CN117120628A - Capture constructs and methods for detecting multiple analytes - Google Patents

Abstract

A capture construct (100, 1000) for capturing a plurality of analytes (402,504,604,706) of a biological sample (1300,1408) is provided. The capture construct (100, 1000) comprises a nanostructure scaffold (102), at least one first orientation indicator (104) and at least one second orientation indicator (106), and at least a plurality of first capture regions (108 a-108 f) on the nanostructure scaffold (102), each capture region (108 a-108 f) comprising at least one affinity capture reagent (400,502,602,702,704), the affinity capture reagent (400,502,602,702,704) being configured to capture one of the analytes (402,504,604,706). In another aspect, a method of detecting a plurality of analytes (402,504,604,706) of a biological sample (1300,1408) is provided.

Description

Capture constructs and methods for detecting multiple analytes

Technical Field

The present invention relates to a capture construct comprising a nanostructure scaffold and a method of detecting a plurality of analytes of a biological sample by the capture construct.

Background

Human proteomes include proteins that are mostly secreted by cells, which are collectively referred to as the cell secretome. For basic and transformation studies and diagnostic applications, there is considerable interest in obtaining secretion profiles, in particular from single cells. A recent study by uhlen et al (Science Signaling,26Nov 2019:Vol.12,Issue 609,DOI:10.1126/sciignal. Aaz 0274) found that most secreted proteins were actually retained in the cell, i.e. sorted into intracellular organelles, rather than released from the cell. It is therefore important to be able to distinguish between proteins that enter the secretory pathway but remain in the cell and proteins that are actually secreted from the cell into the extracellular space or blood stream.

Fluorescence-based assays are commonly used in life science research and diagnostic applications to detect the presence or absence of a target analyte. Such target analytes may also be referred to as molecular markers (markers). The molecular markers of interest may belong to the group of proteins (proteomic levels), RNAs, in particular mRNA (transcriptomic levels), DNA (genomic levels), metabolites (metabolomic levels), secreted molecules (secretome levels), neurotransmitters, hormones and other small molecules of interest.

"Fluorescent Cell Barcode (FCB) is a multiplexing technique for high-throughput Flow Cytometry (FCM). Although powerful in minimizing staining variability, it remains a subjective FCM technique "due to variability between operators and differences in data analysis, cited Tsai et al 2020 (Tsai et al j immunomethods.2020feb; 477:112667.Doi:10.1016/j. Jim.2019.112667.). In FCB, up to three dyes are used in four different concentrations to label cells in different wells of a microplate, for example by coupling the dye to reactive amine groups on the cell surface, which produce different intensity and color combinations. The subjectivity of the technique and the inter-operator variability of the method are both inherently related to the fact that part of the information in the color of the dye is based on coding (i.e. intensity variations in light green, dark green for example), which severely limits the applicability of the technique.

However, no technology is available that allows capturing large amounts of molecular markers, especially for single cell analysis. Furthermore, the subsequent techniques for detecting and analyzing these large numbers of molecular markers are not known, for example for secretome analysis.

Disclosure of Invention

It is an object of the present invention to provide a capture construct for capturing a plurality of analytes of a biological sample and a method for capturing a plurality of analytes of a biological sample, which are capable of capturing a large amount of analytes in a particularly compact space.

The above object is achieved by the subject matter of the independent claims. Advantageous embodiments are defined in the dependent claims and in the following description.

In a first aspect, a capture construct for capturing a plurality of analytes of a biological sample is provided. The capture construct comprises a nanostructure scaffold, at least one first orientation indicator and at least one second orientation indicator, and at least a plurality of first capture regions in the nanostructure scaffold, each capture region comprising at least one affinity capture reagent configured to capture one of the analytes. The capture construct enables capture of a plurality of analytes at predetermined locations of the capture region of the nanostructure scaffold. Furthermore, the capture construct enables capture of analytes at a particularly high density. Capturing the plurality of analytes enables subsequent analysis of the plurality of analytes.

The analyte may be a series of molecules. For example, the analyte may be a chemical substance, such as a metabolite of the biological sample, or a cell signaling molecule of the biological sample. In addition, the analyte may be a protein or peptide of a biological sample, such as a specific enzyme. In addition, the analyte may be a hormone or neurotransmitter. In addition, the analyte may be a cell that expresses a cell surface protein or that expresses a specific combination of cell surface proteins. Further, the analyte may be a cell expressing a certain cell surface protein or a specific combination of cell surface proteins with a specific glycosylation pattern. Furthermore, the analyte may be a bacterium, archaea, fungus (e.g., yeast) or virus. Furthermore, the analyte may be a toxin or a heavy metal. Still further, the analyte may be a nucleic acid molecule having a specific nucleic acid sequence, such as DNA or RNA. In particular, the analyte may be secreted by the biological sample, thereby enabling capture of at least a portion of the secreted group of biological samples. Each affinity capture reagent may be configured to bind to a particular one of the analytes. Each capture region of the plurality of first capture regions may comprise at least one affinity capture reagent attached to the nanostructure scaffold in the capture region and configured to specifically bind or capture one of the analytes. In other words, the capture construct may be configured such that each analyte is captured in a particular one of the first capture areas. The capture area may also be referred to as a capture zone. The capture construct may also be referred to as a nanoarray.

The biological sample may be a multicellular structure (e.g., a living cell cluster), particularly spheroids or single cells, to enable single cell analysis.

The first and second orientation indicators may be, for example, fluorescent dyes attached to the nanostructure scaffold. The orientation indicator is capable of determining the spatial orientation or directionality of the capture construct, in particular the nanostructure scaffold. In this way, the azimuth indicator enables spatial encoding. This means that different positions on the nanostructure scaffold can be assigned as capture bands or capture regions that are reactive to different analytes.

Preferably, the nanostructure scaffold comprises a nucleic acid. For example, the nanostructure scaffold may include DNA, RNA, and/or LNA. In particular, the nanostructure scaffold may comprise DNA origami (origami). These DNA origami structures may range in size from a few nanometers to a micrometer. To make such a DNA-based paper folding structure, longer DNA molecules (scaffold strands) are folded at precisely identified positions by so-called staple strands (staple strands). The DNA fold may be designed to provide a self-assembled nanostructured scaffold of a specific predetermined shape. This makes the synthesis and assembly of the scaffold simple and repeatable. The binder chain may be position-selectively functionalized. In this case, the position resolution is limited by the nucleotide size, which is in the range of nanometers or less. This has been used in the prior art to generate fluorescent standards in which fluorescent dyes are attached to precisely positioned bands on a DNA fold. These standards are known as "nano-meters" and are used for calibration of imaging systems, e.g. as disclosed in US2014/0057805A1, such as confocal or super-resolution microscopes (e.g. STED).

DNA folding provides a scaffold for affinity capture reagents. Preferably, the DNA origami structure comprises at least one scaffold strand and a plurality of binding strands, wherein the binding strands are complementary to at least a portion of the scaffold strand and are configured to form the scaffold strand into a predetermined conformation. In particular, the strand is an oligonucleotide. This enables the generation of self-assemblable nanostructured frameworks having a predetermined two-dimensional or three-dimensional shape. Furthermore, this enables site-specific placement of the capture region on the scaffold. Preferably, the affinity capture reagent of the capture zone may be attached to the binding chain of the nanostructure scaffold at a predetermined position. The binding chain allows spatially accurate functionalization of the DNA fold at various locations on the DNA fold. Thus, each capture region may be positioned at a particular one of the binding chains or at a group of binding chains that are in close proximity along the nanostructure skeleton. Since the binding chain is located at a predetermined position, the position of the capturing area may be equivalent to predetermined.

It is particularly preferred that the affinity capture reagent is selected from the group consisting of antibodies, antibody fragments (e.g. single domain antibodies), aptamers, peptides, oligonucleotides, aptamers, drugs and/or toxins. This enables capture of a wide variety of analytes with affinity capture reagents. For example, the analyte may be a protein and the affinity capture reagent may be an antibody. In this case, the antibody may capture the protein by binding to a specific binding site or epitope of the protein. In general, an affinity capture reagent for a particular capture region can bind to at least one particular binding site in each analyte.

In a preferred embodiment, the capture construct comprises a plurality of first affinity reporter reagents, each comprising a first reporter label and configured to be attached to one of the analytes, wherein the first reporter label is readable to determine whether the respective analyte is captured by the respective affinity capture reagent. This enables the presence of a particular analyte to be determined by capturing the construct. Since the capture construct comprises a plurality of first capture regions, a plurality of specific analytes can be captured and their presence determined. Similar to the affinity capture reagents, each affinity reporter reagent may be configured to capture or specifically bind one of the analytes. Thus, each first capture zone may comprise at least one affinity reporter reagent configured to specifically bind one of the analytes associated with one of the plurality of first capture zones. Thus, for each target analyte, a capture zone with a specific affinity capture reagent and a specific affinity reporter reagent is provided.

Preferably, the affinity reporter reagent is selected from the group consisting of an antibody, an antibody fragment (e.g., a single domain antibody), an aptamer, a peptide, an oligonucleotide, an aptamer, a drug and/or a toxin. This enables a wide variety of analytes to be captured and reported with the capture construct. Typically, an affinity reporter reagent for a particular capture region can bind to at least one particular binding site in each analyte.

In a particularly preferred embodiment, the reporting tag, in particular the first reporting tag, is optically readable. For example, the reporting tag may include an optically detectable fluorescent dye. This enables the determination by microscopy, in particular by imaging with a microscope, of whether an analyte is captured by the respective affinity capture reagent, and whether the various affinity reporter reagents and their associated reporter labels bind to the analyte.

Preferably, the reporter tag, in particular the first reporter tag, is an oligonucleotide and is readable by sequencing. This enables a determination of whether an analyte is captured by the respective affinity capture reagent and whether the various affinity reporter reagents and their associated reporter labels bind to the analyte.

Preferably, the affinity capture reagent is bound to the nanostructure scaffold, or the affinity capture reagent of one particular one of the capture regions comprises an oligonucleotide, and each capture region comprises an oligonucleotide that is complementary to the oligonucleotide to bind the affinity capture reagent.

In a preferred embodiment, the affinity capture reagent is covalently bound to the nanostructure scaffold, or the affinity capture reagent of one specific one of the capture regions comprises an oligonucleotide, and each capture region comprises an oligonucleotide that is complementary to the oligonucleotide to bind the affinity capture reagent. This makes assembly of the capture construct particularly easy.

The complementary oligonucleotide may be part of a nanostructure scaffold, preferably a binding strand, especially when the nanostructure scaffold comprises a nucleic acid. For example, each capture region of the nanostructure scaffold may have a complementary oligonucleotide that is complementary only to the oligonucleotide of the affinity capture reagent of the respective capture region. When an affinity capture reagent is added to the nanostructure scaffold, the affinity capture reagent then binds only to the corresponding complementary oligonucleotide of each capture region.

Examples of direct attachment or linking of the affinity capture reagent to the binding chain (which enables site-selective attachment to the nanostructure scaffold) include direct chemical coupling by, for example, click chemistry reactions (e.g., azide-alkynes) or by high affinity interactions (e.g., biotin-streptavidin). In the latter case, biotinylated binding chains and streptavidin-conjugated affinity capture reagents, such as antibodies, may be used.

Preferably, the nanostructure scaffold comprises at least a plurality of second capture regions, each second capture region comprising at least one affinity capture reagent configured to capture one of the analytes. This enables the capture areas on the capture construct to be particularly closely arranged.

In a preferred embodiment, the capture construct further comprises a plurality of second affinity reporter reagents, each affinity reporter reagent comprising a second reporter label and configured to attach to one of the analytes, wherein the second reporter labels are readable to determine whether the respective analytes are captured by the respective affinity capture reagents. This enables the capture areas on the capture construct to be particularly closely arranged. In particular, the second reporter label is optically readable or readable by sequencing. Furthermore, the plurality of second affinity reporter reagents may specifically bind to an analyte that binds to one of the plurality of second capture areas.

Preferably, the nanostructure scaffold extends linearly in one dimension and the first and second orientation indicators are spaced apart from each other or arranged at opposite ends of the nanostructure scaffold. This enables the orientation of the capture construct to be determined. The orientation indicator may be, for example, a fluorescent dye. In particular, the first and second orientation indicators have different properties, such as excitation wavelength, fluorescence emission wavelength and/or fluorescence lifetime.

In a particularly preferred embodiment, the nanostructure scaffold extends in two or three dimensions and the nanostructure scaffold comprises at least one third-orientation indicator. This enables the orientation of the capture construct to be determined.

Preferably, the maximum spatial limit of the nanostructure scaffold is in the range of 1nm to 10000nm, preferably in the range of 0.1 μm to 5 μm, more preferably in the range of 0.1 μm to 1 μm. This makes the capture construct particularly compact.

Preferably, the capture areas of the plurality of first capture areas are spaced apart from each other in the range of 1nm to 2000nm, preferably in the range of 200nm to 1000 nm. This enables the capture areas to be arranged particularly closely to the nanostructure scaffold.

In a particularly preferred embodiment, the capture areas of the plurality of first capture areas are separated from the capture areas of the plurality of second capture areas by a range of 0.1nm to 500nm, preferably by a range of 1nm to 100 nm. This enables the capture areas to be arranged particularly closely to the nanostructure scaffold.

Preferably, the reporter label comprises fluorophores, in particular having different excitation wavelengths, fluorescence emission wavelengths and/or fluorescence lifetime characteristics. In particular, the excitation wavelength, the fluorescence emission wavelength and/or the fluorescence lifetime characteristics of the first reporting tag and the second reporting tag are different. This enables the capture areas to be arranged particularly close together. In particular, this enables the arrangement of the capture areas of the plurality of first capture areas within a distance from the diffraction limit of the capture areas of the plurality of second capture areas.

In a preferred embodiment, the affinity capture reagent of at least one capture zone is configured to bind one analyte at a single binding site for the analyte. This enables binding of analytes with high sensitivity and high specificity. For example, the analyte may be a protein and the affinity capture reagent may be an antibody. In this case, the antibody may capture the protein by binding to a specific binding site or epitope of the protein.

In a particularly preferred embodiment, the at least one capture zone comprises a first set of affinity capture reagents and a second set of affinity capture reagents, and wherein the first set of affinity capture reagents is configured to bind to said one of the analytes at a first binding site for said one analyte, and the second set of affinity capture reagents is configured to bind to said one of the analytes at a second binding site for said one analyte. This enables the analyte to bind with a particularly high affinity. For example, the capture region may be configured such that the analyte can bind to an associated affinity capture reagent at several different binding sites, the associated affinity capture reagent being specific for one of the binding sites, and the capture region comprising an affinity capture reagent for each of the binding sites. In an alternative example, the analyte may comprise several identical binding sites, and the capture region may be configured such that the analyte may bind through several related affinity capture reagents specific for one binding site.

In a particularly preferred embodiment, the first set of affinity capture reagents comprises a first capture reagent dye and the second set of affinity capture reagents comprises a second capture reagent dye. In particular the capture reagent dye is a fluorescent group.

In a particularly preferred embodiment, the first and second capture reagent dyes are configured to cause proximity when the one analyte is captured by one of the affinity capture reagents of the first set of affinity capture reagents and one of the affinity capture reagents of the second set of affinity capture reagents, and wherein the proximity enables transfer of energy between the respective capture reagent dyes. In particular, this proximity enables FRET (fluorescence resonance energy transfer) pairs or FRET n-tuples (pattern) to be formed between the individual capture reagent dyes. This allows for a particularly high specificity in capturing and detecting analytes.

Preferably, the capture construct and the biological sample are embedded in or attached to a polymeric compound, in particular a hydrogel. This keeps the capture construct and the biological sample very close together and enables particularly easy handling of the biological sample using the capture construct.

In another aspect, a method for detecting a plurality of analytes of a biological sample is provided, the method comprising the steps of: incubating the biological sample in the presence of at least one capture construct; obtaining a readout of at least one capture construct (in particular a capture region); determining whether each of said analytes is captured by a respective one of said affinity capture reagents.

Terminology

In the sense of this document, the following terms are used as follows:

"sample)": in the sense of this document, "sample" may refer to a biological sample, which may also be named as a biological sample (specimen), including, for example, blood, serum, plasma, tissue, bodily fluids (e.g., lymph, saliva, semen, interstitial fluid, cerebrospinal fluid), stool, solid biopsies, liquid biopsies, explants, whole embryos (e.g., zebra fish, drosophila), whole-pattern organisms (e.g., zebra fish larvae, drosophila embryos, caenorhabditis elegans), cells (e.g., prokaryotes, eukaryotes, archaea), multicellular organisms (e.g., algae), suspension cell cultures, monolayer cell cultures, 3D cell cultures (e.g., spheroids, tumor-like (turoroids), organoids derived from various organs (e.g., intestines, brain, heart, liver, etc.), lysates of any of the foregoing, viruses. In the sense of this document, "sample" may also refer to the volume surrounding a biological sample. For example, in assays that study secreted proteins (e.g., growth factors, extracellular matrix compositions), the extracellular environment surrounding a cell that reaches a certain assay-dependent distance may also be referred to as a "sample. Specifically, the affinity reagent brought into the surrounding volume may be referred to as "sample introduced (introduced into the sample)".

"affinity reagent": in the sense of this document, the term "affinity reagent" and/or "affinity capture reagent" may particularly be an antibody, a single domain antibody (also called nanobody), a combination of at least two single domain antibodies, an aptamer, an oligonucleotide, a morpholino, a PNA complementary to a predetermined RNA, a DNA target sequence, a ligand (e.g. a drug or a drug-like molecule), or a toxin (e.g. phalloidin), a toxin that binds to actin filaments. In the sense of this document, an affinity reagent may be configured to bind a target molecule or analyte with a certain affinity and specificity, such that it may be said that the affinity reagent is substantially specific for the target molecule or predetermined target structure only.

"analyte": in the sense of this document, an "analyte" or "predetermined target structure" may refer to a target molecule or target structure, or to an analyte, which may be, for example, a protein (e.g., a protein), an RNA sequence (e.g., mRNA of a gene), a peptide (e.g., somatostatin), a DNA sequence (e.g., genetic locus or element), a metabolite (e.g., lactic acid), a hormone (e.g., estradiol), a neurotransmitter (e.g., dopamine), a vitamin (e.g., cobalamin), a micronutrient (e.g., biotin), a metal ion (e.g., metal and heavy metal ions such as Cd (II), co (II), pb (II), and Hg (II), U (VI)).

"dye": in the sense of this document, the terms "fluorescent dye", "fluorophore", "fluorescent pigment", "dye" are used interchangeably to denote a fluorescent compound or structure, and may in particular be one of the following: fluorescent organic dyes, fluorescent quantum dots, fluorescent binary (dyad), fluorescent carbon dots, graphene quantum dots or other carbon-based fluorescent nanostructures, fluorescent proteins, fluorescent nanostructures based on DNA paper folding. Among the group of organic fluorescent dyes, in particular the following derivatives are referred to by the term "fluorescent dye": xanthenes (e.g. fluorescein, rhodamine, oregon green, texas), cyanines (e.g. cyanine, indocarbocyanine, oxocarbocyanine (oxacarbocyanine), thiocyanine (thiacarbocyanine) and merocyanine), squaraine derivatives, naphthalene, coumarin, oxadiazoles, anthracene (anthraquinone, DRAQ5, DRAQ7, cyTRAK Orange), pyrene (cascade blue), oxazine (nile red, nile blue, cresol purple, oxazine 170), acridine (proavin, acridine yellow), arylmethylamines (gold amine, crystal violet, malachite green), tetrapyrroles (porphine, phthalocyanine, bilirubin), methylenedipyrrole (BODIPY, aza-BODIPY), phosphorescent dyes or luminescent dyes. The following trademark groups specify commercially available fluorescent dyes The materials, which may include Dyes belonging to different chemical families-CF Dyes (Biotium), DRAQ and CyTRAK probes (BioStatus), BODIPY (Invitrogen), everFluor (Setareh Biotech), alexa Fluor (invitrogen), bella Fluor (Setareh Biotech), dyLight Fluor (Thermo Scientific), atto and Tracy (Sigma Aldrich), fluoProbes (Interchim), abberior Dyes (Abberior Dyes), dy and MegaStokes Dyes (Dyomics), sulfo Cy Dyes (cyandyne), hiLyte Fluor (AnaSpec), seta, seTau and Square Dyes (Seta biomedicials), quaar and Cal-Fluor Dyes (Biosearch Technologies), surrelight Dyes (Columbia Biosciences), vio Dyes (Milteny Biotec) [ list modifications self: https:// en.wikipedia.org/wiki/fluorophone]. Among the group of fluorescent proteins, in particular members of the Green Fluorescent Protein (GFP) family comprising GFP and GFP-like proteins (e.g. DsRed, tagRFP) and (monomeric) derivatives thereof (e.g. EBFP, ECFP, EYFP, cerulaen, mTurquoise, YFP, EYFP, mCitrine, venus, YPet, superfolder GFP, mCherry, mPlum) refer to the term "fluorescent dye". Furthermore, from the group of fluorescent proteins, the term "fluorescent dye" may include fluorescent proteins whose absorption or emission characteristics change upon binding to a ligand and to a binding agent like e.g. BFPms1, or change in response to the environment (e.g. redox-sensitive rogp or pH-sensitive variants). Furthermore, from the group of fluorescent proteins, the term "fluorescent dye" may include the derivative small hyper red fluorescent protein smURFP of cyanobacterial phycobiliprotein as well as fluorescent protein nanoparticles derivable from smURFP. For an overview of fluorescent proteins, please refer to Rodriguez et al, 2017 # Trends Biochem Sci.2017 Feb；42(2):111–129). The term "fluorescent dye" may also refer to fluorescent quantum dots. The term "fluorescent dye" may also refer to fluorescent carbon dots, fluorescent graphene quantum dots, carbon-based fluorescent nanostructures, as in Yan et al 2019 at Microchimica Acta (2019) 186:583 and Iravani and Varma 2020Environ Chem Lett2020Mar 10:1-25. The term "fluorescent dye" may also refer to fluorescent polymer dots (pdots) or nanodiamonds. The term "fluorescent dye" may also refer to a fluorescent binary, such as the dinaphtalate described in Kacenauskaite et al 2021J.am.chem.Soc.2021,143,1377-1385Binary bodies of benzene antenna (perene antenna) and triangelium emitter (emitter). The term "fluorescent dye" may also refer to organic dyes, binary, quantum dots, polymer dots, graphene dots, carbon-based nanostructures, DNA paper-folding based nanostructures, nano-rulers, polymer beads containing incorporated dyes, fluorescent proteins, inorganic fluorescent dyes, SMILE, or microcapsules filled with any of the foregoing. The term "fluorescent dye" may also refer to a FRET pair having at least one fluorescent dye as a FRET donor and at least one fluorescent dye as a FRET acceptor, or for use in generating a three component FRET triplets of resonance energy transfer. In particular, FRET pairs or FRET triplets may be linked by a complementary linker or linker. The term "fluorescent dye" may also refer to a FRET n-tuple of a physically linked dye.

"reader": the term "readout device" may refer to a device for performing fluorescent polychromatic reading or imaging. The readout device typically comprises at least one excitation light source and a detection system comprising at least one detection channel, and may further comprise filters and/or dispersive optical elements, such as prisms and/or gratings, to send the excitation light source to the sample and/or to send the emission light source of the sample to the detector or an appropriate area of the detector. The detection system may comprise several detection channels, either a spectral detector that detects multiple bands of the spectrum in parallel, or a hyperspectral detector that detects successive portions of the spectrum. The detection system comprises at least one detector, which may be a point detector (e.g. photomultiplier, avalanche diode, hybrid detector), an array detector, a camera, a hyperspectral camera. The detection system may record the intensity of each channel as is typical in a cytometer, or may be an imaging detection system that records images as in a plate reader or microscope. Readout devices with one detector (e.g. camera or photomultiplier) channel may generate a readout with multiple detection channels using, for example, different excitation and emission bands.

"oligonucleotide": in the sense of this document, it may refer to DNA, RNA, peptide nucleic acids, morpholino or locked nucleic acids, ethylene glycol nucleic acids, threose nucleic acids, hexitol nucleic acids or other forms of artificial nucleic acids.

"Point spread function": the term "point spread function" may be used to denote the principal maximum of the point spread function, which term, unless otherwise indicated, refers to the effective Point Spread Function (PSF) of an imaging system, which is generally elliptical, i.e. with a lateral resolution that is better than an axial resolution, but may approximate a nearly spherical shape as more views are acquired from a preferably equidistant perspective.

Drawings

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

FIG. 1 shows a schematic diagram of a first embodiment of a capture construct;

FIG. 2 schematically shows different affinity reagents;

FIG. 3 shows an affinity reporter reagent with a directly attached dye;

FIG. 4 shows a schematic overview of the elements of the capture construct;

FIG. 5 shows a schematic view of a first embodiment of a capture area;

FIG. 6 shows a schematic view of a second embodiment of a capture area;

FIG. 7 shows a schematic view of a third embodiment of a capture area;

FIG. 8 shows an illumination and detection point diffusion function series and a corresponding effective point diffusion function series;

FIG. 9 shows a detailed view of the capture construct according to FIG. 1;

FIG. 10 shows a detailed view of a second embodiment of a capture construct with several capture zones;

FIG. 11 schematically shows data read from a capture construct;

FIG. 12 shows capture constructs with different geometries;

FIG. 13 shows a capture construct according to FIG. 1 with a biological sample embedded in hydrogel beads; and

fig. 14 shows a sample container containing a biological sample.

Detailed Description

Fig. 1 shows a linear rod-like nanoarray 100 having a linear nanostructure skeleton 102, a first position indicator 104, a second position indicator 106, and a plurality of first capture regions 108 a-108 f.

Preferably, the nanostructure scaffold 102 comprises a nucleic acid. In particular, the nanostructure scaffold 102 is DNA-based, which allows the creation of arbitrary, stable two-dimensional and three-dimensional shapes.

The orientation indicators 104, 106 may be used to determine the orientation, directionality, or polarity of the nanoarray 100. The orientation indicators 104, 106 may comprise a dye, in particular a fluorescent dye, such as fluorescein or a fluorescent protein. In addition, the dye of the first orientation indicator 104 has different characteristics than the dye of the second orientation indicator 106. The features may include fluorescence emission features, excitation features, or lifetime features. This enables distinguishing between the first and second orientation indicators 104, 106 in an optical readout of the nanoarray 100 (e.g., an optical readout generated by a microscope, a cytometer, or an imaging cytometer). The position indicators 104, 106 are arranged spaced apart from each other. Preferably, each of the orientation indicators 104, 106 is disposed at opposite ends of the skeleton 102. Thus, the first and second orientation indicators 104, 106 enable distinguishing between the first and second ends of the skeleton 102, thereby distinguishing the nanoarray 100. Ultimately, this enables the orientation, directionality, or polarity of the nanoarray 100 to be determined, for example, from a first orientation indicator 104 on a first end to a second orientation indicator 106 on a second end. Based on the directionality, the position indicators 104, 106 generate a relative coordinate system of the nanoarray 100 on which each capture area 108 a-108 f may be placed. In the case of the linear nanoarray 100, each capture region 108 a-108 f is placed at a unique (unique) location on the scaffold 102. Each capture area 108a to 108f may be assigned an index n of n=1, 2, 3, … based on the unique location of the respective capture area 108a to 108f. Further, the position indicators 104, 106 and their corresponding unique dye features may be used to identify a particular capture construct 100 from among a variety of capture constructs having position indicators of different dye features.

Each capture region 108a-108f is configured to capture an analyte of a biological sample. The capture areas 108a-108f contain affinity capture reagents, and each capture area 108a-108f contains an affinity capture reagent that binds a particular analyte. Thus, the nanoarray 100 includes six capture areas 108a-108f to capture six different analytes. In addition, the first and second orientation indicators 104, 106 may act as capture areas, which will cause the nanoarray 100 to capture eight different analytes.

As shown in fig. 1, the capture areas 108a-108f and the position indicators 104, 106 may be placed such that their mutual spacing (D) is in the range of 500nm, which results in the skeleton 102 having a length L of about 3.5 μm. The distance D may be selected depending on the resolution of the readout device used to read out the capture areas 108a to 108f, and may be in the range of 1nm to 5nm, 10nm to 25nm, 50nm to 100nm, 100nm to 250nm, 250nm to 500nm, or 500nm to 1000 nm. Preferred ranges correspond to the lateral resolutions available with different microscope modes, such as, for example, single molecule localization microscopy (1 nm to 25 nm), structured illumination microscopy and STED microscopy (50 nm to 100 nm), high NA (numerical aperture ) optical microscopy (about 200 nm) and low NA optical microscopy (about 500 nm).

To read out the capture areas 108 a-108 f, the position indicators 104, 106 are typically also read out. As described above, this enables identification of unique (differential) capture areas 108a to 108f in the readout based on the index n.

Fig. 2 schematically shows different affinity reagents 200a to 200f. Affinity reagents 200a to 200f are, for example, single domain antibody 200a, dimerized single domain antibody 200b, antibody 200c, aptamer 200d, oligonucleotide-based affinity reagent 200e or small molecule-based affinity reagent 200f. These affinity reagents 200a to 200f may be used as affinity capture reagents for the capture zones of the capture zones 108a to 108f. The capture area 108a is shown here by way of example.

In a preferred embodiment, the affinity capture reagent comprises an oligonucleotide tag 202. Furthermore, the capture regions 108a to 108f may comprise corresponding complementary oligonucleotide tags 204, in particular in case the scaffold 102 is based on DNA folding. In this case, when designing and constructing the DNA origami backbone 102, the oligonucleotide tag 204 may be included in the backbone 102 such that the tag 204 is structurally accessible or protrudes from the structure at a specific location of the capture regions 108a to 108f. For example, a binding chain of DNA origami may comprise a tag 204. Since the binding strand is at known predetermined positions of the DNA fold, the affinity capture reagent can be attached to these known predetermined positions to form a capture region. Complementary tags 202, 204 can be used to assemble the capture construct. For example, the scaffold 102 may be constructed with unique tags 204 for each capture region 108 a-108 f, and the tags 204 are selected such that they correspond to the unique complementary tags 202 of the affinity capture reagents in each capture region 108 a-108 f. Thus, the capture regions 108 a-108 f are one region of the scaffold 102 where the affinity capture reagent is bound to the scaffold.

Alternatively, the affinity capture reagent may be covalently attached to the scaffold 102.

In addition, affinity reagents 200a through 200f may be used to generate affinity reporter reagents. In particular, this may be achieved by attaching the dyes 206a, 206b to the affinity reagents 200a to 200f with complementary oligonucleotide tags 202, 208 as described above. The dyes 206a, 206b may be fluorescent dyes, such as fluorescein or fluorescent proteins. In addition, the dyes 206a, 206b may have different characteristics, such as fluorescent emission characteristics, excitation characteristics, or lifetime characteristics. Dyes 206a, 206b comprise an oligonucleotide tag 208 that can be attached to the complementary oligonucleotide tag 202 of affinity reagents 200 a-200 b to provide a corresponding affinity reporter reagent.

The use of oligonucleotide tags 202, 204 and 208 enables the creation of libraries of affinity reagents 200a to 200b that can be mixed and matched according to the user's requirements to produce the desired affinity capture reagents and affinity reporter reagents. This enables flexible and cost-effective assembly of the affinity capture reagent and the affinity reporter reagent conjugated with a suitable dye in the nanostructure.

Alternatively, fig. 3 shows affinity reporter reagents 300a to 300f with directly attached dyes 206a, 206b. For example, the affinity reporter reagents 300 a-300 f may have covalently attached dyes 206a, 206b.

Fig. 4 shows a schematic overview of the elements of the capture construct 100, in particular the capture regions 108a to 108 f. Each capture region 108 a-108 f has a plurality of affinity capture reagents 400 attached to regions of the scaffold 102. These affinity capture reagents 400 may be attached to the scaffold 102 via linkers (e.g., oligonucleotide tags 202, 204). Affinity capture reagent 400 binds to a respective analyte 402. To analyze the captured analyte 402, affinity reporter reagents 300a-300 b may be attached to the analyte 402, with the reporter reagents 300a-300 b comprising dyes 206a, 206b that may be attached through linkers (e.g., oligonucleotide tags 202, 208). The linkers 202, 204, 208 are optional, and may also be photocleavable or enzymatically cleavable (e.g., with restriction enzymes, recombinases, endonucleases, criSPR/CAS, cre/loxP, and the like). Readout 404 may be achieved by sequencing (next generation sequencing (NGS)) or fluorescence detection to determine whether the analyte binds to a particular one of the capture areas 108 a-108 f. For example, where the reporter reagents 300a-300f comprise a sequencable oligonucleotide, readout may be accomplished by sequencing. The unique elements shown in fig. 4 may be combined, for example, a particular analyte (e.g., protein) may be captured by an antibody capture reagent, and an antibody fragment reporter reagent with a fluorescent dye attached may be used and read by a microscope. Alternatively, the capture reagent may be a small molecule and the reporter reagent may be an antibody fragment.

Fig. 5 to 7 show specific examples of possible configurations of the capture construct 100 (in particular of the capture regions 108a to 108 f).

Fig. 5 shows a schematic view of a capture area 500. A plurality of affinity capture reagents 502 in the form of single domain antibodies are attached to the capture region 500. The single domain antibody specifically binds to a particular analyte of interest 504 at a first binding site. Thus, the capture zone 500 binds to the analyte 504, wherein the capture reagent 502 is attached to the capture zone 500.

An affinity reporter 506 may then be added in order to determine whether the analyte 504 is captured by the capture reagent 502. Affinity reporter 506 in the form of an antibody binds to the analyte at least at the second binding site. Affinity reporter 506 comprises dye 508 (e.g., a fluorescent dye). Thus, the affinity reporter 506 accumulates at the capture zone 500 only when the analyte 504 binds to the capture zone 500. The presence of the affinity reporter 506 and thus the analyte 504 can then be read by a readout device based on the optical signal of the dye 508. The analyte 504 is determined to be captured in the capture zone 500 only if an optical signal of the dye 508 is detected in the capture zone 500.

More specifically, fig. 5 illustrates additional optional features of the capture area 500. The use of small affinity capture reagents 502 in the form of antibody fragments results in a separation of analyte binding sites in the grating of about 15nm, which corresponds approximately to the distance between two binding sites or paratopes of a conventional antibody, and is therefore suitable for producing an affinity effect that can significantly increase the overall sensitivity of the assay. In addition, the three-dimensionally arranged affinity capture reagent 502 along and around the periphery of the rod-like scaffold 102 increases the binding site density of the affinity capture reagent 502, thereby increasing the binding site density of the affinity reporter reagent 506. This increases the signal-to-noise ratio of the optical signal when the capture area 500 is read out. Finally, capturing a given analyte with two different affinity capture reagents and/or two different affinity reporter reagents increases the specificity of the assay and reduces steric problems, preferably each reagent having a different epitope.

Fig. 6 schematically shows a capture region 600 with an affinity capture reagent 602 in the form of an oligonucleotide. The affinity capture reagent 602 is configured to bind to an oligonucleotide analyte 604 comprising a complementary nucleic acid sequence. The analyte 604 bound to the capture reagent 602 can be determined by reading the presence of an affinity reporter 606, which affinity reporter 606 binds to the analyte 604 and comprises a nucleic acid sequence complementary to the analyte 604. Reagents 602, 606 and analyte 604 may comprise DNA, RNA and/or LNA nucleotides. This enables detection of a nucleic acid sequence with high sensitivity. This is particularly advantageous for many applications, as the nucleic acid sequences are present in body fluids or can be released from the cells after DNA cleavage and potential cleavage. This embodiment is also particularly advantageous for liquid biopsies in the case of diagnostic examinations and their use for detecting the presence of cancer. In this case, a circulating tumor DNA (ctDNA) target sequence may be detected. Furthermore, this embodiment is particularly advantageous for diagnostic tests (including sepsis tests) of pathogen infection (e.g. viral or bacterial infection). Further fields of application are pathogen detection in food and water quality detection and monitoring.

Fig. 7 schematically shows a capture region 700 having a first set of affinity capture reagents 702 in the form of oligonucleotides and a second set of affinity capture reagents 704 in the form of oligonucleotides. The affinity capture reagents 702, 704 are configured to bind the oligonucleotide analyte 706 at either the first complementary sequence or the second complementary sequence. In addition, each of the affinity capture reagents 702, 704 has a respective attached first dye 708 or second dye 710. When an analyte binds to one of the affinity capture reagents of the first set of affinity capture reagents 702 and one of the affinity capture reagents of the second set of affinity capture reagents 704, the first and second dyes 708, 710 of the respective affinity capture reagents 702, 704 will be brought into close proximity. When the dyes 708, 710 are in close proximity, they form a FRET pair and the corresponding optical signal can be detected by a readout device. FRET refers to fluorescence resonance energy transfer. This increases the specificity of analyte detection.

Fig. 8 to 10 show options for reading out the capture construct 100, in particular the capture regions 108a to 108f, 500, 600, 700.

Fig. 8 shows an illumination and detection Point Spread Function (PSF) column 800a and a corresponding effective PSF column 800b. Unless otherwise indicated, PSF refers to the primary maximum of PSFs in this document. Most microscopes illuminate and examine samples through the same objective. In this case, both the illumination PSF 802a and the detection PSF 804 are elliptical. For example, in the case of an optical sheet fluorescence microscope, the illumination PSF 802b may be sheet-like and the detection PSF 804 may be elliptical, which still produces an elliptical PSF, assuming that the detection PSF 802 is fully illuminated.

In the case of multi-view imaging with multiple detected PSFs (placed at angles 804a-804 f), which may or may not be combined with light sheet illumination, significant improvements in effective PSFs 806c, 806d over elliptical PSFs 806a, 806b may be achieved. In general, an isotropic (PSF) improves the ability of the nanoarray to resolve different capture areas 108a through 108f, 500, 600, 700 and makes it substantially invariant to the orientation of the nanoarray. In other words, if an imaging system having an elliptical effective PSF 806a is used, the resolving power in the axial direction (a) is lower than that in the lateral direction (l). In the case of PSF 806d and PSF 806c, the resolving power is comparable in all spatial directions (room direction), which is not required for reading out the nanoarrays, in particular the capture areas 108a to 108f, 500, 600, 700, but may be preferred.

Fig. 9 shows a detailed view of capture construct 100. The capture areas 108a-108f are 500nm apart from each other (as described above). The capture areas 108a-108f may be read by a readout device having a PSF 806a or a PSF 806d (as described in fig. 8). Importantly, the capture areas 108a-108f are spaced apart from one another so that the readout device can resolve the capture areas 108a-108f, respectively. Thus, all affinity reporter reagents of capture construct 100 may comprise the same dye.

Fig. 10 shows a detailed view of a capture construct 1000 with several capture regions. The capture construct comprises a plurality of first 1002a, a plurality of second 1002b, a plurality of third 1002c, a plurality of fourth 1002d, and a plurality of fifth 1002e capture regions. Reference numerals 1002a to 1002e refer to one of the respective plural constituent capturing areas. The capture areas 1002a-1002e are grouped, wherein each of the groups 1004a, 1004b contains a respective one of the capture areas 1002a-1002 e. The groups 1004a, 1004b are spaced apart from each other along the skeleton 102 by a distance (D) of 500nm. Each capture region 1002a-1002e is approximately 25nm along the width (d) of the skeleton 102.

The capture areas 1002a-1002e may be read by a readout device having a PSF 806a or a PSF 806d (as described in fig. 8 and 9). However, in order to distinguish between the capture regions 1002a-1002e of each set 1004a, 1004b using a readout device, the affinity reporter reagent of the capture construct 1000 comprises a different dye. Specifically, the affinity reporter reagent of the plurality of capture areas 1002a-1002e comprises a dye having a characteristic unique to each of the plurality of capture areas 1002a-1002 e. This enables readout of the individual capture areas 1002a to 1002e of the individual groups 1004a, 1004 b.

This results in an increase in the density of capture areas 1002a-1002e of capture construct 1000, and is accompanied by a substantial increase in the number of capture areas 1002a-1002e as compared to capture construct 100 in FIG. 1.

Fig. 11 schematically illustrates the readout of data from capture constructs 100 and 1000. The optical signals determined by reading out the capture areas 108a to 108f, 1002a to 1002e can be classified as binary codes of "0" and "1", with the fluorescent signal therefrom appearing as "1" when the analyte is present and with no fluorescent signal appearing as "0" when the analyte is not present. For example, a given sequence 0101010 may be interpreted or decoded for a given capture construct with known affinity reagents at each position of the capture region, and the directionality of that capture construct interpreted or decoded based on the orientation indicators 104, 106. This means that the identity of the analyte can be calculated from a given sequence or simply looked up in a memory file or database. In addition to providing a binary answer to the question whether an analyte is detected, the method provides intensity information that can be used for relative quantification (i.e. analyte 1 has a 5x higher signal than analyte 2) or absolute quantification (i.e. the intensity of the reading of analyte 1 corresponds to 10 dye molecules, which for example corresponds to 5 analyte molecules).

FIG. 12 shows capture constructs with different geometries. The sheet capture construct 1200 may be a large linear DNA molecule or an assembly of multiple DNA molecules. The platelet-shaped nanoarray can significantly increase the number of available capture areas. To be able to determine the position of the capture construct 1200, a third position indicator 1202 is provided.

Other geometries are possible, such as tetrahedral capture construct 1204, cubic capture construct 1206, or polyhedral capture construct 1208. They may contain a fourth orientation indicator 1210 to determine their orientation.

Fig. 13 shows capture construct 100 and biological sample 1300 embedded in hydrogel bead 1302. Hydrogel bead 1302 is a multi-phase hydrogel bead having a phase for culturing biological sample 1300 (e.g., single cell or multiple cells in 3D cell culture) and at least one additional layer that may be inside or around the culture phase/layer. The at least one further layer may be of the same or different material. Alternatively, biological sample 1300 and capture construct 100 can be embedded in single-phase hydrogel beads 1304.

Biological sample 1300 encapsulated in hydrogel beads 1302 may be cultured and secreted proteins. For example, immune cells may be isolated after liquid biopsies of tumor patients or patients with infectious diseases to study the patient's immunophenotype, and/or immune repertoire and/or immunocompetence, and to determine an optimal treatment regimen. In this respect are of great interest to the molecules to be separated (e.g. as cytokines) and indicate the activation status of certain immune cells. The present invention utilizes a nanoarray carrying a capture region for capturing an analyte of interest, which may preferably be DNA-based folded paper.

The readout device may include a flow channel 1306 through which the hydrogel beads 1302, 1304 may flow in the liquid 1308. The flow cell 1306 contains an optical window 1310 through which the hydrogel beads 1302, 1304 (particularly the capture constructs 100, 1000) can be imaged by an optical imaging device (e.g., a microscope).

Fig. 14 shows a sample container, such as a microplate 1400 having sample wells 1402 and a culture dish 1404, containing a biological sample (e.g., single cells embedded in a hydrogel). The capture construct 100, 1000 may be suspended in a liquid or embedded in the hydrogel 1406 within the sample well 1402 along with the biological sample 1408 (e.g., single-cell or multi-cell structure in culture). The readout device can image the contents of the aperture 1402 through the optical window 1409 and determine the centroid 1410 of the biological sample 1408 and the capture construct 100 that is within the predetermined radius 1412 of the centroid 1410 of the biological sample 1408.

Thus, to capture and detect target analytes secreted by biological sample 1300, 1408 by capture construct 100, 1000, biological sample 1300 and 1408 are incubated in the presence of capture construct 100, 1000 or multiple capture constructs 100, 1000, comprising a capture region having an affinity capture reagent that binds to target analytes. This enables the analyte secreted by the biological sample 1300, 1408 to be captured by the capture construct 100, 1000, in particular the corresponding affinity capture reagent. Subsequently, at least the capture constructs 100, 1000, in particular the capture areas and the readouts of the orientation indicators, are acquired. The readout may be acquired by an optical device (e.g., a microscope or an imaging flow cytometer). Based on the reads, the capture regions are then evaluated to determine whether each target analyte is captured by the capture region of the capture construct.

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 apparatus, these aspects also clearly represent descriptions of corresponding methods in which a module or apparatus corresponds to a method step or a feature of a method step. Similarly, aspects described in the context of method steps also represent descriptions of features of a corresponding module or item or corresponding apparatus.

List of reference numerals

100. 1000 Capture construct

102. Nano-structure skeleton

104. 106, 1202, 1210 azimuth indicators

108a、108b、108c、

108d、108e、108f、

500、600、700、1002a、

1002b、1002c、1002d、

1002e capture area

200a, 200b, 200c, 200d, 200e, 200f affinity reagents

202. 204, 208 oligonucleotide tag

206a, 206b, 508, 708, 710 dyes

300a、300b、300c、

300d、300e、300f、

506. 606 affinity reporter reagent

400. 502, 602, 702, 704 affinity capture reagents

402. 504, 604, 706 analytes

404. Readout of

800a Lighting and detection Point diffusion function array

800b effective point diffusion function array

802a, 802b illumination point spread function

804、804a、804b、

804c、804d、804e、

804f detection point spread function

806a, 806b elliptic effective point spread function

806c,806d isotropic point spread function

1004a, 1004b capture groups of regions

1300. 1408 biological sample

1302. 1304 hydrogel beads

1306. Flow groove

1308. Liquid

1310. 1409 optical window

1400. Microplate

1402. Sample well

1404. Culture dish

1406. Hydrogel or 3D cell culture matrix

1410. Centroid of mass

Claims (22)

1. A capture construct (100, 1000) for capturing a plurality of analytes (402,504,604,706) of a biological sample (1300,1408), comprising:

a nanostructured framework (102),

at least one first orientation indicator (104) and at least one second orientation indicator (106), and

at least a plurality of first capture regions (108 a-108 f) on the nanostructure scaffold (102), each capture region (108 a-108 f) comprising at least one affinity capture reagent (400,502,602,702,704), the affinity capture reagent (400,502,602,702,704) configured to capture one of the analytes (402,504,604,706).

2. The capture construct of claim 1, wherein the nanostructure scaffold (102) comprises a nucleic acid.

3. The capture construct according to any of the preceding claims, wherein the affinity capture reagent (400,502,602,702,704) is selected from antibodies, antibody fragments, oligonucleotides, aptamers, peptides, drugs and/or toxins.

4. The capture construct of any of the preceding claims, further comprising a plurality of first affinity reporter reagents (300 a-300f,506, 606), each affinity reporter reagent (300 a-300f,506, 606) comprising a first reporter tag (206 a,206b, 508) and configured to be attached to one of the analytes (402,504,604,706), wherein the first reporter tag (206 a,206b, 508) is readable to determine whether each of the analytes (402,504,604,706) is captured by the respective affinity capture reagent (400,502,602,702,704).

5. The capture construct of claim 4, wherein the affinity reporter reagent (300 a-300f,506, 606) is selected from the group consisting of an antibody, an antibody fragment, an oligonucleotide, an aptamer, a peptide, a drug and/or a toxin.

6. The capture construct of claim 4 or 5, wherein the reporting tag (206 a,206b, 508) is optically readable.

7. The capture construct according to any one of claims 4 to 6, wherein the reporter tag (206 a,206b, 508) is an oligonucleotide and is readable by sequencing.

8. The capture construct of any one of the preceding claims, wherein the affinity capture reagent (400,502,602,702,704) binds to the nanostructure scaffold, or wherein the affinity capture reagent (400,502,602,702,704) of one particular of the capture regions (108 a-108 f) comprises an oligonucleotide and each of the capture regions (108 a-108 f) comprises a complementary oligonucleotide that binds to the oligonucleotide of the affinity capture reagent (400,502,602,702,704).

9. The capture construct of any of the preceding claims, wherein the nanostructure scaffold (102) comprises at least a plurality of second capture regions (1002 a), each second capture region (1002 a) comprising at least one affinity capture reagent (400,502,602,702,704), the affinity capture reagent (400,502,602,702,704) being configured to capture one of the analytes (402,504,604,706).

10. The capture construct of any of the preceding claims, further comprising a plurality of second affinity reporter reagents (300 a-300f,506, 606), each affinity reporter reagent (300 a-300f,506, 606) comprising a second reporter tag and being configured to be attached to one of the analytes, wherein the second reporter tag is readable to determine whether each of the analytes is captured by the respective affinity capture reagent (400,502,602,702,704).

11. The capture construct of any of the preceding claims, wherein the nanostructure scaffold (102) extends linearly in one dimension and the first orientation indicator (104) and the second orientation indicator (106) are spaced apart from each other or arranged at opposite ends of the nanostructure scaffold (102).

12. The capture construct of any of the preceding claims 1 to 10, wherein the nanostructure scaffold (102) extends in two dimensions or three dimensions and the nanostructure scaffold (102) comprises at least one third position indicator (1202,1210).

13. The capture construct according to any of the preceding claims, wherein the maximum spatial limit of the nanostructure scaffold (102) is in the range of 1nm to 10000nm, preferably in the range of 0.1 μιη to 5 μιη.

14. The capture construct of any of the preceding claims, wherein the capture regions (108 a-108 f) of the plurality of first capture regions (108 a-108 f) are spaced apart from each other by a range of 1nm to 2000nm, preferably by a range of 200nm to 1000 nm.

15. The capture construct of claim 9, wherein the capture regions (108 a-108 f) of the plurality of first capture regions (108 a-108 f) are spaced apart from the capture regions (1002 a-1002 e) of the plurality of second capture regions (1002 a-1002 e) by a range of 0.1nm to 500nm, preferably by a range of 1nm to 100 nm.

16. The capture construct according to any of the preceding claims, wherein the reporter label comprises a fluorescent group, in particular the fluorescent group has different excitation wavelength, fluorescence emission wavelength and/or fluorescence lifetime characteristics.

17. The capture construct of any of the preceding claims, wherein the affinity capture reagent (400,502,602,702,704) of at least one of the capture regions (108 a-108 f) is configured to bind one of the analytes at a single binding site for the analyte.

18. The capture construct of any of the preceding claims, wherein at least one capture region (108 a-108 f) comprises a first set of affinity capture reagents (400,502,602,702,704) and a second set of affinity capture reagents (400,502,602,702,704), and wherein the first set of affinity capture reagents (400,502,602,702,704) is configured to bind to the one of the analytes at a first binding site of the one analyte, and the second set of affinity capture reagents (400,502,602,702,704) is configured to bind to the one of the analytes at a second binding site of the one analyte.

19. The capture construct of claim 18, wherein the first set of affinity capture reagents (400,502,602,702,704) comprises a first capture reagent dye (708) and the second set of affinity capture reagents (400,502,602,702,704) comprises a second capture reagent dye (710), in particular the capture reagent dye is a fluorescent group.

20. The capture construct of claim 19, wherein the first capture reagent dye (708) and the second capture reagent dye (710) are configured to cause proximity when the one analyte is captured by one of the affinity capture reagents (400,502,602,702,704) of the first set of affinity capture reagents (400,502,602,702,704) and one of the affinity capture reagents (400,502,602,702,704) of the second set of affinity capture reagents (400,502,602,702,704), and wherein the proximity enables transfer of energy between the respective capture reagent dyes (708, 710).

21. The capture construct according to any of the preceding claims, wherein the capture construct (100, 1000) and the biological sample (1300,1408) are embedded in or attached to a polymeric compound, in particular a hydrogel.

22. A method of detecting a plurality of analytes (402,504,604,706) of a biological sample (1300,1408), the method comprising the steps of:

incubating the biological sample in the presence of at least one capture construct (100, 1000) according to any of the preceding claims;

obtaining a readout of the at least one capture construct (100, 1000), in particular of the capture region (108 a-108f,1002a-1002 e); and

determining whether each of said analytes is captured by a respective one of said affinity capture reagents (400,502,602,702,704).