Method for analyzing biological samples

Abstract

There is provided a method of analyzing a biological sample comprising the steps of: a) providing a biological sample having a plurality of target analytes; b) introducing into the biological sample at least a first label, the first label comprising a first affinity reagent and a first optically detectable label bound to the first affinity reagent, where the first affinity reagent is configured to specifically bind to one of the target analytes and configured to be degraded by the degrading agent; c) generating a first optical readout of the biological sample having the first marker; d) degrading the at least first affinity reagent with a degrading agent; e) introducing into the biological sample at least a second label, the second label comprising a second affinity reagent and a second optically detectable label bound to the second affinity reagent, where the second affinity reagent is configured to specifically bind to one of the target analytes and configured to be degraded by the degrading agent; f) generating a second optical readout of the biological sample having a second marker.

Description

Technical Field

[0001] The present invention relates to a method for analyzing biological samples by staining. Background Art

[0002] In the art, it is known to analyze biological samples (such as tissue sections) to determine the presence or spatial distribution of target analytes in biological samples, tissue sections, environmental soil or water samples or liquid biopsies. This is usually dependent on staining with optically detectable (especially fluorescence), specific markers for the corresponding analytes. These markers usually include affinity reagents and optically detectable fluorescent dyes that are specifically attached to the target analyte, and the fluorescent dyes are either directly conjugated with the affinity reagent or attached to the affinity reagent by a second affinity reagent. Generally, antibodies are used as affinity reagents in these applications because there are a large number of antibodies with different specificities. Meanwhile, there are only a limited number of distinguishable optically detectable dyes. This requires that these samples be repeatedly stained, especially when the biological sample contains a large amount of target analytes. For example, fluorescence microscopy and more specifically multiple fluorescence microscopy or space biology applications (wherein a large amount or a very large amount of analytes to be analyzed, such as up to 50,000 analytes) usually rely on a cyclic staining process. In each cycle, different analytes can be marked.

[0003] The shortcoming that often encounters in these cyclic dyeing methods is, for example, to avoid background noise, often need to remove a circular marker or label from sample before next dyeing cycle.Especially, often be difficult to remove affinity reagents such as antibodies from sample.The other shortcoming of using antibody comprises cross reaction with multiple target analytes.This is particularly a problem for the application of detecting a large amount of different analytes.In addition, large affinity reagents, such as antibodies are usually difficult to penetrate, and are equally difficult to remove from sample, such as tissue sections. Summary of the invention

[0004] An object is to provide a method for analyzing a biological sample, which method is capable of efficiently analyzing the biological sample by iterative staining.

[0005] The aforementioned objects are achieved by the subject-matter of the independent claims. Advantageous embodiments are defined in the dependent claims and in the following detailed description.

[0006] A method for analyzing a biological sample, in particular by iterative staining, is provided, the method comprising the following steps: a) providing a biological sample having a plurality of target analytes; b) introducing at least a first marker into the biological sample, the first marker comprising a first affinity reagent and a first optically detectable label bound to the first affinity reagent, wherein the first affinity reagent is configured to specifically bind to one of the target analytes and is configured to be degraded by a degrading agent; c) generating a first optical readout of the biological sample having the first marker; d) applying a degrading agent to degrade at least the first affinity reagent; e) introducing at least a second marker into the biological sample, the second marker comprising a second affinity reagent and a second optically detectable label bound to the second affinity reagent, wherein the second affinity reagent is configured to specifically bind to one of the target analytes and is configured to be degraded by the degrading agent; f) generating a second optical readout of the biological sample having the second marker.

[0007] The degradation agent causes degradation of the affinity agent, in particular, the degradation agent can digest, split or unfold the affinity agent. Such degradation generally leads to loss of function of the affinity agent, in particular loss of function of the corresponding label. Preferably, the degradation of the affinity agent is irreversible.

[0008] In particular, the readout can be produced by an optical readout device (such as a microscope). For example, the biological sample can be imaged by an optical readout device to produce an image of the biological sample with the corresponding marker. In particular, after the excitation light configured to visualize the corresponding label is directed to the biological sample, the readout can be produced by detecting light at least originating from the corresponding label. Preferably, the method continues by iteratively repeating steps d), e) and f), in particular, the marker used can be changed with each iteration so as to produce an optical readout of the biological sample with different markers, thereby visualizing different target analytes. Target analytes may include proteins, oligonucleotides, hormones, metabolites, vitamins, lipids, metals and neurotransmitters.

[0009] The first affinity reagent and the second affinity reagent can be configured to be degraded by the same degradation agent or by different types of degradation agents (specifically). For example, the first affinity reagent can be configured to be degraded by a chemical degradation agent (such as hydrogen peroxide), while the second affinity reagent can be configured to be degraded by a biochemical degradation agent (such as an enzyme) or a physical degradation agent (such as UV light). The first and second affinity reagents can each be configured to be degraded only by a specific type of degradation agent.

[0010] The biological sample is in particular a tissue section, a cell culture sample, an organoid, a cell co-culture or a liquid biopsy sample which has been immobilized, for example on a solid surface or in a 3-dimensional matrix.

[0011] Preferably, the first and second affinity reagents and the corresponding degradation agents are selected so that the degradation agents substantially only or specifically degrade the first and second affinity reagents, and the degradation agents substantially do not degrade the target analyte. For example, in the case where the target analyte is based on a protein, the affinity reagent can be an oligonucleotide configured to be degraded by a degradation agent (such as an endonuclease, which does not degrade a protein-based target analyte). In another example, the target analyte can be an oligonucleotide of a biological sample, and the corresponding affinity reagent can be based on an oligonucleotide and include a portion configured to be degraded by a degradation agent. For example, the portion configured to be degraded can be a UV-cleavable (non-natural) nucleotide, which has a particularly high susceptibility to degradation by a degradation agent (e.g., UV light) relative to the target analyte.

[0012] Preferably, in step d), after adding the degradation agent, the biological sample is washed in order to remove the first marker, in particular part of the first marker, from the biological sample. This makes it possible to reduce interference or noise caused by the first marker, in particular the corresponding label, in the subsequent optical readout. In addition, the degradation agent can be washed off.

[0013] Preferably, at least the first affinity reagent and the second affinity reagent comprise a nucleic acid backbone. This enables the affinity reagent to be easily degraded by a degradation agent. In particular, the nucleic acid backbone can be an oligonucleotide, for example, comprising deoxyribonucleic acid, ribonucleic acid and / or a heterogeneous nucleic acid. Compared with the remaining affinity reagents, the heterogeneous nucleic acid may increase its susceptibility to the degradation agent.

[0014] In the alternative, the first affinity reagent or the second affinity reagent may comprise a nucleic acid backbone.

[0015] In another preferred embodiment, the affinity reagent may comprise a nucleic acid backbone, particularly nucleotides comprising phosphorothioate bonds, which render the backbone resistant to nucleases but sensitive to silver or mercury ions, which may then act as degradation agents.

[0016] Preferably, at least the first affinity agent and the second affinity agent are aptamers. The use of aptamers enables targeted degradation with degradation agents, especially when the target analyte is based on protein. In addition, this allows the marker to have a particularly high specificity for the target analyte, especially because aptamers with specificity for different epitopes of the target analyte can be produced. Another advantage of using aptamers is that they can be reliably and easily produced with very low variability. In particular, aptamers comprise oligonucleotides. Aptamers can, for example, be produced by the SELEX method. In particular, aptamers are essentially composed of natural and / or non-natural nucleotides.

[0017] Preferably, at least the first affinity agent and the second affinity agent have a maximum spatial extent in the range of 1 nm to 25 nm, more preferably in the range of 1 nm to 10 nm. Alternatively, the affinity agent is preferably in the range of 5 kDa to 200 kDa, more preferably 5 kDa to 70 kDa, even more preferably 5 kDa to 30 kDa. This enables particularly small markers to penetrate further into the biological sample, especially compared to antibody-based markers. For example, the affinity agent can be an aptamer.

[0018] Generally, it is preferred that the target analyte has a different chemical nature from the marker (particularly the corresponding affinity reagent). For example, the target analyte may consist essentially of nucleotides, while the marker, particularly the corresponding affinity reagent, may consist essentially of amino acids, or vice versa. This enables the affinity reagent to be easily targeted for degradation by a degradation agent. It is particularly preferred that the target analyte is a protein.

[0019] Preferably, the degradation agent includes at least one of a chemical degradation agent, a biological degradation agent, and a physical degradation agent. For example, a chemical degradation agent may be hydrogen peroxide; a biological degradation agent may be an enzyme, such as a DNA enzyme or an RNA enzyme, and a physical degradation agent may be UV light or temperature. The temperature may include raising the temperature at which the biological sample is located to a temperature above the melting temperature of the affinity reagent. Another example of a degradation agent is a competitive oligonucleotide, which is configured to cause the unfolding of the affinity reagent. This makes the method generally applicable and enables targeted degradation of the affinity reagent by the degradation agent.

[0020] Preferably, in step a), the marker introduced in step b) and / or e) is selected or determined based on the chemical properties of the target analyte in the biological sample. For example, a marker can be selected whose affinity reagent is configured to be degraded by a specific degradation agent in step d), which does not degrade the target analyte. This enables the affinity reagent to be targeted for degradation by the degradation agent.

[0021] In a particularly preferred embodiment, an aptamer comprising a nucleic acid-based backbone is used, and the degradation agent comprises at least one of the following: a DNA enzyme, an RNA enzyme, or a restriction endonuclease. This allows for rapid degradation of the aptamer with essentially no or very little effect on the protein target analyte, which comprises a polypeptide backbone that is not sensitive to any of the aforementioned enzymes. This is also true for target analytes such as metabolites, hormones, vitamins, trace elements, heavy metals, which may also be of great interest when studying biological samples.

[0022] Preferably, steps b) and / or e) include applying an electric field to the biological sample. This enables the marker to effectively penetrate into the biological sample. In particular, a pulsed electric field may be applied. The application of an electric field to the biological sample may also assist in removing the marker from the sample.

[0023] Preferably, the first affinity reagent and the second affinity reagent are specific for the same target analyte, and wherein the first tag and the second tag have different optical properties. This enables analysis of biological samples with high specificity. The first affinity reagent and the second affinity reagent may be different and have specificity for different epitopes of the same target analyte. Alternatively, the first affinity reagent and the second affinity reagent may be the same and have specificity for the same epitope of the same target analyte. The optical properties may be distinguishable in an optical readout and include emission wavelength, excitation wavelength or fluorescence lifetime.

[0024] Preferably, in step g), in particular after step f), the same target analyte is identified in the first optical readout and the second optical readout based on determining the first label and the second label in the first optical readout and the second optical readout. This enables efficient identification of the target analyte.

[0025] Preferably, the first affinity reagent and the second affinity reagent are specific for different target analytes, and wherein the first tag and the second tag have the same optical properties. This enables identification of a large number of target analytes.

[0026] Preferably, in step b), a third marker is introduced, the third marker comprising a third optically detectable label and a third affinity reagent specific for one of the target analytes. This enables identification of a large number of different target analytes in a biological sample. In particular, the third affinity reagent is specific for a target analyte that is different from the target analyte that the first affinity reagent has specificity for. In particular, the third label may have optical properties different from the first label, and the third affinity reagent is configured to be degraded by a degradation agent. Additional markers may be introduced, preferably, in steps b) and / or e), 50 to 1000 markers specific for different target analytes are introduced.

[0027] Preferably, at least the first label and the second label comprise a nucleic acid backbone configured to be degraded by a degradation agent. This enables effective degradation of the marker. For example, the nucleic acid backbone can be an oligonucleotide, wherein the optically detectable portion of the label is bound to the oligonucleotide, and the oligonucleotide backbone is bound to a corresponding affinity reagent. The nucleic acid backbone of the label is preferably configured to be degraded by a corresponding degradation agent of the affinity reagent. Alternatively, the first label or the second label may comprise a nucleic acid backbone.

[0028] Preferably, the nucleic acid backbone of each tag comprises a unique sequence that is configured to bind to the complementary sequence of the corresponding affinity reagent. This enables efficient and targeted assembly of the markers.

[0029] Preferably, each tag comprises at least one optically detectable moiety, such as a fluorophore, preferably, each tag comprises 1 to 200 optically detectable moieties. This enables the production of bright markers and / or the production of a set of different optically distinguishable markers.

[0030] the term

[0031] In the context of this article, the terms are used in the following manner:

[0032] "Sample" or "biological sample": In the sense of the present invention, "sample" refers to a biological sample, which can also be called 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, whole embryo (e.g., zebrafish, fruit fly), whole model organism (e.g., zebrafish larvae, fruit fly embryo, Caenorhabditis elegans), cell (e.g., prokaryotes, eukaryotes, archaea), multicellular organism (e.g., Volvox), suspension cell culture, monolayer cell culture, 3D cell culture (e.g., spheroids, tumor-like bodies, organoids derived from various organs such as intestine, brain, heart, liver, etc.), lysate of any of the foregoing substances, virus. In the sense of the present invention, "sample" further refers to the volume surrounding the biological sample. For example, in the determination of secreted proteins such as growth factors and extracellular matrix components, the extracellular environment surrounding the cell that reaches a certain determination-dependent distance is also called a "sample". In particular, affinity reagents that are brought into this surrounding volume are referred to as being "introduced into the sample" in the sense of the present invention.

[0033] "Affinity reagent": In the sense of the present invention, the term "affinity reagent" can be, in particular, an antibody, a single domain antibody (also called nanobody), a combination of at least two single domain antibodies, an aptamer, an oligonucleotide, a morpholino, a PNA complementary to a predetermined RNA, a DNA target sequence, a ligand (e.g., a drug or drug-like molecule) or a toxin, such as phalloidin (a toxin that binds to actin filaments). In the sense of the present invention, an affinity reagent is configured to bind to a target molecule or analyte with a certain affinity and specificity, so that it can be said that the affinity reagent is substantially specific for the target molecule or the predetermined target structure. In the sense of the present invention, "a plurality of affinity reagents" (S2) contains affinity reagents (a1, a2, a3, ... an) that are configured to specifically bind to a predetermined target structure within a biological sample or to a predetermined chemical compound or to a predetermined chemical element or to an analyte. At least some of the affinity reagents from the plurality of affinity reagents (A) are "introduced into the sample" so that the affinity reagent can attach to the corresponding predetermined target structure in the sample. In the context and meaning of this document, and as described above, "introduced into a sample" may mean physically introduced into the sample volume or into the volume surrounding the sample and distributed to the sample. Examples of the latter case may be, for example, determinations of secreted molecules that are best assessed in the extracellular space where they may be located outside the sample but in or near a specific spatial context of the sample.

[0034] "Apptomer": In the sense of this article, "aptamer" refers to a small affinity reagent, 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 this article, "aptamer" also refers to a derivative of an aptamer, such as SOMAmer or aptabody, which is modified by modifications (such as glycosylation) commonly found on proteins. An aptamer is usually a short nucleic acid sequence folded into a 3-dimensional structure that can bind to a target or analyte with high affinity and specificity. Aptamers exhibit KD in the range of pM to μM, and are therefore viable alternatives to much larger antibodies. The residence time of an aptamer on its target varies from a few seconds to a few hours and is determined by the dissociation rate kOFF. Aptamers are usually selected from an oligonucleotide library in an iterative process called SELEX. Therefore, the discovery and development of aptamers is entirely an in vitro process, in contrast to the discovery and production of antibodies. For this reason, aptamers can be developed faster and at a lower cost than antibodies. Similarly, the production of aptamers is cheap and results in lower batch-to-batch variability. Aptamers are typically 20 to 80 nucleotides in length, corresponding to about 6 kDa–30 kDa, and thus can be about 10 times smaller than conventional antibodies, which puts them in a similar size range to nanobodies, which are antibody fragments derived from llama, alpaca, or shark antibodies. For example, in tissue staining for microscopy, a small size is desirable because antibodies are severely limited in penetrating tissues due to their large size.

[0035] "Predetermined target structure" or "target" or "analyte": In the sense of the present invention, "predetermined target structure" refers to a target molecule or target structure or analyte, which can be, for example, a protein (e.g. a certain protein), an RNA sequence (e.g. mRNA of a certain gene), a peptide (e.g. somatostatin), a DNA sequence (e.g. a locus or element), a metabolite (e.g. lactate), a hormone (e.g. estradiol), a neurotransmitter (e.g. dopamine), a vitamin (e.g. cobalamin), a micronutrient (e.g. biotin), a metal ion (e.g. metal and heavy metal ions, such as Cd(II), Co(II), Pb(II), Hg(II), U(VI)).

[0036] "Dye": In the sense of this article, the terms "fluorescent dye", "fluorophore", "fluorochrome", "dye" are used interchangeably to refer to a fluorescent compound or structure, and in particular can be one of the following: fluorescent organic dyes, fluorescent quantum dots, fluorescent dyads, fluorescent carbon dots, graphene quantum dots or other carbon-based fluorescent nanostructures, fluorescent proteins, fluorescent DNA origami-based nanostructures. The term "fluorescent dye" is intended to mean an organic fluorescent dye, in particular a derivative of a xanthene (e.g. fluorescein, rhodamine, Oregon green, Texas red), a cyanine (e.g. cyanine, indocarbocyanine, oxacarbocyanine, thiacarbocyanine, merocyanine), a derivative, a squarylium rotaxane derivative, a naphthalene, a coumarin, an oxadiazole, anthracene (anthraquinone, DRAQ5, DRAQ7, CyTRAK Orange), a pyrene (Cascade Blue), an oxazine (Nile Red, Nile Blue, Cresyl Violet, Oxazine 170), an acridine (proflavine, acridine orange, acridine yellow), an arylmethylene (auramine, crystal violet, malachite green), a tetrapyrrole (porphine, phthalocyanine, bilirubin), a dipyrromethene (BODIPY, aza-BODIPY), a phosphorescent dye or a luminescent dye. The following trademark groups designate commercially available fluorescent dyes, which may include dyes belonging to different chemical families: CF dyes (Biotium), DRAQ and CyTRAK probes (BioStatus), BODIPY (Invitrogen), EverFluor (Setareh Biotech), Alexa Fluor (Invitrogen), Bella Fluore (Setareh Biotech), DyLight Fluor (Thermo Scientific), Atto and Tracy (Sigma-Aldrich), FluoProbes (Interchim), Abberior dyes (AbberiorDyes), Dy and MegaStokes dyes (Dyomics), Sulfo Cy dyes (Cyandye), HiLyte Fluor (AnaSpec), Seta, SeTau, and Square dyes (SETA BioMedicals), Quasar and Cal Fluor dyes (Biosearch Technologies), SureLight dyes (Columbia Biosciences), Vio dyes (Milteny Biotec) [List modified from: https: / / en.wikipedia.org / wiki / Fluorophore].In the sense of this article, the term "fluorescent dye" means a fluorescent protein group, in particular a member of the green fluorescent protein (GFP) family, including GFP and GFP-like proteins (e.g., DsRed, TagRFP) and (monomerized) derivatives thereof (e.g., EBFP, ECFP, EYFP, Cerulaen, mTurquoise2, YFP, EYFP, mCitrine, Venus, YPet, superfolded GFP, mCherry, mPlum). In addition, in the fluorescent protein group, the term "fluorescent dye" in the sense of this article may include fluorescent proteins whose absorbance or emission characteristics change when bound to a ligand (e.g., BFPms1) or in response to environmental changes (e.g., redox-sensitive roGFP or pH-sensitive variants). In addition, in the fluorescent protein group, the term "fluorescent dye" in the sense of this article may include derivatives of the cyanobacterial phycobiliprotein small super red fluorescent protein smURFP and fluorescent protein nanoparticles that can be derived from smURFP. An overview of fluorescent proteins can be found in Rodriguez et al. 2017 Trends Biochem Sci. 2017 Feb;42(2):111–129. The term “fluorescent dye” in the sense of this article may further refer to fluorescent quantum dots. The term “fluorescent dye” in the sense of this article may further refer to fluorescent carbon dots, fluorescent graphene quantum dots, fluorescent carbon-based nanostructures, as described in Yan et al. 2019 Microchimica Acta (2019) 186:583 and Iravani and Varma 2020 Environ Chem Lett. 2020 Mar 10:1–25. The term “fluorescent dye” in the sense of this article may further refer to fluorescent polymer dots (Pdots) or nanodiamonds. The term "fluorescent dye" in the sense of the present invention may further refer to a fluorescent dyad, such as the dyad of a perylene antenna and a triangular emitter described in Kacenauskaite et al. 2021 J. Am. Chem. Soc. 2021, 143, 1377-1385.

[0037] The term "fluorescent dye" in the sense of this article may further refer to organic dyes, dimers, quantum dots, polymer dots, graphene dots, carbon-based nanostructures, DNA origami-based nanostructures, nanorulers, polymer beads doped with dyes, fluorescent proteins, inorganic fluorescent dyes, SMILEs, or microcapsules filled with any of the foregoing.

[0038] The term "fluorescent dye" in the sense of the present invention may further refer to a FRET pair having at least one fluorescent dye as a FRET donor and at least one fluorescent dye as a FRET acceptor, or a FRET triplet for generating a three-component Resonance Energy Transfer. In particular, the FRET-pairs or FRET-triplets are connected via complementary linkers or connecting elements.

[0039] The term "fluorescent dye" in the sense of the present invention may further refer to a FRET n-tuple of physically linked dyes.

[0040] "Code", "code exchange": In the sense of this article, a "reporter gene" or "combination tag" contains a unique "dye combination", which is similar to the "code" that encodes the identity of a specific affinity reagent and thus the identity of a specific marker (marker = affinity reagent + combination tag), and thus encodes the identity of a specific analyte (e.g., mRNA of gene X or protein X). In the sense of this article, "subsets of unique dye combinations" and "subsets of combination tags" are assigned to specific affinity reagents, forming "marker subsets", in which one affinity reagent is assigned to multiple "dye combinations" or "codes". In the sense of this article, "code exchange" is performed between each round of the cyclic staining, imaging, inactivation process ("inter-round code exchange") and is performed by degrading the affinity reagent of the n-th round marker, which contains the corresponding code assigned to the n-th round of the marker.

[0041] "Readout device": In the sense of this document, "readout device" refers to a device for performing fluorescent multicolor reading or imaging. A readout device typically comprises at least one excitation light source, a detection system comprising at least one detection channel, and may also contain filters and / or dispersive optical elements, such as prisms and / or gratings, to direct the excitation light to the sample and / or to direct the emission light from the sample to the detector or appropriate areas of the detector. In the sense of this document, the detection system may comprise several detection channels, may be a spectral detector that detects multiple bands of the spectrum in parallel, or a hyperspectral detector that detects continuous parts of the spectrum. The detection system comprises at least one detector, which may be a point detector (e.g. a photomultiplier, an avalanche diode, a hybrid detector), an array detector, a camera, a hyperspectral camera. The detection system may record the intensity of each channel, as is typically the case in a hemocytometer, or may be an imaging detection system that records an image, as is the case in a plate reader or a microscope. A readout device (e.g. a camera or a photomultiplier) with one detector channel may produce a readout with multiple detection channels, using, for example, different excitation and emission bands. The readout device allows a certain number of dyes to be analyzed from a given biological sample in a given run. A "run" can refer to an "iteration" or "round", i.e., at least one readout is generated for a given set of dye combinations and a given mapping of affinity reagents to dye combinations, wherein the affinity reagents are attached to the analyte. This number typically depends on the number n of detection channels that the readout device is configured to provide (i.e., capable of performing) spectral resolution. In the case of microscopes, the number of detection channels is typically 4-5 in the case of camera-based wide-field detection (e.g., wide-field epi-fluorescence microscopy, spinning disk microscopy, light-sheet fluorescence microscopy), and 5-12 in the case of microscopes using spectral detection concepts that typically rely on excitation or emission fingerprints and (spectral / linear) unmixing. In contrast, hyperspectral imaging, which can distinguish a large number of dyes by providing very fine spectral resolution over a wide and continuous spectral range, has not yet been widely deployed in microscopes. In addition to spectral properties, the lifetime of fluorescent dyes can also be used to discriminate between multiple dye species and distinguish them from autofluorescence, effectively increasing the number of detection channels y, which may also correspond to the maximum number of dyes that can be reliably separated in a group (i.e., that can be excited by the same excitation light).

[0042] "Oligonucleotide" or "nucleic acid": in the sense herein refers to DNA, RNA, peptide nucleic acid, morpholino or locked nucleic acid, glycol nucleic acid, threose nucleic acid, hexitol nucleic acid or another form of artificial nucleic acid or heterologous nucleic acid.

[0043] "Readout": In the sense of this document, the term "readout" refers to an image-based readout, which can be obtained on a microscope (such as a point scanning confocal or a camera-based / wide-field imaging system, such as a spinning disk microscope, a light sheet fluorescence microscope, a light field microscope, a stereo microscope). In addition, the term "readout" refers to a non-image-based readout, such as in a hemocytometer or a flow-based readout device with at least one point detector or line detector. The readout can consist of a discrete readout such as a single acquisition of an emission spectrum or an image stack, and the readout can be a stream of readout data, such as in a substantially continuous point scanning confocal or hemocytometer. In addition, the readout can be a sequence of images, such as a spectral or hyperspectral image stack, where a different band of fluorescence emission is recorded in each image.

[0044] "Readout volume": As used herein, the term "readout volume" refers to the volume that is effectively detected by an optical system (e.g., a microscope or cytometer) at a given moment in time. For systems with a continuous data stream, the "readout volume" is determined by a clock, such as a "pixel clock," that divides the continuous data stream into chunks that are then assigned to a point in time or a spatial location. The readout volume may depend on the effective point spread function of the imaging system, which may define or limit the maximum range of the readout volume, for example. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] In the following, specific embodiments are described with reference to the accompanying drawings, in which:

[0046] Figure 1 is a schematic diagram of a flow chart of a first embodiment of a method for analyzing a biological sample,

[0047] Figure 2 is based on Figure 1 Schematic diagram of an alternative approach,

[0048] Figure 3 is a schematic diagram of a flow chart of a second embodiment of a method for analyzing a biological sample,

[0049] Figure 4 is based on Figure 3 Schematic diagram of alternative approaches, and

[0050] Figure 5 is a schematic representation of markers used iteratively in a method for analyzing a biological sample. DETAILED DESCRIPTION

[0051] Figure 11 is a schematic diagram of a flow chart of a first embodiment of a method for analyzing a biological sample. The biological sample comprises at least one target analyte 106, in particular a target protein. In step S100, the target analyte 106 is stained or labeled with a first marker 108 specific to the target analyte 106 by introducing the first marker 108 into the biological sample. The first marker 108 comprises an affinity reagent 110, which is configured to specifically bind to the target analyte 106, in particular to a specific epitope of the target analyte 106. The affinity reagent 110 is preferably an aptamer with high affinity for a target analyte 106 comprising a nucleic acid (such as DNA, RNA and / or XNA). Preferably, the aptamer is substantially composed of natural and / or non-natural nucleotides.

[0052] The first marker 108 further comprises a tag 112 having at least one optically detectable moiety, such as a fluorophore, that binds to the affinity reagent 110. The tag 112 can be attached to the affinity reagent 110 via an oligonucleotide 114 that is at least partially complementary to a corresponding oligonucleotide 116 of the affinity reagent 110.

[0053] Furthermore, in step S100, a first optical readout of the biological sample is generated with the first marker 108. In particular, an image of the biological sample with the first marker 108 can be generated, and the position of the first marker 108, and thus the position of the attached target analyte 106, can be determined within the image. The optical readout can be generated by an optical reading device, such as a microscope. The first marker 108 determined in the first optical readout is schematically represented in Figure 1 and indicated by reference numeral 122.

[0054] In step S102, the marker 108 is degraded by applying at least one degradation agent to the biological sample. At least the affinity agent 110 is configured to be degraded by the degradation agent. Therefore, the degradation agent can cause at least the affinity agent 110 to degrade, thereby causing the affinity agent 118 to degrade.

[0055] The degradation agent may be a biodegradation agent, a chemical degradation agent, or a physical degradation agent. The degradation of the affinity reagent 110 may include digesting, breaking, or unfolding the affinity reagent 110. Generally, the degradation of the affinity reagent 110 may result in a loss of functionality of the affinity reagent 110, for example, the affinity reagent 110 may lose its specificity and / or affinity for the target analyte 106 and no longer specifically bind the first marker 108 to the target analyte 106.

[0056] For example, the affinity reagent 110 is degraded by a chemical degradation agent (e.g., hydrogen peroxide), thereby producing oligonucleotide fragments of the degraded affinity reagent 118. In step S102, the hydrogen peroxide concentration 120 is increased to degrade the affinity reagent 110. The aptamer of the affinity reagent 110 may include non-natural nucleotides that are particularly susceptible to the degradation agent (such as hydrogen peroxide) so as to effectively degrade the affinity reagent 110. After the affinity reagent 110 is degraded, the application of the degradation agent is stopped. This may include washing the biological sample to remove the hydrogen peroxide. In addition, the biological sample may be washed to remove the degraded affinity reagent 118 and / or other components of the marker, such as the tag 112. The tag 112 may be additionally configured to be at least partially degraded by the degradation agent. Alternative degradation agents may include: enzymes such as endonucleases (biological degradation agents), UV light, or temperature (physical degradation agents).

[0057] The affinity reagent 110 is configured to be degraded only by the degradation agent added in step S102. In addition, it is preferred that the degradation agent applied in step S102 does not substantially degrade the target analyte 106. Therefore, before step S100, the affinity reagent 110 and the corresponding degradation agent may be selected based on the target analyte 106, in particular its chemical properties, so that the degradation agent that does not substantially degrade the target analyte 106 is selected. Figure 1 In the example of, the target analyte 106 is a protein, which is not or substantially not affected or degraded by the hydrogen peroxide degradation agent. Instead, only the affinity reagent 110 is degraded by the degradation agent. The same is true for enzymes that degrade nucleic acids, such as DNA enzymes, RNA enzymes, or restriction endonucleases (endonucleases), which can be used as degradation agents. Such enzymes can also be used in combination with each other as enzyme mixtures, and further combined with other degradation agents, such as hydrogen peroxide, to achieve particularly rapid and effective degradation. In a particularly preferred embodiment, DNA enzyme I or RNA enzyme A is used in a temperature range of 20°C-50°C, preferably in combination with a Tris-based buffer containing 0.5mM-5mM Mg2+ and EDTA.

[0058] In step S104, a second label 124 is introduced into a biological sample containing a target analyte 106. The second label 124 includes an affinity reagent 110 configured to specifically bind to the target analyte 106, in particular to a specific epitope of the target analyte 106 as in step S100. Alternatively, the second label 108 may include an affinity reagent different from the affinity reagent 110, which binds to a different epitope of the target analyte 106.

[0059] As explained for the first label 108, the second label 124 further includes a label 126 having at least one optically detectable portion, such as a fluorophore, that binds to the affinity reagent 110. The label 126 can be attached to the affinity reagent 110 via an oligonucleotide 114 that is at least partially complementary to the corresponding oligonucleotide 116 of the affinity reagent 110. The labels 112, 126, in particular their respective optically detectable portions, differ in their optical properties. For example, the labels 112, 126 can differ in fluorescence emission wavelength, fluorescence excitation wavelength, or emission duration.

[0060] Furthermore, in step S104, a second optical readout of the biological sample is generated with the second marker 124. In particular, an image of the biological sample with the second marker 124 can be generated, and the position of the second marker 124, and thus the position of the attached target analyte 106, can be determined within the image. Figure 1 A second marker 124 determined in the second optical readout is schematically represented in FIG. 1 and indicated by reference numeral 128 .

[0061] After step S104, the method may continue by iteratively repeating steps S102 and S104 to produce further optical readouts with further labels.

[0062] In order to determine whether the target analyte 106 is present in the biological sample, in particular, whether the target analyte 106 is present at a specific location in the biological sample, the order 130 in which the markers 108, 124 are determined or identified in the optical readout (refer to the reference numerals 122, 128) can be used to identify the specific target analyte at that location in the biological sample.

[0063] Figure 2 is based on Figure 1 Schematic diagram of an alternative approach to the method of . The target analyte 106 can be labeled with a first label 108 in a first step and an initial optical readout is generated, such as in the case of Figure 1 In step S100, similar to step S102, the first marker 108 is then degraded. Figure 1 In an alternative to the hydrogen peroxide degradation agent, the first label 108, in particular the affinity agent 110, is degraded by the biodegradation agent 200 (such as an endonuclease) to produce a degraded affinity agent 118. In a further step, similar to step S104, the target analyte 106 is labeled with a second label 124 and an additional optical readout is produced, such as for Figure 1 described.

[0064] Alternatively, similar to the second marker 124, a different second marker 202 may be introduced into the biological sample after the first marker 108 is degraded. Similar to the markers 108, 124, the marker 202 specifically binds to the target analyte 106, however, the marker 202 specifically binds to a different epitope of the target analyte 106 than the markers 108, 124. To this end, the marker 202 includes an affinity agent 204, such as an aptamer, which is specific for a different epitope of the target analyte 106. This means that the affinity agent 204 specifically binds to a specific region or surface of the target analyte 106 that is different from the region or surface of the target analyte 106 to which the affinity agent 110 specifically binds. By using an affinity agent 204 that is specific for a different epitope of the target analyte 106, the specificity or selectivity of the method can be increased, and the target analyte 106 can be identified, quantified, or its location in the optical readout can be determined with a particularly high degree of confidence. Figure 2 The strategy schematically depicted in is particularly preferred because it allows the design of a (spatial) proteomics assay that is compatible with analyzing a large number of target analytes ranging from 100 to 50,000 target analytes (e.g., 100 to 500, 500 to 1,000, 1,000 to 10,000, 1,000 to 50,000) while increasing the selectivity or specificity of the assay and providing a means to probe another layer of information, which is post-translational modifications (PTMs).

[0065] Figure 3 is a schematic diagram of a flow chart of a second embodiment of a method for analyzing a biological sample. The biological sample comprises at least a target analyte 106 and another target analyte 306, in particular both of which are target proteins. In step S300, the target analyte 106 is stained or labeled with a first marker 108 specific for the target analyte 106 by introducing the first marker 108 into the biological sample. The first marker 108 comprises an affinity reagent 110, which is configured to specifically bind to the target analyte 106. Figure 1 As described, first marker 108 further comprises a tag 112 having at least one optically detectable portion, such as a fluorophore, that binds to affinity reagent 110. Tag 112 can be attached to affinity reagent 110 via an oligonucleotide 114 that is at least partially complementary to a corresponding oligonucleotide 116 of affinity reagent 110.

[0066] Furthermore, in step S300, a first optical readout of the biological sample is generated using the first marker 108. In particular, an image of the biological sample with the first marker 108 can be generated, and the position of the first marker 108, and thus the position of the attached target analyte 106, can be determined within the image. The optical readout can be generated by an optical reading device, such as a microscope. The first marker 108 determined in the first optical readout is schematically represented in Figure 1 and indicated by reference numeral 122.

[0067] In step S302, the marker 108 is degraded by applying at least one degradation agent to the biological sample. At least the affinity agent 110 is configured to be degraded by the degradation agent. Therefore, the degradation agent can cause at least the affinity agent 110 to degrade, thereby causing the affinity agent 118 to degrade.

[0068] The degradation agent may be a biodegradation agent, a chemical degradation agent, or a physical degradation agent. The degradation of the affinity reagent 110 may include digesting, breaking, or unfolding the affinity reagent 110. Generally, the degradation of the affinity reagent 110 may result in a loss of function of the affinity reagent 110, for example, the affinity reagent 110 may lose its specificity for the target analyte 106 and no longer specifically bind the first marker 108 to the target analyte 106.

[0069] For example, the affinity reagent 110 is degraded by a chemical degradation agent (e.g., hydrogen peroxide), thereby producing oligonucleotide fragments of the degraded affinity reagent 118. In step S302, the hydrogen peroxide concentration 120 is increased to degrade the affinity reagent 110. The aptamer of the affinity reagent 110 may include non-natural nucleotides that are particularly susceptible to the degradation agent (such as hydrogen peroxide) so as to effectively degrade the affinity reagent 110. After the affinity reagent 110 is degraded, the application of the degradation agent is stopped. This may include washing the biological sample to remove the hydrogen peroxide. In addition, the biological sample may be washed to remove the degraded affinity reagent 118 and / or other components of the marker, such as the label 112. The label 112 may be further configured to be at least partially degraded by the degradation agent. Alternative degradation agents may include: enzymes such as DNA enzymes, RNA enzymes, endonucleases (biological degradation agents), UV light or temperature (physical degradation agents).

[0070] The affinity reagent 110 is configured to be degraded only by the degradation agent added in step S302. In addition, it is preferred that the degradation agent applied in step S302 does not substantially degrade the target analyte 106, 306. Therefore, before step S300, the affinity reagent 110 and the corresponding degradation agent can be selected based on the target analyte 106, 306, in particular its chemical properties, so that the degradation agent that does not substantially degrade the target analyte 106, 306 is selected.

[0071] In step S304, another label 308 is introduced into the biological sample containing the target analyte 106, 306. The another label 308 includes another affinity reagent 310, which is configured to specifically bind to the target analyte 306.

[0072] As explained for the first marker 108, the other marker 308 further comprises a label 126 having at least one optically detectable portion, such as a fluorophore, that is bound to the affinity reagent 310. The label 126 can be attached to the affinity reagent 310 via an oligonucleotide 114 that is at least partially complementary to the corresponding oligonucleotide 116 of the affinity reagent 310. The labels 112, 126, in particular their respective optically detectable portions, differ in their optical properties. For example, the labels 112, 126 can differ in fluorescence emission wavelength, fluorescence excitation wavelength, or emission duration.

[0073] Furthermore, in step S304, a second optical readout of the biological sample is generated using the further marker 308. In particular, an image of the biological sample with the further marker 308 can be generated, and the position of the further marker 308, and thus the position of the attached target analyte 306, can be determined within the image. The further marker 308 determined in the second optical readout is schematically represented in Figure 3 and indicated by reference numeral 312.

[0074] After step S304, the method may continue by iteratively repeating steps S302 and S304 to produce further optical readouts with further labels.

[0075] In order to determine whether the target analyte 106, 306 is present in the biological sample, in particular, whether the target analyte 106, 306 is present at a specific location in the biological sample, the marker 108, 308 is determined or identified in the corresponding optical readout (refer to reference numerals 122, 312).

[0076] Figure 4 is based on Figure 3 Schematic diagram of an alternative method of the present invention. The marker 108 can be partially introduced into the biological sample. For example, the affinity reagent 110 can be introduced first, and the tag 112 can be introduced later. The complementary oligonucleotides 114, 116 of the affinity reagent 110 and the tag 112 enable the tag 112 to specifically bind to the affinity reagent 110. Then, the marker 108, especially the affinity reagent 110, can be degraded by the biodegradation agent 200, such as a biodegradation agent directed against Figure 3In addition, the biological sample may include a target analyte 306. By repeating the method for another iteration, the target analyte 306 can be labeled with a marker 400. Similarly, the marker 400 can be partially introduced into the biological sample. Initially, an affinity reagent 310 is introduced, followed by a tag 112 that specifically binds to the affinity reagent 310 through complementary oligonucleotides 114, 116. This produces the marker 400 in the biological sample. The marker 400 (particularly the affinity reagent 310) can be sequentially degraded into a fragmented affinity reagent 402. Since a single portion of the marker 108, 400 is smaller than the corresponding entire marker 108, 400, the single portion can penetrate deeper into the biological sample before the marker 108, 400 is produced in situ in the biological sample. Additionally or alternatively, the marker 108, 124, 202, 308, 400 or a corresponding portion of the marker 108, 124, 202, 308, 400 can be introduced into the biological sample by electrophoresis. For example, an electric field can be applied to the biological sample. This is particularly preferred when the marker 108, 124, 202, 308 includes a charged portion (such as an oligonucleotide).

[0077] Figure 5 It is a schematic diagram of a marker 500, 502, 504 used iteratively in a method for analyzing a biological sample. The markers 500, 502, 504 each include an affinity reagent 506 to specifically attach the marker to the target analyte of the biological sample. The markers 500, 502, 504 further include a label with at least two optically detectable parts 508, 510, 512, 514, 516, 518 (such as fluorophores). Each of the optically detectable parts 508, 510, 512, 514, 516, 518 has different optical properties, such as emission wavelength, excitation wavelength or fluorescence lifetime. The markers 500, 502, 504 can be used in one of the above methods. For example, the markers 500, 502, 504 can be used iteratively in steps S100, S104, S300, S304 to identify or distinguish the target analytes marked respectively. By providing labels 500, 502, 504 with different optical properties, multiple unique target analytes can be labeled and distinguished in a particular step S100, S104, S300, S304.

[0078] In particular, a readout device equipped with spectral excitation and emission detection and a software-based unmixing algorithm can be used to detect markers 500, 502, 504 comprising a plurality of the above optically detectable moieties. In particular, this enables, for example, during steps S100, S104, S300 or S304, a plurality of different markers 500, 502, 504 comprising a plurality of optically detectable moieties to be introduced simultaneously into a biological sample in order to detect a plurality of different target analytes. At the same time, this enables the individual markers of the plurality of different markers 500, 502, 504 to be distinguished in an optical readout based on the optical properties of the respective optically detectable moieties 508, 510, 512, 514, 516, 518.

[0079] The ability to distinguish a large number of markers introduced into a biological sample in one of the above disclosed methods can be further improved by applying the method disclosed in document PCT / EP2021 / 073819. In particular, the document discloses labeling a target analyte in a biological sample with a marker comprising a combination of tags, the markers being exchanged from one round of staining to the next, and accepting the presence of the marker in the read volume based on a statistical analysis, which is a measure of statistical confidence.

[0080] In all drawings, the same or similar 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 " / ".

[0081] Although some aspects have been described in the context of an apparatus, it is clear that these aspects also represent a description of the corresponding method, where a block or apparatus 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 feature of a corresponding apparatus. Reference numerals 106、306 Target analytes 108、124、202、308、400、500、502、504 Markers 110、204、310、506 Affinity reagents 112、126 Label 114 Tag complementary oligonucleotide 116 Complementary oligonucleotides to affinity reagents 118、402 Fragmentation affinity reagents 120 Degradant concentration over time 122 First optical readout of labeled 128、312 Second optical readout with markers 130 Reading order of markers 200 Biodegradation Agents 508、510、512、514、516、518 Optically detectable part .

Claims

1. A method for analyzing a biological sample, comprising the following steps: a) providing a biological sample having a plurality of target analytes (106, 306), b) introducing at least a first marker (108, 124, 202, 308, 400, 500, 502, 504) into the biological sample, the first marker comprising a first affinity reagent (110, 204, 310, 506) and a first optically detectable label (112, 126) bound to the first affinity reagent (110, 204, 310, 506), wherein the first affinity reagent (110, 204, 310, 506) is configured to specifically bind to one of the target analytes and is configured to be degraded by the degrading agent (120, 200), c) generating a first optical readout (122) of said biological sample having said first marker (108, 124, 202, 308, 400, 500, 502, 504), d) applying the degradation agent to degrade at least the first affinity reagent, e) introducing at least a second marker (108, 124, 202, 308, 400, 500, 502, 504) into the biological sample, the second marker comprising a second affinity reagent (110, 204, 310, 506) and a second optically detectable label (112, 126) bound to the second affinity reagent (110, 204, 310, 506), wherein the second affinity reagent (110, 204, 310, 506) is configured to specifically bind to one of the target analytes (106, 306) and is configured to be degraded by the degrading agent (120, 200), and f) generating a second optical readout (128, 312) of said biological sample having said second marker (108, 124, 202, 308, 400, 500, 502, 504).

2. The method according to claim 1, wherein in step d), after adding the degradation agent (120, 200), the biological sample is washed to remove the first marker (108, 124, 202, 308, 400, 500, 502, 504) from the biological sample.

3. The method according to any of the preceding claims, wherein at least the first affinity reagent (110, 204, 310, 506) and the second affinity reagent (110, 204, 310, 506) comprise a nucleic acid backbone.

4. The method according to any one of the preceding claims, wherein at least the first affinity agent (110, 204, 310, 506) and the second affinity agent (110, 204, 310, 506) are aptamers.

5. A method according to any of the preceding claims, wherein at least the first affinity reagent (110, 204, 310, 506) and the second affinity reagent (110, 204, 310, 506) have a maximum spatial extent in the range of 1 nm to 25 nm, more preferably in the range of 1 nm to 10 nm.

6. The method according to any of the preceding claims, wherein the target analyte (106, 306) is a protein.

7. The method according to any one of the preceding claims, wherein the degradation agent (120, 200) comprises at least one of a chemical degradation agent, a biological degradation agent, and a physical degradation agent.

8. The method according to any one of the preceding claims, wherein said steps b) and / or e) comprise applying an electric field to the biological sample.

9. A method according to any of the preceding claims, wherein the first affinity reagent (110, 204, 310, 506) and the second affinity reagent (110, 204, 310, 506) are specific for the same target analyte (106, 306), and wherein the first label (112, 126) and the second label (112, 126) have different optical properties.

10. The method of claim 9, wherein in step g), the same target analyte (106, 306) is identified in the first optical readout and the second optical readout based on determining the first label (110, 204, 310, 506) and the second label (110, 204, 310, 506) in the first optical readout and the second optical readout.

11. A method according to any one of the preceding claims 1 to 8, wherein the first affinity reagent (110, 204, 310, 506) and the second affinity reagent (110, 204, 310, 506) are specific for different target analytes (106, 306), and wherein the first label (112, 126) and the second label (112, 126) have the same optical properties.

12. A method according to any of the preceding claims, wherein in step b), a third marker (108, 124, 202, 308, 400, 500, 502, 504) is introduced, wherein the third marker comprises a third optically detectable label (112, 126) and a third affinity reagent (110, 204, 310, 506) specific for one of the target analytes (106, 306).

13. The method according to any of the preceding claims, wherein at least the first tag (112, 126) and the second tag (112, 126) comprise a nucleic acid backbone configured to be degraded by the degradation agent (120, 200).

14. The method of claim 13, wherein the nucleic acid backbone of each tag (112, 126) comprises a unique sequence (114) configured to bind to a complementary sequence (116) of a corresponding affinity reagent (110, 204, 310, 506).

15. The method according to any of the preceding claims, wherein each tag (112, 126) comprises at least one optically detectable portion.

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