Markers and methods for analyzing biological samples

Abstract

In a first aspect, a marker (100, 200) for analyzing a biological sample is provided. The marker (100, 200) comprises an affinity reagent (104) comprising an affinity reagent oligonucleotide (108) having a barcode sequence (110). The marker (100, 200) further comprises a marker (112) comprising a marker oligonucleotide (114) having a complementary barcode sequence (116) that is at least partially complementary to the barcode sequence (110) of the affinity reagent oligonucleotide (108). The marker (100, 200) further comprises at least one blocking oligonucleotide (102, 202) having a blocking sequence (118, 204) that is only partially complementary to the barcode sequence (110) of the affinity reagent oligonucleotide (108) or to the complementary barcode sequence (116) of the marker oligonucleotide (114).

Description

Technical Field

[0001] This invention relates to biomarkers and corresponding markers, affinity reagents, and kits. In another aspect, a method for analyzing biological samples is provided. Background Technology

[0002] Background fluorescence is a significant drawback in fluorescence imaging because it obscures the signal originating from the target fluorescent molecules, thereby reducing the contrast and sharpness of the resulting image. The appearance of background fluorescence in fluorescence imaging is caused by a variety of factors, including autofluorescence from the biological sample and non-specific binding of fluorescent markers. When a fluorescent marker attaches to an unexpected site within the sample, non-specific binding occurs, producing additional fluorescence signals that overlap with the fluorescence signal originating from the target. This overlap reduces the signal-to-noise ratio, making accurate detection and quantification of the target challenging. Therefore, unwanted fluorescence can lead to inaccurate measurements, data misinterpretation, and difficulty in distinguishing the target from the background, thus impairing the overall quality and reliability of the imaging results. Similar problems exist with markers not based on fluorescence detection, such as those read from sequencing and those labeled with metal tags, radioactive labels, enzyme tags (e.g., horseradish peroxidase), Raman labels, or gold particles.

[0003] This non-specific interaction can be influenced by a variety of factors, such as the concentration and chemical properties of the marker, as well as the composition and preparation of the sample or marker. The combination of these background fluorescence sources makes obtaining high-quality, high-contrast fluorescence images a persistent challenge, especially for applications using a large number of fluorescent markers (e.g., multiplex applications). Summary of the Invention

[0004] The purpose of this invention is to provide a biomarker that can reduce background fluorescence when analyzing biological samples, especially biological samples with a large number of target analytes.

[0005] The above-mentioned objective is achieved by the technical subject matter according to the independent claims. Advantageous embodiments are defined in the dependent claims and the following description.

[0006] In a first aspect, a biomarker for analyzing biological samples is provided. The biomarker comprises an affinity reagent, which comprises an affinity reagent oligonucleotide having a barcode sequence. The biomarker further comprises a marker oligonucleotide having a complementary barcode sequence, the complementary barcode sequence being at least partially complementary to the barcode sequence of the affinity reagent oligonucleotide. The biomarker further comprises at least one blocking oligonucleotide having a blocking sequence, the blocking sequence being only partially complementary to either the barcode sequence of the affinity reagent oligonucleotide or the complementary barcode sequence of the marker oligonucleotide.

[0007] At least one blocking oligonucleotide, with only at least partial complementarity, can block a barcode sequence or a complementary barcode sequence. For example, when components of a marker, such as an affinity reagent with an affinity oligonucleotide and a marker oligonucleotide, are introduced into a biological sample for analysis, these components can be introduced individually over time. Specifically, the affinity reagent with the affinity oligonucleotide can first be introduced into the biological sample to bind to a specific target analyte of the biological sample. Subsequently, the marker oligonucleotide can be introduced. In this case, the blocking oligonucleotide prevents the affinity oligonucleotide and / or the marker oligonucleotide from inadvertently and nonspecifically hybridizing to random nucleic acids in the biological sample. The random nucleic acid can be an endogenous nucleic acid molecule or sequence, the sequence of which may have inadvertent similarity or complementarity to the marker oligonucleotide.

[0008] Similarly, the proposed markers can reduce the binding of marker oligonucleotides to off-target affinity oligonucleotides (i.e., affinity oligonucleotides assigned to affinity oligonucleotides that were not assigned to marker oligonucleotides). This unintended interaction that can occur between marker oligonucleotides and endogenous nucleic acids, as well as "off-target" affinity oligonucleotides, can also be termed cross-hybridization.

[0009] Specifically, the proposed marker is capable of reducing unwanted nonspecific binding and / or reducing background fluorescence. Therefore, in a specific embodiment, the marker may comprise two blocking oligonucleotides: a first blocking oligonucleotide having a first blocking sequence, the first blocking oligonucleotide being partially complementary to the barcode sequence of the affinity oligonucleotide, and a second blocking oligonucleotide having a second blocking sequence, the second blocking oligonucleotide being partially complementary to the complementary barcode sequence of the marker oligonucleotide.

[0010] Furthermore, the complementarity between the barcode sequence and its complementary barcode sequence enables highly specific and stable binding between them, and thus also enables highly specific and stable binding between the affinity reagent and the marker. This avoids erroneous binding between the affinity reagent and the marker. This is particularly relevant when using a large number of different markers, including affinity reagents specific to different target analytes, to identify a large number of target analytes.

[0011] The affinity reagent is preferably configured to specifically bind to a target analyte, such as a target protein or target nucleic acid, in a biological sample. For example, the affinity reagent may be an antibody, an antibody fragment, an amino acid- or nucleic acid-based aptamer, or a linear single-stranded nucleic acid. More preferably, the affinity reagent oligonucleotide is covalently attached to the affinity reagent.

[0012] The label comprises at least one labeled portion, and in particular, at least one labeled portion can be configured to be optically detectable. The labeled portion may include a fluorophore, such as a fluorescent protein, an organic or inorganic fluorescent molecule, or fluorescent nanoparticles. This makes the label detectable by optical microscopy, such as fluorescence microscopy, and in particular, confocal scanning microscopy.

[0013] For example, the marker oligonucleotide can be used as the backbone of the marker, the backbone containing complementary barcode sequences, particularly complementary barcode sequences oriented toward the 3' or 5' end. The marker moiety can preferably be (covalently) attached to the marker oligonucleotide.

[0014] Preferably, at least two of the blocking sequence, barcode sequence, and complementary barcode sequence contain the same number of nucleotides. Therefore, they preferably have the same length.

[0015] Specifically, a blocking sequence that is only partially complementary to the blocking oligonucleotide results in a lower melting temperature for the blocking sequence compared to the melting temperature of the corresponding barcode sequence of the affinity oligonucleotide or the complementary barcode sequence of the marker oligonucleotide. This is especially true if the corresponding sequences are of the same length. For example, due to sequence mismatch, the blocking sequence may only be partially complementary to the corresponding barcode sequence or the complementary barcode sequence.

[0016] Therefore, hybridization between barcode sequences and complementary barcode sequences is preferred over hybridization between blocking oligonucleotides (especially blocking sequences) and corresponding barcode sequences or complementary barcode sequences.

[0017] For example, the number of mismatches between the blocking sequence and the corresponding barcode sequence or complementary barcode sequence can be in the range of 1 to 10 mismatched nucleotides per 20 nt of blocking sequence. The mismatched nucleotides are preferably evenly distributed along the length of the blocking sequence.

[0018] Preferably, at least one blocking oligonucleotide is separate from the affinity oligonucleotide and the labeling oligonucleotide. In this case, the blocking oligonucleotide is not covalently linked to the affinity oligonucleotide or the labeling oligonucleotide. The blocking oligonucleotide binds to the corresponding affinity oligonucleotide or the labeling oligonucleotide only through hybridization. This simplifies the generation and assembly of the marker.

[0019] Preferably, at least one blocking oligonucleotide is part of an affinity oligonucleotide or a marker oligonucleotide. Therefore, the blocking oligonucleotide and the affinity oligonucleotide or marker oligonucleotide can be a continuous nucleotide sequence or nucleic acid molecule. This allows the blocking oligonucleotide, particularly the blocking sequence, to readily approach the affinity oligonucleotide or marker oligonucleotide, and enables regular and correct hybridization or blocking of the corresponding affinity oligonucleotide or marker oligonucleotide, even with only partial complementarity or mismatch.

[0020] Preferably, the blocking sequence of the blocking oligonucleotide is a partial inverse complementary sequence of the following: a barcode sequence of an affinity oligonucleotide, or a complementary barcode sequence of a labeling oligonucleotide, particularly the corresponding labeling oligonucleotide or affinity oligonucleotide, wherein the blocking oligonucleotide is a part thereof. This enables the formation of a hairpin loop between the blocking oligonucleotide and the corresponding barcode sequence or complementary barcode sequence. When the marker comprises two blocking oligonucleotides, both blocking oligonucleotides can form a hairpin with the corresponding barcode sequence or complementary barcode sequence. Further, the blocking sequences of the two blocking oligonucleotides can be complementary to each other. Preferably, the first and second barcode sequences can have the same distance (i.e., number of nucleotides) from the corresponding barcode sequence or complementary barcode sequence. When the two blocking oligonucleotides are respectively linked to the affinity oligonucleotide or labeling oligonucleotide, this enables the two blocking sequences to bind effectively to each other.

[0021] Preferably, the complementary barcode sequence of the marker oligonucleotide is perfectly complementary to the barcode sequence of the affinity reagent, particularly at least with respect to consecutive common complementary barcode sequences. Therefore, there is no mismatch between the barcode sequence and the complementary barcode sequence, and preferably the two barcode sequences have the same number of nucleotides. This allows the marker to bind specifically and stably to the biomarker.

[0022] Preferably, the melting temperature of the hybridization complex or duplex of the blocking sequence with the barcode sequence of the affinity oligonucleotide or with the complementary barcode sequence of the marker oligonucleotide is lower than the melting temperature of the hybridization complex or duplex of the barcode sequence of the affinity oligonucleotide and the complementary barcode sequence of the marker oligonucleotide. This allows for preferential hybridization of the barcode sequence with the complementary barcode sequence compared to hybridization of the blocking sequence with the corresponding barcode sequence or complementary barcode sequence.

[0023] Preferably, the affinity reagent oligonucleotide comprises a plurality of barcode sequences. This enables multiple markers to bind to the affinity reagent. Each barcode sequence in the plurality of barcode sequences can be identical to each other. Preferably, the plurality of barcode sequences are distinct from each other, specifically each marker can bind to a specific predetermined barcode sequence within the plurality of barcode sequences. For example, each barcode sequence in the plurality of barcode sequences can be unique. In a specific embodiment, the specific barcode sequence can encode detectable properties of the labeled portion of a marker to which the specific barcode sequence can be attached. Detectable properties can include, for example, the excitation and emission wavelengths of the fluorophore and the emission lifetime.

[0024] Preferably, two adjacent barcode sequences of the affinity oligonucleotide, particularly all adjacent barcode sequences, are separated by a cleavage site configured to be cleaved, particularly selectively, by a cleavage agent. This enables the removal of the barcode sequence from the affinity oligonucleotide. Any markers bound to the corresponding barcode sequence are removed, along with the barcode sequence itself. This is particularly relevant to cyclic staining methods, in which (different) markers are iteratively linked to the same affinity reagent, particularly between subsequent (optical) readouts. Therefore, the marker can combine highly specific and stable binding of the marker to the affinity reagent, and the marker can be easily removed from the affinity reagent.

[0025] Specifically, each cleavage site can be cleaved only by a specific cleavage agent. For example, each cleavage site can be cleaved by a different enzyme (e.g., a restriction enzyme) to enable subsequent and specific cleavage of a specific or selected cleavage site. Examples of cleavage sites or cleavage agents include cleavage sites or nucleotides cleavable by ultraviolet (UV) light, restriction sites, and restriction enzymes. Preferably, the marker comprises at least one marker moiety. This enables easy detection and identification of the marker and the corresponding target analyte. Preferably, the marker moiety may be covalently attached to the marker oligonucleotide. Alternatively, the marker moiety may comprise an attached oligonucleotide capable of hybridizing with the marker oligonucleotide.

[0026] Preferably, the marker portion is at least one of a fluorophore, a polyacetylene, and a metal ion. This allows for the detection and identification of the marker using various detection methods. Furthermore, this enables the generation of a larger number of distinguishable markers.

[0027] Preferably, the barcode sequence contains between 8 and 60 nucleotides. Similarly, the complementary barcode sequence and the blocking sequence may contain the same number of nucleotides as the barcode sequence.

[0028] On the other hand, a system with multiple markers, particularly those as described above, is provided. The barcode sequence of each marker in the multiple markers is different from the barcode sequence of any other marker. Specifically, each marker in the multiple markers has a unique barcode sequence, allowing the marker to be specifically attached to the marker's affinity reagent. For example, each marker in the multiple markers has a marker with detectable properties, which is any remaining marker distinguishable from the remaining markers in the multiple markers. In this case, it is desirable to attach each distinguishable marker to a predetermined specific affinity reagent in the affinity reagents of the multiple markers so that the detection of a specific marker can be associated with a specific target analyte.

[0029] In another aspect, a marker for generating a biomarker, particularly a biomarker as described above, is provided. The marker comprises a marker oligonucleotide having a complementary barcode sequence, the complementary barcode sequence being at least partially complementary to the barcode sequence of the affinity oligonucleotide of the affinity reagent. The marker further comprises at least one blocking oligonucleotide having a blocking sequence, the blocking sequence being partially complementary to the barcode sequence of the marker oligonucleotide. The marker is configured to be linked to an affinity reagent by hybridizing the barcode sequence of the affinity oligonucleotide with the complementary barcode sequence of the marker oligonucleotide. The marker is preferably used for analyzing a biological sample containing a target analyte, and the affinity reagent is configured to specifically bind to the target analyte.

[0030] In another aspect, an affinity reagent is provided for generating a biomarker, particularly a biomarker as described above. The affinity reagent comprises an affinity reagent oligonucleotide having a barcode sequence that is at least partially complementary to the complementary barcode sequence of a biomarker oligonucleotide of the biomarker. The affinity reagent further comprises at least one blocking oligonucleotide having a blocking sequence that is partially complementary to the barcode sequence of the affinity reagent oligonucleotide. The affinity reagent is configured to link to a biomarker by hybridizing the barcode sequence of the affinity reagent oligonucleotide to the complementary barcode sequence of the biomarker oligonucleotide. The affinity reagent is preferably used to analyze a biological sample containing a target analyte, and the affinity reagent is configured to specifically bind to the target analyte.

[0031] In another aspect, a kit for analyzing biological samples is provided. The kit comprises a marker for generating a biomarker, particularly a biomarker as described above; and an affinity reagent having an affinity reagent oligonucleotide having a barcode sequence. The marker is configured to form a biomarker if the barcode sequence of the affinity reagent oligonucleotide hybridizes with the complementary barcode sequence of the marker oligonucleotide. Alternatively, the kit comprises an affinity reagent for generating a biomarker, particularly a biomarker as described above; and a marker having a marker oligonucleotide having a complementary barcode sequence. The affinity reagent is configured to form a biomarker if the complementary barcode sequence of the marker oligonucleotide hybridizes with the barcode sequence of the affinity reagent oligonucleotide.

[0032] On the other hand, a method for analyzing biological samples is provided. The method includes introducing the following substances into the biological sample: at least one biomarker, particularly a biomarker as described above; or a system having multiple biomarkers, particularly biomarkers as described above; or at least one biomarker, particularly a biomarker as described above, and at least one affinity reagent, said at least one affinity reagent having an affinity reagent oligonucleotide having a barcode sequence, the barcode sequence being at least partially complementary to the complementary barcode sequence of the biomarker oligonucleotide; or at least one affinity reagent, particularly an affinity reagent as described above, and at least one biomarker, said at least one biomarker having a biomarker oligonucleotide, the biomarker oligonucleotide having a complementary barcode sequence, the complementary barcode sequence being at least partially complementary to the barcode sequence of the affinity reagent oligonucleotide. The method further includes the step of generating an initial readout of the biomarker in the biological sample. Readouts are typically generated to detect the biomarker, particularly a biomarker of the biomarker. For example, if the biomarker is optically detectable and contains, for example, a fluorophore as a labeling component, the readout can be an optical readout. The readout can be generated by means of, for example, a microscope, such as a fluorescence microscope.

[0033] Preferably, during the step of introducing at least one marker, the marker portion of at least one marker is added separately. Specifically, the marker portions are introduced sequentially in time. For example, affinity reagents may be added first and bound to their respective target analytes. Subsequently, the corresponding markers may be added. The marker portions may be, for example, affinity reagents, markers, and blocking oligonucleotides.

[0034] Preferably, after generating the initial readout, another marker is introduced into the biological sample and another readout is generated. The additional marker may similarly comprise a marker oligonucleotide having a complementary barcode sequence, which is complementary to a barcode sequence of an affinity reagent of one of the markers previously introduced into the biological sample. Before introducing the additional marker, the previous marker can be lysed and removed from the biological sample. After introducing the additional marker into the biological sample, another readout can be generated. This enables a cyclic staining method.

[0035] Typically, multiple biomarkers can be introduced into a biological sample. These biomarkers are specific to their corresponding target analytes or pairs of target analytes to simultaneously identify a large number of (different or identical) target analytes. Preferably, the target analytes are identified and / or located within the biological sample based on markers, particularly marker portions, associated with the target analytes being read out. The biomarkers can be physically constructed prior to their introduction into the biological sample. Alternatively, affinity reagents can be barcode-encoded with barcode sequences and introduced into the biological sample first. In subsequent steps, biomarkers containing complementary barcode sequences can be introduced into the biological sample and then attached to their corresponding affinity reagents, thereby forming the corresponding biomarkers within the biological sample.

[0036] The systems, markers, affinity reagents, kits, and methods for the markers have the same advantages as the markers themselves. Furthermore, the systems, markers, affinity reagents, kits, and methods for the markers can be supplemented with the features of the markers described herein, and in particular the features of the dependent claims of the markers. Attached Figure Description

[0037] The following description, with reference to the accompanying drawings, details specific embodiments of which: Figure 1 This is a schematic diagram of a biomarker containing blocking oligonucleotides used to analyze biological samples; Figure 2 It is based on Figure 1 A schematic diagram of the sign; and Figure 3 This is a schematic diagram of a marker with two blocking oligonucleotides. Detailed Implementation

[0038] Figure 1 and Figure 2This is a schematic diagram of a biomarker 100 used for analyzing biological samples (not shown). Biomarker 100 contains a blocking oligonucleotide 102. The biomarker further contains an affinity reagent 104, which is configured to specifically bind to a target analyte 106 of the biological sample. Affinity reagent 104 contains an affinity reagent oligonucleotide 108 attached to it. Affinity reagent oligonucleotide 108 contains three barcode sequences 110.

[0039] Marker 100 further includes marker 112, which contains blocking oligonucleotide 102. Marker 102 further includes marker oligonucleotide 114. Marker oligonucleotide 114 contains a complementary barcode sequence 116, which is complementary to the barcode sequence 110 of affinity oligonucleotide 108. Blocking oligonucleotide 102 and marker oligonucleotide 114 are a continuous nucleic acid molecule. Blocking oligonucleotide 102 contains a blocking sequence 118, which is partially complementary to the complementary barcode sequence 116. Therefore, blocking sequence 118 may contain mismatches, such that the blocking sequence does not completely hybridize with the complementary barcode sequence 116. Therefore, barcode sequence 110 and complementary barcode sequence 116 preferentially hybridize with each other. In contrast, blocking sequence 118 and complementary barcode sequence 116 hybridize with each other only in a subordinate manner. However, if barcode sequence 110 is absent or not close to complementary barcode sequence 116, then blocking sequence 118 can freely hybridize with complementary barcode sequence 116.

[0040] The marker 112 further comprises a plurality of marker portions 120. The marker portions 120 are, for example, (different or the same) fluorophores. The marker portions 120 may be attached to the marker oligonucleotide 114 by means of an attachment oligonucleotide 122, the attachment oligonucleotide being complementary to the corresponding portion of the marker oligonucleotide 114.

[0041] The affinity oligonucleotide 108 may include a cleavage site 124, which is cleavable by means of a cleavage agent (not shown). For example, the cleavage site 124 may be a restriction site that can be cleaved by a restriction enzyme. Preferably, each cleavage site 124 can be cleaved by only one specific restriction enzyme. Alternatively, the cleavage site 124 may be a UV-sensitive nucleotide that can be cleaved by ultraviolet (UV) light. Cleavage of one of the cleavage sites 124 removes the marker 112 (and the corresponding barcode sequence 110 of the affinity oligonucleotide 108) from the affinity 104.

[0042] Marker 100 comprises multiple barcode sequences 110. Therefore, multiple markers in marker 112 can be attached to affinity reagent 104. Alternatively, these barcode sequences 110 can be different from each other in their specific nucleotide sequences. This enables the generation of markers 112 containing complementary barcode sequences 116 that are complementary to one of the barcode sequences in the different barcode sequences 110, and the attachment of these markers 112 to specific barcode sequences in the barcode sequences 110. These different markers 112 can be different from each other through this set of label portions 120 attached to the marker oligonucleotide 114. For example, each different marker 112 can contain label portions 120 with different detectable properties. An iterative staining method can be performed using marker 100 by attaching these markers 112 to affinity reagent 104, cleaving cleavage site 124, and attaching additional markers 112 to another barcode sequence 110.

[0043] exist Figure 1 In this configuration, marker 100 is shown to be in a state where the blocking sequence 118 has hybridized with the complementary barcode sequence 116 and therefore marker 112 (not yet) has not been bound to affinity reagent 104. The blocking sequence 118 is preferably the inverse complementary sequence of the complementary barcode sequence 116. Therefore, according to... Figure 1 In this state, blocking oligonucleotide 102 and labeled oligonucleotide 114 can form a hairpin loop. For example, this prevents off-target hybridization of labeled 112, particularly blocking oligonucleotide 102 or labeled oligonucleotide 114, with the biological sample. This increases the likelihood that labeled 112 will correctly bind to affinity reagent 104, particularly its affinity reagent oligonucleotide 108, after being added to the biological sample.

[0044] exist Figure 2 In this configuration, marker 100 is shown in a state where complementary barcode sequence 116 hybridizes with barcode sequence 110, and thus marker 112 is bound to affinity reagent 104. When marker 112 and affinity reagent 104 are close together, barcode sequence 110 and complementary barcode sequence 116 preferentially hybridize with each other. Furthermore, barcode sequence 110 can substitute blocking sequence 118 from complementary barcode sequence 116. Figure 2 Marker 100 is shown, which is in a state where the marker can be read when analyzing biological samples.

[0045] In an alternative embodiment, marker 100 may comprise blocking oligonucleotide 102, particularly blocking sequence 118, which is linked to affinity oligonucleotide 108 instead of marker oligonucleotide 114 (e.g., ...). Figure 1 and Figure 2 (As shown).

[0046] Figure 3 This is a schematic diagram of marker 200. Compared to marker 100, marker 200 contains a second blocking oligonucleotide 202 having a second blocking sequence 204. The second blocking sequence 204 may be partially complementary to the barcode sequence 110. Preferably, the second blocking sequence 204 is completely complementary to the blocking sequence 118.

[0047] Therefore, for marker 200, when affinity reagent 104 separates from marker 112, the second blocking sequence 204 can hybridize to barcode sequence 110. Blocking sequence 118 hybridizes to complementary barcode sequence 116, as for marker 200. Figure 1 and Figure 2 As described.

[0048] When affinity reagent 104 and marker 112 of marker 200 become close, for example, when both are introduced (sequentially) into a biological sample, barcode sequence 110 and complementary barcode sequence 116 preferentially hybridize with each other due to their complementarity. Similarly, blocking sequence 118 and second blocking sequence 204 can preferentially hybridize with each other due to their complementarity. Therefore, not only do barcode sequence 110 and complementary barcode sequence 116 replace the corresponding blocking sequence 118 or second blocking sequence 204, but blocking sequence 118 and second blocking sequence 204 also preferentially hybridize with each other. This enables highly specific and stable binding between marker 112 and affinity reagent 104. Specifically, this avoids off-target hybridization of marker 112 (especially blocking oligonucleotide 102 or marker oligonucleotide 114) and affinity reagent 104 (especially affinity oligonucleotide 108) with the biological sample. This further increases the probability that affinity reagent 104 binds to its intended target analyte 106 in the biological sample, and the probability that marker 112 correctly binds to affinity reagent 104, and in particular its affinity reagent oligonucleotide 108, after marker 112 is added to the biological sample.

[0049] In all the accompanying drawings, elements that have the same or similar function are represented by the same element symbols. As used herein, the term "and / or" includes any and all combinations of one or more of the relevant listed items, and may be abbreviated as " / ".

[0050] Although some aspects have been described in the context of the apparatus, it is clear that these aspects also represent descriptions of the corresponding methods, where blocks or devices correspond to method steps or features of method steps. Similarly, aspects described in the context of method steps also represent descriptions of corresponding blocks, entries, or features of the corresponding apparatus.

[0051] Although the present invention has been described using preferred embodiments, the invention is not limited thereto and can be modified in various ways.

[0052] List of reference numerals 100, 200 markers 102, 202 blocking oligonucleotides 104 Affinity Reagent 106 Target Analytes 108 affinity reagent oligonucleotides 110 barcode sequence 112 Markers 114 Marker Oligonucleotides 116 Complementary Barcode Sequences 118, 204 blocking sequences 120 Marking Section 122 Attached oligonucleotides 124 cleavage sites

Claims

1. A biomarker (100, 200) for analyzing biological samples, wherein, The marker includes: Affinity reagent (104), said affinity reagent comprising an affinity reagent oligonucleotide (108) having a barcode sequence (110). A marker (112) comprising a marker oligonucleotide (114) having a complementary barcode sequence (116) that is at least partially complementary to the barcode sequence (110) of the affinity oligonucleotide (108), and At least one blocking oligonucleotide (102, 202), the at least one blocking oligonucleotide having a blocking sequence (118), the blocking sequence being partially complementary to the barcode sequence (110) of the affinity oligonucleotide (108) or the complementary barcode sequence (116) of the marker oligonucleotide (114).

2. The marker according to claim 1, wherein the at least one blocking oligonucleotide (102, 202) is separate from the affinity oligonucleotide (108) and the marker oligonucleotide (114).

3. The marker according to claim 1, wherein the at least one blocking oligonucleotide (102, 202) is part of the affinity oligonucleotide (108) or the marker oligonucleotide (114).

4. The marker according to claim 3, wherein the blocking sequence (118, 204) is a barcode sequence (110) of the corresponding oligonucleotide (108, 114) or a partial inverse complementary sequence of the complementary barcode sequence (116), which is part of the blocking oligonucleotide (102, 202).

5. The marker according to any one of the preceding claims, wherein the complementary barcode sequence (116) of the marker oligonucleotide (114) is completely complementary to the barcode sequence (110) of the affinity reagent oligonucleotide (108).

6. The marker according to any one of the preceding claims, wherein the melting temperature of the hybridization complex of the blocking sequence (118, 204) with the barcode sequence (110) of the affinity oligonucleotide (108) or with the complementary barcode sequence (116) of the marker oligonucleotide (114) is lower than the melting temperature of the hybridization complex of the barcode sequence (110) with the complementary barcode sequence (116).

7. The marker according to any one of the preceding claims, wherein the affinity reagent oligonucleotide (108) comprises a plurality of barcode sequences (110).

8. The marker according to claim 7, wherein two adjacent barcode sequences (110) of the affinity oligonucleotide (108) are separated by a cleavage site (124) configured to be selectively cleaved by a cleavage agent.

9. The marker according to any one of the preceding claims, wherein the marker (112) comprises at least one marking portion (120).

10. The marker according to claim 9, wherein the marking portion (120) is at least one of a fluorophore, a polyacetylene, and a metal ion.

11. The marker according to any one of the preceding claims, wherein the barcode sequence (110) comprises between 10 and 30 nucleotides.

12. A system having a plurality of markers according to any one of the preceding claims, wherein the barcode sequence (110) of each of the plurality of markers (100, 200) is different from the barcode sequence (110) of any other marker (100, 200).

13. A marker (112) for generating markers (100, 200), said marker comprising: A marker oligonucleotide (114) having a complementary barcode sequence (116), said complementary barcode sequence being at least partially complementary to the barcode sequence (110) of the affinity oligonucleotide (108) of the affinity reagent (104), and At least one blocking oligonucleotide (102) having a blocking sequence (118), said blocking sequence being partially complementary to the barcode sequence (116) of said marker oligonucleotide (114), The marker (112) is configured to be linked to the affinity reagent (104) by hybridization of the barcode sequence (110) of the affinity reagent oligonucleotide (108) with the complementary barcode sequence (116) of the marker oligonucleotide (114).

14. An affinity reagent (104) for generating markers (100, 200), said affinity reagent comprising: An affinity oligonucleotide (108) having a barcode sequence (110) that is at least partially complementary to the complementary barcode sequence (116) of the marker oligonucleotide (114) of the marker (112). At least one blocking oligonucleotide (202) having a blocking sequence (204), said blocking sequence (204) being partially complementary to the barcode sequence (116) of said affinity oligonucleotide (114), The affinity reagent (104) is configured to be linked to the marker (114) by hybridization of the barcode sequence (110) of the affinity reagent oligonucleotide (108) with the complementary barcode sequence (116) of the marker oligonucleotide (114).

15. A kit for analyzing biological samples, the kit comprising: According to claim 13, the marker (112) for generating markers (100, 200) and the affinity reagent (104), the affinity reagent having an affinity reagent oligonucleotide (108) having a barcode sequence (110), wherein the marker (112) is configured to form the markers (100, 200) if the barcode sequence (110) of the affinity reagent oligonucleotide (108) is hybridizable with the complementary barcode sequence (116) of the marker oligonucleotide (114), or According to claim 14, the affinity reagent (104) and the marker (112) for generating markers (100, 200) are configured to form the markers (100, 200) if the complementary barcode sequence (116) of the marker oligonucleotide (114) is capable of hybridizing with the barcode sequence (110) of the affinity reagent oligonucleotide (108).

16. A method for analyzing biological samples, the method comprising the following steps: The following substances were introduced into the biological sample: At least one marker (100, 200) according to any one of claims 1 to 11, or a system having multiple markers according to claim 12, or at least one marker (112) according to claim 13, and at least one affinity reagent (104), said at least one affinity reagent having an affinity reagent oligonucleotide (108), said affinity reagent oligonucleotide (108) having a barcode sequence (110), said barcode sequence being at least partially complementary to the complementary barcode sequence (116) of said marker oligonucleotide (114) of said marker (112), or At least one affinity reagent (104) according to claim 14 and at least one marker (112), said at least one marker having a marker oligonucleotide (114) having a complementary barcode sequence (116) that is at least partially complementary to the barcode sequence (110) of the affinity reagent oligonucleotide (108) of said affinity reagent (104); and The initial readout of the markers (100, 200) in the biological sample is generated.

17. The method of claim 16, wherein during the step of introducing the at least one marker (100, 200), the marker portion (104, 112) of the at least one marker (100, 200) is added separately.

18. The method according to claim 16 or 17, wherein after generating the initial readout, another marker (112) is introduced into the biological sample and another readout is generated.

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