WO2025168224A1 - Marker and method for analysing a biological sample - Google Patents

Abstract

The invention relates to a marker (100, 200, 202, 204, 312, 500, 614, 806, 904, 1004, 1112) and method for analysing a biological sample with a plurality of target analytes. The marker comprises at least a first marker part (104, 104a, 104b, 104c, 301, 610) and at least a second marker part (112, 112a, 112b, 112b, 112c, 112d, 306, 308, 310, 608). The first marker part (104, 104a, 104b, 104c, 301, 610) comprises a first affinity reagent (106) and at least a first oligonucleotide label (108), the at least a first oligonucleotide label comprises a first reactive group (110). The second marker part (112, 112a, 112b, 112b, 112c, 112d, 306, 308, 310, 608) comprises a second affinity reagent (114) and a second oligonucleotide label (116, 313, 314, 316), the second oligonucleotide label comprises a second reactive group (118). The first and second reactive group (110, 118) are configured to react with each other. The reaction generates a bond (120) between the first oligonucleotide label and the second oligonucleotide label.

Description

Marker and method for analysing a biological sample

Technical field

The invention relates to a marker and method for analysing a biological sample with a plurality of target analytes.

Background

Labels and markers are frequently used when analysing biological samples, for example in fluorescent microscopy. Markers for fluorescent microscopy generally comprise affinity reagents such as antibodies and labels attached to the affinity reagents in order to enable specifically attaching the labels- in particular via the affinity reagents - to a target analyte in a biological sample. This allows the identification, localisation, and/or quantification of the target analyte in the biological sample. Such a target analyte may hold predictive value and may be used to diagnose, stratify, or monitor patients or healthy subjects and in this case may be referred to as a biomarker.

Detection and identification of robust and significant new biomarkers, especially of interactional biomarkers, remains a constant target in the field.

Summary

It is an object to provide markers for analysing a biological sample that are flexible and reliable in use as well as a respective method for analysing biological samples.

The aforementioned object is achieved by the subject-matter of the independent claims. Advantageous embodiments are defined in the dependent claims and the following description. Marker and chemical proximity ligation assay

In a first aspect, a marker for analysing a biological sample with a plurality of target analytes is provided. The marker comprises at least a first marker part and at least a second marker part.

The first marker part comprises a first affinity reagent and at least a first oligonucleotide label, the at least a first oligonucleotide label comprises a first reactive group.

The second marker part comprises a second affinity reagent and a second oligonucleotide label, the second oligonucleotide label comprises a second reactive group.

The first and second reactive group are configured to react with each other. The first and second reactive group may be selected such that the reaction between them is spontaneous or such that the reaction between them occurs only under a catalytic condition, for example the application of a catalyst or an external stimulus (e.g. UV light, temperature, pH change). A well-known example of the former may be the spontaneous click chemistry reaction of azide with DBCO or other groups that react spontaneously with azide, e.g. Strain-promoted alkyne-azide click chemistry or SpAAC. A particular preferred example of the latter is the click reaction between azide and alkyne groups, which is catalysed by copper ions (Copper-dependent alkyne azide cycloaddition, or CuAAC). Other reactive groups and reactions may be equally suited and may include without intent to be limiting N-hydroxysucciminde ester (NHS), maleimide, aldehyde, photo-crosslinking, phosphoramidite, disulfide bond formation, amide bong formation, or thiol-ene chemistries. Finally, the reactive groups may simply be the naturally occurring 3' hydroxy and 5'phosphate groups and the reaction may be native ligation using a ligase like T4 DNA ligase. In this case, the ligation may be facilitated by a ligation oligonucleotide configured to bind to the first and second oligonucleotide label.

The reaction between the first oligonucleotide label and the second oligonucleotide label generates a bond, in particular a covalent bond, between the first oligonucleotide label and the second oligonucleotide label. The unit resulting from the reaction may be termed a ligate, which comprises the first oligonucleotide label and the second oligonucleotide label. For example, the first oligonucleotide label may comprise an azide as first reactive group and the second oligonucleotide label may comprise an alkyne as second reactive group. In this case, the reaction between the first and second reactive group will lead to the formation of a triazole. This is further described in the following. Other reactive groups may be equally suited and may yield different types of covalent bonds.

The first affinity reagent and the second affinity reagent are each configured to bind specifically to one of the target analytes of the biological sample.

An affinity reagent in the sense of this document may be an antibody, nanobody, an antibody or nanobody-fragment, an aptamer, an aptabody, a polymeric binder, a drug, a toxin, a drug-like molecule, or other small molecule so as long as it is configured to bind a target analyte with high affinity and specificity. The aforementioned affinity reagents may be used to detect the presence of proteins, metabolites, neurotransmitters, peptides, hormones, vitamins, or trace elements, for example.

Further an affinity reagent in the sense of this document may be an oligonucleotide probe configured to bind to a nucleic acid target sequence like a sequence stretch of an RNA or DNA target via in situ hybridization (ISH). Such a probe is referred to as an ISH probe in the following. In this case the marker or method may be used to improve the specificity in detecting nucleic acid targets, to detect interactions amongst nucleic acids as well as between nucleic acids and proteins for example. Likewise, in this case the marker or method may be used to validate the integration of gene expression cassette or viruses into the genome at specific locations as well as to discern them from integrations that are not in the intended locus. This embodiment of the present invention is particularly well suited to monitor the quality of cell therapy products and cell lines in general during development and manufacturing.

Similarly, to the detection of a higher number of protein analytes like for example 20-200 (e.g. multiplex proteomics) or 200-10,000 (e.g. high-plex proteomics) the high-plex detection of RNA is hampered by inspecific binding of affinity reagents. In the context of proteins and antibodies this is referred to as cross-reactivity and in the context of nucleic acids and nucleic acid/ISH probes as cross-hybridization. In both cases the limited specificity results mostly from the presence of closely related homologous sequences and cannot be resolved in principle by either improving of an affinity reagent or by even elaborate "validation" efforts.

The present invention by means of the marker provides a robust assay for proximity of at least two target analytes, which may be for example two distinct proteins, in which case the proximity may be taken as a proxy for interaction between the molecules, or they may be for example two epitopes on the same target protein, or they may be for example two nucleic acid sequence targets that are part of one nucleic acid target (e.g. the mRNA of gene pax7, or the genomic pax7 locus). The latter is particularly interesting in cases, when the specificity of the assay shall be strongly improved. Owing to the cross-reactivity inherent to most if not all affinity reagents probing the same target with two affinity reagents that become part of one marker is a good strategy to render the assay more specific and reduce false-positive rate. This is not only of interest in life science research for multi-plex assays, but also for diagnostic tests which should discern closely related proteins from pathogenic and non-pathogenic strains for example.

For this reason, the present invention relates to both the analysis of liquid samples as well as to the analysis of solid samples. A liquid sample may in particular be a liquid biopsy like a blood, serum, sputum, urine, interstitial fluid, saliva, cerebrospinal fluid sample or a lysate prepared from a solid sample. Lysates may be prepared for example from tissue biopsies by tissue enzymatic and/or mechanic tissue disintegration and cell lysis. Frequently, such lysates may derive from a sorted populations of cells from a tissue sample or even from a single cell.

The present invention essentially allows the detection of paired and n-tupeled binding of markers to target pairs or target n-tupels as stated before. In contrast to similar assays known from prior art like the proximity ligation assay described in US10465235B2, proximity extension assay described in US9777315B2, the marker and method disclose herein do not require predetermined marker-part-specific sequences that mediate duplex formation between oligonucleotide sequences. While the marker and method disclose herein rely on oligonucleotide-labelled affinity reagents like the aforementioned it differs markedly in this regard from the former. As the present design does not depend on duplex formation and therefore not on predetermined sequences, it allows not only for the interrogation of already known target pairs or target n-tupels. In practice this means for example that while prior art methods can be used to detect a known interaction of two proteins in a tissue sample, the method and marker disclosed herein in addition allows for the identification of unknown interactions and/or unknown posttranslational modifications.

This is particularly relevant in the context of what may be referred to as a "interactional biomarker", An "interactional biomarker" in the sense of this document is a molecular interaction e.g. a protein-protein or protein-RNA, RNA-RNA, protein-DNA, RNA-DNA interaction, that has prognostic or predictive value. Of particular interest in this regard are the interactions of cell surface proteins and secreted molecules found on immune cells with both healthy and diseased cells as well as pathogens. This aspect is of great importance in the context of spatial biology and the stratification of patient populations along key "interactional biomarkers" like PD1-PDL1 or CTLA-4/CD80 interaction, which were found to hold predictive value by Sanchez-Magraner er al. Cancer Res (2020) 80 (19): 4244-4257. In this study the authors used i FRET a two-site labeling assays, which is based on detecting antibody pairs, wherein in antibody is directly dye-conjugated to a FRET-donor ATTO488 and the other to horseradish peroxidase (HRP), which is used with tyramide signal amplification to deposit Alexa594 dye. While this assay provides certain advantages with respect to signal amplification, it is limited in terms of plexing and not suited to identify unknown interactions. Likewise, catalyzed reporter-deposition (CARD) as a detection strategy suffers from the fact that the soluble reporter diffuses away from the site of production and hence it is hard to control the spatial stringency of CARD- based assays. Furthermore, this method is not compatible with cyclic methods and/or high-plex readout. The method disclosed in the present invention, however, can be precisely tuned for spatial stringency, allows but does not necessitate amplification, and is compatible with optical readout (intensity-based or imagingbased microscopic) as well next generation sequencing (NGS)-based readout, digital PCR-based readout, or cytometric readout. For this reason, the present marker and method offers great flexibility as a detection platform in terms of choosing the readout platform and is equally well-suited to analyse a single interaction or target as well as to screen for thousands of interactions and targets.

Aside from these aspects, the key improvement of the marker and method disclosed herein over the prior art is that it allows the identification of previously unknown interactional biomarkers that hold high predictive value. Importantly, the marker and method may be used to interrogate large numbers of samples from healthy subjects and disease cohorts. It is especially noteworthy that existing biobanks offer a vast amount of available serum samples as well of FFPE tissue sections, and that the marker and distinct method embodiments disclosed in this document are ideally suited to leverage these pre-existing samples in retrospective studies. It is clear that this pertains to both basic research, translational, clinical research as well as to diagnostics and companion diagnostic application of the method.

A further important aspects, of the present invention is related to the possibility to combine it with nucleic acid amplification methods (e.g. PCR, RCA, LAMP, RPA) which achieve very high amplification factors and hence the method offers high sensitivity and is suited to analyze minute quantities of samples (i.e. in the nl to pl range). This is particularly important for large retrospective studies of plasma or serum for example.

Despite the great value for liquid and solid multi-, and high-plex multi-omics, the marker and method disclosed herein also provide differentiated value for low-plex analysis, which may be the more predominant use in diagnostic applications.

A particular feature of the marker and method, which may also be referred to as a "proximity detection platform", is the ability to combine this detection platform with a variety of readout platforms. While this may most typically be next generation sequencing and microscopy, it is clear that this proximity detection platform can also be used in conjunction with cytometry, mass spectrometric readout, digital PCR, or colorimetric readout. In essence the "proximity detection platform" can be combined easily with any detectable label for downstream readout depending on the needs of the user. An optional amplification step is recommended and improves both the robustness as well as signal-to-noise ratio, but is not necessary in all cases.

In case each marker part binds to entirely different target analytes, the marker enables determining the proximity or the distance between these different target analytes. In case the marker parts bind to the same target analyte but different areas or epitopes of that target analyte, the marker enables determining the presence or location of that target analytes with high precision or reliability or in other words strongly improves the specificity of the assay. Likewise, this may be used to detect the presence of different proteoforms, which may result from alternative splicing or proteolytic processing for example.

Further, in case the marker parts bind to the same target analyte but different areas or epitopes of that target analyte, one of the areas or epitopes may be a posttranslational modification (PTM).

Posttranslational modifications (PTM) constitute important layer for regulating the function of proteins and include for example phosphorylation, ubiquitination, acetylation, methylation, prenylation, glycosylation.

Thus, the first affinity reagent may be specific to a first target analyte of the biological sample and the second affinity reagent may be specific to a second target analyte of the biological sample, for example. In a further example, the first affinity reagent may be specific to a first epitope of the first target analyte of the biological sample and the second affinity reagent may be specific to a second epitope of the first target analyte.

In a further example, the first affinity reagent may be specific to a first sequence of the first target nucleic acid analyte of the biological sample and the second affinity reagent may be specific to a second sequence of the first target nucleic acid analyte. Thus, the marker parts may bind to the same target analyte at the same time. This enables flexible use of the marker and strongly improves the specificity of the assay and thereby mitigates the detrimental effects of cross-reactivity or cross-hybridization on the quality of the assay.

The at least one first and at least one second oligonucleotide labels may comprise a naturally occurring or artificial nucleic acid (xeno nucleic acid) like for example D-DNA, L-DNA, LIMA, PIMA, PT-DNA. The at least one first and at least one second oligonucleotide label preferably comprises a unique molecular identifier (UMI) that identifies the marker part and/or the affinity reagent of the marker part and/or the target to which said affinity reagent is configured to bind. The at least one first and at least one second oligonucleotide label may be directly conjugated to the first or second affinity reagent or indirectly via for example hybridization of barcoded oligonucleotides or indirectly via a secondary antibody for example. The at least one first and at least one second oligonucleotide label may thus be physically connected to the first and second affinity reagent before, during, after introduction to the biological sample. In a particularly preferred embodiment, the at least one first and at least one second oligonucleotide label comprise predetermined sequences or barcodes, which the first and second affinity reagent are labelled with a first and second attachment oligonucleotide that comprise the respective reverse complementary barcode sequence to allow flexible attachment of the at least one first and at least one second oligonucleotide label. In other words, the at least one first and at least one second oligonucleotide label may be directly or indirectly connected to the first or second affinity reagent. Likewise, they may be connected to the first or second affinity reagent prior to, concomitant with, or following to the introduction of the first and the second affinity reagent into the biological sample.

Spatial stringency of the assay

The first and second reactive group preferably bind to the respective other one when in immediate proximity to each other, in particular. Preferably, each first and second reactive group binds to a single one of the respective other one. The length of the at least a first and at least a second oligonucleotide label as well as the physical sizes of the first and second affinity reagent therefore dictate the spatial stringency of the assay, in other words the maximal distance that two target analytes may have, which still allows the first and second reactive group to bind to and react with each other. For example, an antibody has approximately 15nm wide and thus the lateral distance between the variably region a coupling site of an oligonucleotide label may be up to 7nm away. An oligonucleotide label may for example comprise 20 nucleotides (nt), which corresponds roughly to another 7nm. The spatial stringency of the resulting assay in this case would be in the range of 14nm. Smaller affinity reagents like for example nanobody and aptamers, which are typically around 10-times smaller than antibodies with a size of around 3-4nm, may be used to improve the spatial stringency to about lOnm. Likewise shorter oligonucleotide labels may be used to improve spatial stringency. The minimal length in this case depends on whether an amplification step shall be performed via for example LAMP, PCR, RCA nucleic acid amplification. In cases where detection is performed without amplification, for example, when the detection is based on binding the chemical ligate of the first and second oligonucleotide label with a fluorescent in situ hybridization (FISH) probe or ISH probe comprising another form of detectable label, then the first and second oligonucleotide label may be as short as 4-5nt or 9-llnt. In this case the first and second oligonucleotide label each basically comprise a half to the binding site for said FISH probe. For example, a 4nt half site and a 5nt will yield a 9nt chemically ligated binding site for a FISH probe, which corresponds to 9nt binding site length typically used for DNA-PAINT. This is a particularly preferred embodiment, as it allows combining the method disclosed in this document with a readout method that offers both super resolved readout as well as high-plex readout. In this case the association with a FISH probe with the binding site will be only transient leading to a blinking, which will allow the localization of the marker with very high resolution, e.g. few nanometers lateral resolution. The combination of such a nanobody- or aptamer-based first and/or second marker part with such short first and/or second oligonucleotide labels will yield spatial stringencies in the range of 6-7nm. The use of longer first and/or second oligonucleotide labels, like for example 9-llnt, may yield chemical ligates of 18-22nt in length, which corresponds well to the typical length of probe binding site used for stable association of the FISH probe with the binding site.

A detectable label in the sense of this document may be detectable by sequencingbased readout, may be optically detectable, may be detectable by electrical readout, may be magnetic, may be an enzyme (e.g. HRP, AP)., may comprise a metal or masstag for mass spectrometry-based readout, may comprise a radioactive tag. An optically detectable label in the sense of this document may comprise a fluorescent dye or fluorophore (e.g. ATTO425, ATTO430LS, ATTO465, ATTO488, ATTO514, ATTO532, ATTO565, ATTO594, ATTO643, ATTO647N, ATTO680, ATT0700, Alexa Fl uor488™, BODIPY dye, Cy3/Cy5/Cy5.5 dye, a rhodamine or rhodamine derivative, a coumarinederivative, an oxazine-derivative, fluorescein or a fluorescein-derivative, a CF dye, a Janelia Fluor dye, a polymer dye, SuperNova v428, SuperNova v605, SuperNova786, a Brilliant Violet™ dye), a fluorescent structure (e.g. a quantum dot, a polymer dot, as disclosed in WO 2023/198291 Al, the contents of which is incorporated herein by reference, a DNA-origami or DNA nanostructure-based fluorescent structure), a combinatorial fluorescent label comprising at least two distinct dyes), a polyyne, CARS, SERS, or other Raman label, a gold or other nanoparticle. An optically detectable label may be readout in brightfield, by measuring or registering color, by detecting fluorescence (e.g. spectral properties, lifetime, bleaching kinetic, blinking behaviour), by measuring absorption.

If a downstream detection is preceded by an amplification step, which may be performed using a variety of nucleic acid amplification methods including but not limited loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA), polymerase chain reaction (PCR), or rolling-circle amplification (RCA), it may be necessary to include further sequence elements like primer binding sites into the at least one first and/or second oligonucleotide label.

In some cases, it may be desirable to substantially decrease the spatial stringency of the assay, which may be achieved by using longer at least one first and/or second oligonucleotide labels.

Preferably, the first reactive group and the second reactive group are configured to bind to each other - or in other words react with each other - under a catalytic condition, in particular, only under the catalytic condition. This enables control over the binding of the marker parts to each other and therefore control over the generation of the marker, preferably after the introduction of the first oligonucleotide label and the second oligonucleotide label parts into the biological sample. In particular, the first reactive group and the second reactive group are configured to form the covalent bond under the catalytic condition. The catalytic condition may be an environmental condition. For example, the catalytic condition maybe characterised by the presence of a particular catalyst, such as (UV-)light or copper ions. Thus, the first reactive group and the second reactive group may react with each other. In particular, this may form a covalent bond between one of the at least a first oligonucleotide label and one of the at least a second oligonucleotide label.

Preferably, the at least a first oligonucleotide label is attached to the first affinity reagent by a first cleavable linker and/or the second oligonucleotide label is attached to the second affinity reagent by a second cleavable linker. This enables easy inactivation of the marker. In particular, the cleavable linker may be configured to be specifically cleaved by a cleaving agent. The cleavage site may be for example a restriction site and the cleavage agent a restriction enzyme. The cleavage site may also be a specific modification of the nucleic acid backbone of the first oligonucleotide label and/or the second oligonucleotide label.

Preferably, the at least a first oligonucleotide label is attached to the first affinity reagent by a first secondary affinity reagent (e.g. antibody, nanobody, or aptamer) and/or the second oligonucleotide label is attached to the second affinity reagent by a second secondary affinity reagent (e.g. antibody, nanobody, or aptamer).

Preferably, the at least a first oligonucleotide label is attached to the first affinity reagent by hybridisation and/or the at least a second oligonucleotide label is attached to the second affinity reagent by hybridisation. This allows introducing at least some of the first and second affinity reagents to the sample and bind them to their target analytes before at least some of the respective at least one first and second oligonucleotide labels are introduced to the sample and allowed to bind to their respective first and second affinity reagents thereby forming first and second marker parts. This may be beneficial, when a large number or markers shall be generated and analyzed in a cyclic process.

Alternatively, the at least one first oligonucleotide label and/or the at least one second oligonucleotide label are attached to a first secondary affinity reagent and/or a second secondary affinity reagent (e.g. antibody, nanobody, or aptamer) via hybridization. This enables easy assembly of the marker. For example, the respective affinity reagent may comprise an oligonucleotide with a sequence complementary like a unique oligonucleotide sequence barcode to a part of the respective backbone. The aforementioned linker may be part of the oligonucleotide of the affinity reagent.

The marker parts may be used to analyse a biological sample. A biological sample or "sample" refers to a biological sample which may also be named a biological specimen including, for example blood, serum, plasma, tissue, bodily fluids (e.g. lymph, saliva, semen, interstitial fluid, cerebrospinal fluid), feces, solid biopsy, liquid biopsy, explants, cells (e.g. prokaryotes, eukaryotes, archea), suspension cell cultures, monolayer cell cultures, 3D cell cultures (e.g. spheroids, tumoroids, organoids derived from various organs such as intestine, brain, heart, liver, etc.), a lysate of any of the aforementioned, a virus. A biological sample or sample may further be an environmental sample, like a water sample.

In the sense of this document sample further refers to a volume surrounding a biological sample. Like for example in assays, where secreted proteins like growth factors, extracellular matrix constituents are being studied the extracellular environment surrounding a cell up to a certain assay-dependent distance, is also referred to as the sample. Specifically, affinity reagents brought into this surrounding volume are referred to in the sense of this document as being "introduced into the sample".

The biological sample may be analysed using different assay formats. For example the biological sample may be a liquid biopsy or lysate and analysed in a liquid format. For example the biological sample may be a liquid biopsy or lysate and may be analysed by immobilizing the analytes of the biological sample on a solid support. A solid support may be a particle (e.g. microsphere, polystyrene bead, magnetic bead, nanoparticle, or a NanoArray as described in WO 2022/207832 Al the contents of which is incorporated herein by reference), a coverslip, glass slide, semiconductor or integrated circuit, the (transparent) bottom of a flow cell]. The biological sample may also be a solid sample like a tissue section. In particular an FFPE tissue section, which are widely available in biobanks, and provide a rich source for studies that are trying to find ways to stratify patient populations for example.

In another aspect, a method for analysing a biological sample is provided. The method comprises the steps of: Introducing at least one marker, in particular a plurality of markers, as described above into the biological sample, optionally performing a nucleic acid amplification step and generating a readout of the biological sample with the marker. The readout may be based on next generation sequencing (NGS) of the amplica or based on optical readout. Optical readout may be intensity-based (e.g. cytometry, plate reader) or imaging-based (e.g. microscopy, digital PCR).

In particular, the biological sample comprises a plurality of target analytes. The at least one marker, in particular the respective affinity reagent, may be specific to one of the target analytes of the biological sample. The marker may be generated prior to introduction to the biological sample. Alternatively, the marker parts, specifically, the first and second marker parts may be introduced into the biological sample simultaneously or separately and subsequently. Preferably, during or prior to the step of introducing the marker, a non-catalytic condition is applied to the biological sample. This enables efficient penetration of the marker, in particular its individual parts, into the biological sample and avoids aggregation of markers, in particular its individual parts, in solution. In particular, the step of introducing the marker is carried out under the non-catalytic condition. Preferably, the introduced marker only forms under the catalytic condition by the binding of the first and second reactive groups to each other, which forms the covalent bond between the at least a first and at least a second oligonucleotide label. The resulting ligated product is referred to as the chemical ligate in this document. When subsequent enzymatic amplification is intended the covalent bond formed by the first and second reactive group is configured such that it is tolerated by the polymerase enzyme used for the nucleic acid amplification. For example, several different types of triazole linkages are known to be compatible with amplification using GoTaq® DNA Polymerase (Promega, Madison, Wisconsin, USA) as described by Shivalingam J. Am. Chem. Soc. 2017, 139, 4, 1575-1583. Furthermore triazol linkages resulting from CuCAAC either single or multiple have been shown to be compatible with DNA synthesis with different polymerases as well as with transcription in bacterial and human cells.

Preferably, after the step of introducing the marker, a catalytic condition is applied to the biological sample. This enables generating the marker in the biological sample, in particular the binding of the first and second reactive group to each other and forming the covalent bond and thereby chemical ligate.

Preferably, the method comprises the following steps, in particular in their ascending order:

(1) optionally, immobilizing the biological sample on a solid support

(2) optionally, encapsulating the biological sample in droplets or hydrogel beads (3) optional ly, applying a fixative (e.g. 4% PFA or glutaraldehyde) to the biological sample

(4) optionally, blocking the unspecific binding sites with a blocking buffer

(5) introducing the first and second marker parts are introduced to the biological sample,

(6) allowing the first and second marker parts to bind to their target analytes, preferably for a sufficient amount of time,

(7) washing away unbound first and second marker parts,

(8) applying the catalytic condition or external stimulus and allowing - preferably for a sufficient amount of time - for the formation of the markers

(9) optionally, performing a second strand synthesis to generate a duplex of the at least a part of the ligate covering at least a part of the at least one first and second oligonucleotide label

(10) optionally, amplifying the at least a part of the ligate covering at least a part of the at least one first and second oligonucleotide label

(11) optionally, removing the amplicon

(12) optionally, fixing the amplicon place

(13) optionally, sequencing the amplicon

(14) optionally, analysing the reads of the amplicon to identify the markers

(15) optionally, labelling the ligate with an ISH probe comprising a detectable label

(16) optionally, reading the sequence of the ligate or the amplicon using a microscope by performing an in situ sequencing-by-synthesis reaction

(17) optionally, binding the marker to microelectrodes of an electrical circuit, integrated circuit or transistor of an electrical readout device to detect the marker

Preferably, prior to and/or after applying the catalytic condition the biological sample is washed in order to remove markers not bound to a target analyte. Removing unbound marker parts by washing prior to application of the catalytic conditions helps to avoid the possibility of a marker forming involving an unbound marker part, which would lead to false-positive results. Depending on the specific format of the assay and required performance of the assay for a given application this may or may not be a strictly necessary step.

Preferably, the first affinity reagent of the marker is configured to bind to a first target analyte of the biological sample and the second affinity reagent of the marker is configured to bind to a second target analyte of the biological sample, and wherein an optical property, in particular fluorescent intensity, of the marker is determined in the generated optical readout in order to determine a proximity, in particular a distance, between the first target analyte and the second target analyte.

In order to use the marker and the method in conjunction with an optically detectable label, said optically detectable label may comprise an ISH probe complementary to a sequence stretch of the ligate of the marker that covers at least a part of the first oligonucleotide label and the second oligonucleotide label. This allows stable binding of said optically detectable label to the marker. Optionally, a part of the ligate may be amplified by an enzymatic amplification method (e.g. LAMP, PCR, RCA) before the detectable label comprising a complementary ISH probe is bound to the marker. This is further detailed with reference to Figure 10. In essentially the same way, the marker may be labelled with non-fluorescent labels like for example non-fluorescent dyes for colorimetric readout, enzymes e.g. HRP for reporter deposition, metal tags for imaging mass cytometry, or gold-particles for electron microscopy. This enables efficiently determining the proximity or distance between the first target analyte and the second target analyte by means of an optical readout device. It is important to note that a first marker part may typically comprise 2 to 8 first oligonucleotide labels and therefore may form covalent bonds with a plurality of second marker parts, wherein the plurality of second marker parts may comprise different second affinity reagents binding to different second target analytes. In this way, the proximity of one first target analyte to n second target analytes may be efficiently determined. In this way protein-protein proximities may be determined as a proxy for protein interaction and complex formation for example.

Preferably, the first affinity reagent of the marker is configured to bind to a first binding site of a target analyte of the biological sample and the second affinity reagent of the marker is configured to bind to a second binding site of the same target analyte of the biological sample. In particular, an optical property, in particular fluorescent intensity, of the marker is determined in the generated optical readout in order to determine the presence and/or quantity, and/or location of the target analyte in the biological sample. The optical property may be determined as described in the paragraph above. This enables determining the presence and/or the location of the target analyte accurately. In particular, the sequence of the ligate, of the marker is determined in the generated sequencing readout in order to determine the presence, and/or quantity, and/or location of the target analyte in the biological sample.

In another aspect, a readout device is provided that is configured to carrying out the method, in particular as described above. The readout device comprises at least one of the following: a field effect transistor (FET), metal-oxide-semiconductor field effect transistor (MOSFET), bipolar junction transistor (BJT) complementary metal-oxide- semiconductor (CMOS), high electron mobility transistor (HEMT), insulated gate bipolar transistor (IGBT) configured to bind at least one marker. By means of the readout device, a target analyte binding to the marker may be detected.

In a further aspect, a labelling kit is provided comprising at least a marker with a first oligonucleotide label and a second oligonucleotide label, the at least one first oligonucleotide label comprising a first reactive group, and the at least one second oligonucleotide label comprising a second reactive group. The kit further comprises moieties or linkers as well as buffers, catalysts, and enzymes required to conjugated the at least one first or second oligonucleotide label to an affinity reagent like an antibody for example. The first reactive group and the second reactive group are configured to bind to the respective other one to form a covalent bond and chemically ligate the at least one first and at least one second oligonucleotide label for example via a triazol linkage, thereby forming a ligate. Further, the at least one first oligonucleotide label is configured to be attached to the first affinity reagent and the at least one second oligonucleotide label is configured to be attached to the second affinity reagent, in particular, in order to form a marker as described above.

The labelling kit further comprises a bifunctional linker configured to conjugate a fourth reactive group to an affinity reagent or protein and, preferably, at least one of the following: a column, a buffer, and a catalyst required for coupling and/or purifying the first or second marker part. The first oligonucleotide label and second oligonucleotide label of the marker are configured to be activated by a third reactive group allowing the covalent conjugation of the first oligonucleotide label and the second oligonucleotide label to a respective affinity reagent or protein. The third reactive group is configured to couple spontaneously to the fourth reactive group.

The labelling kit and the method have the same advantages as the marker. Further, the labelling kit and the method may be supplemented with the features of the marker described in this document, in particular, the features of the dependent claims of the marker.

In the sense of this document the term "affinity reagent" may in particular be an antibody, a single-domain antibody (also known as nanobody), a combination of at least two single-domain antibodies, an aptamer, an oligonucleotide, a morpholino, a PNA complementary to a predetermined RNA, DNA target sequence, a ligand (e.g. a drug or a drug-like molecule), or a toxin, e.g. Phalloidin a toxin that binds to an actin filament. Further an affinity reagent may be a polymeric binder, i.e. any polymeric molecule designed to or found to bind a target analyte with affinity and specificity, comprising a naturally occurring backbone (e.g. peptide, nucleic acid) or non-naturally occurring backbone (e.g. PEG). An affinity reagent may be monovalent or multivalent. An affinity reagent may be a monomer or multimer, like for example a multimerized aptamer. Multimerization may serve to incorporate an avidity effect. In the sense of this document an affinity reagent is configured to bind a target molecule or to an analyte with a certain affinity and specificity such that it can be said that the affinity reagent is substantially specific to the target molecule or predetermined target structure. An affinity reagent used for the method disclosed in this document may bind to several target analytes, i.e. be cross-reactive and bind to its cognate target or "ON-target" as well as so called "OFF- targets". Information about the cross-reactivity of a given affinity reagent may be used to analyse data obtained with the method disclosed in the present invention as described by the European patent application having the application number EP 23190568.8, the complete content thereof is incorporated herein by reference.

In another aspect diagnostic kit is provided, which comprises a marker, at least one cartridge configured to receive a biological sample, and the at least one first marker part of the marker, and the at least one second marker part of the marker. Preferably, the diagnostic kit further comprises a catalyst or catalytic buffer, a wash buffer, and an ISH probe configured to bind to at least one ligate and comprising an optically detectable label.

In a further aspect, a cartridge is provided, preferably a microfluidic cartridge, configured to perform the method, in particular as described above. The cartridge comprises at least one flow cell comprising a transparent window configured for a microscopic observation, at least one waste reservoir, at least one pressure connector configured to be connectable to a - preferably external - pressure source, at least one buffer reservoir connectable to the pressure connector via a channel configured to allow pressurization of the buffer reservoir, and at least one fluid connection configured to allow moving liquids from the buffer reservoir through the flow cell into the waste reservoir. The cartridge preferably comprises at least one unidirectional valve, and preferably at least one well comprising lyophilized solids of the first and second marker part of a marker. The cartridge further comprises at least one catalytic condition buffer reservoir connectable to the pressure connector via a channel configured to allow pressurization of the buffer reservoir, at least one fluid connection configured to allow moving liquids from the catalytic condition buffer reservoir through the flow cell into the waste reservoir, and a readout device, preferably as described above.

Short Description of the Figures

Hereinafter, specific embodiments are described referring to the drawings, wherein:

Figure 1A is a schematic view of a marker with a first marker part and a second marker part,

Figure IB is a schematic view of the marker according to Fig. 1A and ligation oligonucleotide,

Figure 1C is a schematic view of different embodiments of the marker according to Fig. 1,

Figure ID is a schematic view of further embodiments of the marker according to Fig. 1,

Figure 2 is a schematic view of markers according to Fig. 1A and unique molecular identifiers (UMIs),

Figure 3 is a schematic view of a markers comprising one first marker part and a plurality of distinct second marker parts, Figure 4 is a schematic view of the placement of priming sequences relative to the covalent bond,

Figure 5 is a schematic view of second strand synthesis and nucleic acid amplification,

Figure 6 is a schematic view of an "2D array" assay format wherein analytes are immobilized on the (transparent) bottom of a flow cell,

Figure 7 is a schematic view of distinct "3D array" assay formats wherein analytes are immobilized on a polymer and/or within a gel matrix,

Figure 8 is a schematic view of different detection and readout options compatible with the method,

Figure 9 is a schematic view of tissue section and a cyclic detection, readout, inactivation process,

Figure 10 is a schematic view of different in situ hybridization-based detection strategies involving optically detectable labels,

Figure 11 is a schematic view of an electrical readout device based on the marker,

Figure 12 is a schematic view of different cartridge designs, and

Figure 13 is a schematic view of a (single) cell encapsulation workflow, which may be combined with digital PCR-based readout or an in situ hybridizationbased detection strategy. Detailed Description

Figure 1 is a schematic view of a marker 100 with a first marker part 104 and a second marker part 112. The first marker part 104 and the second marker part 112, in particular a first reactive group 110 of a respective first oligonucleotide label 108 and a second reactive group 118 of a second oligonucleotide label 116 of the marker 100, are configured to react with each other to form a covalent bond 120 and thereby a nucleic-acid based ligate 122 in order to generate the marker 100.

The first marker part 104 comprises a first affinity reagent 106 configured to specifically bind to a first target analyte 102a of a biological sample (not shown). Further, the first marker part 104 further comprises the at least one first oligonucleotide label 108 that is attached to the first affinity reagent 106. The at least one first oligonucleotide label 108 comprises the first reactive group 110 configured to react with the second reactive group 118 to form the covalent bond 120 thereby forming the ligate 122.

Similarly, the second marker part 112 comprises a second affinity reagent 114 configured to specifically bind to a second target analyte 102b of the biological sample. Further, the second marker part 112 comprises the at least one second oligonucleotide label 116 that is attached to the second affinity reagent 114. The at least one second oligonucleotide label 116 comprises the second reactive group 118 configured to react with the first reactive group 110 to form the covalent bond 120 thereby forming the ligate 122.

Preferably, the first and second reactive groups 110, 118 are configured to form a triazole linkage. This may for example be the case when the first reactive group 110 is an alkyne and the second reactive group 120 is an azide. The first and second reactive groups 110, 128 may be conjugated to different positions on the ribose backbones of the at least one first and second oligonucleotide labels 108, 116. Alternatively, they may be conjugated to positions on the nucleobases. Several options for placing the reactive groups exists, which will lead to a triazole linkage that is compatible with downstream nucleic acid amplification or transcription. Triazole linkages are therefore particularly preferred forms of the covalent bond in the sense of this document. Other types of chemical linkages resulting from other reactions, i.e. other pairings of first and second reactive groups 110, 118 may be equally suited to perform the method disclosed in this document.

The first and second reactive group 110, 118 may be configured such that they react only with each other in the presence of a catalyst, as is the case with the copperdependent azide-alkyne chemistry (CuAAC), which is a well-known click chemistry. Azide- or alkyne-conjugate oligonucleotides are readily commercially available for example from Biomers (Ulm, Germany). This enables control over the formation of the marker 100 from the first and second marker parts 104, 112 depending on whether the catalytic condition is present. For example, the catalytic condition may be the presence of a particular catalyst or a specific stimulus. Examples of a suitable catalyst include UV-light, pH changes, certain temperatures or ions such as copper, or enzymes. This enables workflows, wherein the marker parts 104, 112 are first allowed to bind to the target analytes 102a, 102b for a sufficient amount of time and preferably to remove unbound marker parts by washing before the catalytic condition is applied. This may help to avoid aggregation of marker parts and reduce false-positive marker formation, i.e. formation of markers from unbound marker parts.

Different samples and/or different classes of affinity reagents and/or target analytes may need distinct conditions and timings for effective binding of the marker parts. In this context a sufficient amount of time is dependent on the affinity reagents used in the marker parts, the class of target analyte, the type, size, and preparation of the biological sample. Likewise, the catalytic condition is applied for a sufficient amount of time, which depends on the aforementioned factors and the choice of the first 110 and second reactive group 118. For example, l-3h at RT may typically suffice to bind proteins in cells or on solid supports. Whereas the binding of proteins in tissues such as tissue sections or more 3- dimensional samples like spheroids or organoids or whole-mounts for example is generally hampered by antibody penetration into the sample and may require several hours, overnight, or multiday incubation. Electrophoresis, the application of pressure or raising the temperature may help to reduce the time that marker parts need to penetrate the sample. When nucleic acid probes are used to detect endogenous nucleic acid targets such as genomic DNA loci or mRNA target analyte binding may typically require several hours to overnight incubation.

The first and second reactive group may be configured such that they react spontaneously with each other, as is the case with strain-promoted azide-alkyne cycloaddition (SpAAC). SpAAC may be performed for example using azide- and DBCO- conjugated oligonucleotides that are commercially available for example from Biomers (Ulm, Germany).

The first and second reactive groups may be 3'OH and 5'phosphate groups and the catalyst may be a ligase.

The chemical ligation may be performed in the absence of a template or absence of duplex formation between the at least one first and second oligonucleotide. Natural ligation does occur under such circumstances, but only with extremely low efficiency.

Both chemical and natural ligation may be facilitated by holding the ends of the at least one first and second oligonucleotide in place using a suitably configured ligation oligonucleotide 124. Such a ligation oligonucleotide 124 comprises complementary sequence stretches 126a, 128a and binds to respective complementary sequences 126b, 128b of the at least one first and second oligonucleotide 108, 116 as shown in Figure IB. The at least one first and second oligonucleotide 108, 116 may be covalently coupled 130 to the respective affinity reagents 106, 114 of the respective marker parts 104, 112. This may also be referred to as direct coupling 130 as shown in Figure 1C. To this end bifunctional linkers may be used such as a NHS-PEG-azide. Suitable homo- or heterobifunctional linkers can be procured from Creative PEG Works (Chapel Hill, USA) and Broadpharm (San Diego, CA). Such linkers can be procured from multiple vendors and may be conjugate to an antibody for example to lysine residues via N-hydroxy succinimide coupling chemistry (NHS) for example, which generally yields degrees of labelling in the range of 4-8 and achieves good efficiencies in the coupling reactions. In a subsequent step the now "activated" antibody may be coupled with for example a "DBCO-activated" first 108 or second oligonucleotide 116, which will lead to a spontaneous coupling of the oligonucleotide to the antibody.

A number of other suitable chemistries exist and are known to someone skilled in the art. For example, Abeam (Cambridge, UK) offers antibody oligonucleotide conjugation kits. Likewise, there are site-specific chemistries like SiteClick™ THERMO (USA), and GlyGlick® Genovis (Kaplinge, Sweden). The latter offer precise control overthe number of oligonucleotides that are conjugated, which may be desirable in some situations. The choice of the coupling chemistry may also depend on the desired degree of labelling, i.e. whether a single, 2, or more oligonucleotide labels shall be coupled to the affinity reagent.

Alternatively, the coupling may be indirect and mediated via hybridization by means of an attachment oligonucleotide 132 comprising a suitable barcode sequence complementary to a barcode 134 on the at least one first or second oligonucleotide label 108, 116 or, alternatively, via a first 136 and second secondary affinity reagentl38, which in turn may be directly or indirectly connected to the at least one first 108 and second 116 oligonucleotide label respectively. In particular, the first and second affinity reagents 106, 114 may each comprise the attachment oligonucleotide 132. The attachment oligonucleotides 132 may be attached to the respective affinity reagent 106, 114 covalently, or via a biotinstreptavidin linker, orvia another affinity interactor pairfor example high affinity guest molecule-host pair. The at least one first and second oligonucleotide labels 108, 116 may also be conjugated to an affinity interactor and be used in conjunction with the flexible connector like the one described in EP 4 092 414 Al and/or EP 4 220 162 A2 the contents of which are incorporated herein by reference, which can be used to quickly generate a multitude of marker parts in a process that can be automated and is thus suited for high-throughput screening. Such a flexible connector may be based on a nanobody 136 for example or may be an aptamer-based flexible connector 138.

The at least one first and second oligonucleotide labels 108, 116 may be attached to the respective attachment oligonucleotide 132 via hybridisation. For example, each at least one first and second oligonucleotides label 108, 116 may comprise an oligonucleotide with the attachment sequence 134 complementary to at least a part of the sequence of the respective attachment oligonucleotide 132. In particular, the at least one first oligonucleotide label 108 and the at least one second oligonucleotide label 116 and the respective attachment oligonucleotides 132 may have unique sequences that are complementary and that enable the specific attachment of the at least one first oligonucleotide label 108 and the at least one second oligonucleotide label 116 to the respective affinity reagent 106, 114. This enables efficient assembly of the marker parts 104, 112.

The affinity reagent 106, 114 are antibodies, for example. Alternatively, the affinity reagents 106, 114 may be one of the following: an antibody, a Fab fragment, a nanobody, a polymeric binder, an aptamer, an aptabody, a nucleic acid probes, a toxin, a drug, a drug-like molecule. The target analytes 102a, 102b may be different proteins of the biological sample, for example. Alternatively, the target analytes 102a, 102b may be one of the following: a protein, a peptide, a hormone, a neurotransmitter, a metabolite, a vitamin, a metal, a nucleic acid sequence, an mRNA, a siRNA, a rRNA, genomic DNA, mitochondrial DNA, a sugar, a fat, a lipid, a phospholipid, a sterol.

The at least one first and second oligonucleotide 108, 116 may be short in the range of 4-20nt. Alternatively, they may be longer and comprise additional sequence elements such as sites for priming a second strand synthesis or sites for primer binding for a subsequent nucleic acid amplification of the ligate 122 or parts thereof.

As shown in Figure ID several different configurations of markers disclosed in this document may be used for a variety of assays. For example, the first marker part 104a, in particular the respective affinity reagent, may be configured to bind to a first target analyte 102a and the second marker part 112a, in particular the respective affinity reagent, may be configured to bind to second target analyte 102b. This embodiment of the present marker and method may be used to assess the proximity of the target analytes 102a, 102b as a proxy for molecular interactions, i.e. a proximity-based interaction assay 142. In other words, the marker 100 may therefore indicate that the first target analyte 102a and the second target analyte 102b are in proximity, when the marker 100 forms from the marker parts 104a, 112a. The detection of marker formation may be performed in situ of the biological sample or in the supernatant. The detection of the marker formation may resolve the individual marker or it may analyse marker formation in bulk or on the level of the population of markers that are formed in the sample.

The marker 100 may also be used to improve the specificity with which a particular target analyte can be detected, in what may be called proximity-based high specificity assays 144. In this case two affinity reagents are configured to bind the same target analyte 102c at different positions 162a, 162b, i.e. different sequences or different epitopes or surface areas. The two affinity reagents are comprised by respective first and second marker parts 104b, 112b. The resulting increase in specificity is of particular importance for multiplex or high-plex assays, which are severely limited by the cross-reactivity of antibodies and other affinity reagents. For example, when a liquid biopsy is analyzed the level of expression of certain biomarkers, the presence or absence of rare circulating tumor cells, or exosomes may be of great interest to diagnose a certain disease or to define a best course of action for a treatment. In this case, the level of expression of a given biomarker may be correlated to the number of reads obtained from amplica of the corresponding ligate 122 in a next generation sequencing run. Likewise, in the case of diagnosing an infection with a virus, it may be very important to have high confidence in the ability of the test to differentiate a pathogenic strain from a harmless or non-pathogenic one. The embodiment described above and indicated by reference sign 144 is particularly relevant to these use cases.

The marker and method may further be used to interrogate protein-DNA, or protein- RNA, or nucleic acid-non-nucleic acid interactions as shown in the embodiment indicated by reference sign 146. Most typically, this may be the interactions of transcriptions factors 148, polymerase, and histones for example with specific DNA sequences or target sequences that may be part of a genetic locus or target. The method may then be used to test the post-translational modification of genomic DNA (e.g. methylation) or histones at specific loci (e.g. phosphorylation, ubiquitination, methylation). This is of great relevance in the context of epigenetic regulation. Using a plurality of suitably configured markers the binding of a given transcription factor 148 to certain gene regulatory elements 150 may be assessed and correlated to certain histone modifications. The marker may for example form from a first marker part 104c comprising the ISH probe 154a as first affinity reagent binding to target sequence 164 and a second marker part 112c comprising an antibody binding to transcription factor 148, that is itself binding the gene regulatory element 150, which is part of the genetic locus that is the target analyte 102d. In this regard, it is important to point out that the marker and method can also be used to interrogate multiple interactions in a complex. This is further described in Figure 3. In this way it would be possible to detect the binding of a transcription to a genetic locus, measure a post-translational modification on the same transcription molecule and determine the resulting epigenetic make-up at the said genetic locus in a single assay.

Afurther embodiment is particularly preferred, as it holds great value forthe detection of mRNA using in situ hybridization especially when a large number of mRNA molecules shall be analysed. This embodiment is referred to as proximity-based nucleic acid detection assay indicated by reference sign 152.

Detecting a large number such for example several hundred or thousands of mRNA molecules is hampered by cross-hybridization of nucleic acid probes, i.e. their binding to OFF-target nucleic acid stretches. In fact, it is quite difficult to design in situ probes that do not have such OFF-targets. As the present method generates markers from paired binding events, this can also be used to improve mRNA detection. In this case first and second marker parts comprise the first 154b and second in situ probes 156 as affinity reagents. A marker thus only forms when both first and second probes 154b, 156 bind to the same target mRNA 102e, in particular complementary binding sites 158, 160. This effectively reduces the false-positives that would otherwise result from cross-hybridization. More detail on the use of the method for RNA detection and different readout strategies is provided in Figure 8.

As shown in Figure 2, the at least one first and second oligonucleotide 108, 116 of marker 200 may comprise unique molecular identifiers (UMIs) 206a, 206b, which are unique sequence stretches that identify the affinity reagent 106, 114 of the respective marker part 104, 112 and thereby the respective target analyte 102a, 102b.

The UMIa 206a for example identifies or is assigned to target analyte a 102a, whereas the UMIt 206c for example identifies or is assigned to target analyte 1 102f. This concept is Figure 2 exemplifies this concept with markers 202, 204, each comprising a first marker part comprising an affinity reagent 106 specific to target analyte a 102a. The marker 200 for detecting the proximity of analytes a 102a and k 102b, marker 202 for detecting the proximity of analytes a 102a and j 102f, and a marker 204 for detecting the proximity of analytes a 102a and 1 102g are shown as an example. It is also important to remark that markers 200, 202, 204 may alternatively comprise marker parts comprising affinity reagents of distinct classes. For example, a first marker part may comprise an antibody and second marker part may comprise an aptamer. Alternatively, or in addition a first marker part may comprise a nanobody and a second marker part may comprise a drug as affinity reagents.

As mentioned before the marker and method is particularly suited to not only detect the presence of pairs of target analytes, epitopes, nucleic acid targets, but also to detect n:l relationships. This is shown schematically in Figure 3. A target analyte a 102a is shown bound by the first marker part 301, which comprises 7 first oligonucleotide labels 108 each comprising a UMIa 206a and a first reactive group 110. Within the proximity, i.e. the spatial stringency limit, are also target analytes t 300, g 302, I 304 each specifically bound by a second affinity reagent of a respective second marker part 306, 308, 310. The second marker parts 306, 308, 310 further comprise at least one second oligonucleotide label 313, 314, 316 further comprising a UMI specific to the respective target analyte 300, 302, 304. In this case the marker 312 resulting from the formation of the covalent bond comprises 4 covalent bonds belonging to 3 distinct ligates a:t, a:h, and a:j. The marker 312 thereby detects the proximity of target analyte a 102a, t 300, h 302, and j 304.

As stated before the target analytes 102a-g, 300, 302, 304 may be present in solution, in a suspension, may be immobilized on a solid support, gel or polymer or may be expressed on the surface of cells or within cells in a cell culture or a tissue sample. The method may thus be used to analyse molecular neighbourhoods as well as cellular neighbourhoods. The length of the at least one first and second oligonucleotide labels may be adapted to tune the spatial stringency of the assay to the desired point and range form 4-20, 20-50, 50-200, 200-1,000 nt for example.

With reference to Figure 4, top panel, the at least one first and second oligonucleotide labels 108, 116 may comprise a primer binding site for second strand DNA synthesis 400, primer binding sites for a forward and reverse primer 402a, 402b for subsequent polymerase chain reaction, as well as preferably a primer binding site for the sequencing 404. Other amplification methodologies like for example LAMP may require additional sequence elements, which can be easily incorporated.

A part of the ligate 122 may be amplified by an optional nucleic acid amplification step indicated in Fig. 4 by reference sign 410. Such amplification may be performed using polymerase chain reaction (PCR), rolling circle amplification (RCA), loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA), or bridge amplification. The choice of the amplification method depends on numerous factors and the ideal amplification strategy is sample-, assay-, and applicationdependent choice. When for examples liquid samples are analysed it may be desirable to perform the amplification via PCR in a PCR cycler using typical cycling protocols and temperatures for denaturing, annealing, and synthesis. In this case the resulting amplica are freely diffusible in the supernatant, which may be taken into an NGS run for the readout by sequencing. When the downstream detection and readout shall be performed in a tissue section for example, i.e. in situ, then this precludes the use of standard PCR as the conditions would be too harsh and lead to the deterioration of the sample and build up of strong autofluorescence. In this case RCA or alternatively LAMP or RPA are superior choices.

In cases where the detectable signal is an optical signal and low abundance targets or even single molecules shall be analyzed or as well as in cases where the readout may be performed at high speeds, using low NA objectives, or less sensitive, cheaper detectors/cameras, it may be desirable to amplify the signal. Amplification may be important and may happen via nucleic acid amplification of the linker using one methods mentioned. Alternatively, or in addition the optically detectable label connected to the ISH probe used for detection may comprise a secondary amplification mechanism based on one of the following: dendrimeric structures, hybridization chain reaction (HCR), catalyzed-reporter deposition.

Typically, the amplification will be configured such that the resulting amplicon or amplica 406 include(s) the UMIs of both the at least one first and second oligonucleotide label. Depending on the choice of downstream detection strategy and readout modality, it may be desirable to generate amplica which are diffusible or to generate amplica which are less mobile or even conjugated to the marker. In the former case, PCR may be well suited and in this case the supernatant may be taken into a next generation sequencing run for readout.

A schematic of an analysed NGS readout is shown in the bottom panel of Figure 4. In this hypothetical example the number of reads of a given UMI pair e.g. a:a, a:c, a:k is shown in a histogram comparing healthy control subjects with a cohort of patients. In this hypothetical example the presence of a:k and the reduction in the number of a:c reads points to an increase in the interaction of target analyte a with target analyte k and a decrease of the interaction of target analyte a with target analyte c. This exemplifies what may be called an "interactional biomarker" 408 and how the method can be used to detect for known and screen for unknown interactional biomarkers. As protein-protein interactions are key to biological function, such interactional biomarkers hold great promise for the stratification of patient populations and are expected to have higher predictive value than simple biomarker expression levels. Such interactional biomarkers may of course also be correlated to further multi-omic data and to phenotypic characterization.

Figure 5 shows the second strand synthesis and nucleic acid amplification steps in relation to a marker 500. If amplification is performed in situ, like for example in a tissue section it may be advantageous to use an amplification method such as RCA or LAMP that generates a large concatenate of amplicon. This reduces the mobility of the amplicon and ensures that the spatial relationships of the target analytes, markers and other structures of the sample are maintained. If a liquid sample is analysed, immobilization of the amplicon, if desired, may be a result of bridge amplification or alternatively may be achieved in a different way. For example, an anchoring sequence may be incorporated into the at least one first or second oligonucleotide, which can later be used to anchor the amplicon to a complementary anchoring oligonucleotide, which may be present on the bottom of the flow cell, on the surface of the solid support or on present on a gel/polymer-matrix brought into or to the sample.

Figure 6 is a schematic showing a 2D array assay format and steps of a corresponding method which may be used for the analysis of a liquid biological sample, like a liquid biopsy (e.g. serum) or a lysate. In this case it may be desirable to immobilize the target analytes on a solid support 600. In Figure 6 this is a planar support, like a glass slide, coverslip, or (transparent) bottom of a flow cells. Preferably, the planar support has optical quality and is configured to allow high numerical aperture imaging. The planar support in the example is functionalized with attachment sites 602 for example one of biotin and streptavidin or one of cucu bit [7] u ril and adamantane-/ferrocence-derived high affinity guest and the target analytes are conjugated to a corresponding attachment group 604 for example the other one of one of biotin and streptavidin or the other one of cucubit[7]uril and adamantane-/ferrocence-derived high affinity guest. Alternatively, the attachment site 602 and attachment group 604 may be reactive groups configured to mediate a covalent coupling between the analyte and the solid support. In this case the care has to be taken to use an orthogonal chemistry to the one used for generating the covalent bond of the marker.

Figure 6 further shows the steps of immobilizing analytes, binding first marker parts 610 and second marker parts 608 of a marker 614, removing unbound marker parts, applying the catalytic condition (i.e. "chemical ligation"), and the optional step of amplification of the ligate or parts thereof. The target analytes 612 in this case are shown interspersed between high abundance proteins which are frequently found in serum samples. This may for example be proteins like albumins, fibronectin, or coagulation factors. These high abundance proteins may be present in concentrations to up to 10¹² of times higher than the low abundance targets, which may be of highest interest. For this reason, it may be desirable to not immobilize all proteins, but only the target analytes 612 of interest. This can be done by including a capturing step, wherein one of the first and second marker parts 608, 610 is immobilized on a microsphere, a nanoparticle or a NanoArray as described in WO 2022/207832 Al, the content of which is incorporated herein by reference, or on a polymer or within a gellike matrix as shown in Figure 7. Such 2D arrays combine well with widefield (WF) microscope-based readouts including WF deconvolution, structured illumination super resolution, and TIRF microscopes. Likewise, the are well suited for single molecule localization-based detection strategies, such as for example DNA-PAINT and related technologies.

The assay formats in Figure 7 may be referred to as 3D arrays and may be particularly suited for fast volumetric readout systems like OPM/SCAPE microscopes, light field microscopes, spinning disk imaging systems or confocal microscopes. Target analytes may for example be immobilized by simple embedding in a hydrogel 700 or may be attached to a gel-matrix or polymer-backbone 702 in a flow cell 704. Protein A or G may for example be used as an attachment agent 706 configured to capture an antibody of a first marker part 610. In this case the second marker part 608 would typically be introduced following to capturing the first marker part 610 with the target analyte 612 and washing out the unbound marker parts and other components of the biological sample incl. high abundance proteins. The second marker part 608 would be introduced and allowed to bind in a subsequent step before the catalytic condition is applied. These different embodiments may each be combined with different detection strategies as shown in Figure 8 including the detection of the amplica 406 resulting from the amplification 410 of the ligate or the unamplified ligate directly using

• in situ hybridization (ISH) like for example fluorescent in situ hybridization (FISH), catalyzed-reporter deposition CARD-ISH,

• in situ sequencing via sequencing-by-synthesis (SBS)

For example the detection of a marker 806 may be performed by either direct detection of a ligate 122 with an ISH probe 800 comprising a detectable label 802, wherein the ISH probe 800 is configured to bind to a sequence stretch of the ligate 122 comprising at least a part of the at least one first oligonucleotide 108, which may be referred to as a first half binding site 808 of the binding site 812, and at least a part of the at least one second oligonucleotide 116, which may be referred to as a second half binding site 810 of the binding site 812. The ISH probe 800 may be configured such that the association with binding site 812 is substantially stable and the association with either half binding site 808, 810 is only transient. This may be the generally preferred way of performing detection of a marker by binding an ISH probe 800 with a detectable label in both cases (A) "ISH detection of a ligate" (indicated by reference sign 814) and (B) "ISH detection of an amplicon" (indicated by reference sign 816). Similarly, when an amplicon 406 is detected with an ISH probe 800 comprising a detectable label 802 the ISH probe 800 is configured to bind to a binding site 812 comprising a first and second half binding site 808, 810. Thus, the stable binding of the ISH probe 800 to the binding site 812 faithfully detects the respective marker. In some cases the ISH probe 800 may be configured to bind to the binding site 812 transiently. For example, when single RNA molecules shall be imaged (compare Figure 10) transient association of an ISH probe 800 comprising a fluorescent dyes 802 may lead to blinking, which allows super-resolved detection providing resolution in the range of few nanometers laterally, while also opening the way to high-plex encoding and detection for example as described in the international patent application with the application number PCT/EP2023/075470, the content of which is incorporated herein by reference.

Alternatively, the ligate 122 may be amplified and the amplicon 406 separated from the immobilized target analytes 102, i.e. supernatant or flow-through for example. It is clear that in the sense of this document a ligate may refer to a plurality of distinct types and to a plurality of ligates of the same as well as to a plurality of ligates of distinct types. The same may be said about the amplicon, which in the context of this document may refer to single amplicon molecule, or a plurality of amplica of the same sequence or of a plurality of sequences.

In the latter case performing a next generation sequencing (NGS) of the supernatant for example is a powerful readout strategy that offers great depth and coverage or plexity. While high plexity is a native strength of NGS-based readout, it is important to note that also imaging-based readouts, are capable of offering high-plex readout. For example combinatorial labelling may be used in particular with the method disclosed in the European patent application having the application number EP23192184.2 and/or WO 2022/242887 Al and WO 2022/242895 Al, the contents of which are incorporated herein by reference.

It is important to note that the combination of the method with ISH-based detection renders the assay deterministic as the ISH probe sequences need to configured to bind to the ligates 122 or their amplika 406. In this case the ISH probes 800 further comprise an optically detectable label 802 or an enzyme configured to generate an optically detectable label through a (e.g. CARD-FISH).

An optically detectable label may for example comprise a dye to allow detection of a marker using brightfield microscopy, a fluorescent dye for fluorescence microscopy, cytometry, or plate reading, an enzyme like HRP to catalyze the deposition of a dye, which may be fluorescent or non-fluorescent. An optically detectable label may also be a gold particle or any other metal or non-metal particle, which can be optically detected by light or electron microscopy. A fluorescent dye may for example be AlexFluor488, ATTO 390, ATTO 425, ATTO 465, ATTO 488, ATTO 565, ATTO 590, ATTO 643, ATTO 647N, ATTO 700, a Bodipy dye, polymer dye, a QDot, a PDot, a SMILE (dyemacrocycle co-crystal). An optically detectable label may comprise a nucleic acid backbone and multiple dyes. An optically detectable label may comprise a tag or hapten configured to be detected by a secondary affinity reagent, like for example a secondary dye-conjugated antibody.

If detection is based on sequencing, the assay remains unbiased and explorative, i.e. allows the detection of markers that mark unknown proximity pairs like for example unknown protein-protein interactions. Sequencing may be performed in situ by sequencing-by-synthesis 804 as described in the European patent application having the application number EP 23161816.6, the content of which is incorporated herein by reference, using a microscope as a readout device, which will maintain the spatial relationships of the analytes and markers in the biological sample. Alternatively, sequencing may be performed in bulk using NGS for example by sequencing supernatant or may be performed following to microfluidic encapsulation of single cells. In the latter case the information obtained from the markers may be related back to single cells similar to scRNAseq, this would allow single profiling of protein-protein or protein-nucleic acid interaction for example.

Figure 9 is a schematic showing a tissue section 900, e.g. an FFPE tissue section, and a cyclic staining, imaging, inactivation process 902. This embodiment of the present invention is used for spatial analysis of for example protein-protein interaction in the tissue context. In this case, the user may desire to analyse a large number of interactions, which significantly exceeds the single round plex that a microscope typically provides, e.g. 5-10 channels. Say for example the user wanted to test 200 interactions or perform an unbiased screen involving 200 target analytes, wherein the number of interactions that may occur may be in the range of several hundreds to thousands. In this case all markers may be generated in a first step and subsequently a cyclic process may be performed to stain the markers with optically detectable labels, image the labels, inactivate labels attached to the markers and repeat the process until all markers 904 1,1 to n,m have been imaged. Staining in this case may be performed directly on the ligates or on the amplica of the ligates. Staining may be achieved using for example ISH probes comprising a detectable label 802a, 802b like for example fluorescent dyes, fluorescent structures, polyynes or other Raman labels, combinations of dyes, or Raman labels or even hybrids of fluorescent and Raman labels. Inactivation may be performed enzymatically using restriction enzymes or by using cleavage sites such as UV orTCEP cleavable sites, which can be incorporated into the labels. This enables iterative staining methods using the marker 100. Under certain circumstances it may be desirable to completely remove the ligates of all markers for example, when a subsequent staining shall be performed. In this case, a nuclease like Dnase I may be used to remove all markers and blank the sample.

In a further embodiment the detection and readout may be performed by quantitative PCR in a suitable readout device.

In a further embodiment, the samples may be (single) cells or cell-free lysates that are encapsulated into droplets and the readout is performed using digital PCR.

Figure 10 shows different ISH-based detection modes and compares binding and readout schematically for a cognate target analyte versus an OFF-target. In the case of nucleic acid targets 1006, which may be for example an mRNA of a given gene for example pax7 mRNA or a genomic locus for example the pax7 promoter region the method may be used with markers that either completely nucleic acid-based or ISH probe-based markers 1004, i.e. first and second affinity reagents are a first 1008 and second ISH probe 1010, which are configured to bind to a first 1012 and second 1014 nucleic acid target sequence, respectively. Typically, such a target sequence may comprise in the range of 9-40nt, whereas the entire mRNA or genomic locus may encompass several kb in extreme cases, like the pax7 promoter region >120kb. Due to 3D architecture of the genome target sequences as far distant as >120kb may found in close spatial proximity. As such the method is of great use to the study of the 3D architecture of the genome and how it relates to epigenetic modifications and gene regulation.

For example, when standard ISH like FISH is performed with probes binding directly to the target, they may label the target but also OFF-targets. If a time series of such a binding is recorded it will show a continuous signal in both cases, i.e. the target to OFF- target binding cannot be differentiated. In other words, in a smFISH experiment for example, a fluorescent spot corresponds to a single RNA molecule. If only one probe is used for labelling, than a target and OFF-target may generated fluorescent spots that are indiscernible. The same would be true, when landing probes would be used and the labelled probes are bound to the landing probes.

The present invention allows to significantly improve upon this problem in two ways: First, it is possible to use at least two probes for two target sequences on the same mRNA or genomics locus as part of the first and second marker part. In this case a marker is only formed, when both probes bind to target mRNA or target genomic locus. An OFF-target would only be bound by either the first or second marker part comprising either the at least one first oligonucleotide label or at least one second oligonucleotide label. This means that a complete binding site for the landing probe or direct ISH probe is not formed. The resulting association with a half binding site 1000 would in this case not be stable but rather transient. For example a half binding may comprise 8-10nt and a ligated complete binding site 16-20nt. In this case a time series would show some spots that are persistently fluorescing corresponding to the targets and some that are blinking 1002 due to the repeated association and dissociation of the label, which would correspond to the OFF-targets. Alternatively, OFF-target binding may be removed or strongly reduced by washing. Further the detection of a marker used to mark mRNA or genomic targets with very high specificity may employ an amplification as described above. In this case the amplicon will only form when a marker is formed, but not from either a first or second marker part as either the forward or reverse priming site would be missing. In this case only the targets that are bound by both first and second marker parts, will be marked by markers and form amplicons during the amplification step, which may then be detected by an ISH method (e.g. FISH, CARD-FISH, smFISH, combinatorial FISH labelling) or by in situ sequencing using SBS (compare also Figure 8).

In other words, for in situ hybridization (ISH)-based detection ISH probes are used that either comprise a detectable label for example an enzyme, a hapten, a tag, a metal tag, a dye, a fluorescent dye, a fluorescent label (e.g. QDot, PDot, SMILE) or are configured to be bound by at least one secondary ISH probe that comprises a detectable label. In the latter case the first ISH probe that is configured to bind to said ligated complete binding site that is formed due to the formation of the covalent bond of the marker

Figure 11 is a schematic showing a method and readout device. In this case the first and second marker part each comprise nucleic acid-based affinity reagents for example two aptamers, two nucleic acid probes, an aptamer and a nucleic acid probe. The example shows an embodiment, wherein target analytes 1101 are immobilized. Further this example shows an aptamer-based marker 1112, wherein both the first and second affinity reagent are aptamers. The embodiment shown in Figure 11 exploits the electrical properties of DNA and combines the marker and method with an electrical readout device, which may be a circuit, integrated circuit (IC), a transistor or CMOS. Further the electrical readout device may be based on one of the following: a field effect transistor (FET), metal-oxide-semiconductor field effect transistor (MOSFET), bipolar junction transistor (BJT) complementary metal-oxide- semiconductor (CMOS), high electron mobility transistor (HEMT), insulated gate bipolar transistor (IGBT). In this embodiment the marker is detected and readout by measuring one or a combination of the following electrical properties: voltage, current, resistance, conductance, impedance, reactance, capacitance, inductance, power, susceptance, admittance, frequency, phase angle, quality factor, noise figure, gain.

In the embodiment shown in Figure 11 both nucleic acid-base affinity reagents 1100a, 1100b comprise an additional contact oligonucleotide 1102a and 1102b configured to bind to complementary contact oligonucleotides 1104a and 1104b that are configured to establish a connection between elements of an electrical circuit 1106, integrated circuit, or transistor 1108 and the marker parts. This connection maybe via hybridization or maybe established via ligation using a ligase. In the latter case a third oligonucleotide may be used to bind to both contact oligonucleotides to facilitate ligation.

For example a chip may contain a high number of FETs wherein each gate voltage source and gate are conjugated to contact oligonucleotides configured to bind to complementary contact oligonucleotides that are conjugated to the first and second marker part respectively. As a marker provides a continuous nucleic acid backbone, the binding of the marker to the contact oligonucleotides closes the circuit. Most typically the gate voltage source and the gate may be conjugated to a plurality of contact oligonucleotides. In the absence of a marker the gate voltage source and gate are disconnected as more and more marker bind to the gate voltage source and gate via the contact oligonucleotide duplexes the conductivity increases and the gate voltage changes, which leads a measurable response of the FET. This is a different mechanism of operation as compared to aptamer-FETs of the prior art which operate by binding of an aptamer to the target. In the present case, it is not the binding of the target analyte to the gate-conjugated aptamer that is detected, but the binding of the marker to the transistor. This opens the way to inexpensive readout devices for the markers described in this disclosure comprising any of the following field effect transistor (FET), metal-oxide-semiconductor field effect transistor (MOSFET), bipolar junction transistor (BJT) complementary metal-oxide-semiconductor (CMOS), high electron mobility transistor (HEMT), insulated gate bipolar transistor (IGBT).

The advantage of such readout device is that the signal is electrical and depends on both the quality and quantity of the markers. In other words such devices may be able to assess millions of distinct markers on a single chip. For example a modern CMOS chip may have more than 4 Megapixels, which could be used to build a readout device that measures 4 million markers in a single "shot".

Contact oligonucleotides may in situ synthesized onto a chip or fixed to the correct pixel in a sequential process as described in Caillat et al. 2000 Sensors and actuators, B; Volume 61, Issues 1-3, 14 December 1999, Pages 154-162. Two-photon lithography using a confocal microscope may likewise be used for either in situ synthesis or fixation of contact oligonucleotides by localized deprotection. In addition it may be possible to spot nanodrops using acoustic droplet ejection onto chips with sufficient accuracy.

The advantage of such a chip-based readout device may be low cost, high sensitivity, high speed readout. In combination with the advantages of the marker and method disclosed in this document, this may also open the way for highly specific, cost- effective diagnostic testing at point-of-care or even in the field or at home by the user. A chip and plurality of first and second marker parts may be configured such that they test a panel of common respiratory viruses.

Importantly, the contact oligonucleotide on the chip may comprise a nuclease- resistant backbone, which will render them more stable and allow regeneration of the chip by adding nuclease to digest the markers or marker part-conjugated contact oligonucleotides. In this case, a user may have a chip-based device at home and procure kits of marker parts that comprise panels against certain indication groups, like for example respiratory infections, skin diseases, sexually transmitted diseases, microbiome testing panels. Following to taking a (mouth) swab the sample would be added the marker parts, which be stored lyophilized for extended periods of time.

A cartridge 1200 for use with the method is schematically depicted in Figure 12 in a top view and a side view. The cartridge 1200 may comprise at least one buffer reservoir 1204 that is in communication with a pressure connector 1206. Such pressure connectors 1206 may be used to allow air pressure-controlled operation 1208 of the cartridge like for example they may be used to move liquids or control pressure-controlled valves. The cartridge may comprise several buffer reservoirs 1204, in particular for wash buffer and a catalytic condition buffer, which may for example contain copper ions. Buffer reservoirs may be in direct communication with the flow cell 1210. Different embodiments of the cartridge can be used to adapt to different readout devices like a microscope, a NGS device, or the electrical readout device described in Figure 11. For example this may be a closed flow cell cartridge 1212 with an optical-grade window configured for microscopic imaging, an open flow cell 1214 cartridge configured to form a leak-tight assembly with a glass slide 1216 comprising suitable holding devices 1218, an open flow cell cartridge 1220 configured to form a leak-tight assembly with an electrical readout device 1222, like a readout device comprising a chip 1224 like for example a functionalized CMOS chip. The cartridge may be pressed against the electrical readout device by a clamping or piston mechanism applying the required force 1226 to generate the leak-tight assembly.

The cartridge 1200 may further comprise at least one well 1228 comprising lyophilized solids 1230 of marker parts. In addition, the cartridge 1200 may further comprise additional wells with further detection reagents 1232 such as for example lyophilized solids of ISH probes comprising optically detectable labels.

The top middle panel of Figure 12 shows a cartridge for microscopic readout in a side view and a microscope objective 1228, which can be used to image the contents of the flow cell through the bottom, which may be glass of around 170pm thickness or a polymer like COC, polystyrene, or PMMA.

The lower middle panel of Figure 12 provides examples of ways in which a flow cell or in general a solid support may be functionalized to bind marker parts or analytes in an unspecific or specific fashion. For example, the panel on the left depicts poly-L-lysine coating, the middle panel shows analyte captures via affinity interactions or covalent coupling, the panel on the right the capture of a first or second marker part using a secondary affinity reagent.

It is important to note that during the readout on the electrical device the marker may or may not still be bound to the target analyte or target analyte pair. In some cases, it may be desirable and improve the signal, when the target analyte(s) are released from the markers prior to hybridization to the chip. In this case, release may be achieved by heating which unfolds the aptamers or unbinds the nucleic acid probes from their target analytes.

The markers, methods, and readout device described above may be applied in a method for analysing biological samples. Such biological samples may be for example without any intent to be limiting: monolayer cell cultures, 3D cell cultures, spheroids, organoids, organ-on-a-chip and microphysiological systems samples, tissue biopsies (whole-mount) or tissue sections (cryosections, FFPE sections), cleared samples, embryos, water samples, environmental samples, as well as liquid biopsies and liquid samples prepared from any of the aforementioned.

Specifically, the markers may be introduced into the biological sample in a first step. Each marker may be specific to a particular target analyte or a set of two target analytes. Further, multiple copies of each marker may be introduced. Subsequently, an optical readout, NGS-based readout, cytometry-based readout, digital PCR-based, or qPCR-based readout of the biological sample with the markers may be generated. This enables detecting the target analytes in the biological sample, in particular, their presence or location in the biological sample.

In a preferred embodiment of the method, the markers 100, 404, 500 are introduced into the biological sample under a non-catalytic condition, which do not cause or enable the binding of the first and second reactive group to each other. In a subsequent step, the catalytic condition is applied to the markers 100. This enables formation of the covalent bond and thereby the ligate, in case the at least one first and second oligonucleotide label are in close proximity. In a subsequent step, the readout may be generated.

As described above, the optical readout may comprise optical signals from at least one detectable label. This may be an indication of the presence of a single target analyte or a measure of the proximity of two target analytes.

Figure 13 is a schematic showing steps of analysing an encapsulated biological sample 13OO.This may be done in the case of single cell analysis or when digital PCR shall be performed. A microfluidic chip may be used to encapsulate cells or cell-free samples into droplets or hydrogel beads 1301. When hydrogel beads are used the method as disclosed in WO 2022/207125 Al, the content of which is incorporated herein by reference, may be used for indexing of the hydrogel beads, which serve as sample containers. Other ways of encapsulation such emulsification may be equally suited. The analysis of large populations of single cells is important in many areas as interesting or desired phenotypes are often rare. For example, a clone 1300 secreting a certain antibody or protein 1302 may be present in frequencies ranging from 1 in 10,000 cells to 1 in 10,000,000 cells. In some cases, it may be important to find such a clone and isolate it live, in other cases it suffices to isolate the clone and a specific genetic sequence. This may for example be the case in antibody discovery. In the context of cell therapy and immune cel Is, it may be desirable to find super killer cells, which are particular effective in killing tumor cells for example. Such cells may be T-cells or NK-cells that secrete specific combinations of cytokines. The present invention may in this case be used to measure such cytokine profiles with very high specificity.

Further, in the context of cell therapy monitoring, it is very important to regularly take blood samples and monitor therapy progress and the development of the cellular therapeutic. The present invention may in this case be used to characterize the clone by secretome profiling with very high specificity. This may be performed in bulk and readout by an NGS device or performed on the single cell level and readout by digital PCR, microscopy, or cytometry.

Further, in the context of translational research and patient stratification in particular with respect to immuno-oncology the present invention allows the faithful detection of molecular interactions and post-translational modifications as well determination of the epigenetic markup at key genetic loci to be analysed. This may be of great utility for basic, translational, and clinical research, but also of great use in diagnostic procedures or companion diagnostics.

Identical or similarly acting elements are designated with the same reference signs in all Figures. 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

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 device corresponds to a method step or a feature of a method step.

Analogously, 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 signs

100, 200, 202, 204, 312, 500, 614, 806, 904, 1004, 1112 Marker

102a, 102b, 102c, 102d, 102e, 102f, 612, 1101 Target analytes

104, 104a, 104b, 104c, 301, 610 First marker part

106 First affinity reagent

108 First oligonucleotide label

110 First reactive group

112, 112a, 112b, 112c, 112d, 306, 308, 310, 608 Second marker part

114 Second affinity reagent

116, 313, 314, 316 Second oligonucleotide label

118 Second reactive group

120 Covalent bond

122 Chemical ligate or ligate

124 Ligation oligonucleotide

126a, 128a Complementary sequence of ligation oligonucleotide

126b Complementary sequence of first oligonucleotide label

128b Complementary sequence of second oligonucleotide label

130 Covalent coupling or covalent conjugation

132 Attachment oligonucleotide

134 Attachment sequence

136 Nanobody

138 second secondary affinity reagent

140 Aptamer-based flexible connector

142 Proximity-based interaction assay

144 Proximity-based high specificity assay

146 Further embodiment

148 Transcription factor 150 Gene regulatory element

152 Proximity-based nucleic acid detection assay

154a, 154b, 1008 First in situ hybridization (ISH) probe as 1^st affinity reagent

156, 1010 Second in situ hybridization (ISH) probe as 2^nd affinity

158, 1012 First nucleic acid target sequence

160, 1014 Second nucleic acid target sequence

162a, 162b Epitopes

200 Marker for detecting the proximity of analytes a and k

202 Marker for detecting the proximity of analytes a and j

204 Marker for detecting the proximity of analytes a and t

206a Unique molecular identifier

300 Target analyte t

302 Target analyte g

304 Target analyte I

306 Second marker part configured to bind 300

308 Second marker part configured to bind 302

310 Second marker part configured to bind 304

400 Second strand synthesis primer binding site

402a, b Forward and reverse primer binding sites

404 Nucleic acid sequencing step

408 Interactional biomarker

410 Nucleic acid amplification step

600 Solid support

602 Attachment sites

604 Attachment group

700 Hydrogel or polymer matrix

702 Polymer backbone

704 Flow cell 706 Attachment agent

800 ISH probe with detectable label

802, 802a, 802b Optically detectable label (e.g. fluorophore, Raman label)

804 in situ sequencing by synthesis (SBS)

806 Antibody/aptamer-based marker

808 First half binding site

810 Second half binding site

812 Binding site for ISH probe on ligate or amplicon

814 ISH detection of a ligate

816 ISH detection of an amplicon

900 (FFPE) tissue section

902 Cyclic process

904 Further marker

1000 Half-binding site

1002 Blinking

1004 Nucleic acid-based marker, nucleic acid probe, ISH probe-based

1006 Nucleic acid target, e.g. mRNA or genomic locus

1100a, b Nucleic acid-based affinity reagent, aptamer, ISH probe

1102a, b Contact oligonucleotide

1104a, b Complementary contact oligonucleotide

1106 Electrical circuit with bound marker

1108 Transistor with bound marker

1110 Transistor with bound marker part

1112 Nucleic acid-based marker, aptamer-based

1200 Cartridge

1204 Buffer reservoir

1206 Pressure connector

1208 Air pressure 1212 Closed flow cell cartridge

1214 Open flow cell cartridge for glass slides

1216 Glass slide

1218 Holding device 1220 Open flow cell cartridge for electrical readout devices

1222 Electrical readout device

1224 Chip

1226 External force

1228 Well 1230 Lyophilized solids

1232 Detection reagents

1234 Waste reservoir

1236 Fluid connection or channel

1238 Unidirectional valve or pressure-controlled valve 1300 Biological sample

1301 Droplet or hydrogel bead

1302 Secreted protein

Claims

Claims

1. A marker (100, 200, 202, 204, 312, 500, 614, 806, 904, 1004, 1112) for analysing a biological sample (1300) with a plurality of target analytes (102, 102a, 102b, 102d, 102e, 102f, 612, 1101) comprising: a first marker part (104, 104a, 104b, 104c, 301, 610) comprising a first affinity reagent (106) and at least one first oligonucleotide label (108), the at least one first oligonucleotide label (108) comprising a first reactive group (110), and a second marker part (112, 112a, 112b, 112b, 112c, 112d, 306, 308, 310, 608) comprising a second affinity reagent (114) and at least one second oligonucleotide label (116, 313, 314, 316), the second oligonucleotide label (116, 313, 314, 316) comprising a second reactive group (118), wherein the first reactive group (110) and the second reactive group (118) are configured to react with the respective other one (110, 118) to form a - preferably covalent - bond (120) and thereby a ligate (122) of the at least one first oligonucleotide label (108) and at least one second oligonucleotide label (116, 313, 314, 316), wherein the first affinity reagent (106) and the second affinity reagent (114) are each configured to bind specifically to one of the target analytes (102a, 102b, 102c, 102d, 102e, 102f,612, 1101) of the biological sample (1300).

2. The marker according to claim 1, wherein the first reactive group (110) and the second reactive group (118) are configured to react with each other under a catalytic condition or wherein the first reactive group (110) and the second reactive group (118) are configured to react with each spontaneously.

3. The marker according to one of the preceding claims, wherein the at least one first oligonucleotide label (108) is attached to the first affinity reagent (106) by a first - preferably cleavable - linker and/or the at least one second oligonucleotide label (116) is attached to the second affinity reagent (114) by a second - preferably cleavable - linker.

4. The marker according to one of the preceding claims, wherein the at least one first oligonucleotide label (108) is attached to the first affinity reagent (106) via hybridization and/or by a first secondary affinity reagent (136) or wherein the at least one second oligonucleotide label (116, 313, 314, 316) is attached to the second affinity reagent (114) via hybridization and/or by a second secondary affinity reagent (138).

5. The marker according to one of the preceding claims, comprising a ligation oligonucleotide (124) configured to hybridize to respective predetermined sequences (126b, 128b) on the at least one first oligonucleotide label (108) and on the second oligonucleotide label (116, 313, 314, 316) in order to position the first reactive group (110) and the second reactive group (118) in close proximity.

6. The marker according to any of the preceding claims, comprising the first marker part (104, 104a, 104b, 104c, 301, 610) with the first affinity reagent (106) and a plurality of the second marker part (112, 112a, 112b, 112b, 112c, 112d, 306, 308, 310, 608), each second marker part (112, 112a, 112b, 112b, 112c, 112d, 306, 308, 310, 608)comprising the second affinity reagent (114) configured to bind to a particular one of the target analytes (102a, 102b, 102c, 102d, 102e, 102f, 612, 1101).

7. The marker according to any of the preceding claims, wherein the at least one first oligonucleotide label (108) and the at least one second oligonucleotide label (116, 313, 314, 316) comprise unique molecular identifier sequences (UMI; 206a) that identify the target analyte (102a, 102b, 102c, 102d, 102e, 102f, 612, 1101) the respective first affinity reagent (106) or the second affinity reagent (114) of the respective first marker part (104, 104a, 104b, 104c, 301, 610) and second marker part (112, 112a, 112b, 112b, 112c, 112d, 306, 308, 310, 608) is configured to bind to.

8. The marker according to one of the preceding claims, wherein the first oligonucleotide label (108) and/or the second oligonucleotide label (116, 313, 314, 316) comprise a priming sequence (400) configured to be bound by a primer and thereby allow the synthesis of a second DNA or XNA strand.

9. The marker according to one of the preceding claims, wherein the first oligonucleotide label (108) and/or the second oligonucleotide label (116, 313, 314, 316) comprise binding sites for forward and reverse primers (402a, 402b) for amplifying at least a part of the ligate (122).

10. The marker according to any of the preceding claims, wherein one of the first marker part (104) and the second marker part (112) is configured to bind to a protein target analyte and the other one of the first marker part (104) and the second marker part (112) is configured to bind to a nucleic acid target analyte or to a posttranslational protein modification group.

11. The marker according to any of the preceding claims, wherein one of the first marker part (104, 104a, 104b, 104c, 301, 610) and the second marker part (112, 112a, 112b, 112b, 112c, 112d, 306, 308, 310, 608) comprises an affinity reagent with a peptide-based backbone and the other one of the first marker part (104, 104a, 104b, 104c, 301, 610) and the second marker part (112, 112a, 112b, 112b, 112c, 112d, 306, 308, 310, 608) comprises an aptamer.

12. The marker according to any of the preceding claims, wherein the first affinity reagent (106) or the second affinity reagent (114) comprises a toxin, a drug, a drug-like molecule.

13. The marker according to any of the preceding claims, wherein one of the first marker part (104, 104a, 104b, 104c, 301, 610) and the second marker part (112, 112a, 112b, 112b, 112c, 112d, 306, 308, 310, 608) comprises an aptamer or a nucleic acid-based affinity reagent or a nucleic acid probe.

14. The marker according to any of the preceding claims, wherein the first marker part (104, 104a, 104b, 104c, 301, 610) and the second marker part (112, 112a, 112b, 112b, 112c, 112d, 306, 308, 310, 608) each comprise an aptamer or a nucleic-acid probe.

15. A method for analysing a biological sample comprising the steps: introducing at least one marker (100, 200, 202, 204, 312, 500, 614, 806, 904, 1004, 1112) according to one of the preceding claims into the biological sample, and generating a readout of the biological sample with the marker (100, 200, 202, 204, 312, 500, 614, 806, 904, 1004, 1112).

16. The method according to claim 15, wherein the biological sample is immobilized on a solid support (600) prior or after the at least one marker (100, 200, 202, 204, 312, 500, 614, 806, 904, 1004, 1112) is introduced into the biological sample.

17. The method according to claim 15 or 16, wherein the biological sample is a tissue section immobilized on a glass slide (600) or on a transparent window of a flow cell (1210), which is configured to allow microscopic observation of contents of the flow cell.

18. The method according to any of the claims 15 to 17, wherein the target analytes (102, 102a, 102b, 102d, 102e, 102f, 612, 1101) are immobilized on a solid support (600).

19. The method according to any of the claims 15 to 18, wherein the target analytes (102, 102a, 102b, 102d, 102e, 102f, 612, 1101) are biotinylated or conjugated to a guest molecule with high affinity to a host molecule or another first affinity interactor (602) in order to allow their subsequent immobilization on a solid support that is functionalized with streptavidin, cucurbit[n]uril, or another corresponding second affinity interactor (604).

20. The method according to any of the claims 15 to 19, wherein the target analytes (102, 102a, 102b, 102d, 102e, 102f, 612, 1101) are conjugated to a bifunctional linker configured to allow the subsequent covalent coupling of the target analytes to a suitably configured solid support (600).

21. The method according to any of the claims 15 to 20, wherein at least one of the first marker part (104, 104a, 104b, 104c, 301, 610) and the second marker part (112, 112a, 112b, 112b, 112c, 112d, 306, 308, 310, 608) is immobilized on a solid support (600).

22. The method according to any of the claims 15 to 21, wherein at least one of the first marker part (104, 104a, 104b, 104c, 301, 610) and the second marker part (112, 112a, 112b, 112b, 112c, 112d, 306, 308, 310, 608) is immobilized on a microbead, a nanoparticle, or a NanoArray.

23. The method according to any of the claims 15 to 22, wherein at least one of the first marker part (104, 104a, 104b, 104c, 301, 610) and the second marker part (112, 112a, 112b, 112b, 112c, 112d, 306, 308, 310, 608) is immobilized on a DNA-origami-based structure.

24. The method according to any of the claims 15 to 23, wherein at least one of the first marker part (104, 104a, 104b, 104c, 301, 610) and the second marker part (112, 112a, 112b, 112b, 112c, 112d, 306, 308, 310, 608) or the target analytes (102, 102a, 102b, 102d, 102e, 102f, 612, 1101) are immobilized on a polymeric matrix (702) or within a gel-like matrix (700).

25. The method according to any of the claims 15 to 24, wherein a blocking buffer comprising at least one of bovine serum albumin, serum, dextran sulfate, a polyanionic competitor, formamide, a blocking oligonucleotide, and sheered Salmon sperm DNA is added to the biological sample in orderto block unspecific binding sites of the biological sample.

26. The method according to any of the claims 15 to 25, wherein the first marker part (104, 104a, 104b, 104c, 301, 610) and the second marker part (112, 112a, 112b, 112b, 112c, 112d, 306, 308, 310, 608) are allowed to bind to their respective target analytes (102, 102a, 102b, 102d, 102e, 102f, 612, 1101), preferably for a sufficient amount of time. 1. The method according to any of the claims 15 to 26, wherein unbound first marker parts (104, 104a, 104b, 104c, 301, 610) and unbound second marker parts (112, 112a, 112b, 112b, 112c, 112d, 306, 308, 310, 608) are washed out by at least one washing step before a catalytic condition is applied.

28. The method according to any of the claims 15 to 27 , wherein during the step of introducing the at least one marker (100, 200, 202, 204, 312, 500, 614, 806, 904, 1004, 1112), a non-catalytic condition is applied to the biological sample.

29. The method according to any of the claims 15 to 28, wherein after the step of introducing the at least one marker (100, 200, 202, 204, 312, 500, 614, 806, 904, 1004, 1112), a catalytic condition is applied to the biological sample.

30. The method according to any of the claims 15 to 29, wherein after formation of the covalent bond (120) and the ligate (122), a nucleic acid amplification is performed generating at least one amplicon of at least one part of at least one ligate (122) sequence covering at least a part of the at least one first oligonucleotide label (108) and the at least one second oligonucleotide label (116, 313, 314, 316) of the at least one marker (100, 200, 202, 204, 312, 500, 614, 806, 904, 1004, 1112).

31. The method according to claim 30, wherein the at least one amplicon of the at least one ligate (122) comprises the unique molecular identifier (UMI) sequences of the at least one first oligonucleotide label (108) and the second oligonucleotide label (116, 313, 314, 316).

32. The method according to any of the claims 30 to 31, wherein nucleic acid amplification is performed using at least one of the following: an isothermal amplification method, a polymerase chain reaction (PCR), a quantitative polymerase chain reaction, a rolling circle amplification (RCA), a bridge amplification, a loop-mediated isothermal amplification, and a recombinase polymerase amplification.

33. The method according to any of the claims 15 to 32, wherein in order to detect the at least one marker (100, 200, 202, 204, 312, 500, 614, 806, 904, 1004, 1112), a nucleic acid probe or ISH probe (1004) comprising an optically detectable label (802) is added to the biological sample for binding to either the ligate (122) or an amplicon (406) of said marker, wherein the corresponding binding site, to which the ISH probe (1004) is configured to bind, covers at least a part of the respective at least one first oligonucleotide labels (108) and the at least one second oligonucleotide labels (116, 313, 314, 316).

34. The method according to claim 33, wherein the markers (100, 200, 202, 204, 312, 500, 614, 806, 904, 1004, 1112) are detected using a cyclic staining, imaging, inactivation process (902).

35. The method according to claim 34, wherein inactivation is performed by removing, degrading, or cleaving the nucleic acid probe or ISH probe (1004) and/or optically detectable label and/or cleavable linker.

36. The method according to any of the claims 34 to 35, wherein inactivation is performed using a nuclease or restriction endonuclease.

37. The method according to any of the claims 33 to 36, wherein the nucleic acid probe comprises at least one cleavage site that can be cleaved by either one of the following: UV light, TCEP, Dnase I, and a restriction enzyme.

38. The method according to any of the claims 15 to 37, wherein the detection of the markers is performed using nucleic acid probes or ISH probes (1004) comprising an optically detectable label (802), which are configured to bind to the ligate (120) of the marker transiently.

39. The method according to any of the claims 15 to 38, wherein the first affinity reagent (106) of the marker (100, 200, 202, 204, 312, 500, 614, 806, 904, 1004, 1112) is configured to bind to a first target analyte (102a) of the biological sample and the second affinity reagent (114) of the marker is configured to bind to a second target analyte (102b) of the biological sample, and wherein a property of the marker is determined in the readout in order to determine a proximity between the first target analyte (102a) and the second target analyte (102b).

40. The method according to any of the claims 15 to 38, wherein the first affinity reagent (106) of the marker (100, 200, 202, 204, 312, 500, 614, 806, 904, 1004, 1112) is configured to bind to a first binding site (132a, 158, 1012) of a particular target analyte (106b, 1006) of the biological sample and the second affinity reagent (114, 156, 1010) of the marker is configured to bind to a second binding site (132b, 160, 1014) of the particular target analyte (405, 506) of the biological sample.

41. The method according to any of the claims 15 to 40, wherein the step of generating the readout is performed by DNA sequencing (404) or by next generation sequencing, in particular of the ligate (120) or the amplicon.

42. The method according to any of the claims 15 to 41, wherein a supernatant is used for the sequencing step.

43. The method according to any of the claims 15 to 42, wherein the sequencing is performed in situ.

44. The method according to any of the claims 15 to 43, wherein the method comprises a step of encapsulating the biological sample.

45. The method according to claim 44, wherein the method comprises a step of sorting the encapsulated biological sample based on the generated readout.

46. The method according to any of the claims 15 to 45, wherein the readout is generated by determining an electrical property of an electrical circuit (1106) or a transistor (1108), which is configured to bind at least one marker (100, 200, 202, 204, 312, 500, 614, 806, 904, 1004, 1112).

47. The method according to claim 46, wherein the electrical property is at least one of the following: voltage, current, resistance, conductance, impedance, reactance, capacitance, inductance, power, susceptance, admittance, frequency, phase angle, quality factor, noise figure, gain.

48. The method according to any of the claims 46 to 47, wherein each marker parts comprises an affinity reagent comprising a nucleic acid-backbone (1104a, 1104b, 1112) and an additional contact oligonucleotide (1102a, 1102b) configured to bind to complementary contact oligonucleotides (1104a, 1104b) that are attached to an electrical circuit (1106), integrated circuit, or transistor (1108), wherein following to the formation of the covalent bond (120) the markers are bound to said electrical circuit (1106), integrated circuit, or transistor (1108) to readout an electrical property and thereby determine the binding of a marker to said electrical circuit (1106), integrated circuit, or transistor (1108).

49. The method according to any of the claims 46 to 48, wherein the electrical circuit comprises a field effect transistor (FET), metal-oxide-semiconductor field effect transistor (MOSFET), bipolar junction transistor (BJT) complementary metal-oxide-semiconductor (CMOS), high electron mobility transistor (HEMT), or insulated gate bipolar transistor (IGBT) configured to bind at least one marker (100, 200, 202, 204, 312, 500, 614, 806, 904, 1004, 1112).

50. A readout device configured to carrying out the method of any of the claims 46 to 49 comprising at least one of the following: a field effect transistor (FET), metal-oxide-semiconductor field effect transistor (MOSFET), bipolar junction transistor (BJT) complementary metal-oxide-semiconductor (CMOS), high electron mobility transistor (HEMT), insulated gate bipolar transistor (IGBT) configured to bind at least one marker (100, 200, 202, 204, 312, 500, 614, 806, 904, 1004, 1112).

51. The readout device according to claim 50 configured to bind at least one marker (100, 200, 202, 204, 312, 500, 614, 806, 904, 1004, 1112) thereby establishing an electrical connection between a gate-voltage source and a gate.

52. A labelling kit comprising: a marker according to one of the claims 1 to 14, a bifunctional linker configured to conjugate a fourth reactive group to an affinity reagent or protein and, preferably, at least one of the following: a column, a buffer, and a catalyst required for coupling and/or purifying the first (104, 154, 1008) or second marker part (112, 156, 1010), wherein the first oligonucleotide label (108) and second oligonucleotide label (116) of the marker are configured to be activated by a third reactive group allowing the covalent conjugation of the first oligonucleotide label (108) and the second oligonucleotide label (116) to a respective affinity reagent or protein, wherein the third reactive group is configured to couple spontaneously to the fourth reactive group.

53. A diagnostic kit comprising: at least one marker (100, 200, 202, 204, 312, 500, 614, 806, 904, 1004, 1112) according to any of the claims 1 to 14, at least one cartridge (1200) configured to receive a biological sample, preferably, further comprising a catalyst or catalytic buffer, wash buffer and, preferably, further comprising an ISH probe (800) configured to bind to at least one ligate (122) comprising an optically detectable label (802).

54. A cartridge (1200), preferably a microfluidic cartridge (1200), configured to perform the method according to any of the claims 15 to 49, the cartridge (1200) comprising: at least one flow cell (1210) comprising a transparent window configured for a microscopic observation, at least one waste reservoir (1234). at least one pressure connector (1206) configured to be connectable to a - preferably external - pressure source (1208), at least one buffer reservoir (1204) connectable to the pressure connector via a channel configured to allow pressurization of the buffer reservoir, at least one fluid connection (1236) configured to allow moving liquids from the buffer reservoir through the flow cell into the waste reservoir, preferably at least one unidirectional valve (1238), preferably at least one well comprising lyophilized solids (1232) of the first and second marker part of a marker (100, 200, 202, 204, 806, 904, 1004, 1112) according to any of the claims 1 to 14, at least one catalytic condition buffer reservoir connectable to the pressure connector via a channel configured to allow pressurization of the buffer reservoir, at least one fluid connection (1236) configured to allow moving liquids from the catalytic condition buffer reservoir through the flow cell into the waste reservoir, and a readout device, preferably according to one of the claims 50 and 51.

55. The cartridge (1200) according to claim 54, further comprising at least one well in fluid communication with at least one buffer reservoir comprising lyophilized solids of optically detectable labels configured to bind to markers.

56. The cartridge (1200) according to claim 54 or 55, wherein externally applied pressure, preferably air pressure, is used to control at least one valve and thereby to control a movement of at least one liquid in the cartridge.

57. The cartridge (1200) according to any of the claims 54 to 56, further comprising at least one of the following: a microfluidic cell encapsulation unit; an open flow cell configured to form a leak-tight assembly with a glass slide; a mixing chamber configured to allow mixing of liquids and biological sample; a port configured to allow loading of a biological sample; a port configured to allow loading of a biological sample via insertion of a cotton swab; a port configured to allow loading of a biological sample via attachment of a syringe; a mixing chamber; and a mixing chamber comprising a rotatable stirring device, which can be moved by an external device or manually.