Markers, markers and methods for analyzing biological samples

Abstract

A marker for analyzing a biological sample is provided. The marker includes a first marker moiety and a second marker moiety, the first marker moiety including a first nucleic acid strand (106), the second marker moiety including a second nucleic acid strand (110). The first nucleic acid strand (106) and the second nucleic acid strand (110) are configured to form a duplex. The marker further includes at least one first marker moiety (108, 710) and at least one second marker moiety (112), and the marker further includes at least one blocking nucleic acid strand (126, 128). In other aspects, markers (100, 500, 600, 700, 1000) including the marker and corresponding methods are provided.

Description

Technical Field

[0001] This invention relates to a marker for analyzing biological samples, said marker comprising at least one blocking nucleic acid strand. Alternatively, it provides a marker and method for analyzing biological samples. Background Technology

[0002] Labels and markers are frequently used in the analysis of biological samples, such as in fluorescence microscopy. Markers used in fluorescence microscopy typically consist of affinity reagents (such as antibodies) and a marker attached to the affinity reagent, enabling the marker to specifically (particularly via the affinity reagent) attach to a target analyte in the biological sample. This allows for the identification and / or localization of the target analyte within the biological sample.

[0003] The various applications of markers and biomarkers include multi-method approaches that require a large number of distinguishable markers and biomarkers, while biomarkers with bright markings are needed to detect analytes present in small quantities. In particular, when dealing with a large number of biomarkers (which can be assembled from individual components such as dyes and affinity reagents), it is important to reduce processing complexity while maintaining flexibility and reliability. Summary of the Invention

[0004] The purpose of this invention is to provide a marker and a biomarker to enable efficient processing when analyzing biological samples.

[0005] The above objectives are achieved through the subject matter of the independent claims. Advantageous embodiments are defined in the dependent claims and the following description.

[0006] In a first aspect, a marker for analyzing biological samples is provided. The marker includes a first marker portion and a second marker portion, the first marker portion having a first nucleic acid chain and the second marker portion having a second nucleic acid chain. The first and second nucleic acid chains are configured to form a double strand. The marker further includes at least one first marker portion and at least one second marker portion. The marker also includes at least one blocking nucleic acid chain, said at least one blocking nucleic acid chain being at least partially complementary to one of the first and second nucleic acid chains.

[0007] The blocking nucleic acid strand prevents unnecessary or uncontrolled hybridization between the first and second labeling portions. Accordingly, this enables efficient processing of the labeling portions, for example, in preparing biological samples for analysis, such as when analyzing the proximity between target analytes in biological samples using the label.

[0008] The label is preferably configured to be optically detectable at least when the first and second label portions are in close proximity. Specifically, the label portions are (covalently) attached to one of the first and second nucleic acid chains. For example, the first and second label portions can be a fluorescence resonance energy transfer (FRET) pair, which is optically detectable when they are in close proximity (particularly within 1 to 10 nm). This can be used for FRET-based proximity detection, where the FRET efficiency between different label portions of the marker can be measured by reading in the presence and absence of one or more blocking strands (i.e., before and after the removal of the blocking strands). FRET can be measured by reading changes in intensity and / or fluorescence lifetime (e.g., by confocal microscopy).

[0009] Specifically, the first nucleic acid chain may include a first nucleotide sequence that is complementary to a second nucleotide sequence of the second nucleic acid chain. Preferably, the first and second nucleotide sequences are at least partially complementary to each other, particularly based on Watson-Crick base pairing. Therefore, the first and second sequences can form a double-stranded structure. This enables stable hybridization between the first and second nucleotide sequences. The double-stranded structure can be a double helix, such as B-type DNA.

[0010] In one embodiment, the marker includes (first) a plurality of blocking nucleic acid strands, all of which are complementary to a corresponding nucleotide sequence of the first or second nucleic acid strand. This efficiently blocks hybridization between the first and second nucleic acid strands, thus preventing random or uncontrollable binding of the marker portions to each other. Each of the plurality of blocking nucleic acid strands of the marker can hybridize with a different portion of the first nucleic acid strand or with a different portion of the second nucleic acid strand.

[0011] Preferably, the blocking nucleic acid strands of the plurality of blocking nucleic acid strands are not connected to each other, but can be individual and / or separate nucleic acid molecules.

[0012] In one embodiment, each of the at least one blocking nucleic acid chain, particularly each of the plurality of blocking nucleic acid chains, comprises a plurality of nucleotides, the number of which is 5 to 15, preferably 5 to 12, and more preferably 6 to 9. For each of the blocking nucleic acid chains, this enables it to individually have a lower melting temperature or a reduced hybridization free energy (ΔG), thereby achieving lower hybridization stability. The nucleotides of each of the blocking nucleic acid chains can be linked together by phosphodiester bonds to form the corresponding nucleic acid chain.

[0013] Preferably, the melting temperature of the at least one blocking strand can be in the range of 28°C to 42°C, particularly between 28°C and 36°C. The at least one blocking strand may have a sequence that mismatches with its corresponding sequence on the first or second nucleic acid strand. Therefore, compared to the corresponding sequence on the first or second nucleic acid strand, the at least one blocking strand may have one or more mismatched nucleotides. This allows for modulation of the hybridization stability of the at least one blocking strand.

[0014] In one embodiment, the first nucleic acid chain and / or the second nucleic acid chain comprises, in particular, each comprising a plurality of nucleotides, the number of said nucleotides being 50 to 150.

[0015] In one embodiment, the at least one blocking nucleic acid strand is complementary to a portion of the first nucleic acid strand or a portion of the second nucleic acid strand (wherein a portion of the first nucleic acid strand and a portion of the second nucleic acid strand are complementary to each other), particularly to a portion (wherein this portion is complementary to the corresponding other of the first or second nucleic acid strand). This effectively blocks hybridization between the first and second nucleic acid strands.

[0016] In one embodiment, the marker includes a second plurality of blocking nucleic acid strands, wherein the blocking nucleic acid strands of the second plurality of blocking nucleic acid strands are complementary to the corresponding nucleotide sequences of the second nucleic acid strand. This enables the use of dedicated blocking nucleic acid strands to block both the first and second nucleic acid strands. Preferably, in this case, the blocking nucleic acid strands of the first plurality of blocking nucleic acid strands may be complementary to the corresponding nucleotide sequences of the first nucleic acid strand.

[0017] In one embodiment, the at least one first labeling portion and / or the at least one second labeling portion is optically detectable. This enables efficient detection of the label, for example, by a microscope, particularly a fluorescence microscope. For instance, the first labeling portion and / or the second labeling portion may comprise a fluorophore, such as a fluorescent protein, an organic or inorganic fluorescent molecule, or fluorescent nanoparticles.

[0018] In one embodiment, the at least one first marking portion and / or the at least one second marking portion have the same optical characteristics, or the at least one first marking portion and / or the at least one second marking portion have different optical characteristics. The optical characteristics may include, for example, excitation or emission wavelength, or fluorescence lifetime.

[0019] In one embodiment, the at least one first labeled portion and the at least one second labeled portion are configured for nonradiative energy transfer between them, particularly when they are in close proximity. This enables efficient determination of whether the labeled portions are in close proximity to each other. For example, the first labeled portion and the second labeled portion can be configured to form an FRET pair with each other, one being an FRET donor and the other an FRET acceptor. FRET between the first labeled portion and the second labeled portion can typically occur when the labeled portions hybridize with each other and are therefore in close proximity.

[0020] In one embodiment, both the at least one first labeling portion and the at least one second labeling portion are attached, preferably covalently attached, to the first or second nucleic acid chain. For example, they may be attached to the phosphate backbone of the respective nucleic acid. In particular, the labeling portions may be attached to opposite ends of the respective first or second nucleic acid chains. For example, the at least one first labeling portion may be attached toward the 3' end of the respective nucleic acid chain, and the second labeling portion may be attached toward the 5' end of the respective nucleic acid chain.

[0021] In one embodiment, the at least one first labeling portion is preferably covalently attached to the first nucleic acid chain, for example, to the phosphate backbone of the nucleic acid chain, and the at least one second labeling portion may (covalently) be attached to the second nucleic acid chain, for example, to the phosphate backbone of the nucleic acid chain. In this case, when the first and second nucleic acid chains hybridize with each other, the optical properties of the first and second labeling portions may similarly change due to their proximity.

[0022] In one embodiment, the first nucleic acid chain extends along a first direction, and a plurality of first labeling portions are disposed on the first nucleic acid chain along the first direction; and / or the second nucleic acid chain extends along a second direction, and a plurality of second labeling portions are disposed on the second nucleic acid chain along the second direction. This enables the generation of an optically detectable signal that is proportional to the distance between the first labeling portions and the second labeling portions. Specifically, the corresponding labeling portions are disposed sequentially along the respective directions.

[0023] In one embodiment, the marker includes a third marker portion comprising a third nucleic acid strand, wherein the third nucleic acid strand is configured to form a triplet with the first and second nucleic acid strands. Preferably, the marker portion is disposed on or attached only to the third nucleic acid strand, and not on the other nucleic acid strands.

[0024] In particular, where the marker includes the third marker portion, the marker may include a third blocking nucleic acid strand that is at least partially complementary to the third nucleic acid strand. The third nucleic acid strand and the third blocking nucleic acid strand may additionally incorporate features described for the first nucleic acid strand or the second nucleic acid strand, or the first blocking nucleic acid strand or the second blocking nucleic acid strand. For example, the third blocking nucleic acid strand may include a cleavable linker. Specifically, there may be multiple third blocking nucleic acid strands, wherein the third blocking nucleic acid strands of the multiple blocking nucleic acid strands are connected to each other by cleavable linkers, particularly to form a cleavable linear strand of the blocking nucleic acid strand. The connected third blocking nucleic acid strands of the multiple third blocking nucleic acid strands can form a third cleavable linear strand of the blocking nucleic acid strand. The cleavable linker can be selectively cleaved by applying a cleavage agent.

[0025] Specifically, providing the marker having the third nucleic acid strand requires the three nucleic acid strands to specifically hybridize with each other to generate the marker. In particular, when the marker portion is attached to the third nucleic acid strand, the marker can only be detected if the three nucleic acid strands have hybridized with each other and formed the triploid structure. Furthermore, the triploid structure provides a robust and rigid structure to which the marker portion can attach.

[0026] Specifically, the third nucleic acid strand may have a third nucleotide sequence. This third nucleotide sequence may be at least partially complementary to the first and second nucleotide sequences in the hybridization, particularly based on Hoogsteen base pairing, to form the triplet structure. Therefore, the third nucleotide sequence can hybridize with the duplex of the first and second nucleotide sequences, particularly with the major groove of the duplex. This enables a stable triplet structure.

[0027] In one embodiment, each marker portion is equidistant from any adjacent marker portion. This enables the generation of an optically detectable signal that is proportional to the distance between the first and second marker portions. When the marker comprises multiple marker portions, the spacing between each first marker portion and any adjacent first marker portion is substantially equal, and / or the spacing between each second marker portion and any adjacent second marker portion is substantially equal. Preferably, the substantially equal spacing can be in the range of 0.33 to 33 nm.

[0028] On the other hand, a biomarker for analyzing biological samples having multiple target analytes is provided. The biomarker includes a marker, specifically, as described above, the marker includes a first marker portion and a second marker portion. The biomarker further includes: a first marker portion having a first affinity reagent and the first marker portion; and a second marker portion having a second affinity reagent and the second marker portion. Each of the first affinity reagent and the second affinity reagent is configured to specifically bind to one of the target analytes of the biological sample. Optionally, the biomarker may include a third marker portion, the third marker portion including the third marker portion having the third nucleic acid chain.

[0029] The biomarker can efficiently determine the distance or proximity between two target analytes. The biomarker can also accurately determine the presence and / or location of two or a single target analyte in a biological sample. Furthermore, the at least one blocking nucleic acid strand can process the first and second biomarker portions without the risk of unwanted uncontrolled hybridization between the first and second nucleic acid strands, for example, when preparing to analyze a biological sample containing the biomarker.

[0030] The biological sample may be a cell sample, such as a tissue section. Other examples include biopsy samples, such as liquid biopsy samples or tissue biopsy samples. The target analyte may be, for example, a protein or nucleic acid of the biological sample. The affinity reagent for the marker may be an antibody, an antibody fragment, an amino acid-based or nucleic acid-based aptamer, or a linear nucleic acid. This enables the detection of a wide variety of target analytes. In particular, the nucleic acid strands of the first and second marker portions are attached to the corresponding affinity reagent, preferably covalently attached to the corresponding affinity reagent.

[0031] The affinity reagents of the markers can bind to a single target analyte or different target analytes. In a first case, the first affinity reagent can be configured to specifically bind to a first region or epitope of the single target analyte, and the second affinity reagent can be configured to specifically bind to a second region or epitope of the single target analyte. By requiring both markers to partially bind to the single target analyte, the presence and / or location of the single target analyte can be precisely determined, particularly with improved specificity. In a second case, the first affinity reagent can be configured to specifically bind to a first target analyte, and the second affinity reagent can be configured to specifically bind to a second target analyte. This enables the determination of the distance or proximity between the first and second target analytes.

[0032] In particular, when the biomarker includes the third biomarker portion and the biomarker portion is attached to the third nucleic acid strand, the biomarker is thus detectable only if the third biomarker portion is present and the triplet is formed. This enables improved specificity in the detection of the single target analyte. Similarly, the third biomarker portion enables improved specificity in determining the proximity of the first and second target analytes.

[0033] On the other hand, a method for analyzing biological samples is provided. The method includes introducing at least one biomarker (particularly a biomarker as described herein) into the biological sample. Optionally, a first optical readout of the biological sample having the biomarker can be generated, particularly prior to the step of removing the blocking nucleic acid chain from the biomarker. In another step, the at least one blocking nucleic acid chain is removed from the biomarker. In a subsequent step, another optical readout of the biological sample having the biomarker is generated. In a subsequent optional step, the first optical readout and the other optical readout are compared, particularly to determine changes in the optical properties of the biomarker. Specifically, a fold change between the first and other optical readouts, particularly a fold change in the fluorescence emission of the biomarker, can be calculated.

[0034] For example, the optical readout can be generated using a microscope, such as a fluorescence microscope. The optical readout can determine the presence and / or location of a target analyte or the proximity of two target analytes.

[0035] The step of introducing the at least one biomarker may optionally include waiting for a predetermined amount of time to allow the at least one biomarker to bind to one or more corresponding target analytes of the biological sample. After the introduction of the at least one biomarker, any biomarkers not bound to the target analytes may optionally be removed from the biological sample, for example, by washing. When the at least one biomarker is introduced, the biomarker comprises the blocking nucleic acid strand, preferably hybridizing with a first or second nucleic acid strand corresponding to a marker of the biomarker.

[0036] The step of introducing the at least one marker may include adding individual portions of the at least one marker to the sample, particularly individually over time. In this case, the marker is assembled in situ in the sample. Alternatively, the individual portions of the marker may be added to the sample together or simultaneously.

[0037] Following the introduction of the at least one marker, preferably after the at least one marker has bound to the corresponding target analyte of the biological sample, and particularly after any markers not bound to the target analyte have been removed from the biological sample (e.g., by washing), the first optical readout of the biological sample containing the marker can be generated. This enables the optical readout of the individual marker or label portion (e.g., the first and second markers or label portions) before proceeding to the next method step (i.e., removal of the blocking nucleic acid chain from the marker). While not always necessary, it is generally recommended to perform at least two optical readouts of the biological sample: one in which the first and / or second label portion is still hybridized with the blocking nucleic acid chain, and another after the blocking chain has been removed. This allows for the calculation of the fold change or proportion of fluorescence emission.

[0038] In embodiments of the method, during the step of introducing the marker, an excess portion of the blocking nucleic acid strand of the marker is introduced relative to the first and second nucleic acid strands. Specifically, the excess portion can be 5 to 1000 times higher than the molar concentration of the at least one or more blocking nucleic acid strands. Preferably, the excess portion can be 5 to 100 times higher than the molar concentration. More preferably, the excess portion can be 10 to 100 times higher than the molar concentration. For example, the first and second nucleic acid strands can be introduced at concentrations in the range of 1 nM to 100 nM, particularly 10 nM to 25 nM. In contrast, the at least one blocking nucleic acid strand can be introduced at a concentration in the range of 10 nM to 100 µM, particularly 10 nM to 1 µM. Therefore, multiple copies of the at least one blocking nucleic acid strand or multiple copies of each blocking nucleic acid strand can be introduced into the biological sample.

[0039] In certain embodiments, the at least one biomarker may first be provided at the concentration and / or in excess as described above. Specifically, the at least one biomarker may be stored under these conditions. This enables the first and second nucleic acid strands to prevent hybridization before being placed in the biological sample or when the biological sample is prepared for analysis using the biomarker.

[0040] Therefore, excessive introduction of the at least one blocking nucleic acid chain can prevent the first and second nucleic acid chains from replacing the blocking nucleic acid chain and hybridizing with each other.

[0041] The step of removing the at least one blocking nucleic acid chain may include washing the biological sample to dilute and / or wash away the excess blocking nucleic acid chain. Reducing the number of blocking nucleic acid chains allows the first and second nucleic acid chains to replace the blocking nucleic acid chain and hybridize with each other. Therefore, controlling the concentration of the blocking nucleic acid chain allows control over the point at which the first and second nucleic acid chains hybridize.

[0042] In another embodiment of the method, in the step of removing the blocking nucleic acid strands, the temperature of the sample is raised to a temperature in the range of 37°C to 42°C, particularly in the range of 40°C to 42°C. Raising the temperature of the sample to near or above the average melting temperature of the duplexes of the respective blocking nucleic acid strands can remove the blocking nucleic acid strands from the first or second nucleic acid strand.

[0043] Specifically, after removing the excess blocking nucleic acid strands, the individual blocking nucleic acid strands can be replaced by the first / second nucleic acid strand. Specifically, compared to the first and second nucleic acid strands used for hybridization, the individual blocking nucleic acid strands have a lower melting temperature or a reduced hybridization free energy (ΔG). The lower hybridization stability of the blocking nucleic acid strands allows them to replace the individual blocking nucleic acid strands from the first or second nucleic acid strand.

[0044] On another front, a kit for analyzing biological samples is provided. The kit includes a biomarker as described herein. Relative to the first and second nucleic acid chains, the kit also includes an excess portion of at least one blocking nucleic acid chain of the biomarker, particularly an excess portion of the plurality of blocking nucleic acid chains. Specifically, the excess portion may be 5 to 1000 times higher than the molar concentration of the at least one or the plurality of blocking nucleic acid chains. Preferably, the excess portion may be 5 to 100 times higher than the molar concentration. More preferably, the excess portion may be 10 to 100 times higher than the molar concentration. For example, the first and second nucleic acid chains may be introduced at concentrations in the range of 1 nM to 100 nM, particularly 10 nM to 25 nM. In contrast, the at least one blocking nucleic acid chain may be introduced at a concentration in the range of 10 nM to 100 µM, particularly 10 nM to 1 µM.

[0045] The method for analyzing the biological sample, the biomarker, and the kit described herein have the same advantages as the biomarker. Furthermore, the method, the biomarker, and the kit can be supplemented with features of the biomarker described herein, particularly the features in the dependent claims of the biomarker. Attached Figure Description

[0046] The specific implementation scheme will be described below with reference to the attached drawings, in which: Figure 1 This is a schematic diagram of a marker that includes markers for analyzing biological samples. Figure 1a It is a schematic diagram of a marker based on an alternative implementation scheme. Figure 2 It is based on Figure 1 and 1a A schematic diagram of the logo. Figure 3 It is based on Figure 1 and 1a A schematic diagram of the logo. Figure 4 It is based on Figure 1 and 1a A schematic diagram of the logo. Figure 5 It is a schematic diagram of the marker according to the second implementation scheme. Figure 6 It is a schematic diagram of the marker according to the third implementation plan. Figure 7 It is a schematic diagram of the marker according to the fourth implementation plan. Figure 8 It is based on Figure 7 A schematic diagram of the logo. Figure 9 A flowchart of a method for analyzing biological samples. Figure 10 This is a schematic diagram illustrating the use of biomarkers in flow cytometry. Figure 11 This is a schematic diagram illustrating the use of proximity hybridization assays (PHAs) for evaluating gene modifications. Figure 12 This is a schematic diagram illustrating the use of neighboring hybridization (PHA) analysis for quality control and monitoring in gene and cell therapy. Detailed Implementation

[0047] Figure 1 This is a schematic diagram of a biomarker 100 used for analyzing biological samples (not shown). Biomarker 100 includes a first biomarker portion 102 and a second biomarker portion 104. The first biomarker portion 102 may include a first label portion (having a first nucleic acid chain 106) and a plurality of first label portions 108. The first label portions 108 may (covalently) attach to the first nucleic acid chain 106.

[0048] The second marker portion 104 includes a second marker portion (having a second nucleic acid chain 110) and a plurality of second marker portions 112 (attached to the second nucleic acid chain 110). For example, the first nucleic acid chain 106 and the second nucleic acid chain 110 can be polynucleotides.

[0049] The first nucleic acid strand 106 and the second nucleic acid strand 110 are at least partially complementary to each other. This enables the first nucleic acid strand 106 and the second nucleic acid strand 110 to hybridize, particularly to form a double strand based on Watson-Crick base pairing.

[0050] The first biomarker portion 102 also includes a first affinity reagent 114 configured to specifically bind to the first target analyte 116. Similarly, the second biomarker portion 104 also includes a second affinity reagent 118 configured to specifically bind to the second target analyte 120.

[0051] The first labeled portion attaches to the first affinity reagent 114 via barcoded oligonucleotide 122. Similarly, the second labeled portion attaches to the second affinity reagent 118 via barcoded oligonucleotide 124. Each barcoded oligonucleotide 122, 124 (particularly its nucleotide sequence) is preferably specific to the corresponding affinity reagent 114, 118 and / or the corresponding labeled portion, particularly to the first and second nucleic acid chains 106, 110. In particular, the first and second affinity reagents 114, 118 are antibodies.

[0052] The first marker portion 102 binds to the first target analyte 116 via the first affinity reagent 114. Similarly, the second marker portion 104 binds to the second target analyte 120 via the second affinity reagent 118.

[0053] The marker 100 may also include a first plurality of blocking nucleic acid chains 126 and / or a second plurality of blocking nucleic acid chains 128.

[0054] Figure 1 and Figure 1a Two different configurations of marker 100 are shown. Figure 1 In the diagram, marker 100 includes the first multiple blocking nucleic acid strands 126. Figure 1a In this embodiment, marker 100 includes a first plurality of blocking nucleic acid strands 126 and a second plurality of blocking nucleic acid strands 128. The blocking nucleic acid strands 126 and 128 are configured to hybridize with one of the first nucleic acid strand 106 or the second nucleic acid strand 110.

[0055] The presence of one or both of the blocking nucleic acid strands 126 and 128, and their hybridization with the corresponding nucleic acid strands 106 and 110, means preventing the first nucleic acid strand 106 and the second nucleic acid strand 110 from directly hybridizing with each other. Therefore, when processing marker portions 102 and 104, for example when preparing to analyze biological samples, marker portions 102 and 104 do not hybridize with each other due to the presence of one or both of the blocking nucleic acid strands 126 and 128.

[0056] In cases where marker 100 includes only one of the first or second blocking nucleic acid chains 126, 128, for example, Figure 1 The diagram shows only the first blocking nucleic acid chain 126. These individual multiple blocking nucleic acid chains 126 and 128 can still achieve the above effect, namely, preventing the hybridization of the first nucleic acid chain 106 and the second nucleic acid chain 110.

[0057] Specifically, the marker 100 may include excess of a first plurality of blocking nucleic acid chains 126 and / or a second plurality of blocking nucleic acid chains 128. For example, excess portions 126a, 128a of the first plurality of blocking nucleic acid chains 126 and / or the second plurality of blocking nucleic acid chains 128 may be present. In a specific embodiment, the excess portion may be relative to the corresponding first or second nucleic acid chains 106, 110.

[0058] Specifically, the excess portions 126a, 128a of the first plurality of blocking nucleic acid chains 126 and / or the second plurality of blocking nucleic acid chains 128 can be 5 to 1000 times higher than the molar concentration of the plurality of blocking nucleic acid chains 126, 128, for example, relative to the molar concentration of the corresponding first or second nucleic acid chains 106, 110. Preferably, the excess portions can be 5 to 100 times higher than this molar concentration. More preferably, the excess portions can be 10 to 100 times higher than this molar concentration.

[0059] The excess portions 126a and 128a of the blocking nucleic acid strands 126 and 128 may include multiple copies of each of the blocking nucleic acid strands 126 and 128, wherein each blocking nucleic acid strand is complementary to the corresponding sequence along the first or second nucleic acid strand 106 and 110. Each nucleic acid strand in the excess portions 126a and 128a of the blocking nucleic acid strands 126 and 128 can dynamically hybridize and dissociate with the corresponding first or second nucleic acid strand 106 and 110. However, by providing the excess portions 126a and 128a of the blocking nucleic acid strands 126 and 128, this balance favors hybridization of the blocking nucleic acid strands 126 and 128 with the corresponding first or second nucleic acid strand 106 and 110.

[0060] exist Figure 1 and 1aIn an exemplary embodiment, target analytes 116 and 120 are in contact with each other, for example, due to mutual affinity between target analytes 116 and 120. This causes marker portions 102 and 104 to come into proximity once bound to target analytes 116 and 120. As described above, due to the blocking nucleic acid chains 126 and 128 (particularly the excess portions 126a and 128a of the corresponding blocking nucleic acid chains 126 and 128), the first nucleic acid chain 106 and the second nucleic acid chain 110 do not hybridize with each other despite their proximity. Therefore, marker 100, particularly its marker portions 102 and 104, can be easily processed during the analysis of biological samples containing target analytes 116 and 120. For example, if when marker 100 is introduced into a biological sample, marker portions 102 and 104 have not yet bound to target analytes 116 and 120, blocking nucleic acid chains 126 and 128 prevent unnecessary and uncontrolled hybridization of the first nucleic acid chain 106 and the second nucleic acid chain 110.

[0061] The first labeled portion 108 and the second labeled portion 112 can be fluorophores used as FRET donors or FRET acceptors, respectively. Therefore, each first labeled portion 108 can form an FRET pair with one second labeled portion 112. Because the first nucleic acid strand 106 and the second nucleic acid strand 110 are in... Figure 1 In the indicated state, no hybridization occurs, therefore the first nucleic acid strand 106 and the second nucleic acid strand 110 are not in close proximity to each other. Specifically, the first labeled portion 108 and the second labeled portion 112 are not within the distance that would allow FRET to occur. This distance is typically in the range of 1 to 10 nm. Therefore, the first labeled portion 108 and the second labeled portion 112 could be optically detectable (single) ordinary fluorescent dyes, but would not meet the characteristics of FRET.

[0062] In particular, Figure 1 and 1a A step in a method for analyzing biological samples is shown, according to which a marker 100 is introduced into the biological sample to be analyzed. Before the marker 100 is introduced into the biological sample and before affinity reagents 114, 118 have bound to their respective target analytes 116, 120, blocking nucleic acid strands 126, 128 prevent unwanted hybridization of the first and second nucleic acid strands 106, 110.

[0063] Figure 2This is a schematic diagram of marker 100, wherein blocking nucleic acid chains 126, 128 (particularly their respective excess portions 126a, 128a) have been removed from the corresponding first and second nucleic acid chains 106, 110. This can be achieved by first removing the excess portions 126a, 128a of the blocking nucleic acid chains 126, 128. For example, the excess portions 126a, 128a can be washed off the sample, wherein marker 100 (particularly its affinity reagents 116, 120) is bound to the sample.

[0064] Generally, hybridization between shorter nucleic acids and longer nucleic acids is less thermodynamically stable compared to hybridization between longer nucleic acids. Therefore, hybridization between each blocking nucleic acid strand 126, 128 and the first or second nucleic acid strand 106, 110 is less thermodynamically stable compared to hybridization between the first and second nucleic acid strands 106, 110. Preferably, the first and second nucleic acid strands 106, 110 are longer than each blocking nucleic acid strand 126, 128.

[0065] Preferably, each of the blocking nucleic acid strands 126, 128 has a plurality of nucleotides, the number of which is 5 to 15. The 5 to 30 blocking nucleic acid strands 126, 128 can hybridize with corresponding adjacent sequences of the first or second nucleic acid strands 106, 110. Therefore, the 5 to 30 blocking nucleic acid strands 126, 128 can hybridize with each of the first and second nucleic acid strands 106, 110, particularly with complementary portions of the first and second nucleic acid strands 106, 110. Thus, the excess portions 126a, 128a can include multiple copies of these 5 to 30 individual blocking nucleic acid strands.

[0066] Therefore, by removing the excess portions 126a and 128a of the blocking nucleic acid chains 126 and 128, the balance between hybridization and dissociation of the blocking nucleic acid chains 126 and 128 with the first or second nucleic acid chains 106 and 110 is altered, resulting in fewer blocking nucleic acid chains 126 and 128 hybridizing with the first or second nucleic acid chains 106 and 110. Conversely, the thermodynamically more stable hybridization between the first and second nucleic acid chains 106 and 110 can replace the blocking nucleic acid chains 126 and 128 from the first and second nucleic acid chains 106 and 110.

[0067] In addition, in order to further destabilize the double strands formed between each blocking nucleic acid strand 126, 128 and the corresponding nucleic acid strands 106, 110, the temperature of the marker can be increased, especially to near or above the average melting temperature of the double strands between each blocking nucleic acid strand 126, 128 and the corresponding nucleic acid strands 106, 110.

[0068] Figure 3 This is a schematic diagram showing that marker 100 has been removed from blocking nucleic acid chains 126 and 128, for example. Figure 2 As described. In the absence of blocking nucleic acid strands 126 and 128, the first and second nucleic acid strands 106 and 110 can freely hybridize with each other to form a double strand. Figure 3 The image shows the first and second nucleic acid chains 106 and 110 hybridizing with each other.

[0069] Hybridization of the first nucleic acid chain 106 with the second nucleic acid chain 110 brings the first labeled portion 108 and the second labeled portion 112 closer together, thereby allowing FRET to occur between the first labeled portion 108 and the second labeled portion 112. Therefore, the proximity of target analytes 116 and 120 can be determined, for example, by optical readout using a fluorescence microscope, through FRET between the first labeled portion 108 and the second labeled portion 112. Figure 3 In this context, FRET can occur between all the first marking portions 108 and the second marking portions 112.

[0070] In a method for analyzing biological samples, markers 100, particularly marker portions 102 and 104, can be initially introduced into the biological sample in the presence of blocking nucleic acid strands 126 and 128, such as... Figure 1 and 1a As shown. After a suitable period of time (for the biomarker portions 102 and 104 to bind), any biomarker portions 102 and 104 that remain unbound to the corresponding target analytes 116 and 120 can be removed, for example, by washing the biological sample. Similarly, excess portions 126a and 128a of the blocking nucleic acid chains 126 and 128 can be removed by washing the biological sample.

[0071] This correspondingly enables hybridization between the first nucleic acid strand 106 and the second nucleic acid strand 110, such as Figure 3 As shown. Subsequently, an optical readout of the biological sample with marker 100 can be generated. The optical readout can be an image, such as an image generated by a fluorescence microscope.

[0072] Optical readout can determine whether target analytes 116 and 120 are in close proximity to each other. In particular, by observing the corresponding changes in their optical properties caused by FRET, optical readout can be used to determine whether FRET has occurred between the first marker portion 108 and the second marker portion 112.

[0073] Figure 4 This is a schematic diagram of marker 100 with target analytes 116 and 120, and... Figures 1 to 3 In comparison, the target analytes 116 and 120 are more widely spaced. Figure 3Similarly, there are no blocking nucleic acid strands 126 and 128, and the first nucleic acid strand 106 and the second nucleic acid strand 110 can hybridize with each other. However, the greater distance between the target analytes 116 and 120 results in only partial hybridization between the first nucleic acid strand 106 and the second nucleic acid strand 110, especially with... Figure 3 Compared to the state shown (where target analytes 116 and 120 are in contact with each other), the degree of hybridization is lower.

[0074] Partial hybridization of the first nucleic acid strand 106 and the second nucleic acid strand 110 results in a greater distance between some of the labeled portions in the first labeled portion 108 and the second labeled portion 112. Specifically, the distance between these first labeled portions 108 and the second labeled portions 112 can prevent FRET from occurring between them. Therefore, the overall observable FRET efficiency is related to... Figure 3 The state shown is reduced compared to the previous state. This reduced FRET efficiency can be observed when generating optical readout of a biological sample with marker 100. Therefore, the first labeled portion 108 and the second labeled portion 112 that do not form FRET pairs (e.g., the labeled portions 108, 112 between the hybridization portions of barcode oligonucleotides 122, 124 and the first nucleic acid chain 106 and the second nucleic acid chain 110) may be optically detectable as (single) ordinary fluorescent dyes based on their fluorescent dye (spectral) emission characteristics; while the first labeled portion 108 and the second labeled portion 112 that form FRET pairs (e.g., the labeled portions 108, 112 between the hybridization portions of the first nucleic acid chain 106 and the second nucleic acid chain 110) can be detected based on the FRET characteristics of the FRET pairs, and therefore based on the different spectral characteristics of the FRET pairs compared to the spectral characteristics of individual fluorescent dyes. The ratio between two different spectral features (the spectral features of the FRET pair and the spectral features of a single fluorescent dye) can be used as a quantitative measure of the corresponding proximity of affinity reagents 114 and 118, and thus as a measure of the corresponding proximity of target analytes 116 and 120.

[0075] In short, and specifically refer to Figure 3 and Figure 4 It can be seen that the distance between target analytes 116 and 120 directly affects the degree of hybridization between the first nucleic acid chain 106 and the second nucleic acid chain 110, and therefore directly affects the overall FRET efficiency between the first labeled portion 108 and the second labeled portion 112. Thus, the FRET efficiency between the first labeled portion 108 and the second labeled portion 112 is proportional to the distance between target analytes 116 and 120, and can be used to determine the distance or proximity between target analytes 116 and 120.

[0076] Figure 5This is a schematic diagram of marker 500. Marker 500 includes a first marker portion 502 having an affinity reagent 503, which is a linear nucleic acid. Marker 500 also includes the second marker portion 104 described above. For simplicity, Figure 5 The marking portion of marker 500 is not shown. As mentioned above, marker 500 may also include blocking nucleic acid chains 126 and 128. Figure 5 The image shows marker 500, which does not have blocking effect on nucleic acid chains 126 and 128.

[0077] The first marker portion 502 specifically binds to the nucleic acid target analyte 504. The second marker portion 104 specifically binds to the second target analyte 120, which may be a protein. Target analytes 504 and 120 may interact with or bind to each other. As described with respect to marker 100, such interaction or proximity between target analytes 504 and 120 can be determined by marker 500 in the same manner. In particular, the proximity of the marker portions can be determined by FRET occurring between the corresponding labeled portions (not shown) of marker portions 502 and 104, which serves as a measure of the distance between target analytes 504 and 120.

[0078] Figure 6 This is a schematic diagram of marker 600. Marker 600 includes a first marker portion 502 and a second marker portion 602, which respectively have first and second affinity reagents 503 and 604, which are linear nucleic acids. For simplicity, Figure 6 The marking portion of marker 600 is not shown. As mentioned above, marker 600 may also include blocking nucleic acid chains 126 and 128. Figure 6 The image shows marker 600, which does not have blocking effect on nucleic acid chains 126 and 128.

[0079] Both affinity reagents 503 and 604 are configured to specifically bind to specific target sequences. Nucleic acid target analyte 504 includes these target sequences. The proximity between target sequences along target analyte 504 can be determined using marker 600, or the presence of target analyte 504 can be determined with high confidence due to the use of two affinity reagents (which need to bind to target analyte 504 simultaneously). Marker 600 can be detected as described above for markers 100 and 500. Specifically, the proximity of the corresponding labeled portions (not shown) of marker portions 502 and 602 can be determined by FRET occurring between the corresponding labeled portions (not shown) of marker portions 502 and 602, which serves as a measure of the distance between target sequences or as a measure of the presence of target analyte 504 in a biological sample.

[0080] Figure 7 and Figure 8 This is a schematic diagram of a marker 700 including a first marker portion 702, a second marker portion 704, and a third marker portion 706. The first marker portion 702 includes a first nucleic acid strand 106, and the second marker portion 704 includes a second nucleic acid strand 110. The third marker portion 706 includes a third nucleic acid strand 708, which is configured to form a triplet with the first and second nucleic acid strands 106 and 110, specifically based on Hoogsteen base pairing. Two marker portions 710 are attached to the third nucleic acid strand 708.

[0081] exist Figure 7 The diagram illustrates a marker 700 having a second plurality of blocking nucleic acid strands 128 and corresponding excess portions 128a, wherein these blocking nucleic acid strands hybridize with a second nucleic acid strand 110. This prevents hybridization between marker portions 702, 704, and 706. Specifically, double strands between the first and second nucleic acid strands 106 and 110, and triple strands between the third nucleic acid strand 708 and the first and second nucleic acid strands 106 and 110, cannot be formed.

[0082] exist Figure 8 The diagram shows marker 700, in which blocking nucleic acid chains 126 and 128 have been removed, for example by washing away blocking nucleic acid chains 126 and 128 and excess portion 128a. This enables the formation of a triplet among the first, second, and third nucleic acid chains 106, 110, and 708. Specifically, this associates the labeled portion 710 with affinity reagents 114 and 118, and thus with target analytes 116 and 120. In optical readout of the sample, this enables the determination of the presence and / or location of target analytes 116 and 120. Simultaneously, close proximity of target analytes 116 and 120 is required to enable the formation of a triplet. The labeled portion 710 may have the same or different optical properties.

[0083] Figure 9 This is a schematic flowchart of a method for analyzing biological samples. The method begins at step S900.

[0084] In step S902, at least one biomarker 100, 500, 600, 700 as described herein is introduced into the biological sample. Specifically, multiple biomarkers can be introduced into the sample, each biomarker including an affinity reagent configured to specifically bind to a different target analyte. In this step, blocking nucleic acid strands 126, 128 are bound to the corresponding nucleic acid strand of the biomarker portion. This enables the introduction of the biomarker portion into the biological sample without the risk of hybridization between the biomarker portions before they bind to their respective target analytes. Subsequently, step S902 may include removing biomarker portions that are not bound to any target analyte, for example, by washing the sample.

[0085] When a biomarker is introduced into a biological sample, the blocking nucleic acid chains 126 and 128 are preferably introduced as excess portions 126a and 128a of the blocking nucleic acid chains 126 and 128. In particular, the molar concentration of the excess portions 126a and 128a can be 5 to 1000 times higher than that of the first and second nucleic acid chains 102 and 110 of at least one biomarker introduced into the sample.

[0086] In optional step S904, a first optical readout of the sample with the marker is generated, for example, by microscopy. This is particularly important when marker 100 is introduced into the sample in step S902. The first optical readout can determine the fluorescence of the labeled portions 108 and 112 when the marker portions 102 and 104 are prevented from forming double strands due to the presence of blocking nucleic acid chains 126 and 128. Therefore, the labeled portions 108 and 112 retain their inherent optical properties without undergoing FRET.

[0087] In step S906, the blocking nucleic acid chains 126 and 128 are removed. In particular, this includes removing the corresponding excess portions 126a and 128a of the blocking nucleic acid chains from the biological sample. This can be achieved by washing the biological sample and thereby reducing or removing the excess portions 126a and 128a.

[0088] Step S906 may optionally include altering the conditions under which the sample is preserved to further destabilize the duplexes between the blocking nucleic acid strands 126, 128 and the corresponding nucleic acid strands 102, 110 of the marker moiety. These altered conditions may be physical or chemical, such as the temperature of the sample or the concentration of the buffer / salt used to preserve the sample. Specifically, the sample temperature may be increased to a range of 37°C to 42°C.

[0089] Removing the blocking nucleic acid strands allows the nucleic acid strands in the marker portion to form double strands.

[0090] In step S908, another optical readout of the sample with the markers is generated, for example, by a microscope. For example, when marker 100 is in close proximity to target analytes 116 and 120, marker portions 102 and 104 (specifically their corresponding nucleic acid chains 106 and 110) now hybridize with each other. This results in the formation of FRET pairs by the marker portions 108 and 112, which can be optically detected in the optical readout.

[0091] In step S910, the presence and / or location of the target analyte in the sample can be determined based on the optical signal of the marked portion present in at least one other optical readout. For example, in the case of marker 700, the optical signal of the marked portion 710 can be determined in another optical readout, and based on this, the presence and / or location of the target analytes 116 and 120 can be determined. Additionally, it can be determined that the target analytes 116 and 120 are close to each other.

[0092] Optionally, and in the case of marker 100, the first optical readout can be compared with another optical readout, and the differences in the optical properties of the marker portions 108, 112 can be used to determine the presence, location and / or proximity of the target analytes 116, 120.

[0093] The method ends at step S912.

[0094] Method steps S902 to S910 can be repeated iteratively, with each iteration using a different set of markers. In this case, the marked portion of the marker can preferably be removed after each iteration to remove the marked portion of the corresponding marker.

[0095] Figure 10 This is a schematic diagram illustrating the use of marker 1000 in flow cytometry. Marker 1000 includes marker portions 1002 and 1004, which bind to corresponding targets 1006 and 1008 present or expressed on the cell surface 1010. If both targets 1006 and 1008 are present on the cell surface 1010, both marker portions 1002 and 1004 bind to them, and marker 1000 generates an optically detectable signal 1012. If only one target 1008 is present on the cell surface 1010, only marker portion 1004 binds to it, and no optically detectable signal is generated. Therefore, prior to flow cytometry, cells can be contacted with marker 1000 to distinguish cells that have one or both targets 1006 and 1008.

[0096] Marker 1000 can also be used to determine whether two types of cells (each with target 1006 or target 1008) are closely adjacent, especially whether targets 1006 and 1008 are close to each other on the surface of the cells.

[0097] Figure 11 This is a schematic diagram of PHA analysis, used to assess the integration of gene cassettes for gene and cell therapy. These analyses are based on the use of oligonucleotides as affinity reagents and single-molecule fluorescent in situ hybridization (smFISH) technology. Figure 11 In the diagram, the first and second marker portions are schematically shown as semi-circular shapes. The corresponding markers may include blocking nucleic acid strands to prevent the first and second marker portions from hybridizing with each other.

[0098] Figure 11 The top row of the diagram shows the integration of the gene cassette (white arrow) into the target site next to the exon (black rectangle), with the rest of the genomic DNA represented by black lines. Figure 11 The bottom row of the top figure shows random or ectopic integration. PHA analysis allows for accurate differentiation of different types of integration because it can detect target site integration with only the two labeled portions 1100 and 1102, which are the markers, whereas in random integration, only the first labeled portion 1100 or the second labeled portion 1102 is present. After removal of the blocking strand, target site integration is detectable as a fluorescent spot, which is brighter than the fluorescent spot produced by ectopic integration when using dequenching-based PHA analysis, and / or has higher FRET efficiency or different fluorescence lifetimes when using FRET-based PHA analysis.

[0099] Figure 11 The bottom diagram illustrates a schematic of a PHA analysis used to assess genomic sequence deletions. For example, in cell lines, partial genomic deletions can be induced to correct gene defects. This can be achieved, for example, using CRISPR / Cas9 or similar systems and utilizing homologous recombination. For example, if patient-derived cells are modified to delete specific portions of the genome, it may be necessary to assess the appropriate frequency of deletion occurrence. This can be achieved using a PHA analysis schematically shown in the bottom diagram. Due to the deletion of the target site, the two probes of the first and second marker portions are brought close to each other, and they can be read as corresponding smFISH spots, which are brighter than spots at sites without deletions when using dequenching-based PHA analysis, and / or have higher FRET efficiency or different fluorescence lifetimes when using FRET-based PHA analysis.

[0100] Figure 12 yes Figure 11This is a schematic diagram of the hypothetical readout of the described analysis. Analysis is performed on various cell types 1200a, 1200b, 1200c, 1200d, and 1200e, which may be derived, for example, from blood samples, to monitor the evolution of the cell therapy product in a patient. Such monitoring can be performed to assess how different clones within the cell therapy product evolve in the patient during cell therapy. Such clones may not include genetic modifications (e.g., no insertion / integration or deletion), they may include only the desired target site modification, they may include only ectopic integration and / or deletion, and they may include a mixture of target site and ectopic modifications. Cells are stained and readouted before and after removal of the blocking chain, the spots are segmented, and the before-and-after images are registered to identify spots that change after removal of the blocking chain; changes in the spots represent a paired detection event.

[0101] As used herein, the term “and / or” includes any and all combinations of one or more of the associated listed items and may be abbreviated as “ / ”.

[0102] Although some aspects have been described in the context of the device, these aspects clearly 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, items, or features of the corresponding device.

[0103] Figure Labels

Claims

1. A marker for analyzing biological samples, comprising: The first marker portion includes a first nucleic acid chain (106), and The second marker portion includes a second nucleic acid strand (110), and The first nucleic acid strand (106) and the second nucleic acid strand (110) are configured to form a double strand. The marker further includes at least one first marking portion (108, 710) and at least one second marking portion (112), and The marker further includes at least one blocking nucleic acid strand (126, 128) that is at least partially complementary to one of the first nucleic acid strand (106) and the second nucleic acid strand (110).

2. The marker according to claim 1, comprising a plurality of blocking nucleic acid strands (126, 128). All of the plurality of blocking nucleic acid chains (126, 128) are complementary to the corresponding nucleotide sequence of the first nucleic acid chain (106) or the second nucleic acid chain (110).

3. The marker according to claim 1 or 2, wherein each of the at least one blocking nucleic acid strand (126, 128) comprises a plurality of nucleotides, the number of said nucleotides being in the range of 5 to 15, preferably 5 to 12, more preferably 6 to 9.

4. The marker according to any one of the preceding claims, wherein the first nucleic acid chain (106) and / or the second nucleic acid chain (110) comprises a plurality of nucleotides, the number of said nucleotides being in the range of 50 to 150.

5. The marker according to any one of the preceding claims, comprising a second plurality of blocking nucleic acid chains (128), wherein the blocking nucleic acid chain (128) of the second plurality of blocking nucleic acid chains is complementary to the corresponding nucleotide sequence of the second nucleic acid chain (110).

6. The marker according to any one of the preceding claims, wherein the at least one blocking nucleic acid chain (126, 128) is complementary to a portion of the first nucleic acid chain (106) or a portion of the second nucleic acid chain (110), wherein a portion of the first nucleic acid chain (106) and a portion of the second nucleic acid chain (110) are complementary to each other.

7. The marker according to any one of the preceding claims, wherein the at least one first marking portion (108, 710) and / or the at least one second marking portion (112) is optically detectable.

8. The marker according to any one of the preceding claims, wherein the at least one first marking portion (108, 710) and / or the at least one second marking portion (112) have the same optical characteristics, or The at least one first marking portion (108, 710) and / or the at least one second marking portion (112) have different optical characteristics.

9. The marker according to any one of the preceding claims, wherein the at least one first marker portion (108, 710) and the at least one second marker portion (112) are configured for nonradiative energy transfer between them.

10. The marker according to any one of the preceding claims, wherein the at least one first marker portion (108, 710) and the at least one second marker portion (112) are both attached to the first nucleic acid chain (106) or the second nucleic acid chain (110).

11. The marker according to any one of claims 1 to 9, wherein the at least one first marker portion (108, 710) is attached to the first nucleic acid chain (110).

12. The marker according to any one of the preceding claims, wherein the first nucleic acid chain (106) extends along a first direction, and a plurality of the first marker portions (108, 710) are disposed on the first nucleic acid chain (106) along the first direction, and / or The second nucleic acid chain (110) extends along a second direction, and a plurality of the second label portions (112) are arranged on the second nucleic acid chain (110) along the second direction.

13. The marker according to any one of the preceding claims, comprising a third marker portion, the third marker portion comprising a third nucleic acid strand (708), wherein the third nucleic acid strand (708) is configured to form a triplet with the first nucleic acid strand (106) and the second nucleic acid strand (110).

14. The marker according to any one of the preceding claims, wherein the spacing between each marker portion (108, 112, 710) and any adjacent marker portion (108, 112, 710) is equal.

15. A biomarker (100, 500, 600, 700, 1000) for analyzing biological samples having multiple target analytes (116, 120), comprising: The marker according to any one of the preceding claims comprises a first marker portion and a second marker portion. The first marker portion (102, 502, 702, 1002, 1100) includes a first affinity reagent (114, 503) and the first marker portion, and The second marker portion (104, 602, 704, 1004, 1102) includes a second affinity reagent (118, 604) and the second marker portion, and The first affinity reagent (114, 503) and the second affinity reagent (118, 604) are both configured to specifically bind to one of the target analytes (116, 120, 504, 1006) of the biological sample.

16. A method for analyzing biological samples, comprising the following steps: Introducing at least one biomarker (100, 500, 600, 700, 1000) according to claim 15 into the biological sample, Optionally, a first optical readout of the biological sample having the aforementioned markers (100, 500, 600, 700, 1000) is generated. Remove blocking nucleic acid chains (126, 128) from the markers (100, 500, 600, 700, 1000), and Generate optical readout or another optical readout of the biological sample having the aforementioned markers (100, 500, 600, 700, 1000), and Optionally, the multiple change between the first and second optical readouts is calculated, or the first optical readout is compared with the other optical readout.

17. The method of claim 16, wherein, In the step of introducing the marker, an excess portion (126a, 128a) of the blocking nucleic acid chain (126, 128) of the marker (100, 500, 600, 700, 1000) is introduced relative to the first nucleic acid chain (106) and the second nucleic acid chain (110).

18. The method according to claim 16 or 17, wherein, In the step of removing the blocking nucleic acid chains (126, 128), the temperature of the sample is increased to a temperature in the range of 37°C to 42°C.

19. A kit for analyzing biological samples comprising biomarkers (100, 500, 600, 700, 1000) according to claim 15, wherein the kit comprises an excess portion (126a, 128a) of a blocking nucleic acid chain (126, 128) of the biomarker (100, 500, 600, 700, 1000) relative to a first nucleic acid chain (106) and a second nucleic acid chain (110).

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