some embodiments, for example, the oxidizing agent is H2O2. Various other substances can also be used.

[0094] Furthermore, it should be noted that in some embodiments, the deposition of labeled species 218 (or its derivatives) in sample 202 is irreversible, but in certain embodiments, the deposition of labeled species 218 in sample 202 is reversible, and the labeled species 218 can be removed from sample 202 after deposition by methods such as washing, one or more chemical reactions to release the labeled species 218, and physical methods such as heating and exposure to radiation (e.g., photocutting, photoionization, or sputtering).

[0095] Labeled species 218 can stably bind to sample 202 for 48 hours or more at room temperature. In some embodiments, a sample labeled with labeled species 218 refrigerated at 4°C may be stable for at least 4 weeks, and a sample frozen at -20°C or -80°C may remain stably bound to labeled species 218 for 4 months or more.

[0096] The stability of the bond between the labeled species 218 and the sample 202 may vary depending on the properties of both the labeled species 218 and the sample 202. Generally, if the sample 202 is stored at or near room temperature, the bond is stable for at least 48 hours. For example, in a particular embodiment, if the sample 202 is maintained at room temperature within approximately 5°C, the bond is stable for at least 48 hours. In a certain embodiment, sample 202 is found to be at a temperature between 0°C and 40°C (for example, between 10°C and 40°C, between 15°C and 40°C, between 20°C and 40°C, between 25°C and 40°C, between 30°C and 40°C, between 35°C and 40°C, between 0°C and 35°C, between 5°C and 35°C, between 10°C and 35°C, between 15°C and 35°C, between 20°C and 35°C, between 25°C and 35°C, between 30°C and 35°C, between 0°C and 30°C, between 5°C and 30°C, between 10°C and 30°C, between 15°C and 30°C) The bond is stable for at least 48 hours when maintained at temperatures between 0°C, between 20°C and 30°C, between 25°C and 30°C, between 0°C and 25°C, between 5°C and 25°C, between 10°C and 25°C, between 15°C and 25°C, between 0°C and 20°C, between 5°C and 20°C, between 10°C and 20°C, between 15°C and 20°C, between 0°C and 15°C, between 5°C and 15°C, between 10°C and 15°C, between 0°C and 10°C, between 5°C and 10°C, and between 0°C and 5°C.

[0097] Additional methods and embodiments of TSA are described, for example, in Faget et al., Methods Mol. Biol. 1318: pp. 161-1672 (2015) (the full contents of which are incorporated herein by reference).

[0098] Multiple analysis Referring again to Figures 1 and 2C, after the labeled species 218 has been detected in the sample, the second substance 216 can optionally be removed from the sample. In particular, since the first oligonucleotide 208 and the second oligonucleotide 212 are hybridized, the removal of the second substance 216 involves dehybridizing the first and second oligonucleotides. As discussed above, dehybridization can generally be achieved under significantly milder conditions than antibody removal from a sample that occurs in certain immunohistochemical methods.

[0099] Dehybridization can also be used to control the amount of labeled species 218 deposited in sample 202 (i.e., during amplification). More specifically, dehybridization of the first and second oligonucleotides can be used to terminate the reaction between reactant 214 and labeled species 218 (e.g., a catalytic reaction such as enzyme-mediated deposition of labeled species 218), thereby controlling the amount of time during which deposition of labeled species 218 occurs in the sample.

[0100] Various methods can be used to achieve dehybridization of the first and second oligonucleotides. In some embodiments, for example, dehybridization of oligonucleotides can be achieved by exposing the oligonucleotides to one or more chaotropic reagents such as dimethyl sulfoxide (DMSO) and formamide, in which case the molar concentration of the chaotropic reagent in the solution is 60% or higher (e.g., 70%, 80%, 90%). Alternatively, dehybridization can be carried out by washing the sample 202, by heating the sample 202, and by a combination of the aforementioned techniques.

[0101] Dehybridization of the first and second oligonucleotides in sample 202, followed by a washing step to remove the second substance 216 freed after dehybridization, results in sample 202 in which the first substance 204 remains bound to the target analyte 210 through the binding species 206, and the labeled species 218 remains bound to sample 202 in close proximity to the target analyte 210. In effect, dehybridization returns sample 202 to a state similar to that shown in Figure 2A, with the added presence of the labeled species 218. Figure 2D shows sample 202 in schematic form after dehybridization of the first and second oligonucleotides and subsequent removal of the second substance 216 freed from the sample.

[0102] Some or all of the steps shown in flowchart 100 can be optionally repeated to selectively identify and quantify a second (and subsequent) target analyte 210 in sample 202. Specifically, sample 202 can be contacted with another first substance containing a binding species 206 that selectively binds to a different target analyte 210 in sample 202, and a first oligonucleotide different from the first oligonucleotide of the previous first substance. The sample can then be contacted with another second substance containing a reactant (e.g., any of the reactants described above), and a second oligonucleotide that is at least partially complementary to and hybridizes with the first oligonucleotide of the newly added first substance.

[0103] Following the addition of the additional second substance, a new labeling species that reacts with the reactant can be introduced to deposit the new labeling species (or its derivative) in the sample at a location close to the newly added first and second substances, and therefore close to the second target analyte 210 to which the newly added first substance selectively binds. The measured signal corresponding to the newly added labeling species can be used to identify and quantify the second target analyte 210 in the sample.

[0104] The newly added second substance can again be removed from the sample by dehybridization of the first and second oligonucleotides, as described above, and additional cycles can be repeated to selectively identify and quantify multiple different target analytes 210 in sample 202.

[0105] Each iteration of part or all of the steps in flowchart 100 is called an analysis cycle, and generally any number of cycles can be performed to selectively identify and quantify different target analytes 210 in the sample 202. In some embodiments, for example, the number of such cycles is N, where N is 2 or greater (e.g., 3 or greater, 4 or greater, 5 or greater, 6 or greater, 7 or greater, 8 or greater, 10 or greater, 12 or greater, 15 or greater, or more).

[0106] Generally, for each different combination of first and second substances targeting different target analytes 210 in a sample for analysis, a different label species 218 selectively deposits in the sample in close proximity to the specific target analyte 210. By selecting different label species, different target analytes can be selectively matched by isolating and optionally quantifying the measured synchrotron, reflected, or transmitted light contributions from the sample that specifically arise from the different label species. Because each species effectively "maps" to different target analytes, the identification and quantification of a specific analyte can be achieved by isolating the measurement signals corresponding to their associated label species.

[0107] Multiple target analytes 210 can also be analyzed by multiplexing the addition of different first and second substances to the sample. Figure 3A is a flowchart 350 showing an example of a series of steps for analyzing N different target analytes in a sample. Generally, N can be 2 or more (e.g., 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 10 or more, 12 or more, 15 or more, or even more).

[0108] In the first step 352, the sample comes into contact with N different first substances. Each of the N different first substances contains a binding species that specifically binds to one of the N different target analytes, and a unique first oligonucleotide conjugated to that binding species. In other words, the binding species and conjugated first oligonucleotide of each of the N first substances are different from the binding species and conjugated first oligonucleotide of the other first substances among the N first substances.

[0109] Next, in step 354, one of the n target analytes is selected for analysis, and the sample is brought into contact with a second substance containing a reactant conjugated to a second oligonucleotide that is at least partially complementary to and hybridizes with a first oligonucleotide of a first substance that selectively binds the nth target analyte. Thus, the second oligonucleotide binds to the sample at a position corresponding to the nth target analyte (and the corresponding nth first substance).

[0110] Subsequently, in step 356, the sample is brought into contact with the nth labeled species, which contains a labeled portion different from the labeled portions of the other (n-1) labeled species. The labeled species reacts with the nth reactant of the second substance, depositing the nth labeled species in the sample in close proximity to the nth target analyte.

[0111] Next, in step 360, the nth second substance is removed from the sample by dehybridization and washing, as previously described. If all N target analytes have been analyzed in step 362, the procedure ends in step 366. Otherwise, another nth analyte among the N target analytes is selected for analysis, and the procedure returns to step 354.

[0112] The procedure described above is schematically illustrated in Figures 3A to 3E for a sample containing N=3 target analytes for analysis. Figure 3B is a schematic diagram showing a sample 302 containing three different target analytes 310a to c. In the first step of the analysis of sample 302, three different first substances are brought into contact with the sample, each having different binding species 306a to c that specifically bind to one of the three target analytes 310a to c. Each first substance contains different first oligonucleotides 308a to c. As shown in Figure 3A, bringing each of the first substances into contact with the sample results in a sample in which the first substances selectively bind only to the corresponding target analytes 310a to c whose binding species 306a to c are matched.

[0113] In the next step of the analysis, a second substance containing reactant 314a and a second oligonucleotide 312a that is at least partially complementary to the first oligonucleotide 308a comes into contact with the sample. As shown in Figure 3C, the second oligonucleotide 312a hybridizes with the first oligonucleotide 308a, selectively binding this second substance to the first substance bound to the target analyte 310a. Due to the lack of complementarity between the second oligonucleotide 312a and the first oligonucleotides 308b and 308c, the second substance does not bind to the first substance bound to the target analyte 310b or the first substance bound to the target analyte 310c.

[0114] Subsequently, as shown in Figure 3C, the labeled substance 318a comes into contact with the sample and reacts with the reactant 314a in the manner described above, selectively depositing the labeled substance 318a (or its derivative) in the immediate vicinity of the target analyte 310a. The reaction and deposition of the labeled substance 318a are schematically illustrated by the dashed arrow 320a in Figure 3C.

[0115] After the deposition of the labeled substance 318a, the second substance (i.e., the second oligonucleotide 312a and the reactant 314a) is removed from sample 302 by dehybridization and washing as described above. The resulting sample 302 is schematically shown in Figure 3D. Each of the different first substances remains bound to the corresponding different target analytes 310a, and the labeled substance 318a deposited in close proximity to the target analyte 310a also remains bound to sample 302.

[0116] The cycle illustrated in Figure 3C and described in steps 354-360 of flowchart 350 is then repeated, first with a second substance containing a second oligonucleotide that selectively hybridizes through at least partial complementarity with the first oligonucleotide 308b of the first substance bound to the target analyte 310b, and then with a second substance containing a second oligonucleotide that selectively hybridizes through at least partial complementarity with the first oligonucleotide 308c of the first substance bound to the target analyte 310c. In each cycle, a different labeled substance comes into contact with the sample and is deposited in close proximity to the corresponding target analyte.

[0117] After three complete labeling cycles, sample 302 appears as schematically shown in Figure 3E, with each of the three different primary substances remaining bound to their corresponding target analytes 310a-c, and the three different labeling substances 318a-c remaining deposited in close proximity to each of the three different target analytes 310a-c.

[0118] Generally, each of the different labeling substances 318a-c may correspond to any of the labeling substances described above. To perform the analysis of multiple target analytes in a sample, the labeling substances are generally selected so that they produce different measurement signals. For example, in embodiments in which each labeling substance includes a fluorescent or chromogenic dye moiety, the substance is selected so that each dye moiety has different spectral properties (e.g., absorption, emission), and as a result, the measurement light emitted from, transmitted through, or reflected from the sample can be separated into contributions from each dye and used to separately identify and quantify each of the target analytes 310a-c in the sample.

[0119] Report material Reporting materials can be used in connection with the methods described above. Generally, reporting materials include oligonucleotides directly or indirectly conjugated with a labeling moiety. Reporting materials used in the methods described herein include oligonucleotides that are at least partially complementary to and hybridize with the corresponding first oligonucleotide of the first material described above. Thus, the oligonucleotides of the reporting material may generally include any features of the first and second oligonucleotides discussed above. Furthermore, reporting materials may include one or more of the different types of labeling moieties described above.

[0120] The use of reporting substances can be incorporated into the procedures described herein in various ways and for various purposes. For example, in some embodiments, reporting substances can be used to verify the presence of a specific target analyte in a sample. Figure 4A shows a schematic diagram of sample 402 containing three different target analytes 410a-c, similar to sample 302 in Figure 3A. The three different first substances are selectively bound to sample 302 such that one of each binds to each of the different target analytes. Each of the first substances contains binding substances 406a-c and first oligonucleotides 408a-c.

[0121] Before introducing the second substance into sample 402 (for example, in step 354 of flowchart 350), the sample is brought into contact with a reporting substance 450 to verify the presence of the target analyte 410a in sample 402. As shown in Figure 4A, the reporting substance 450 contains a labeling portion 454 conjugated to oligonucleotide 452. Oligonucleotide 452 is at least partially complementary to oligonucleotide 408a and hybridizes with oligonucleotide 408a when the reporting substance 450 is introduced into the sample. The nucleotide sequence of oligonucleotide 452 is sufficiently discomplementary to the sequences of the first oligonucleotides 408b and 408c, and as a result, oligonucleotide 452 does not hybridize with either of these first oligonucleotides. After washing away the unbound reporting substance 450, the remaining reporting substance 450 in sample 402 is selectively bound to the first substance that binds the target analyte 410a.

[0122] The presence of the target analyte 410a in sample 402 can be verified by measuring the light emitted from, reflected from, or transmitted through sample 402. In particular, the presence of the target analyte 410a in sample 402 can be verified by detecting a portion of the measured light corresponding to the labeled portion 454. Optionally, the amount of the target analyte 410a in sample 402 can also be quantified based on the measured signal contribution that may be attributable to the labeled portion 454.

[0123] The location of the target analyte 410a in the sample 402 can be used to determine the region of interest for further measurements and / or delivery of the substance to the sample 402. For example, if the target analyte 410a is localized to only a portion of the sample 402, subsequent emission, reflection, or transmission measurements of sample light and / or delivery of the substance to the sample 402 can optionally be limited to that region of the sample.

[0124] Alternatively, if the target analyte 410a is not present in the sample, the first substance that selectively binds to the target analyte 410a (i.e., having binding species 406a and the first oligonucleotide 408a) will not bind to the sample 402. As a result, the reporting substance 450 will also not bind to the sample 402 due to the absence of the first oligonucleotide 408a, and the light measured from the sample 402 will not include a contribution from the labeled portion 454, indicating the absence of the target analyte 410a in the sample. Consequently, the step of contacting the sample with the second substance and the labeled species to specifically deposit the labeled portion in the immediate vicinity of the target analyte 410a can be omitted.

[0125] In the example described above, a single report substance 450 is used to selectively identify and optionally quantify a single target analyte 410a in the sample. However, similar labeling and measurement can be performed using multiple different report substances, each of which contains a different oligonucleotide and a different labeling moiety that selectively hybridizes with only one of the first oligonucleotides 408a-c in Figure 4A, so that each report substance 450 targets a specific target analyte 410a-c. Each report substance can be sequentially contacted, measured, and removed from the sample, or alternatively, a group of multiple report substances can be applied to the sample. Parallel analysis of two or more (e.g., three, four, five, six, eight, ten, or more) different target analytes in the sample can be performed, provided that the measured synchrotron, transmitted, or reflected light from the sample can be decomposed into contributions from the respective labeling moieties of the individual report substances.

[0126] Reporting substances can also generally be used in any intermediate step in the analytical procedures described herein. For example, referring to the procedures described in relation to Figures 3A-3E, reporting substances can be used to verify the presence of any of the target analytes 310a-c (and the corresponding target analytes 410a-c in Figure 4A). They can be introduced at any stage of the procedure after contact of the sample with one or more of the first substances, as described above.

[0127] In some embodiments, the reporting substance can be used together with or as a substitute for a labeling species to identify and quantify the target analytes in the sample. Figure 4B shows a schematic diagram of a sample 402 containing five different target analytes 410a-e. The sample is in contact with five different first substances, each of which has different binding species 406a-e and different first oligonucleotides 408a-e that selectively bind each of them to one of the five different target analytes.

[0128] The three analytical cycles were also performed on sample 402 in Figure 4B, as generally described above, depositing the labeled portion in close proximity to the three different target analytes. As a result, three different label species 418a-c are deposited in the sample in close proximity to their respective target analytes 410a-c.

[0129] Furthermore, two different reporting substances are introduced into the sample. The first reporting substance contains oligonucleotide 452d and a labeled moiety 454d. Oligonucleotide 452d is at least partially complementary to the first oligonucleotide 408d and hybridizes with the first oligonucleotide 408d. Oligonucleotide 452d is not sufficiently complementary to any of the other first oligonucleotides in the sample to significantly hybridize with any of the other first oligonucleotides. Similarly, oligonucleotide 452e of the second reporting substance is at least partially complementary to the first oligonucleotide 408e and hybridizes with the first oligonucleotide 408e. Oligonucleotide 452e is not sufficiently complementary to any of the other first oligonucleotides in sample 402.

[0130] The labeled portions in the labeled species 418a-c and the reporting material (i.e., labeled species 454d-e) are selected so that each labeled species is different and therefore has different spectral absorption, reflection, or emission properties. By analyzing the synchrotron, transmitted, or reflected light from sample 402, the contribution from each of the different labeled portions can be identified and selectively attributed to the target analytes 410a-e. In this way, each of the target analytes 410a-e in sample 402 can be identified and quantified.

[0131] Sample 402 in Figure 4B contains a mixture of a labeled species and a labeled portion derived from the reporting substance. However, in some embodiments, the reporting substance can be used to label all of the target analytes in a biological sample and to detect the corresponding signals.

[0132] For example, Figure 4C is a schematic diagram of a sample 402 containing M different target analytes 410a…410M. M can generally be 2 or greater (e.g., 3 or greater, 4 or greater, 5 or greater, 6 or greater, 8 or greater, 10 or greater, 15 or greater, 20 or greater, 30 or greater, 40 or greater, 50 or greater, or even greater). The sample is brought into contact with a set of first substances 410a…410M as described above, to selectively bind one of each of the first substances to one of the M different target analytes.

[0133] Subsequently, the reporting substances 450a…450M are selectively hybridized with the first substance to label M target analytes. Each reporting substance contains different labeling portions such that each of the M target analytes is labeled with a different labeling portion. In some embodiments, the reporting substances are applied one at a time (i.e., sequentially) to the sample 402, and the light emitted, reflected, or transmitted from the sample in which only one reporting substance is present at a time is measured to identify and quantify the target analyte corresponding to that one reporting substance.

[0134] However, in certain embodiments, the sample is brought into contact with a group of reporting substances (e.g., a group of two or more reporting substances, a group of three or more reporting substances, a group of four or more reporting substances, a group of five or more reporting substances, a group of six or more reporting substances, a group of eight or more reporting substances, or even more reporting substances), and each reporting substance selectively hybridizes with only one of the first substances bound to one of the target analytes in the sample. Light emitted, reflected, or transmitted from the sample having multiple reporting substances of the group present is measured and decomposed into contributions from each of the labeling portions of the reporting substances. These contributions can then be used to identify and quantify each of the target analytes corresponding to the reporting substances of the group.

[0135] Subsequently, the group of reporting substances can be removed from the sample by dehybridization, as previously discussed, and a new group of reporting substances is introduced that selectively hybridizes with different sets of the first substance bound to different sets of target analytes. The new group may contain the same number of reporting substances or a different number of reporting substances compared to the previous group.

[0136] The analysis of target analytes in the sample continues in this manner, with a successive group of reporting substances hybridizing, being detected, and removed until all M target analytes have been analyzed.

[0137] In some embodiments, a hybrid analytical workflow can be performed that uses both a reporting substance and deposition of a labeled portion (e.g., by TSA) to analyze the target analyte. Such a procedure can be used, for example, for samples containing a relatively large number of target analytes, where a few of these analytes (e.g., between one and eight) are particularly important. The hybrid procedure is also useful for samples containing a relatively large number of target analytes, where some of these analytes are weakly expressed or generate post-labeled signals that are difficult to measure by other methods. Amplification of measurement signals corresponding to particularly important or weakly expressed target analytes can be used to achieve highly multiplexed sample characterization with particular attention to certain analytes.

[0138] An example of such sample 402 is shown in Figure 4D. The sample includes target analytes 410a…410M, as well as 410u, 410v, and 410w. Of these target analytes, 410u to 410w are particularly important.

[0139] An example of a hybrid analysis workflow is as follows: Sample 402 is brought into contact with the first substances 404a…404M and 404u~404w. Each of these first substances contains a different binding group that selectively binds to one of the target analytes, and a different first oligonucleotide.

[0140] After the first substances selectively bind to their respective target analytes and localize to their corresponding positions in sample 402, the target analytes 410a…410M are analyzed using report substances 450a…450M in the manner described above with respect to Figure 4C. Specifically, the report substances hybridize with the first substances in sample 402, either in groups or individually. Each report substance contains different oligonucleotides that are at least partially complementary to only one of the first oligonucleotides of the first substance 404a…404M, and different labeling moieties. The report substances 450a…450M can hybridize with the sample one by one (e.g., sequentially) or in groups of two or more. To detect the report substances after hybridization, light emitted, reflected, or transmitted from the sample is measured and, if it includes contributions from multiple labeling moieties, is decomposed into contributions from each labeling moiety. The target analytes corresponding to each group of reporting substances are identified and optionally quantified, and then the groups of reporting substances are removed from sample 402 by dehybridization and washing. Analysis of the remaining target analytes 410a…410M is carried out in a similar manner by hybridizing one or more groups of selective reporting substances with the corresponding first substance 404a…404M, measuring the signal corresponding to the reporting substances, and then removing the reporting substances in preparation for another analytical cycle.

[0141] Next, each of the very important target analytes 410u to 410w is analyzed similarly. As schematically shown in Figure 4E, the sample comes into contact with a second substance containing a second oligonucleotide 412u that selectively hybridizes with the first oligonucleotide 408u. The reaction species 414u is conjugated to the second oligonucleotide 412u. After hybridization of the second substance, the labeled species 418u comes into contact with the sample and reacts with the reaction species 414u, depositing the labeled species 418u (or its derivative) at a location in the sample close to the target analyte 410u. After the deposition of the labeled species 418u, the second substance (e.g., containing the second oligonucleotide 412u and the reaction species 414u) is removed from the sample by dehybridization and washing.

[0142] To analyze the target analyte 410u, the light emitted, reflected, or transmitted from the sample is measured, and the contribution of the labeled species 418u to the measured signal is determined to identify and optionally quantify the target analyte 410u.

[0143] Subsequently, the above series of steps for analyzing target analytes 410u is repeated to analyze target analytes 410v and 410w, using a second substance containing a different second oligonucleotide that specifically hybridizes with a first substance that specifically binds to target analytes 410v and 410w. In the examples shown in Figures 4D and 4E, two additional cycles of the series of steps described for target analyte 410u are performed to analyze target analytes 410v and 410w, respectively.

[0144] Kit and composition The substances, species, and parts described herein may be included in various kits featuring compositions comprising the substances, species, and parts. Generally, a kit is a package of one or more reagents, each in the form of a composition. Compositions featuring any of the different substances, species, and parts described herein can be prepared and used for target analyte analysis as described herein. These compositions may be included in a product kit along with other features such as instructions for preparing the composition and using it for sample analysis. Product kits may be sealed or otherwise contained in various different containers.

[0145] Measurement of the optical signal corresponding to the marked portion. Figure 6 is a schematic diagram showing a system 600 for obtaining multiple spectrally resolved images of a sample. The system 600 can measure light emitted, transmitted, and / or reflected from a sample containing one or more of the labeled portions described herein. The measured light generally includes contributions from each of the labeled portions present in the sample, and the system 600 can analyze coded multispectral image information in the measured light and resolve the image information to isolate the contribution of each labeled portion in the sample to the measured light. The resolution yields a set of amplitude or intensity measurements for each labeled portion in the sample as a function of its position in the sample. The amplitude or intensity measurements can be used to quantify the amount of each labeled portion at each position in the sample, and therefore the amount of each target analyte.

[0146] The light source 602 provides light 622 to the light-tuning optical element 604. Light 622 may be incoherent light, such as light generated from a filament source, or it may be coherent light, such as light generated by a laser. Light 622 may be either continuous wave (CW) or time-gated (i.e., pulsed) light. Furthermore, light 622 may be provided in a selected portion of the electromagnetic spectrum. For example, light 622 may have a central wavelength of the spectrum and / or a distribution of wavelengths falling within the ultraviolet, visible, infrared, or other regions.

[0147] The light-tuning optical element 604 can be configured to transform the light 622 in several ways. For example, the light-tuning optical element 604 can spectrally filter the light 622 to provide output light in a selected wavelength range of its spectrum. Alternatively or in addition, the light-tuning optical element can adjust the spatial distribution and temporal characteristics of the light 622. The incident light 624 is generated from the light 622 by the action of the elements of the light-tuning optical element 604.

[0148] The incident light 624 is directed to project onto a sample 608 mounted on an illumination stand 606. The stand 606 may provide means for securing the sample 608, such as mounting clips or other fastening devices. Alternatively, the stand 606 may include a movable track or belt to which multiple samples 608 are attached. The driver mechanism may be configured to move the track to sequentially move multiple samples one by one through the illumination area on the stand 606, with the incident light 624 acting on that illumination area. The stand 606 may further include a translation axis and mechanism for translating the sample 608 relative to a fixed position on the illumination stand 606. The translation mechanism may be operated manually (e.g., by a screw rod) or be automatically moved by electric drive (e.g., by a motor-driven driver, piezoelectric actuator).

[0149] In response to the incident light 624, synchrotron radiation 626 emerges from the sample 608. Synchrotron radiation 626 can be generated in several ways. For example, in some embodiments, synchrotron radiation 626 corresponds to a portion of the incident light 624 that has passed through the sample 608. In other embodiments, synchrotron radiation 626 corresponds to a portion of the incident light 624 that has been reflected from the sample 608. In yet another embodiment, the incident light 624 may be absorbed by the sample 608, and synchrotron radiation 626 corresponds to fluorescence emission from the sample 608 (e.g., from a fluorescent component in the sample 608) in response to the incident light 624. In yet another embodiment, the sample 608 may be luminescent and may produce synchrotron radiation 626 even in the absence of incident light 624. In some embodiments, synchrotron radiation 626 may include light generated by two or more of the mechanisms described above.

[0150] The optical collecting element 610 is positioned to receive synchrotron radiation 626 from the sample 608. The optical collecting element 610 may be configured to parallelize the synchrotron radiation 626, for example, if the light 626 is diverging. The optical collecting element 610 may also be configured to spectrally filter the synchrotron radiation 626. Filtering operations may be useful, for example, to isolate a portion of the synchrotron radiation 626 produced by one of the mechanisms discussed above from light produced by other processes. For example, the methods described herein are used to determine accurate estimates of the fluorescence spectra of one or more labeled portions in a sample. The optical collecting element 610 may be configured to filter out non-fluorescent components of the synchrotron radiation 626 (e.g., components corresponding to transmitted and / or reflected incident light). Furthermore, the optical collecting element 610 may be configured to modify the spatial and / or temporal characteristics of the synchrotron radiation 626 for specific purposes in the embodiments. The light-collecting optical element 610 converts the synchrotron radiation 626 into output light 628 that is projected onto the detector 612.

[0151] The detector 612 includes one or more elements, such as a CCD sensor, configured to detect the output light 628. In some embodiments, the detector 612 may be configured to measure the spatial and / or temporal and / or spectral characteristics of the light 628. The detector 612 generates an electrical signal corresponding to the output light 628, which is communicated to the electronic control system 614 via the telecommunication line 630.

[0152] The electronic control system 614 includes a processor 616, a display device 618, and a user interface 620. In addition to receiving signals corresponding to the output light 628 detected by the detector 612, the control system 614 transmits electrical signals to the detector 612 to adjust various characteristics of the detector 612. For example, if the detector 212 includes a CCD sensor, the control system 614 can transmit electrical signals to the detector 612 to adjust the exposure time, active area, gain setting, and other characteristics of the CCD sensor.

[0153] The electronic control system 614 also communicates with the light source 602, the light-tuning optical element 604, the illumination stand 606, and the light-collecting optical element 610 via telecommunication lines 632, 634, 636, and 638, respectively. The control system 614 provides electrical signals to each of these elements of the system 600 to adjust various characteristics of that element. For example, an electrical signal provided to the light source 602 can be used to adjust the intensity, wavelength, repetition rate, or other characteristics of the light 622. Signals provided to the light-tuning optical element 604 and the light-collecting optical element 610 may include signals for setting the characteristics of devices that adjust the spatial characteristics of light (e.g., spatial light modulators) and signals for setting spectral filtering devices. A signal provided to the illumination stand 606 may, for example, cause the sample 608 to be positioned relative to the stand 606 and / or to be moved to a position for illumination on the stand 606.

[0154] The control system 614 includes a user interface 620 for displaying system characteristics and parameters, and for displaying captured images of the sample 608. The user interface 620 is provided to facilitate the operator's interaction with and management of the system 600. The processor 616 includes a storage device for storing image data captured using the detector 612, and also includes computer software that embodies instructions to the processor 616, causing the processor 616 to perform control functions, such as those discussed above. Furthermore, the software instructions cause the processor 616 to mathematically manipulate the images captured by the detector 612 and to decompose the images acquired by the system 600 into contributions from specific marker species in the sample.

[0155] In some embodiments, the light-tuning optical element 604 includes an adjustable spectral filter element, such as a filter wheel or a liquid crystal spectral filter. The filter element may be configured to provide illumination of the sample using different wavelength bands. The light source 602 can supply light 622 having a broad distribution of spectral wavelength components. A selected region of this broad wavelength distribution is allowed to pass through the filter element in the light-tuning optical element 604 as incident light 624 and directed to project onto the sample 608. Subsequently, the wavelength of the filter pass-through region in the light-tuning optical element 604 is modified to provide incident light 624 having different wavelengths. Spectrally resolved images can also be recorded by using a light source 602 having multiple source elements that generate light of different wavelengths, and, alternatively, by switching different source elements on and off to provide incident light 624 having different wavelengths.

[0156] The light-collecting optical element 610 may include a configurable spectral filter element similar to those discussed above in relation to the light-tuning optical element 604. Thus, spectral resolution can be provided on the excitation side of the sample 608 (e.g., by the light-tuning optical element 604) and the emission side of the sample 608 (e.g., by the light-collecting optical element 210).

[0157] The result of collecting multiple spectrally resolved images of a sample is an "image stack," where each image in the stack is a two-dimensional image of the sample corresponding to a specific wavelength. Conceptually, the set of images can be visualized as forming a three-dimensional matrix, where two of the matrix dimensions are the spatial length and width of each image, and the third matrix dimension is the spectral index. For this reason, the set of spectrally resolved images can be called an "image spectral cube." As used herein, a "pixel" in such a set of images (or image stack or spectral cube) refers to a common spatial location for each image. Thus, a pixel in the image set contains a value associated with each image at the spatial location corresponding to that pixel.

[0158] Spectral unmixing can be used to isolate the contribution of each of several label species in a sample to the image information contained in a multispectral image stack. Spectral unmixing is a technique for quantitatively separating contributions in an image from spectrally different sources. For example, a sample may contain three different types of target analytes, each labeled with a label species. Each of the three different label species may have a different absorption spectrum. Typically, the individual absorption spectra of the label species are known before they are used or before they can be measured. In most cases, the image of the sample under illumination contains spectral contributions from each of the three label species. A similar situation arises, for example, in a sample containing several different fluorescent label species, each of which contributes to the fluorescence emission being measured.

[0159] Spectral unmixing decomposes one or more images containing contributions from multiple spectral sources into a set of component images ("unmixed images") corresponding to the contribution from each spectral entity in the sample. Therefore, if a sample contains three different labeling species, each specific to a particular target analyte, the image of the sample can be separated into three unmixed images, each reflecting the contribution primarily from only one of the dyes.

[0160] The unmixing procedure essentially corresponds to decomposing an image into a set of spectral eigenstates. In many embodiments, as discussed above, these eigenstates are known beforehand. In other embodiments, the eigenstates may be determined using techniques such as principal component analysis. In any case, once the eigenstates are identified, the image can usually be decomposed by calculating a set of values ​​as a coefficient matrix corresponding to the relative weighting of each eigenstate in the overall image. Subsequently, the contributions of each individual eigenstate can be separated to produce an unmixed image set.

[0161] For example, a series of 2D images having x and y coordinates, one set of different excitation wavelengths λ k By irradiating the sample, measurements can be taken about the sample. As described above, the two-dimensional images can be combined to form a three-dimensional image cube I(x,y,k), where the first two indices of the image cube represent the coordinate directions and the third index is the spectral index corresponding to the wavelength of the illumination light. For simplicity, each image of the sample can be irradiated with two different spectral sources F(λ k ) and G(λ k Assuming that it includes spectral contributions from ), the values ​​in the 3D image cube I(x,y,k) can be given by: S(x,y,k) = a(x,y)·F(λ) k )+b(x,y)·G(λ k ) (1) In the formula, λk This is used to indicate a predetermined wavelength (or wavelength range). Functions a(x,y) and b(x,y) describe the spatial abundance of spectral contributions from two different spectral sources in the sample.

[0162] According to equation (1), the net signal at any position in the 3D image cube (i.e., at any 2D pixel coordinate and at a particular illumination wavelength) is the sum of two contributions weighted by their respective relative abundances. This can be expressed as follows: I(λ k ) = aF(λ k )+bG(λ k ) (2)

[0163] The functions F and G can be named "spectral eigenstates" for the system, because they correspond to the pure spectra of the spectral sources in the sample, and they are combined in various proportions to produce the measured spectral image of the sample. Thus, the sample spectrum is a weighted superposition corresponding to separate contributions from the two spectral sources.

[0164] Spectrum F(λ k ) and G(λ k If ) is known (or can be estimated), then equation (2) is given that the spectrum I contains at least two elements (i.e., at least two wavelengths λ k (Assuming we have data on ), we can reverse the equation to solve for a and b. Equation (2) can be rewritten in matrix form as I=EA, and as a result, we have: A=E -1 I (3) In the formula, A is a column vector having components a and b, and E is a matrix whose columns are spectral eigenstates, i.e., [FG].

[0165] Using equation (3), the measured spectral image of the sample can be used to calculate the contribution to the image purely from source F and the contribution to the image purely from source G at specific pixel locations. This process can be repeated for each pixel location in the selected image (i.e., across the range of values ​​x and y in I) to produce an image of the sample containing only the contribution from source F, and another image of the sample containing only the contribution from source G.

[0166] In the above discussion, the number of spectral sources is 2 (i.e., F and G). However, in general, the unmixing technique is not limited to any particular number of sources. For example, a sample may generally contain m different spectral sources. If the number of wavelengths from which data is collected is n, i.e., k=1…n, then the matrix E is an n×m matrix instead of an n×2 matrix as in the above discussion. The unmixing algorithm can then be used in the same manner as above to isolate the specific contribution from each of the m spectral eigenstates at each pixel position in the image.

[0167] One factor that can limit the ability of an algorithm to distinguish between contributions from different spectral eigenstates is the degree of spectral discrimination between the eigenstates. The correlation between two spectra, such as two spectral eigenstates I1 and I2, can be explained by the following spectral angle θ:

[0168]

number

[0169] A set of spectra where θ is small for two members is not easily separated into its components. Physically, the reason for this is easily understood as follows: if two spectra are only barely different, it is more difficult to determine their relative abundances.

[0170] Several techniques can be used to measure or estimate the pure spectra of spectral sources F and G (and, if the sample contains more than two, other spectral sources). In general, any method that produces spectral eigenstates with sufficient accuracy can be used. Some samples may contain spectral sources such as dyes or other chemical parts for which known spectra are available in published reference materials. Alternatively, it may be possible to directly measure the spectra of the source components using one or more measurement systems. In some samples, it may be known that a particular region of the sample contains only one specific spectral source, and the spectrum of that source can be extracted from measurements performed only on the identified region of the sample.

[0171] Various data analysis techniques can also be used to determine component spectra as spectral unmixing, such as principal component analysis (PCA), which identifies the most numerous orthogonal spectral eigenvectors from an image cube and produces a score image showing the weighting of each eigenvector throughout the image. This may be done in combination with other mathematical processes, and there are other known techniques for identifying low-dimensional spectral vectors, such as projection tracking, for example, the technique described in L. Jimenez and D. Landgrebe, "Hyperspectral Data Analysis and Feature Reduction Via Projection Pursuit," IEEE Transactions on Geoscience and Remote Sensing, Vol. 37, No. 6, pp. 2653-2667, November 1999 (its entire contents are incorporated herein by reference). Other techniques include, for example, independent component analysis (ICA) and end-member detection algorithms.

[0172] These techniques are typically not well-suited for applications in life sciences. For example, some techniques are optimized for spectral image datasets containing spectra with dense spectral shapes and well-defined narrow peaks. In some techniques, the spectral range is large compared to the individual spectral features and peaks used for analysis. Subsequently, the presence or ratio of peaks can be used to classify "end members" to be separated. Unfortunately, components in biological samples typically do not have such well-defined narrow peaks.

[0173] Some of these techniques generate images related to spectra that exist somewhere in their pure form within the initial image cube. In many cases in life sciences, the signal spectra present in an image cube are mixtures of components. If the component of interest does not exist anywhere in its pure form within the initial image cube, these techniques are unlikely to generate an image that accurately represents the abundance of that component.

[0174] There are several techniques, sometimes called "convex-hull" algorithms, that estimate what the true end members are, even if they do not exist in their pure form in the image, but their effectiveness depends on how close the signal spectrum in the image cube is to the end members.

[0175] One technique that can be used to extract spectral eigenstates (or their representations) even when not all empirical knowledge of eigenstates is to extract the signal spectrum I(λ) for a given pixel. k Taking this into consideration, and leaving the remaining signals which are positive definite in all spectral channels, the first spectral source F(λ k This involves subtracting the maximum amount of ) from it. That is, for each pixel, the so-called "remainder spectrum" U is calculated as follows. a (λ k ) define: Ua (λ k )=I(λ k )-aF(λ k ) (5) At that time, U has a non-negative value in all spectral channels. a (λ k Select the maximum value of parameter a that is consistent with ). Then, select the resulting spectrum U a (λ k ) is used as the signal spectrum with the contribution from the first spectral source F eliminated. The determination of parameter a can also be made based on several relevant criteria that incorporate a small negative distribution to account for considerations such as shot noise or detector noise in the measurement system, rather than based on the strict non-negative criteria mentioned above. An example of adding an optimization criterion to remove the maximum amount of spectral source F is to use a different error function.

[0176] Alternatively, one might attempt to extract the contribution of the second spectral source G to the measured spectrum. Similar to equation (5), the residual spectrum can be calculated for each pixel as follows: U b (λ k )=I(λ k )-bG(λ k ) (6) However, U has non-negative values ​​in all spectral channels. b (λ k Select the maximum value of parameter b that is not inconsistent with ).

[0177] Residual techniques can be extended to cases where the spectra of one or more additional components of a sample are known and it is desired to remove their contributions to the signal. In such cases, the residual spectrum is expressed by subtracting the contribution of each such component from the observed signal, based on the additional spectra and without inconsistency with the positive residuals in each spectral channel.

[0178] Additional embodiments of spectral unmixing are described in U.S. Patents 10,126,242 and 7,555,155, and PCT Publication No. WO2005 / 040769 (the full contents of each are incorporated herein by reference).

[0179] Figure 7 shows an example of an electronically controlled system 614 that may be used in conjunction with the systems and methods disclosed herein. The electronically controlled system may include one or more processors 702 (e.g., corresponding to processor 616 in Figure 6), a memory 704, a storage device 706, and an interface 708 for interconnection. The processor 702 can process instructions for execution within the electronically controlled system 614, including instructions stored in the memory 704 or the storage device 706. For example, an instruction may instruct the processor 702 to perform any of the analyses and control the processes disclosed herein.

[0180] Memory 704 can store executable instructions for the processor 702, information about system parameters such as excitation and detection waves, and measured spectral image information. Storage device 706 may be a computer-readable medium such as a floppy disk device, hard disk device, optical disk device, or tape device, flash memory or other similar solid memory device, or a set of devices including devices or other equipment configurations in a storage area network. Storage device 706 can store either the instructions that can be executed by the processor 702 or other information that can be stored by memory 704.

[0181] In some embodiments, the electronic control system 614 may include a graphics processing unit for displaying graphical information (e.g., using a GUI or text interface) on an external input / output device such as a display 716. The graphical information may be displayed by a display device (e.g., a CRT (cathode ray tube) or LCD (liquid crystal display) monitor) for displaying any of the information disclosed herein, such as measured and calculated spectra and images. The user can use an input device (e.g., a keyboard, a pointing device, a touchscreen, a voice recognition device) to provide input to the electronic control system 614.

[0182] The methods disclosed herein can be implemented by the electronic control system 614 (and processors 702 and 616) executing instructions in one or more computer programs executable and / or interpretable in the electronic control system 614. These computer programs (also known as programs, software, software applications, or code) include machine instructions for a programmable processor and can be implemented in a high-level procedural and / or object-oriented programming language and / or assembly / machine language. For example, a computer program may include instructions that can be stored in memory 704, in storage device 706, and / or in a tangible computer-readable medium, and can be executed by processor 702 (processor 616). As used herein, the term “computer-readable medium” means any computer program product, apparatus, and / or device (e.g., magnetic disks, optical disks, memory, programmable logic devices (PLDs), ASICs, and electronic circuits) used to provide machine instructions and / or data to a programmable processor, including a machine-readable medium that receives machine instructions.

[0183] Generally, the electronic control system 614 can be implemented in a computing system to perform the operations described above. For example, the computing system may include backend components (e.g., as a data server), middleware components (e.g., an application server), or frontend components (e.g., a client computer with a graphical user interface), or any combination thereof.

[0184] Reagents and conditions In general, the various procedures described herein can be carried out under a wide range of conditions and with different reagents. Therefore, the reagents and conditions described in this section should be understood to represent only examples of suitable reagents and conditions.

[0185] Typically, the first substance can be stored after preparation in a buffer solution which may contain one or more of PBS, PBS-T, TBS, TBS-T, water, saline, and Krebs buffer. The buffer solution may optionally contain one or more blocking materials. Examples of suitable blocking materials include, but are not limited to, BSA, casein, sheared salmon sperm DNA, oligonucleotides, rat IgG antibodies, and mouse IgG antibodies.

[0186] The second substance can also be stored after preparation in a buffer solution. The buffer may contain one or more of PBS, PBS-T, TBS, TBS-T, water, saline solution, and Krebs buffer. The buffer solution may be the same as or different from the buffer solution used to store the first substance.

[0187] To facilitate hybridization between the first substance and the second substance, and / or between the first substance and the reporting substance, the first and second substances (or the first substance and the reporting substance) may be immersed in a hybridization buffer. A suitable hybridization buffer may contain DNA components, protein components, surfactants, and / or chaotropic reagents at concentrations between 5% and 20%.

[0188] To facilitate dehybridization between the first substance and the second substance, and / or between the first substance and the reporting substance, the first and second substances (or the first substance and the reporting substance) may be immersed in a dehybridization buffer. A suitable dehybridization buffer may contain chaotropic reagents such as DMSO and / or formamide at concentrations between 60% and 90%.

[0189] To promote the binding of the first substance to the target analyte in the sample, the first substance can be layered onto the sample in solution, for example by pipetting, and incubated with the sample. After incubation, any unbound first substance can be washed from the sample using a buffer solution containing one or more of the following: PBS, PBS-T, TBS, TBS-T, water, saline solution, and Krebs buffer.

[0190] The incubation time for any of the hybridization, reaction, binding, and dehybridization steps described herein may be 10 minutes or longer (for example, 20 minutes or longer, 30 minutes or longer, 40 minutes or longer, 60 minutes or longer, 1 hour or longer, 2 hours or longer, 3 hours or longer, 4 hours or longer, 5 hours or longer, 6 hours or longer, 8 hours or longer, 10 hours or longer, 16 hours or longer, 20 hours or longer, 24 hours or longer, 48 hours or longer, 7 days or longer, 30 days or longer). [Examples]

[0191] Several studies were conducted to demonstrate the efficacy of the methods described herein for analyzing multiple target species in biological samples. First, FFPE samples of human tonsil tissue were obtained. In the first labeling and imaging cycle, the tissue samples were labeled with a first substance containing an antibody-conjugating agent that specifically targets the biomarker PD-1. Subsequently, a second substance was incubated with the samples and hybridized with the first substance. The second substance contained an HRP moiety conjugated to an oligonucleotide. The labeling substance, including the labeling moiety (OPAL® dye HX0046, available from Akoya Biosciences, Inc., Menlo Park, CA), was deposited in the tissue samples by HRP-mediated TSA. After removal of the second substance, the samples were imaged to reveal the presence of PD-1.

[0192] Two further analysis cycles were performed. In the second cycle, the sample was labeled with a first substance containing an antibody-conjugating agent that specifically targets the biomarker PDL1, and the label portion (OPAL® dye HX043, available from Akoya Biosciences, Inc.) was deposited with HRP-mediated TSA to label PDL1. In the third cycle, the sample was labeled with a first substance containing an antibody-conjugating agent that specifically targets the biomarker FOXP3, and the label portion (OPAL® dye HX031, available from Akoya Biosciences, Inc.) was deposited with HRP-mediated TSA to label FOXP3.

[0193] Figures 8A to 8C are images showing the distribution and relative concentrations of the biomarkers PD-1, PDL1, and FOXP3 in tissue samples, respectively. Figure 8D is an overlay image showing the distribution of all three markers in the sample. As is clear from the images, each of the different biomarkers could be independently identified and quantified in the sample, and there was little to no cross-channel interference when the applied labeling portion was appropriately selected.

[0194] To investigate the effect of amplification by HRP-mediated TSA, human FFPE sections of tonsil tissue were obtained, and more than 15 different primary substances (each containing a binding substance targeting a different biomarker) were conjugated to the samples. Subsequently, the corresponding reporting substances were hybridized with the primary substances, and images of samples with different subsets of reporting substances were obtained. Then, the reporting substances were removed from the samples by dehybridization as described above, and labeling moieties targeting three different markers, namely PD-1, PDL-1, and FOXP3, were deposited in the samples in three separate HRP-mediated TSA labeling cycles. Subsequently, images of samples with amplified signals corresponding to the markers PD-1, PDL-1, and FOXP3 were obtained.

[0195] Figure 9A shows the distribution and relative concentrations of markers CD8, CD31, CD20, CD45RO, CD4, pancytokeratin, and CD34 in tissue sections, and Figure 9B shows the distribution and relative concentrations of CD11c, Ki67, PDL-1, E-cadherin, CD3, and FOXP3 in tissue sections. The amplified signals for PD-1, PDL-1, and FOXP3 are shown in Figure 9C. The signals corresponding to PDL-1 and FOXP3 in Figure 9C are significantly stronger than the corresponding signals for these markers in Figure 9B. The nucleotide sequences corresponding to the first substance (first oligonucleotide), the reporting substance, and the second substance (second oligonucleotide) are shown in Table 1 below.

[0196] [Table 1]

[0197] To demonstrate the removal of the second substance from the sample, FFPE sections of tonsil tissue were obtained and subjected to three cycles of HRP-mediated TSA to deposit the labeled portion. In the first cycle, OPAL® dye 570 (Akoya Biosciences, Inc.) was deposited in the sample by HRP-mediated TSA. Subsequently, the second substance was removed from the sample, and the second cycle was performed by introducing OPAL® dye 690 (Akoya Biosciences, Inc.) without hybridizing the second substance, and therefore without the presence of an HRP reagent in the sample. In the third cycle, OPAL® dye 690 was deposited by HRP-mediated TSA. After the second and third cycles, sample images were obtained.

[0198] Figures 10A and 10D show nuclear staining images after cycles 2 and 3, Figures 10B and 10E show OPAL® dye 570 images after cycles 2 and 3, and Figures 10C and 10F show OPAL® dye 690 images after cycles 2 and 3. As is clear from Figures 10C and 10F, complete removal of the second substance was achieved between cycles 1 and 2.

[0199] For the aforementioned example, the labeling protocols for OPAL® dyes 570 and 670 were as follows: (a) Wash the tissue three times with 20% DMSO. (b) Incubate the tissue in 200 μl of hybridization buffer for 10 minutes. After 10 minutes, wash the tissue 3× with 20% DMSO, followed by 3× washing with 1× CODEX® assay buffer. (c) Add 200 μl of OPAL® dye (O570 or O670), diluted in ratios of 1:200 and 1:400, respectively, to the 1×plus amplification dilution reagent. Incubate for 20 minutes. (d) After 20 minutes, wash the tissue with 1× CODEX® assay buffer (3×). Wash with DI water (3×) and image with a 20x objective lens.

[0200] The labeling protocol for OPAL® dye 780 was as follows: (a) Wash the tissue three times with 20% DMSO. Incubate the tissue in 200 μl of hybridization buffer for 10 minutes. After 10 minutes, wash the tissue with 20% DMSO (3×), followed by a 3× wash with 1× CODEX® assay buffer. (b) Add 200 μl of TSA dig diluted in a 1:50 ratio to the 1×plus amplification dilution reagent. Incubate for 15 minutes. (c) After 15 minutes, wash the tissue with 1×CODEX® assay buffer. (d) Add 200 µl of OPAL 780 diluted in a 1:50 ratio to the 1×plus amplification dilution reagent. Incubate for 1 hour. Wash the tissue with 1×CODEX® assay buffer (3×). Wash with DI water (3×) and image with a 20x objective lens.

[0201] Other Embodiments This disclosure describes specific executions, but these should be interpreted not as limitations to the scope of this disclosure, but rather as descriptions of features in certain embodiments. Features described in the context of separate embodiments can also generally be executed in combination in a single embodiment. Conversely, various features described in the context of a single embodiment can also be executed separately or in any suitable partial combination in multiple embodiments. Furthermore, although features are described above as existing in a particular combination, and may initially be claimed as such, one or more features from a claimed combination can generally be removed from that combination, and the claimed combination can be directed towards a partial combination or a variation of a partial combination.

[0202] In addition to the embodiments expressly disclosed herein, it will be understood that various modifications to the embodiments described may be made without departing from the spirit and scope of this disclosure. Accordingly, other embodiments are within the scope of the following claims. [Explanation of Symbols]

[0203] 100 flowcharts 202 Biological Samples 204 The first substance 206 Combined species 208 First oligonucleotide 210 Target Analyte 212 Second oligonucleotide 214 Reactants 216 The second substance 218 Marked species 302 Samples 306a~c Combined species 308a~c First oligonucleotide 310a~c Target Analyte 312a Second oligonucleotide 314a Reactant species, reactants 318a~c Labeled substances 320a Dashed arrow 350 flowcharts 402 samples 404a~M, 404u~w: The first substance 406a~e Binding substance, binding species 408a~e, 408u First oligonucleotide 410a~M, 410u~410w target analyte 412u Second oligonucleotide 414u reactant 418a~c, 418u marked species 450 Reporting Substances 450a~M Reporting Material 452 Oligonucleotides 452d oligonucleotide 452e oligonucleotide 454 Sign part 454d~e Mark part, mark species 502 Double-stranded region 504 Single-stranded region 506 Secondary oligonucleotide chains 508 Major Oligonucleotide Chains 510 Intermediate join area 510a Join area 510b Combined area 512a Unbonded region 512b Unbonded region 600 System 602 Light source 604 Light-adjusting optical element 606 Lighting stand 608 samples 610 Light collecting optical element 612 detectors 614 Electronic control systems 616 processors 618 Display Devices 620 User Interfaces 622 light 624 Incident light 626 Synchrotron radiation 628 Output light 630 Telecommunication Lines 632 Telecommunication Lines 634 Telecommunication Lines 636 Telecommunication Lines 638 Telecommunication lines 702 Processor 704 memory 706 Storage device 708 Interface 716 displays

Claims

1. The following steps: (i) A step of contacting a biological sample containing a first target analyte with a first substance, wherein the first substance contains a first binding species that specifically binds to the first target analyte and a first oligonucleotide conjugated to the binding species; (ii) A step of contacting the biological sample with a second substance comprising a first reactant and a second oligonucleotide conjugated to the first reactant, thereby hybridizing at least a portion of the second oligonucleotide to at least a portion of the first oligonucleotide; (iii) A step of contacting the biological sample with a first labeled species, wherein the first labeled species reacts with the first reactant to deposit the first labeled species or a derivative thereof in the biological sample; (iv) After deposition of the first labeled species or its derivative, the second substance is removed from the biological sample by dehybridizing the second oligonucleotide from the first oligonucleotide; (v) A step of contacting the biological sample with a third substance, wherein the third substance comprises a second binding species that specifically binds to a second target analyte in the biological sample, and a third oligonucleotide conjugated to the second binding species; (vi) A step of contacting the biological sample with a fourth substance comprising a second reactant and a fourth oligonucleotide conjugated to the second reactant, thereby hybridizing at least a portion of the fourth oligonucleotide with at least a portion of the third oligonucleotide; (vii) A step of contacting the biological sample with a second labeled species, wherein the second labeled species reacts with a second reactant to deposit the second labeled species or a derivative thereof in the biological sample. A method that includes this.

2. The method according to claim 1, wherein the first reaction species includes a catalytic substance.

3. The method according to claim 1, wherein the first reaction species comprises an enzyme.

4. The method according to claim 3, wherein the enzyme comprises horseradish peroxidase.

5. The method according to claim 1, wherein the first labeled species includes a dye.

6. The method according to claim 4, wherein the first labeled species comprises a conjugate of an inactive tyramide or a derivative thereof and a dye.

7. The method according to claim 6, wherein the step of contacting the biological sample with the first labeled species includes converting the first labeled species into a conjugate of an active tyramide or a derivative thereof and a dye, wherein the active tyramide or a derivative thereof binds to the biological sample in close proximity to the second substance.

8. The method according to claim 1, wherein the first binding species comprises an antibody or an antibody fragment.

9. The method according to claim 1, wherein the first oligonucleotide comprises at least 10 nucleotides.

10. The method according to claim 1, wherein the second oligonucleotide comprises at least 10 nucleotides.

11. The method according to claim 1, wherein the nucleotide sequences of the first and second oligonucleotides are at least 70% complementary.

12. The method according to claim 1, wherein the second oligonucleotide comprises a greater number of nucleotides than the first oligonucleotide.

13. The method according to claim 1, wherein the second oligonucleotide has a nucleotide sequence that is at least 80% complementary to the nucleotide sequence of the first oligonucleotide.

14. The method according to claim 1, wherein the first and second reaction species are the same.

15. The method according to claim 1, wherein each of the first and second reaction species comprises an enzyme.

16. The method according to claim 1, wherein each of the first and second reaction species comprises horseradish peroxidase.

17. The method according to claim 1, wherein the first oligonucleotide and the third oligonucleotide are different.

18. The method according to claim 1, wherein the second oligonucleotide and the fourth oligonucleotide are different.

19. The method according to claim 1, wherein the first labeled species comprises a first dye, and the second labeled species comprises a second dye different from the first dye.

20. The method according to claim 1, wherein the first conjugate comprises a first antibody or a first antibody fragment, the second conjugate comprises a second antibody or a second antibody fragment, and the first and second conjugates selectively conjugate to different first and second target analytes in the biological sample.

21. The method according to claim 1, wherein the first oligonucleotide comprises a nucleotide sequence of RNA bases.

22. The method according to claim 1, wherein the first oligonucleotide comprises a nucleotide sequence of DNA bases.

23. The method according to claim 1, wherein the first oligonucleotide comprises at least one synthetic nucleotide.

24. The method according to claim 1, wherein the first oligonucleotide is completely single-stranded.

25. The method according to claim 1, wherein the first oligonucleotide is partially double-stranded.

26. The method according to claim 5, wherein the dye comprises a chromogenic species or a fluorescent species.

27. A first substance comprising a first binding species that specifically binds to a first target analyte of a biological sample, and a first oligonucleotide conjugated to the first binding species; A second substance comprising a second conjugate species that specifically binds to a second target analyte of the biological sample, and a second oligonucleotide conjugated to the second conjugate species; A third substance comprising a reactant and a third oligonucleotide conjugated to the reactant; A fourth substance comprising a reactant and a fourth oligonucleotide conjugated to the reactant; The first marked species; The second type of marker; and Chaotropic reagents A reagent kit comprising the first and second labeled species, each reacting with the reaction species to deposit the first and second labeled species or derivatives thereof, respectively, in the biological sample.

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