Method and system for analyzing biological samples

Abstract

A method for analyzing a biological sample (200, 308) having at least one target analyte (208) is provided. The method comprises the steps of: adding at least a first marker (202) and a second marker (204) to a biological sample (200, 308) wherein the first marker (202) is configured to specifically bind to a first portion of at least one target analyte (208) and the second marker (204) is configured to specifically bind to a second portion of the at least one target analyte (208); and determining the presence of the target analyte (208) in the biological sample (200, 308) based on detecting the first marker (202) and the second marker (204) in the biological sample (200, 308). In a further aspect, a system (300) configured to perform the method is provided.

Description

Technical Field

[0001] The present invention relates to a method and system for analyzing a biological sample having a target analyte using a first marker and a second marker. Background Art

[0002] A variety of methods for labeling and imaging biological samples are known in the art. Generally, these methods rely on markers comprising affinity reagents, such as labeling to identify antibodies for target analytes in biological samples. This method inherently relies on affinity reagents that have high affinity only for the corresponding target analytes. However, affinity reagents are usually not the case, so there are markers that show at least moderate cross-reactivity to multiple different so-called off-targets. Therefore, the fact that most antibodies show cross-reactivity is a significant challenge in life science research, which produces false positive results, increases costs, and has uncertainty in data interpretation.

[0003] Especially in the case of high throughput and high complexity assays, where a large number of samples are analyzed with a large number of markers, any cross-reactivity may have a large impact on the reliability and reproducibility of these assays. For example, antibodies, especially monoclonal antibodies produced by hybridomas, can regularly have cross-reactivity with several target analytes. The availability of commercial antibodies has increased rapidly in recent years. Antibodypedia.com lists more than 11,000 antibodies against epidermal growth factor receptor (EGFR) from 65 suppliers. Although efforts have been made to improve antibody validation protocols, it is clear that the scientific community has difficulty keeping up with the rapid growth in the availability of commercial antibodies.

[0004] Multi- and high-complexity proteomics are becoming increasingly important in the context of liquid biopsies, which can be derived from blood samples, saliva, lymph or fluid, as well as in the context of spatial proteomics of three-dimensional samples such as tissue sections and organoids. For example, the human blood proteome retains information about physiological and pathophysiological processes in the donor. Therefore, high-complexity plasma proteomics is of considerable interest for stratification of patient populations, early disease detection and prevention. However, the analysis of blood proteins is complicated by the fact that high-abundance plasma proteins, such as serum albumin and globulins, are significantly more frequent than low-abundance proteins, such as e.g. thyroid stimulating hormone, which may be of particular interest. The dynamic range can be as high as 10 12 .

[0005] Similarly, in the context of space biology samples (typically tissue sections), the greatest interest may be related to the discovery of particularly important rare cells.

[0006] For both liquid and solid biological samples, non-imaging based methods exist to analyze the samples. However, imaging based readout is usually time consuming. Summary of the invention

[0007] An object of the present invention is to provide a method and system for reliably and efficiently analyzing a biological sample having a target analyte.

[0008] The above objects are achieved by the subject-matter of the independent claims. Advantageous embodiments are defined in the dependent claims and in the following description.

[0009] A method for analyzing a biological sample having at least one target analyte is provided. The method comprises the steps of: a) adding at least a first marker and a second marker to the biological sample, wherein the first marker is configured to specifically bind to a first portion of the at least one target analyte, and the second marker is configured to specifically bind to a second portion of the at least one target analyte; and b) determining the presence of the target analyte in the biological sample based on detecting the first marker and the second marker in the biological sample.

[0010] By using at least two markers, each marker being specific for a different portion of the target analyte, the method is able to determine the presence of the target analyte in a biological sample with high specificity. In particular, this is able to significantly reduce the risk of misidentifying the target analyte due to cross-reactivity of the markers to other target analytes.

[0011] For example, the biological sample can be a tissue section. Alternatively, the biological sample can be a liquid sample, such as a liquid biopsy, particularly its solid component. The target analyte can be a proteomics component, i.e., a protein of the biological sample, or a genomic component, i.e., a nucleic acid (DNA, RNA) sequence, particularly a sequence folded into a structure. The first part and the second part of the target analyte can also be referred to as different epitopes of the target analyte. Based on, for example, detecting the first marker and the second marker in the biological sample with a microscope to determine the presence of the target analyte in the biological sample, it can preferably include determining the position of the target analyte in the biological sample.

[0012] In the sense of the present invention, "label" is preferably used to denote both a single molecule used as a label and a collection of the same molecules used as labels. A "label" in the sense of the present invention is a combination of affinity reagents that are configured to attach to a predetermined structure (also called target molecule or analyte and / or "reporter"), which may also be referred to as a "detectable label".

[0013] In the sense of this article, the analyte can be at least one of the secondary structures of proteins, peptides, neurotransmitters, hormones, small molecules, nanostructures and nucleic acids. More specifically, the nucleic acid sequence extension that can be combined with the oligonucleotide sequence by hybridization is not an analyte in the sense of this article. Similarly, the affinity reagent in the sense of this article is at least one of the following: antibodies, antibody fragments, nano antibodies, aptamers, wherein aptamers can also refer to modified aptamers modified by changing the main chain or modifying the nuclear base, such as SOMAmer and aptabody. In the sense of this article, aptamers can be used as affinity reagents of markers used in combination with the method to mark the nucleic acid extensions forming the secondary structure, as long as the interaction between the affinity reagent and the target is not mediated by the hybridization of the complementary nucleic acid sequence, but by the interaction mediation of the paratope-epitope type. Therefore, the marker can preferably include such an affinity reagent: the affinity reagent is a nucleic acid aptamer with a complex secondary and / or tertiary structure, and the nucleic acid aptamer is configured to specifically bind to the target analyte due to its complex secondary and / or tertiary structure rather than complementary base pairing.

[0014] "Aptamer": In the sense of the present invention, "aptamer" preferably refers to a small affinity agent, which is usually based on nucleic acids - naturally occurring RNA, DNA or artificial XNA, but in some cases can also be based on a peptide backbone. In the sense of the present invention, "aptamer" also refers to derivatives of aptamers, such as SOMAmers or aptabodies, which are modified by modifications commonly found on proteins (such as, for example, glycosylation).

[0015] "Sample": In the sense of the present invention, "sample" preferably refers to a biological sample, which can also be named as a biological specimen, including, for example, blood, serum, plasma, tissue, body fluid (e.g., lymph, saliva, semen, tissue fluid, cerebrospinal fluid), feces, solid biopsy, liquid biopsy, explant, model organism embryo (e.g., zebrafish, fruit fly), whole model organism (e.g., zebrafish larvae, fruit fly embryo, Caenorhabditis elegans), cell (e.g., prokaryotes, eukaryotes, archea), multicellular model organism (e.g., Volvox), suspension cell culture, monolayer cell culture, 3D cell culture (e.g., spheroids, tumoroids, organoids derived from various organs such as intestine, brain, heart, liver, etc.), lysate of any of the above, virus. In the sense of the present invention, "sample" further refers to the volume surrounding the biological sample. For example, in an assay, the extracellular environment surrounding the cell up to a certain assay-dependent distance at the location where secreted proteins such as growth factors, extracellular matrix components are being studied is also referred to as a "sample". In particular, the affinity reagent brought into this surrounding volume is referred to as being "introduced into the sample" in the sense of the present invention.

[0016] In the step of adding the marker to the biological sample (step a)), the marker can, in particular, be introduced into the biological sample. For example, this can comprise permeabilizing the biological sample.

[0017] Preferably, the marker is detected optically based on the light emitted by the marker. Therefore, the marker may include an optically detectable label, which may include a fluorescent dye. This enables easy detection of the marker. When one of the markers binds to the target analyte, the light emitted by the marker can be detected, attributed to the target analyte and the presence of the target analyte is determined.

[0018] Preferably, before the step of adding the marker, the biological sample is fixed in place. In particular, the components of the biological sample (including the target analyte) are fixed in their positions relative to each other. This makes it possible to effectively determine the position of the target analyte in the biological sample. Specifically, when the biological sample is a liquid sample, being fixed in place can include cross-linking the solid components of the biological sample to each other, embedding the solid components in a hydrogel, or connecting the solid components to a surface. Such a surface can be the surface of a microfluidic device, and the liquid sample flows through the surface so that the sample is fixed or deposited in place.

[0019] In a preferred embodiment, the first marker and the second marker are added to the biological sample simultaneously or successively. This enables the method to be performed flexibly. When the markers are added successively, they are added to the biological sample at separate or different times. When the markers are added simultaneously, they are added at the same time.

[0020] Specifically, in the case where the marker is added to the biological sample successively, preferably, the first marker is removed before adding the second marker. This enables the biological sample to be effectively cyclically stained with a variety of markers to determine the presence of the target analyte. When one of the markers is removed, the entire marker can be removed, or only the optically detectable label of the marker can be removed. The removal of the marker causes the target analyte to no longer associate with at least the optically detectable label, so that it is no longer possible to detect the presence of the target analyte based on the removed marker. The marker can be based on oligonucleotides or peptides and removed by adding a degradation agent (such as a nuclease or protease). Preferably, the degradation agent is specific to a specific marker. Preferably, the marker is removed by degrading the marker with a degradation agent. The degradation agent may include at least one of the following: deoxyribonuclease (DNase), ribonuclease (RNase), endonuclease / exonuclease. Therefore, preferably, the marker is removed by degrading the marker with a degradation agent, and the degradation agent may include at least one of the following: deoxyribonuclease, ribonuclease, endonuclease / exonuclease.

[0021] Preferably, at least one optical readout of the biological sample with the first marker and / or the second marker is generated. This enables the location of the target analyte within the image and within the biological sample to be determined. In particular, the optical readout is generated before the presence of the target analyte is determined. The presence of the target analyte is then based on the generated optical readout. The optical readout is preferably generated over time. The optical readout may include several discrete images or, for example, a continuous stream of images over time.

[0022] The method can preferably be carried out in a cyclic manner, i.e., through multiple cycles of staining, imaging, and blanking, wherein the blanking can be carried out by inactivating the label, for example, by label removal, bleaching or quenching, or by removing the marker (marker removal). In the case of cyclic operation, at least one optical readout of a single time point can be collected in each cycle, or alternatively, an optical readout time series can be collected in each cycle. The former allows multi-complexity or high-complexity proteomics using monochrome labels or combined encodings. The latter allows the same thing, but in addition also enables (A) markers to be located with higher spatial accuracy, and (B) kinetic information can be derived, because the binding of the marker to the target is monitored over time. In this way, for each target, multiple binding events can be monitored, and kinetic information can be derived. As previously mentioned, each target can be bound by at least two and at most n affinity reagents, which increases the specificity of the assay.

[0023] Preferably, the presence of the target analyte in the biological sample is determined based on the detection of the first marker and the second marker over time (e.g., in an optical readout form). For example, when the target analyte is combined with the corresponding marker, the light emitted by the marker is detected over time. This can be achieved in a cyclic manner by obtaining a readout trajectory during multiple rounds of staining, imaging, and marker removal. This enables the presence of the target analyte to be determined with increased specificity and certainty.

[0024] Particularly preferably, the presence of the target analyte in the biological sample is determined based on the frequency and / or duration of the light emitted over time by the first marker and the second marker. This enables the presence of the target analyte to be determined with increased specificity and certainty. Specifically, the presence of the target analyte in the biological sample is determined when the frequency and / or duration of the light emitted by the marker exceeds a predetermined threshold. For example, a marker that binds to the target analyte only briefly or only with low affinity may also bind to the target analyte only for a short time.

[0025] Preferably, the association of the first and second markers with the target analyte (on-rate, k ON) and dissociation (off-rate, k OFF ), and the presence of the target analyte in the biological sample is determined based on the equilibrium constant. Specifically, the determination of the equilibrium constant can be based on the frequency and / or duration of light emitted by the marker over time. Therefore, by determining the kinetics of the binding of the marker to the target analyte, weak off-target binding or non-specific interactions between the marker and the off-target can be identified as background binding or cross-reactivity and distinguished from the binding of the actual target analyte. For example, when the binding constant between a specific marker in the marker and the target analyte is higher than a specific threshold, the presence of the target analyte can be determined.

[0026] Preferably, the environmental conditions where the biological sample is located are set based on at least one physicochemical parameter of the first marker and / or the second marker. The physicochemical parameter may include the pKa or pI of the corresponding marker. The environmental conditions may include the type or concentration of the buffer where the biological sample is located, or the temperature of the biological sample. Specifically, the environmental conditions may be set so that the first marker and / or the second marker bind to the target analyte with high affinity. This makes it possible to determine the presence of the target analyte with high specificity. The environmental conditions may further or alternatively include the concentration of a competitor (such as a polyanion competitor) to prevent weak binding or non-specific binding of the marker to the components of the biological sample.

[0027] Preferably, the environmental conditions are set so that the first marker and / or the second marker only transiently bind to the target analyte. This enables the presence of the target analyte to be determined with high specificity and efficiency, particularly when the first marker and the second marker are added to the biological sample at the same time.

[0028] The methods disclosed herein can therefore utilize affinity reagents that show only moderate affinity and only moderate specificity for their targets. Since the target analyte is identified by combining n affinity reagents with 1 target analyte, i.e., n:1 detection, the method provides an exponential increase in specificity when a higher number of affinity reagents are used to detect the target analyte. This is true even when each affinity reagent does show cross-reactivity, as long as the n affinity reagents used to detect the 1 target analyte do not show exactly the same cross-reactivity. Although most affinity reagents, such as antibodies, nanobodies, and aptamers are cross-reactive, it is generally expected that their cross-reactivity is substantially different, i.e., to substantially different off-target groups.

[0029] Preferably, the first marker and the second marker each comprise an affinity reagent configured to specifically bind to a corresponding portion of the target analyte, and the affinity reagent is an aptamer or an antibody fragment, such as a single domain antibody or a nanobody. This enables the method to be flexibly adapted to different target analytes. Preferably, the first marker is one of the aptamer or the antibody fragment, and the second marker is another of the aptamer or the antibody fragment. This enables the presence of the target analyte to be determined with particularly high specificity.

[0030] Preferably, the first marker and the second marker each include an optically detectable label, such as a fluorophore, and the optically detectable labels of the first marker and the second marker can be optically distinguished from each other. Among other things, this enables the association and dissociation of the marker and the target analyte to be effectively determined. Specifically, the optical properties (such as emission wavelength, excitation wavelength and / or fluorescence lifetime) of the labels of the first marker and the second marker can be different.

[0031] The optically detectable label can, for example, include an oligonucleotide or DNA nanostructure backbone, and can be a single color label or a dye combination (combination label) including a single dye. The optically detectable label can be conjugated directly or indirectly to the barcode sequence of the affinity reagent by hybridization. The backbone of the label and / or aptamer can also be an artificial nucleic acid (XNA).

[0032] Preferably, at least another marker is added to the biological sample. Specifically, another marker can be configured to specifically bind to the target analyte, preferably to another part of the target analyte. This makes it possible to increase the specificity of determining the presence of the target analyte. Another marker can be added after the first marker and the second marker. The use of aptamers is particularly preferred because they can be directly generated to have high specificity for a specific target analyte, for example, by the SELEX method. In an alternative, another marker is configured to specifically bind to another target analyte, so as to determine the presence of the other target analyte in the biological sample. Therefore, the biological sample can be repeatedly stained with at least three different markers to determine the presence of the target analyte. Additional markers can be used to determine the presence of another target analyte.

[0033] The use of aptamers is particularly preferred because their backbones are typically nucleic acids and therefore exhibit different sensitivities to nucleases (e.g., DNase I, RNase I, endonucleases / exonucleases) compared to protein analytes. This allows for particularly gentle cyclic labeling utilizing the removal of markers that substantially degrade aptamers, as previously described, particularly in the appropriate methods for analyzing biological samples described in European patent application No. 23178065.1, the entire contents of which are incorporated herein by reference. The use of aptamers is particularly preferred because, compared to antibodies, aptamers can be selected, particularly designed for selected epitopes, in which case the immune system of the animal producing the antibody randomly selects the epitope. This is important when affinity reagents are generated for specific post-translational modifications to be generated (such as phosphorylation, ubiquitination, acetylation, methylation, glycosylation, isoprenylation, sulfation, disulfide bonds, proteolytic cleavage, and sumoylation).

[0034] Preferably, the optical readout is generated by a microscope, in particular an optical microscope, a fluorescence microscope, a confocal scanning microscope. This enables efficient generation of images of the biological sample and determination of the location of the target analyte in the biological sample.

[0035] In another aspect, a system configured to perform the above method is provided. The system may include a microscope for generating an optical readout. In addition, the system may include a microfluidic device, specifically, a microfluidic device for analyzing a liquid sample. The microfluidic device may include a surface for immobilizing a solid component of the liquid sample. The surface may be observed with a microscope to generate an optical readout of the sample immobilized on the surface. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Specific embodiments are described below with reference to the accompanying drawings, in which:

[0037] Figure 1 is a flow chart of a method for analyzing a biological sample having at least one target analyte,

[0038] Figure 2 is a schematic diagram of a biological sample with markers over time, and

[0039] Figure 3 is configured to execute according to Figure 1 A flowchart of the method is a schematic diagram of the system,

[0040] Figure 4 is a schematic diagram showing a cyclic process, each cycle having a time point acquisition or a time series (continuous) acquisition, and

[0041] Figure 5is a diagram in which the execution can be based on Figure 4 Schematic diagram of different modes of the process. DETAILED DESCRIPTION

[0042] Figure 1 Flow chart of a method for analyzing a biological sample having at least one target analyte. The biological sample can be, for example, a tissue section or a liquid sample, such as a liquid biopsy. The target analyte can be a specific protein or nucleic acid of the biological sample, including mRNA or genomic DNA.

[0043] The method starts with step S100. In step S102, a biological sample with a target analyte is provided. When the biological sample is a tissue section, the biological sample can be provided on a sample holder, such as a slide or a microscope slide, a well plate or a microfluidic chip. In addition, the tissue section can be amplified. In particular, when the biological sample is a liquid sample, the solid components of the liquid sample (such as proteins and nucleic acids, and including the target analyte) can be fixed to, for example, a surface. The surface can be a functionalized slide, so that the solid components can be attached. Additionally or alternatively, the solid components can be cross-linked to each other, or embedded in a hydrogel. The fixing of the components of the biological sample makes them fixed in position relative to each other. The fixing of the solid components can be carried out in a microfluidic device, and the liquid sample flows through the microfluidic device, and the microfluidic device provides an attachment surface or a fixed space where a fixed sample can be visually observed. The biological sample can also be amplified at this stage or at a later stage of the method.

[0044] In step S104, at least a first marker and a second marker are added to the biological sample. The first marker and the second marker are specific to the target analyte of the biological sample. Specifically, the first marker and the second marker each contain an affinity reagent, such as an aptamer or an antibody fragment, which specifically binds to a portion of the target analyte. Therefore, the marker includes an affinity reagent with a high affinity for the corresponding portion of the target analyte. Further, the first marker specifically binds to the first portion of the target analyte. In contrast, the second marker specifically binds to the second portion of the target analyte; the first portion is different from the second portion.

[0045] For example, in the case where the target analyte is a protein, the first portion may be a first epitope to which an antibody fragment of a first marker specifically binds. In this case, the second portion may be a second epitope to which an antibody fragment of a second marker specifically binds.

[0046] Thus, adding the first and second markers in step S104 causes the markers to bind to the target analyte.The biological sample may clearly contain a large number of copies of the target analyte, and similarly, a large number of copies of the first and second markers may be added to the biological sample.

[0047] The first and second markers also each include an optically detectable label, such as a fluorophore. Preferably, the optical property of the optically detectable label of the first marker is different from the optical property of the optically detectable label of the second marker. Therefore, the first marker and the second marker are optically distinguishable. The optical property may include an excitation wavelength, an emission wavelength, or a fluorescence lifetime.

[0048] Optionally, step S104 may include introducing a marker into the biological sample. For example, when the biological sample is a tissue section, the biological sample may be permeabilized to facilitate the introduction of the marker into cells of the biological sample.

[0049] In step S106, an optical readout, in particular an image, of the biological sample with the target analyte and the marker is generated. When the marker is bound to the target analyte, the optically detectable label of the marker is associated with the target analyte, and the target analyte can be visually detected as a point light source. When the marker is not bound to the target analyte, the marker may not be detected or only detected as diffuse non-point-like background noise.

[0050] Preferably, the optical readout is generated over time. Thus, the optical readout may be a sequence of images of the biological sample generated at predetermined time intervals. Alternatively, the optical readout may be a continuous image or stream of images. The optical readout may be generated by a microscope configured to illuminate the biological sample with at least the excitation light. This is particularly useful in Figure 4 and Figure 5 Further details are given in .

[0051] In step S108, the presence of the target analyte in the biological sample is determined based on detecting the first and second markers in the biological sample. Specifically, the presence of the target analyte can be determined based on the light emitted by the first and second markers associated with the target analyte.

[0052] Where the optical readout is an image of a biological sample, detection of a label associated with a target analyte may enable the location of the target analyte to be determined based on the location of light emitted by the label associated with the target analyte within the biological sample.

[0053] The use of a first marker in combination with a second marker results in a higher confidence that the presence of the target analyte is correctly determined. For example, in the case where both markers have cross-reactivity to other components of the biological sample, the presence of the target analyte can only be determined if both markers are associated with the target analyte.

[0054] In the case of generating an optical readout over time, the duration and / or frequency of the association of the first and second markers with the target analyte can be determined. For example, in the case where the marker has cross-reactivity to other components of the biological sample, or in the case where the marker has only a general affinity to the target analyte, the marker can only be briefly bound to the target analyte. In this case, the presence of the target analyte can still be reliably determined by determining the duration and / or frequency of the association of the first and second markers with each other at the target analyte. Therefore, the position where the first and second markers are detected together with an increased duration and / or frequency in the biological sample can be determined as the position of the target analyte. A predetermined threshold value of duration and / or frequency can be set to determine the position of the target analyte in the biological sample.

[0055] Further, based on the duration and / or frequency of the light emitted from the first and second markers, the equilibrium constants of association and dissociation of the first and second markers with the target analyte, particularly the binding constant, can be determined. Based on these determined constants, the presence or position of the target analyte can be determined. For example, in the case where both markers have a high binding coefficient for a compound at a position in a biological sample, the compound can be determined as the target analyte. A predetermined threshold value of the binding coefficient can be set to determine the position of the target analyte in the biological sample.

[0056] In an alternative example of the method, in step S104 after step S102, only the first marker is added to the biological sample. Then in step S106 an optical readout of the biological sample is generated using only the first marker. In step S110, a second marker is added similarly to that described for step S104, and in step S112, a second optical readout is generated similarly to that described for step S106. In step S108, the two optical readouts can then be used to determine the presence of the target analyte based on the first marker and the second marker, similar to that described above for step S108. By separating the addition of the markers in steps S104 and S110, the number of separate markers present in the optical readout is reduced at the same time. This is advantageous in the case where multiple different target analytes are labeled simultaneously with markers in steps S104 and S110, or in the case where the target analyte is present in a very large number of copies in the biological sample.

[0057] The amount of marker present in the biological sample can also be reduced by removing the first marker before step S110. This can be achieved by adding a degradation agent such as a protein or a portion thereof that digests the marker. Removing the marker at least results in the tag of the marker no longer being associated with the corresponding target analyte.

[0058] Optionally, the environmental conditions in which the biological sample is located can be adjusted or set based on the physicochemical parameters of the first and / or second markers. The affinity of the affinity reagent can be affected by the environmental conditions in which it is located. For example, the affinity of the antibody can be affected by the type or concentration of the buffer and the temperature. In another example, when setting the pH in which the biological sample is located, the pI of the affinity reagent of the first marker can be taken into account. This ensures that the biological sample is under conditions in which the marker is at least temporarily bound to the corresponding target analyte. Setting the environmental conditions can be part of steps S102, S104 and / or S110.

[0059] The method may be repeated iteratively after step S108, starting again in step S102, using a further label specific for at least a further target analyte. Prior to adding the further label, the first and second labels are preferably removed.

[0060] Alternatively, additional labels may be added together with the first and second labels. In this case, the additional labels may be specific for additional parts of the target analyte or for additional analytes.

[0061] In a further alternative, the additional label may be added after the second label is added and the optical readout is generated in steps S110 and S112. The optical readout is generated with the additional label before step S108. In this case, the additional label is preferably specific for an additional portion of the target analyte.

[0062] The method ends in step S114.

[0063] Figure 2 is a schematic diagram of a biological sample 200 with markers 202, 204 over time. The biological sample 200, in particular its solid components, is fixed on a surface 206. Arrow P1 is the time line. Therefore, Figure 2 A series of optical readouts of a biological sample 200 over time is shown. A first marker 202 and a second marker 204 are configured to attach with high affinity to a respective first portion and a second portion of a target analyte 208 of the biological sample 200. In three of the four views of the time series, the markers 202, 204 are attached to the target analyte 208. Figure 2In the second figure from the top, the second marker 204 is attached to another component 210 of the biological sample. The second marker 204 may exhibit a low affinity to the component 210 and thus bind to the component 210 briefly. However, by observing the entire time series, the presence and position of the target analyte 208 can be determined based on the duration of the first and second markers 202, 204 being located at the position of the target analyte 208 within the biological sample 200. Even if only the second figure from the top is observed, the difference in the position of the first and second markers 202, 204 will indicate that the presence and position of the target analyte 208 is not and cannot be clearly determined from this view alone. In this context, the position of the term marker 202, 204 can be understood in particular as the maximum distance between the position of the first marker 202 and the position of the second marker 204 in the biological sample 200. The distance can depend on the characteristics and maximum spatial extension of the target analyte 208 and can be set to a predetermined threshold of, for example, 500nm. Thus, by using two labels 202, 204, the presence and location of the target analyte 208 can be robustly determined. Depending on the mode in which the method is used, the labeling of the analyte can be essentially stable during the observation time, or the labeling can be dynamic, i.e., the label can repeatedly associate and dissociate during the monitoring of the sample. In the latter case, this effectively results in "blinking" of the target, which allows localization with super-resolution until single molecules can be imaged and the binding of the label can be assigned to a single target molecule.

[0064] Figure 3 300 is a schematic diagram of a system 300 configured to perform a method for analyzing a biological sample having a target analyte. The system 300 includes a microfluidic device 302 and a microscope 304. The microfluidic device includes a plurality of flow channels 306 for accommodating a biological sample having a target analyte. For example, a liquid sample 308 can flow into the flow channel 306 via an inlet 310. Then, for example, the liquid sample 308 can be fixed to a surface of the flow channel 306. Labels or other reagents can also flow into the flow channel 306 via the inlet 310 so that they are added to the fixed sample 308. The fixed sample 308 can then be observed and an optical readout generated in the flow channel 306 by the microscope 304.

[0065] Figure 4 Schematically depicted are different ways in which the method can be performed. For example, the method can be used with a cyclic staining, imaging, withering process, where the withering can be achieved by, for example, marker removal. During the "imaging" step (optical readout) of the process, discrete images or single time points can be recorded, thereby generating a time series, where time point n corresponds to cycle n, and time point n+1 corresponds to cycle n+1 (exemplarily shown in Figure 4 on the left side of the ).

[0066] Alternatively, each "imaging" step may comprise a time sequence consisting of a plurality of time points (exemplarily shown in Figure 5 ). In this case, the time series n with time points eh (e to h are time point identifiers) corresponds to cycle n, and the time series n+1 with time points mp (m to p are time point identifiers) corresponds to cycle n+1. The latter mode of operation allows the introduction of a marker set that binds to one target analyte in a "staining" step, and then monitoring the association and dissociation of said markers to said marker set over time, allowing not only combinatorial encoding, but also the derivation of kinetic information, such as association / dissociation rates and equilibrium constants.

[0067] This is Figure 5 This is further schematically illustrated in Figure 5 In the example of , the target analyte 508 (pentagon with a dotted pattern) is assigned to a marker set comprising three markers 502, 504, 506. The first marker 502 includes a first aptamer 522 (affinity reagent) for a first epitope and a first tag 542. The second marker 504 includes a first aptamer 522 for a first epitope and a second tag 544, i.e., the same specificity but different tags. The third marker 506 includes a second aptamer 524 for a second epitope and a third tag 546. This effectively allows (A) encoding the recognition of the analyte 508 in a combination code, which can be a combination or arrangement, and (B) detecting the recognition of the analyte 508 with two different aptamers 522, 524, improving the specificity of the assay. Using this configuration of the assay, the method can now be performed so that all markers 502, 504, 506 are introduced in the "staining" step, and then a time series is recorded to continuously monitor the binding and dissociation of the markers to the target. This is advantageous if kinetic information about the binding of labels 502, 504, 506 is sought to distinguish on-target from off-target binding, or when the kinetic information is thus used as an additional carrier to encode information about the identification of the interaction. Figure 5 The top portion shows the time series monitoring of binding for this case.

[0068] exist Figure 5 The bottom of the figure shows a cyclic method, in which each marker 502, 504, 506 in the marker set is introduced independently of each other in three separate cycles (each cycle can still contain many markers, albeit from different sets, i.e. for different target analytes). In this case, the first marker 502 is introduced in the first round, the second marker 504 is introduced in the second round, and the third marker 506 is introduced in the third round. The readout is performed in each round during the "imaging" step or optical readout. The markers are removed before the next round.

[0069] In all drawings, the same or similarly acting elements are indicated by the same reference numerals. 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 " / ".

[0070] Although some aspects have been described in the context of an apparatus, it is clear that these aspects also represent a description of a corresponding method, where a block or device corresponds to a method step or a feature of a method step. Similarly, aspects described in the context of a method step also represent a description of a corresponding block or item or a feature of a corresponding apparatus. Reference numerals 200 biological samples 202, 502 First marker 204, 504 Second marker 506 Third Marker 206 Surface 208, 508 Target analytes 210 Compounds from biological samples 300 Systems for analyzing biological samples 302 Microfluidic Devices 304 Microscope 306 Runner 308 Liquid Samples 310 Liquid Inlet 522, 524 Affinity reagent, aptamer 542, 544, 546 Label, fluorescent dye.

Claims

1. A method for analyzing a biological sample (200, 308) having at least one target analyte (208), the method comprising the following steps: a) adding at least a first marker (202) and a second marker (204) to the biological sample (200, 308), wherein the first marker (202) is configured to specifically bind to a first portion of the at least one target analyte (208), and the second marker (204) is configured to specifically bind to a second portion of the at least one target analyte (208), and b) determining the presence of the target analyte (208) in the biological sample (200, 308) based on detecting the first marker (202) and the second marker (204) in the biological sample (200, 308).

2. The method of claim 1, wherein prior to step a), the biological sample (200, 308) is fixed in place.

3. The method according to any of the preceding claims, wherein the first marker (202) and the second marker (204) are added to the biological sample (200, 308) simultaneously or successively.

4. The method according to any of the preceding claims, wherein the first marker (202) is removed before adding the second marker (204).

5. A method according to any of the preceding claims, wherein at least one optical readout of the biological sample (200, 308) having the first marker (202) and / or the second marker (204) is generated.

6. The method of any of the preceding claims, wherein the presence of the target analyte (208) in the biological sample (200, 308) is determined based on detecting the first marker (202) and the second marker (204) over time.

7. A method according to any of the preceding claims, wherein the presence of the target analyte (208) in the biological sample (200, 308) is determined based on the frequency and / or duration of light emitted by the first marker (202) and the second marker (204) over time.

8. The method according to claim 7, wherein the equilibrium constant of the association and dissociation of the first marker (202) and the second marker (204) with the target analyte (208) is determined based on the frequency and / or duration of light emitted by the first marker (202) and the second marker (204), and the presence of the target analyte (208) in the biological sample (200, 308) is determined based on the equilibrium constant.

9. A method according to any one of the preceding claims, wherein the environmental conditions of the biological sample (200, 308) are set based on at least one physicochemical parameter of the first marker (202) and / or the second marker (204).

10. The method of claim 9, wherein the environmental conditions are configured such that the first marker (202) and / or the second marker (204) only transiently binds to the target analyte (208).

11. A method according to any of the preceding claims, wherein the first marker (202) and the second marker (204) each comprise an affinity reagent configured to specifically bind to a corresponding portion of the target analyte (208), and the affinity reagent is an aptamer or an antibody fragment.

12. A method according to any of the preceding claims, wherein the first marker (202) and the second marker (204) each comprise an optically detectable label, and the optically detectable labels of the first marker (202) and the second marker (204) are optically distinguishable from each other.

13. The method according to any of the preceding claims, wherein at least a further marker is added to the biological sample (200, 308).

14. The method according to any of the preceding claims, wherein the optical readout is generated by a microscope (304).

15. A system (300) configured to perform the method according to any one of the preceding claims.

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