WO2025218883A1

WO2025218883A1 - Analysis system and method for analysing capture constructs

Info

Publication number: WO2025218883A1
Application number: PCT/EP2024/060273
Authority: WO
Inventors: Soeren Alsheimer
Original assignee: Leica Microsystems CMS GmbH
Current assignee: Leica Microsystems CMS GmbH
Priority date: 2024-04-16
Filing date: 2024-04-16
Publication date: 2025-10-23
Anticipated expiration: 2026-10-16

Abstract

An analysis system (1300, 1400, 1500) for analysing capture constructs (100, 1000, 1302) is provided. The analysis system (1300, 1400, 1500) comprises an analysing space with a surface (1304) comprising at least first binding moieties (1306) configured to bind the capture constructs (100, 1000, 1302), and an electrophoretic device (1312, 1314) configured to apply an electric field to the analysing space. In a further aspect a method for analysing capture constructs (100, 1000, 1302) is provided.

Description

Analysis system and method for analysing capture constructs

Technical field

The invention relates to an analysis system and a method for analysing capture constructs. The capture constructs are configured to capture analytes of biological samples.

Background

High-throughput analyses of biological samples, particularly when employing optical methods in combination with fluorescent dyes, encounter substantial challenges related to the inherent disorder and variability of biological specimens. Biological samples are often heterogeneous, with individual cells or molecules exhibiting diverse characteristics even within seemingly homogeneous populations. This heterogeneity can lead to variations in fluorescence intensity, localisation, and other parameters, making it difficult to obtain precise and consistent measurements.

Another significant issue in high-throughput analyses of biological samples is the potential for contamination and interference from extraneous factors. Biological samples are prone to contamination from environmental contaminants, crosscontamination between samples, or unintended interactions with experimental materials. These issues can introduce confounding variables and compromise the accuracy and reproducibility of the analysis.

Summary

It is an object to provide a device and a method for robustly 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.

In a first aspect an analysis system for analysing capture constructs is provided. The analysis system comprises an analysing space with a surface comprising at least first binding moieties configured to bind the capture constructs, and an electrophoretic device configured to apply an electric field to, in particular across, the analysing space.

Thus, the capture constructs may be bound to the analysis surface of the analysing space by means of the binding moieties. The capture constructs enable capturing or binding relevant target analytes from a biological sample. The analysis system in turn enables analysis of the capture reagents, in particular the captured target analytes, for example by aligning the capture constructs for further analysis. This enables efficient and robust analysis of the capture constructs, in particular of the target analytes captured by the capture constructs.

Preferably, the capture constructs have a charge, in particular a net electrical charge. This enables efficiently orientating the capture constructs that are bound to the binding moieties in the applied electric field.

In particular, the capture constructs are for capturing a plurality of analytes, for example of a biological sample. The biological sample may be a multicellular structure such as a cluster of live cells in particular a spheroid, or a single cell, enabling single cell analysis. The biological sample may further be a tissue section or tissue biopsy, a water sample, or an environmental sample. The biological sample may further be a liquid biopsy of blood, plasma, serum, semen, sputum, liquor, lympha, or any other bodily fluid. The biological sample may be a lysate of any of the aforementioned. Such a lysate may be generated by mechanical, chemical, enzymatic tissue and/or cell lysis and may be further purified by filtration. The lysate may further be processed to increase the relative abundance of target analytes, which are frequently of medium- to low abundance with respect to high abundance proteins like for example albumin, fibronectin, or immunoglobulins. The biological sample may be a supernatant of a cell culture for example.

In particular, the capture constructs each comprise a nanostructure backbone, at least a first orientation indicator and a second orientation indicator, and at least a first plurality of capture regions on the nanostructure backbone, each capture region comprising at least one affinity capture reagent configured to capture one of the analytes. The capture construct enables capturing the plurality of analytes at predetermined positions, the capture regions, on the nanostructure. Further, the capture construct enables capturing the analytes at a particularly high density. The capturing of the plurality of analytes enables subsequent analysis of the plurality of analytes.

The analytes may be a range of molecules. For example, an analyte may be a chemical species such as a metabolic product of the biological sample, or a cell signalling molecule of the biological sample. Further, the analyte may be a protein or peptide of the biological sample, such as a particular enzyme. Further, the analyte may be a hormone or a neurotransmitter. Further, the analyte may be a cell expressing a certain cell-surface protein or 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 certain glycosylation pattern. Further, the analyte may be a bacterium, an archaeon, a fungus (e.g. a yeast), or a virus. Further, the analyte may be a toxin or a heavy metal. Even further, the analyte may be a nucleic acid molecule, such as DNA or RNA, with a particular nucleic acid sequence. In particular, the analyte may be secreted by the biological sample, thus, enabling the capture of at least part of the secretome of the biological sample. Each affinity capture reagent may be configured to capture or bind to specifically one of the analytes. Each one of the first plurality of the capture regions may comprise at least one affinity capture reagent attached to the nanostructure backbone in that capture region and configured to specifically bind to or capture one of the analytes. In other words, the capture construct may be configured such that each analyte is being captured in a particular one of the first capture regions.

The orientation indicators of the capture construct are preferably configured to attach to the backbone of the capture construct. The orientation indicators may be for visually determining the orientation of the capture construct in space. The first orientation indicator and the second orientation indicator may be, for example, fluorescent dyes attached to the nanostructure backbone. In particular, the fluorescent dyes may have different optical properties, such as emission wavelength, for each orientation indicator. In particular, the orientation indicators may comprise fluorescent dyes that are either directly or indirectly attached (e.g. via staple strands and hybridization of oligonucleotide-conjugated fluorescent dyes) to the nanostructure backbone. For example a first orientation indicator may be marked with ATTO390 and a second with ATT0700. One or multiple orientation indicators may further comprise a fluorescent barcode of multiple dyes, which encodes the identity of the capture construct, e.g. ATTO390-ATTO488 may be capture construct 1, whereas ATTO390-ATTO647N maybe capture construct 2. In this way readout of the orientation indicators allows identification of the capture construct and orientation in space, which enables identifying the capture regions, e.g. capture region 1, 2, 3,...n on capture construct m.

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

Preferably, the nanostructure backbone of the capture construct extends in two dimensions or three dimensions and the nanostructure backbone comprises at least a third orientation indicator.

Thus, the orientation indicators enable determining the spatial orientation or the directionality of the capture construct, in particular the nanostructure backbone. In this way, the orientation indicators enable spatial encoding. This means, different positions on the nanostructure backbone may be assigned to capture bands or capture regions that have reactivities to distinct analytes.

It is preferred, that the largest spatial extent of the nanostructure backbone of the capture construct is in a range from 1 nm to 10000 nm, preferably in a range from 0.1 pm to 5 pm, more preferably in a range from 0.1 pm to 1 pm. This enables a particularly compact capture construct. In particular, the largest spatial extent of the nanostructure backbone is the extent of a longitudinal axis of the capture construct.

Preferably, the nanostructure backbone of the capture construct comprises nucleic acids. For example, the nanostructure backbone may comprise DNA, RNA and/or LNA. In particular, the nanostructure backbone may comprise DNA origami or a DNA brickbased nanostructure. These DNA origami structures may range in size from a few nanometres into the micron range. For the fabrication of such DNA origami-based structures longer DNA molecules (scaffold strands) are folded at precisely identified positions by so called staple strands. The DNA origami may be designed to provide a self-assembly nanostructure backbone of a particular predetermined shape. This enables an easy and reproducible synthesis and assembly of the backbone. Staple strands may be position-selectively functionalised. The positional resolution in this case is limited by the size of a nucleotide, which is in the range of a nanometre or below. This has been exploited in the prior art to generate fluorescent standards, wherein fluorescent dyes are connected to precisely located bands on the DNA origami. These standards are known as "nanoruler" and are used for the calibration of imaging systems like confocal or super resolution microscopes (e.g. STED), for example, as disclosed by US2014/0057805 Al.

The DNA origami provides a scaffold for the affinity capture reagents. Preferably, the DNA origami structure comprises at least one scaffold strand and multiple staple strands, wherein the staple strands are complementary to at least parts of the scaffold strand and configured to bring the scaffold strand into a predetermined conformation. In particular, the strands are oligonucleotides. This enables generating nanostructure backbones with predetermined two- or three-dimensional shapes that can selfassemble. Further, this enables the site-specific placement of capture regions on the backbone. Preferably, the affinity capture reagents of the capture regions may be attached to staple strands of the nanostructure backbone at predetermined positions. Staple strands allow the spatially precise functionalisation of the DNA origami at their respective locations on the DNA origami. Thus, each capture region may be located along the nanostructure backbone at a particular staple strand or group of staple strands that are in close proximity. Since the staple strands are located at predetermined positions the positions of the capture regions may equally be predetermined.

In a preferred embodiment, the capture construct comprises a first plurality of affinity reporter reagents, each affinity reporter reagent comprising a first reporter tag and configured to attach to one of the analytes, wherein the first reporter tag is readable, to determine whether or not the respective analyte is captured by the respective affinity capture reagent. This enables determining the presence of a particular analyte by means of the capture construct. Since the capture construct comprises the first plurality of capture regions, a plurality of particular analytes may be captured and their presence determined. Similarly to the affinity capture reagents, each affinity reporter reagent may be configured to capture or bind to specifically one of the analytes. Therefore, each first capture region may comprise at least one affinity reporter reagent configured to specifically bind to one of the analytes associated with one of the first plurality of capture regions. Thus, for each target analyte one capture region with specific affinity capture reagents and specific affinity reporter reagents is provided.

Preferably, the affinity reporter reagents are selected from an antibody, an antibody fragment such as a single-domain antibody, an aptamer, a peptide, an oligonucleotide, an aptamer, a drug, and/or a toxin. The affinity reagent may also be an artificial polymeric binder configured to bind a target analyte with high affinity and specificity. This enables capturing and reporting a large variety of analytes with the capture construct. Generally, the affinity reporter reagents of a particular capture region may bind to at least one particular binding site of the respective analyte.

In a particularly preferred embodiment, the reporter tag, in particular the first reporter tag, is optically readable. For example, the reporter tag may comprise a fluorescent dye that is optically detectable. This enables determining by means of a microscope, in particular by imaging with a microscope, whether or not an analyte is captured by the respective affinity capture reagent and the respective affinity reporter reagent with its associated reporter tag is bound to that analyte.

Preferably, the reporter tag, in particular the first reporter tag, is an oligonucleotide and readable by sequencing. This enables determining, whether or not an analyte is captured by the respective affinity capture reagent and the respective affinity reporter reagent with its associated reporter tag is bound to that analyte.

Further details and examples of suitable capture constructs as well as methods for detecting analytes of a biological sample with the capture constructs may be found in the document WO 2022/207832, the contents of which are incorporated herein by reference.

Thus, the analysis system may be used to analyse the capture constructs, in particular the analytes captured by the affinity capture reagents of the respective capture regions. The binding of the capture constructs to the surface of the analysing space of the analysis system enables orientating all bound capture constructs in the electric field. The common orientation of the bound capture constructs enables easy and efficient analysis of the capture constructs, in particular of the captured analytes, in optical readouts of the analysing space. In particular, the orientation indicators of the capture constructs may additionally aid the identification and analysis of capture regions of bound capture constructs.

In a preferred embodiment of the analysis system, the first binding moieties are configured to bind the capture constructs at a first end of the capture constructs. This enables efficient alignment of the capture constructs with the electric field. In particular, the first end of the capture constructs may be an end along a longitudinal axis of each of the capture constructs or an end in a direction of the largest spatial extent of each of the capture constructs. Further, the binding of the capture constructs to the surface of the analysing space enables aligning the capture constructs in a common plane.

Preferably, the first binding moieties (106) comprise at least one of a biotin and a streptavidin. In a particular embodiment, the first binding moieties may comprise one of the biotin and the streptavidin and the capture constructs may comprise the other one of the biotin and the streptavidin. This enables robust binding of the capture constructs to the surface of the analysing space, in particular due to the near-covalent interaction between biotin and streptavidin. In an alternative embodiment, the first binding moieties may comprise both the biotin and the streptavidin, wherein one of the biotin and the streptavidin is, preferably covalently, attached to the surface of the analysing space and the other one of the biotin and the streptavidin is attached, preferably covalently, to an element of the first binding moieties that binds to the capture reagents, for example an antibody. This enables efficient assembly of the analysis system, in particular efficient attachment of particular binding moieties from a set of binding moieties with different affinities to surface of the analysing space.

Preferably, the first binding moieties are configured to bind the capture constructs specifically. This enables attachment of specific capture constructs from a plurality of different capture constructs to the surface of the analysing space. For example, the first binding moieties may comprise biotinylated oligonucleotides attached to a streptavidin on the surface of the analysing space. The biotinylated oligonucleotides may comprise a specific sequence complementary to a complementary oligonucleotide of the capture construct.

Preferably, the surface of the analysing space comprises second binding moieties configured to bind the capture constructs. This enables particularly robust binding of the capture constructs to the surface of the analysing space. In a particular embodiment, the second binding moieties are configured to bind the capture constructs at a second end of each of the capture constructs. The second end is preferably at an end of the capture construct opposing the first end. In particular, the second binding moiety may be a biotinylated oligonucleotide attached to the surface of the analysing space and configured to bind to a complementary oligonucleotide of the capture construct.

Preferably, each of the first binding moieties are arranged on the surface at a distance from an adjacent one of the second binding moieties, the distance being at least the length of the capture constructs along a longitudinal axis, in particular from a first end to a second end of the capture constructs or the length along the largest spatial extent. This avoids overlapping capture constructs on the surface of the analysing space and enables robust detection of the capture constructs in an optical readout of the surface. In particular, the length of the capture constructs is the length or size of their respective nanostructure backbones. Preferably, the distance is a maximum of twice the length of the capture constructs, in particular, to enable sufficiently dense arrangement of the capture constructs on the surface of the analysing space.

Preferably, the capture constructs comprise nucleic acids or nucleic acid analogues. This enables robust capture constructs. In addition, nucleic acids, in particular due to their phosphodiester backbones, generally are charged molecules. Nucleic acids are naturally occurring deoxyribonucleic acids, for example. Nucleic acid analogues are non-naturally occurring xeno nucleic acids, for example.

Preferably, the capture constructs have a negatively charged first region and a positively charged second region. This enables efficient orientation of the capture constructs in the electric field. For example, the first region may be arranged at the first end of the capture constructs and the second region may be arranged at the second end of the capture constructs.

Preferably, each capture construct comprises at least a first plurality of capture regions, each capture region comprising at least one affinity capture reagent configured to capture an analyte. This enables capturing a plurality of analytes, in particular different analytes, on each capture construct.

Preferably, each capture construct comprises a first optically detectable orientation indicator and a second optically detectable orientation indicator. This enables identifying the capture constructs in the analysing space, in particular determining the orientation of the capture constructs. Thus, the orientation indicators are for determining the orientation of the capture constructs. In particular, the orientation indicators are attached to the capture constructs, for example, the orientation indicators may be arranged at respective first and second ends of the capture constructs. Preferably, the analysis system comprises the capture constructs.

Preferably, the analysis system comprises an optical readout device configured to generate an optical readout of the capture constructs bound to the surface of the analysing space. This enables efficient analysis of the bound capture constructs. The optical readout device may be a microscope, for example.

In another aspect a method for analysing capture constructs is provided. The method comprises the following steps: contacting the capture constructs with a surface of an analysing space, the surface comprising at least first binding moieties configured to bind the capture constructs, in particular, this enables binding of the capture constructs to the surface of the analysing space. In another step, an electric field is applied to or along the analysing space. In a further step, an optical readout of the capture constructs is generated. This enables analysing the capture constructs, in particular analytes captured by the capture constructs.

Applying the electric field to the analysing space enables aligning the capture constructs with the electric field, in particular, aligning all bound capture constructs along a common direction. Further, the binding of the capture constructs to the surface of the analysing space enables bringing all capture constructs into a single plane. This enables efficient generation of the optical readout in the subsequent step, due to all capture constructs being a in a single (focus) plane.

Preferably, the capture constructs are contacted with target analytes of a biological sample. This enables capture of the target analytes by the capture constructs, in particular the respective affinity capture reagent. In particular, the capture constructs are contact with the target analytes prior or after contacting the capture constructs with the surface of the analysing space. Preferably, prior to generating the optical readout, a first set of affinity reporter reagents is introduced to the analysing space. This enables efficiently determining the presence of a particular analyte bound to the capture constructs.

Preferably, the first set of affinity reporter reagents is removed after generating an optical readout and a second set of affinity reporter reagents is introduced to the analysing space and a further optical readout is generated. This enables iteratively determining the presence of a particular analyte.

Short Description of the Figures

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

Figure 1 is a schematic view of a first embodiment of a capture construct,

Figure 2 is a schematic view of different affinity reagents,

Figure 3 is a schematic view of affinity reporter reagents with directly attached dyes,

Figure 4 is a schematic view of an overview of the elements of the capture construct,

Figure 5 is a schematic view of a first embodiment of a capture region,

Figure 6 is a schematic view of a second embodiment of the capture region,

Figure 7 is a schematic view of a third embodiment of the capture region, Figure 8 is a schematic view of illumination and detection points spread functions and of corresponding effective point spread functions,

Figure 9 is a schematic view of details of the capture construct according to Fig. 1,

Figure 10 is a schematic view of details of a capture construct with several pluralities of capture regions,

Figure 11 is a schematic view of read out data from a capture construct,

Figure 12 is a schematic view of capture constructs with different geometries,

Figure 13 is a schematic view of an analysis system for analysing capture constructs,

Figure 14 is a schematic view of a further analysis system for analysing capture constructs, and

Figure 15 is a schematic view of a further analysis system and steps of a method for analysing capture constructs.

Detailed Description

In the following Figures 1 to 12 describe embodiments of capture constructs. Figures 13 to 15 describe analysis systems, in particular analysis systems to be used in conjunction with the aforementioned capture constructs.

Figure 1 shows a linear, rod-like capture construct 100 with a linear nanostructure backbone 102, a first orientation indicator 104, a second orientation indicator 106, a first plurality of capture regions 108a to 108f.

Preferably, the nanostructure backbone 102 comprises nucleic acids. In particular, the nanostructure backbone 102 is a DNA-origami based, which allows generating arbitrary, stable two- and three-dimensional shapes.

The orientation indicators 104, 106 may be used to determine the orientation, directionality or polarity of the capture construct 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 characteristics may include fluorescent emission characteristics, excitation characteristics or lifetime characteristics. This enables differentiating between the first and the second orientation indicators 104, 106 in an optical readout of the capture construct 100, for example generated by a microscope, a cytometer, or an imaging cytometer. The orientation indicators 104, 106 are arranged spaced apart from each other. Preferably each orientation indicator 104, 106 is arranged on the backbone 102 at opposite ends. Thus, the first and second orientation indicators 104, 106 enable differentiating between a first end and a second end of the backbone 102 and therefore of the capture construct 100. Ultimately, this enables determining the orientation, directionality or polarity of the capture construct 100, for example from the first orientation indicator 104 on the first end to the second orientation indicator 106 on the second end. Based on the directionality, the orientation indicators 104, 106 generate a relative coordinate system for the capture construct 100, on which each capture region 108a to 108f may be placed. In case of the linear capture construct 100, each capture region 108a to 108f is placed at a unique location on the backbone 102. Each capture region 108a to 108f may be assigned an index n with n=l, 2, 3, ..., based on the unique location of the respective capture region 108a to 108f between the orientation indicators 104, 106. In addition, the orientation indicators 104, 106 and their corresponding unique dye characteristics may be used to identify the particular capture construct 100 from a variety of capture constructs with orientation indicators with different dye characteristics.

Each capture region 108a to 108f is configured to capture an analyte of a biological sample. The capture regions 108a to 108f comprise affinity capture reagents with each capture region 108a to 108f comprising affinity capture reagents that bind a particular analyte. Thus, the capture construct 100 comprises six capture regions 108a to 108f to capture six different analytes. In addition, the first and second orientation indicators 104, 106 may be used as capture regions, which would result in the capture construct 100 capturing eight different analytes.

As indicated in Figure 1, the placing of the capture regions 108a to 108f and orientation indicators 104, 106 may be such that their distance to each other (D) is in the range of 500 nm, which leads to the backbone 102 having a length L of approximately 3.5 pm. The spacing D may be chosen depending on the resolving power of a readout device used to read out the capture regions 108a to 108f and may be in a range of 1 nm to 1000 nm, in particular, in a range of 1 nm to 5 nm, 10 nm to 25 nm, 50 nm to 100 nm, 100 nm to 250 nm, 250 nm to 500 nm, or 500 nm to 1000 nm. The preferable ranges correspond to the lateral resolution achievable with different microscopic modalities such as for example single molecule localization microscopy (1 nm to 25 nm), structured illumination and STED microscopy (50 nm to 100 nm), high NA (numerical aperture) light microscopy (around 200 nm), and low NA light microscopy (around 500 nm).

In order to read out the capture regions 108a to 108f, generally, the orientation indicators 104, 106 are read out as well. This enables identifying the individual capture regions 108a to 108f in the readout based on the index n, as described above. Figure 2 shows schematically different affinity reagents 200a to 200f. The affinity reagents 200a to 200f are, for example, single domain antibodies 200a, dimerised single domain antibodies 200b, antibodies 200c, aptamers 200d, oligonucleotide- based affinity reagents 200e, or small molecule-based affinity reagents 200f. These affinity reagents 200a to 200f may be used as the affinity capture reagents of one of the capture regions 108a to 108f. In Fig. 2, the capture region 108a is exemplarily shown.

In a preferred embodiment, the affinity capture reagents comprise oligonucleotide tags 202. Further the capture regions 108a to 108f may comprise corresponding complementary oligonucleotide tags 204, in particular, in case the backbone 102 is a DNA origami-based. The oligonucleotide tags 204 may in that case be included in the backbone 102 when designing and constructing the DNA origami backbone 102 such that the tags 204 are accessible on the structure or protrude from the structure at the specific locations of the capture regions 108a to 108f. For example, the staple strands of the DNA origami may comprise the tags 204. Since the staple strands are at known predetermined locations of the DNA origami, the affinity capture reagents may be attached to these known predetermined locations to form the capture regions. Complementary tags 202, 204 may be used to assemble the capture construct. For example, the backbone 102 may be constructed with unique tags 204 for each capture region 108a to 108f and the tags 204 chosen such that they correspond to the unique complementary tags 202 of the affinity capture reagents of each capture region 108a to 108f. Thus, the capture regions 108a to 108f are an area of the backbone 102, in which affinity capture reagents are bound to the backbone.

Alternatively, the affinity capture reagents may be covalently attached to the backbone 102.

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

The use of oligonucleotide tags 202, 204, 208 enables creating libraries of affinity reagents 200a to 200b that can be mixed and matched according to a user's requirements to result in required affinity capture reagents and affinity reporter reagents. This enables the flexible and cost-effective assembly of affinity capture reagents on the nanostructures, as well as the assembly of suitable dye-conjugated affinity reporter reagents.

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

Figure 4 shows a schematic overview of the elements of the capture construct 100, in particular of one of the capture regions 108a to 108f. Each capture region 108a to 108f has a plurality of affinity capture reagents 400 attached to the area of the backbone 102. These affinity capture reagents 400 may be attached to the backbone 102 by linkers such as the oligonucleotide tags 202, 204. The affinity capture reagents 400 bind a respective analyte 402. In order to analyse the captured analytes 402, the affinity reporter reagents 300a to 300b may be bound to the analyte 402, the reporter reagents 300a to 300b comprising a dye 206a, 206b, which may be attached by linkers such as the oligonucleotide tags 202, 208. The linkers 202, 204, 208 are optional and may further be photocleavable or enzymatically cleavable, for example, with restriction enzymes, recombinases, endonucleases, CriSPR/CAS, Cre/loxP and similar. The readout 404 may be achieved by (next generation) sequencing (NGS) or fluorescence detection, in order to determine whether or not the analyte is bound to a particular one of the capture regions 108a to 108f. The readout may be achieved by sequencing when the reporter reagent 300a-300f comprises a sequencable oligonucleotide, for example. The individual elements shown in Figure 4 may be combined, for example, a particular analyte, such as a protein, may be captured by an antibody capture reagent and an antibody fragment reporter reagent may be used, with a fluorescent dye attached, to be read out by a microscope. Alternatively, the capture reagent may be a small molecule and the reporter reagent an antibody fragment.

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

Figure 5 shows a schematic view of a capture region 500. To the capture region 500 are attached multiple affinity capture reagents 502 in the form of single domain antibodies. The single domain antibodies specifically bind a particular analyte 504 of interest at a first binding site. Thus, the capture region 500 binds the analyte 504 where a capture reagent 502 is attached to the capture region 500.

In order to subsequently determine whether or not the analytes 504 are captured by the capture reagents 502, affinity reporter reagents 506 may be added. The affinity reporter reagents 506, in the form of antibodies, bind to the analyte at least at a second binding site. The affinity reporter reagents 506 comprise dyes 508, such as a fluorescent dye. Thus, the affinity reporter reagents 506 only accumulate at the capture region 500 when the analyte 504 is bound to the capture region 500. The presence of the affinity reporter reagent 506 and thus the analyte 504 may then be read out by the readout device as an optical signal of the dye 508. Only in the case that an optical signal of the dye 508 is detected in the capture region 500, in particular at the location of the capture region 500 on the nanostructure backbone 102, it is determined that the analyte 504 is captured in the capture region 500.

More specifically, Figure 5 shows additional, optional features of the capture region 500. The use of a small affinity capture reagent 502, in the form of an antibody fragment, enables the spacing of analyte binding sites in a pattern that has approximately 15 nm spacing, which corresponds roughly to the distance between the two binding sites, or paratopes, of conventional antibodies and is thus suited to create an avidity effect, which may increase the overall sensitivity of the assay substantially. Further, the three-dimensional arrangement of the affinity capture reagents 502 along and around the circumference of the rod-like backbone 102 increases the density of the binding sites of the affinity capture reagents 502 and consequently the affinity reporter reagents 506. This increases the signal to noise ratio of the optical signal when reading out the capture region 500. Finally, capturing a given analyte with two distinct affinity capture reagents and/or two distinct affinity reporter reagents, preferably each with different epitopes increases specificity of the assay and reduces sterical problems.

Figure 6 schematically shows a capture region 600 with affinity capture reagents 602 in the form of oligonucleotides. The affinity capture reagent 602 is configured to bind an oligonucleotide analyte 604 comprising a complementary nucleic acid sequence. The analyte 604 bound to the capture reagent 602 may be determined by reading out the presence of an affinity reporter reagent 606 bound to the analyte 604 and comprising a complementary nucleic acid sequence to the analyte 604. The reagents 602, 606 and the analyte 604 may comprise DNA, RNA and/or LNA nucleotides. This enables detection of nucleic acid sequences with high sensitivity. This is particularly advantageous for numerous applications as nucleic acid sequences occur in bodily fluids or can be released from cells following lysis and potentially shearing of the DNA. This embodiment is also particularly advantageous for diagnostic testing in the context of liquid biopsies and their use to detect the presence of cancer. In this case circulating tumour DNA (ctDNA) target sequences may be detected. Further this embodiment is particularly advantageous for diagnostic testing of pathogen infection such as viral or bacterial infections including sepsis testing. Further areas of application are in pathogen detection in food and water quality testing and monitoring.

Figure 7 schematically shows a capture region 700 with 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 an oligonucleotide analyte 706 at either a first complementary sequence or a second complementary sequence. Further the affinity capture reagents 702, 704 each have a corresponding first dye 708 or second dye 710 attached. When the analyte is bound to one of the affinity capture reagents 702 of the first set and one of the affinity capture reagents 704 of the second set, the first and second dyes 708, 710 of the respective affinity capture reagents 702, 704 are brought into close proximity, in particular within their Forster distance. When the dyes 708, 710 are in close proximity they form a FRET-pair and a corresponding optical signal may be detected by the readout device. FRET refers to fluorescence resonance energy transfer. This increases the specificity of the detection of the analyte.

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

Figure 8 on the left shows a column of illumination and detection points spread functions (PSFs) 800a and on the right a column of corresponding effective PSFs 800b. Unless noted otherwise PSF refers to the main maximum of the PSF. Most microscopes illuminate and detect the sample through the same objective. In this case both the illumination PSF 802a and the detection PSF 804 are elliptical. In the case of light sheet fluorescence microscopy for example the illumination PSF 802b may be sheet-like and the detection PSF 804 may be elliptical, which still leads to an elliptical PSF provided that the detection PSF 804 is fully illuminated.

In the case of multi-view imaging with multiple detection PSFs placed at an angle 804a-804f, which may or may not be combined with light sheet illumination, effective PSFs 806c, 806d can be achieved, which are substantially improved over the elliptical PSFs 806a, 806b. Generally, an isotropic PSF improves the ability to resolve distinct capture regions 108a to 108f, 500, 600, 700 on a capture construct and renders this also largely invariant to the orientation of the capture construct. In other words, if an imaging system with an elliptical effective PSF 806a is used, then the resolving power in the axial direction (a) is lower than the in lateral direction (I) (also refer to Fig. 9). In the case of PSF 806d and PSF 806c the resolving power would be comparable in all room directions, which is not required for reading out capture constructs, in particular capture regions 108a to 108f, 500, 600, 700, but may be preferable.

Figure 9 shows a detailed view of the capture construct 100. The capture regions 108a to 108f are at distance from each other of 500 nm, as described above. The capture regions 108a to 108f may be read out by a readout device having the PSF 806a or the PSF 806d, as described for Figure 8. Importantly, the capture regions 108a to 108f are distanced from each other such that the readout device can resolve the capture regions 108a to 108f individually. Thus, all the affinity reporter reagents of the capture construct 100 may comprise the same dye.

Figure 10 shows a detailed view of a capture construct 1000 with several pluralities of capture regions. The capture construct comprises a first plurality 1002a, a second plurality 1002b, a third plurality 1002c, a fourth plurality 1002d and a fifth plurality 1002e of capture regions. The reference signs 1002a to 1002e refer to one of the capture regions of the respective plurality. The capture regions 1002a to 1002e are grouped with each group 1004a, 1004b comprising one of each of the capture regions 1002a to 1002e. The groups 1004a, 1004b are at a distance (D) from each other of 500 nm along the backbone 102. Each capture region 1002a to 1002e is approximately 25 nm wide (d) along the backbone 102.

The capture regions 1002a to 1002e may be read out by the readout device having the PSF 806a or the PSF 806d, as described for Figure 8 and 9. However, in order to differentiate between the capture regions 1002a to 1002e of each group 1004a, 1004b with the readout device, the affinity reporter reagents of the capture construct 1000 comprise different dyes. Specifically, the affinity reporter reagents of the plurality of capture regions 1002a to 1002e comprises a dye with characteristics unique to each of the plurality of capture regions 1002a to 1002e. This enables reading out individual capture regions 1002a to 1002e of a single group 1004a, 1004b. The characteristics of the dyes of the affinity reporter reagents may include fluorescent emission characteristics, excitation characteristics or lifetime characteristics.

This results in an increased density of capture regions 1002a to 1002e of capture construct 1000 and an accompanying vastly increased number of capture regions 1002a to 1002e compared to the capture construct 100 in Figure 1.

Figure 11 schematically illustrates read out data from the capture constructs 100 and 1000. The optical signal determined from reading out the capture regions 108a to 108f, 1002a to 1002e may be categorised in a binary code of "0"s and "l"s, wherein a fluorescent signal from the analyte being present results in a "1" and no fluorescent signal when the analyte is absent results in a "0". A given sequence 0101010 for example can be interpreted or decoded for a given capture construct with known affinity reagents at each location of the capture regions and the directionality of the capture construct based on the orientation indicators 104, 106. This means, that the identities of analytes can be computed 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 a certain analyte was detected or not, the method provides intensity information, which 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 read for analyte 1 corresponds to 10 dye molecules, which correspond to 5 analyte molecules for example).

Figure 12 shows capture constructs with different geometries. A sheet-like capture construct 1200, which may be a large linear DNA molecule or an assembly of multiple DNA molecules. Sheet-like capture constructs may increase the number of available capture regions substantially. In order to be able to determine the orientation of the capture construct 1200, a third orientation indicator 1202 is provided.

Further geometries are possible, for example, a tetrahedral capture construct 1204, a cubic capture construct 1206, or a polyhedral capture construct 1208. These may comprise a fourth orientation indicator 1210 in order to determine their orientation.

Figure 13 is a schematic view of an analysis system 1300 for analysing capture constructs 1302. In particular, the analysis system 1300 may be configured to analyse one of the capture constructs described above, specifically, the capture constructs 100, 1000. While the provided example capture constructs 100, 1000 are rod-shaped and extend linearly in one direction, the method and analysing system 1300 are similarly applicable to and suitable for analysing capture constructs of different geometries. For example, a capture construct may be of sheet-like geometry, or a tetrahedron or have another polyhedric geometry. Even arbitrary shaped capture constructs can be synthesized. A capture constructs nanostructure backbone may also be based on meta-DNA, a DNA nanotechnology concept described in Yao, G., Zhang, F., Wang, F. et al. Meta-DNA structures. Nat. Chem. 12, 1067-1075 (2020). https://doi.Org/10.1038/S41557-020-0539-8.

The top left view of Fig. 13 shows a top view of an analysing space of the analysis system 1300 and a corresponding side view of the analysing space below. To the right is a detailed view of a particular one of the capture constructs 1302. The analysing space of the analysis system 1300 comprises a surface 1304. Attached to the surface 1304 are first binding moieties 1306 (shown in the detailed view) that are configured to bind the capture constructs 1302. The first binding moieties 1306 may be configured to specifically bind the capture constructs 1302. The surface 1304 may be of a glass slide, a coverslip, a window of a flow cell, a bottom of a well of a microwell plate, for example. The surface 1304 may preferably comprise glass or polymers (e.g. COC, polystyrene, Zeonex (COP), Zeonor (COP), polymethylmethacrylate (PMMA), Vinyl, polycarbonate).

For example, the first binding moieties 1306 may each comprise a biotinylated oligonucleotide 1308 and a (monomeric) streptavidin 1310. In particular, the surface 1304 may be coated with streptavidin 1310, entirely or in a particular pattern. The biotinylated oligonucleotide 1308, in particular a respective biotin molecule, may be attached to the streptavidin 1310 in order to attach the biotinylated oligonucleotide 1308 to the surface 1304 of the analysing space. The near-covalent attachment of biotin to streptavidin enables a robust attachment of the first binding moiety 1306 to the surface 1304.

The biotinylated oligonucleotide 1308 of the first binding moiety 1306 may have a sequence that is at least partially complementary to a part of a nucleic acid backbone of the capture constructs 1302. In particular, the part of the nucleic acid backbone of the capture constructs 1302 may be towards a first end of the nucleic acid backbone of the capture constructs. The capture constructs 1302 may have an essentially linear shape and the first end may be an end along a longitudinal axis of each capture construct 1302.

In an alternative embodiment, the first binding moieties 1306 may comprise an (peptide or nucleic acid) aptamer directly or indirectly attached to the surface 1304 of the analysing space. In a further alternative embodiment, the first binding moieties 1306 may comprise at least one click chemistry group. The click chemistry group may be configured to react to a corresponding click chemistry group of the capture constructs 1302, in order to covalently link the capture constructs 1302 with the first binding moieties 1306 and to attach the capture constructs 1302 to the surface 1304. Alternatively or additionally, the first binding moieties 1306 may comprise a click chemistry group for attaching the first binding moieties 1306 to the surface 1304 of the analysing space.

The analysis system 1300 further comprises an electrophoretic device with at least a first electrode 1312 and a second electrode 1314. The first and second electrodes 1312, 1314 are configured to apply an electric field to the analysing space. In the top view of Fig. 13 the electric field is not applied to the analysing space, whereas in the bottom view of Fig. 13 the electric field is applied to the analysing space by means of the first and second electrodes 1312, 1314, as indicated by the "+” and signs.

The application of the electric field causes the capture constructs 1302 to align with the electric field across the analysing space of the analysis system 1300. Since the first ends of the capture constructs 1302 are bound to the surface 1304 by means of the first binding moieties 1306, the opposing free end or second end of the capture constructs 1302 are motile in the analysing space and may be drawn towards one of the electrodes 1312, 1314.

The capture constructs 1302 comprise a nucleic acid backbone, which is negatively charged. Thus, the capture constructs 1302 in the electric field are drawn towards the positively charged second electrode 1314, the anode.

To aid the motility of the capture constructs 1302 in the analysing space, the analysing space may be filled with a liquid, in particular, the capture constructs 1302 may be introduced to the analysing space in the liquid. The liquid may be an aqueous buffer solution, for example. The capture constructs 1302 are preferably introduced to the analysing space prior to application of the electric field.

Thus, by applying the electric field to the analysing space the bound capture constructs 1302 are aligned between the first and second electrodes 1312, 1314 essentially in a common direction. In addition, the application of the electric field causes the capture constructs 1302 to be orientated essentially in parallel to the surface 1304 of the analysing space. Thus, providing the electrical field across the analysing space enables the common alignment of all bound capture constructs 1302.

This significantly improves efficiency of further analysis of the capture constructs 1302. For example, the bound and aligned capture constructs 1302 may be imaged in the analysing space, for example by means of an optical readout device such as a microscope. Using the assumption that all bound capture constructs 1302 are aligned along the electrical field, the analysis, in particular the detection of capture constructs 1302 is improved in generated optical readout data. In particular, the analysis or detection of optically detectable dyes of the orientation indicators and capture regions of capture constructs is improved. Preferably, the analysis system 1300 comprises the optical readout device.

The nucleic acid backbone of the capture construct 1302 may optionally comprise charged moieties. For example, the end of the nucleic acid backbone of the capture construct 1302 not bound to the first binding moiety 1306 may be charged such that it is attracted by the second electrode 1314. The opposing other end of the capture construct 1302 may be charged such that it is attracted by the first electrode 1312. To that end, the nucleic acid backbone of the capture construct 1302 may comprise natural and/or non-natural nucleic acids that carry the specifically desired charge.

Figure 14 is a schematic view of an analysis system 1400 for analysing capture constructs 1302. Similarly to the analysis system 1300, the analysis system 1400 may be particularly configured to analyse one of the capture constructs described above, specifically, the capture constructs 100, 1000.

The top left view of Fig. 14 shows a top view of an analysing space of the analysis system 1400 and a corresponding side view of the analysing space below. To the right is a detailed view of a particular one of the capture constructs 1302.

The analysing space of the analysis system 1400 comprises the surface 1304. Attached to the surface 1304 are the first binding moieties 1306 (shown in the detailed view) that are configured to bind the capture constructs 1302. In contrast to the analysis system 1300 shown in Fig. 13, the analysis system 1400 further comprises second binding moieties 1402.

The second binding moieties 1402 each comprise a biotinylated oligonucleotide 1404 and the (monomeric) streptavidin 1310. In particular, the surface 1304 may be coated with streptavidin 1310, entirely or in a particular pattern. The biotinylated oligonucleotide 1404, in particular a respective biotin molecule, may be attached to the streptavidin 1310 in order to attach the biotinylated oligonucleotide 1404 to the surface 1304 of the analysing space. The near-covalent attachment of biotin to streptavidin enables a robust attachment of the second binding moiety 1402 to the surface 1304.

The biotinylated oligonucleotide 1404 of the second binding moiety 1402 may have a sequence that is at least partially complementary to a part of the nucleic acid backbone of the capture constructs 1302. In particular, the part of the nucleic acid backbone of the capture constructs 1302 may be towards a second end of the nucleic acid backbone of the capture constructs opposing the first end.

In alternative embodiments, the second binding moieties 1404 may comprise an (peptide or nucleic acid) aptamer directly or indirectly attached to the surface 1304 of the analysing space, or click chemistry groups for attaching to the surface 1304 or for attaching of the capture constructs 1302 to the second binding moieties 1404.

The capture constructs 1302, in particular their first ends, are preferably initially bound to the first binding moieties 1306. Subsequently, the electric field is applied to the analysis space by means of the first and second electrodes 1312, 1314 of the analysis system 1400. This aligns the capture constructs 1302 with the electric field. This is followed by the binding of the capture constructs 1302, in particular their second ends, to the second binding moieties 1402. Preferably, after the binding to the second binding moieties 1402, the electric field may be removed from the analysing space.

Preferably, the first and second binding moieties 1306, 1402 each bind specifically to the respective first or second end of the capture constructs 1302. Further, the first and second binding moieties 1306, 1402 may be arranged in pairs on the surface 1304 of the analysis space in a predetermined pattern wherein the first binding moieties 1306 are at a distance from paired adjacent second binding moieties 1402 that is proportional to the length of the capture construct 1302. In particular, the distance is essentially equal to the length of the capture constructs 1302. This enables that the capture constructs 1302 are arranged in a linear manner between the first and second binding moieties 1306, 1402 when they are bound to the binding moieties 1306, 1402.

The analysis systems 1300, 1400 may in particular be used to efficiently detect target analytes of a biological sample. For example, preferably after attaching the capture constructs 1302 to the surface 1304, target analytes of the biological sample may be contacted with the capture constructs in order to capture the target analytes. In a next step the capture constructs may be readout by means of the optical readout device in order to detect the presence of particular target analytes in the biological sample. In particular, affinity reporter reagents specific to the target analytes may be contacted with the target analytes captured by the capture constructs 1302. This enables efficient optical detection of the captured target analytes.

Figure 15 shows a schematic view of an analysis system 1500. The analysis system 1500 comprises the surface 1304 of the analysing space. The surface 1304 may be that of a microscope slide, in particular a glass slide, for example. To the surface 1304 spots 1502 of capture constructs 1302 may be attached as explained for Figs. 13 and 14, in particular with at least the first binding moiety 1306. When attaching the capture constructs 1302 to the surface 1304, the capture constructs 1302 may be aligned with the electric field generated by means of the first and second electrodes 1312, 1314 (not shown in Fig. 15). In this example each spot 1502 on the surface 1304 of the analysis system 1500, may comprise a different type of capture construct or alternatively a different mix of capture constructs. The different types of capture constructs may capture or bind a different set of target analytes, for example.

After generating the spots 1502 of capture constructs 1302, biological samples may be applied generally to the surface 1304 or specifically to each of the spots 1502. In particular, a flow cell cover may be assembled on the surface 1304 in order to generate cavity between the surface 1304 and the flow cell cover, in which the spots 1502 are arranged. The cavity may be flushed with the biological sample and/or with reagents, such as staining reagents after application of the biological samples to the surface 1304. Alternatively, the surface 1304 may be submerged in a bath of biological sample and/or reagents.

When applying the biological samples to surface 1304, the target analytes may be allowed to bind to their respective affinity reagents on the respective capture constructs 1302. In this way a large number of target analytes may be analysed efficiently. For example each spot may comprise 10 capture constructs each comprising 5 capture regions each comprising 5 colors. In this way each spot 1502 may enable analysing the presence of 250 analytes. In order to reduce the amount of biological sample needed, the application of the sample may be performed by spotting or acoustic droplet ejection using for example an ECHO® (Beckman-Coulter, USA).

In a further example, all spots 1502 on the analysis system 1500 may comprise the same capture constructs. This may be useful, if a large number of samples, needs to be analysed quickly. For example, when a large number of plasma samples from a patient cohort shall be analysed to stratify patient populations or to perform a retrospective study, it is desirable to have an analysis system 1500 that offers both high throughput, short time-to-result and very low sample consumption. In this case each spot 1502 may be specifically spotted with a small volume of plasma form a master plate using automated pipetting or acoustic droplet ejection.

Each spot 1502, in particular the capture constructs 1302 of the spot 1502, may be readout by means of a microscope, for example.

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, 1000, 1302 Capture construct

102 Nanostructure backbone

104, 106, 1202, 1210 Orientation indicator

108a, 108b, 108c,

108d, 108e, 108f,

500, 600, 700, 1002a,

1002b, 1002c, 1002d,

1002e Capture region

200a, 200b, 200c,

200d, 200e, 200f Affinity reagent

202, 204, 208 Oligonucleotide tag

206a, 206b, 508, 708,

710 Dye

300a, 300b, 300c,

300d, 300e, 300f,

506, 606 Affinity reporter reagent

400, 502, 602, 702,

704 Affinity capture reagent

402, 504, 604, 706 Analyte

404 Readout

800a Illumination and detection points spread functions

800b Effective point spread functions

802a, 802b Illumination point spread function

804, 804a, 804b,

804c, 804d, 804e,

804f Detection point spread function

806a, 806b Elliptical effective point spread function

806c, 806d Isotropic point spread function 1004a, 1004b Group of capture regions

1300, 1400, 1500 Analysis system

1304 Surface

1306 First binding moiety

1308, 1404 Biotinylated oligonucleotide

1310 Streptavidin

1312 First electrode

1314 Second electrode

1402 Second binding moiety

1502 Spot of capture constructs

Claims

1. An analysis system (1300, 1400, 1500) for analysing capture constructs (100, 1000, 1302) comprising: an analysing space with a surface (1304) comprising at least first binding moieties (1306) configured to bind the capture constructs (100, 1000, 1302), and an electrophoretic device (1312, 1314) configured to apply an electric field to the analysing space.

2. The analysis system according to claim 1, wherein the first binding moieties (1306) are configured to bind the capture constructs (100, 1000, 1302) at a first end of the capture constructs (100, 1000, 1302).

3. The analysis system according to one of the preceding claims, wherein the first binding moieties (1306) comprise at least one of a biotin and a streptavidin (1310).

4. The analysis system according to one of the preceding claims, wherein the first binding moieties (1306) are configured to bind the capture constructs (100, 1000, 1302) specifically.

5. The analysis system according to one of the preceding claims, wherein the surface (1304) of the analysing space comprises second binding moieties (1402) configured to bind the capture constructs (100, 1000, 1302).

6. The analysis system according to claim 5, wherein each of the first binding moieties (1306) are arranged on the surface (1304) at a distance from an adjacent one of the second binding moieties (1402), the distance being at least the length of the capture constructs (100, 1000, 1302) along a longitudinal axis.

7. The analysis system according to one of the preceding claims, wherein the capture constructs (100, 1000, 1302) comprise nucleic acids or nucleic acid analogues.

8. The analysis system according to one of the preceding claims, wherein the capture constructs (100, 1000, 1302) have a negatively charged first region and a positively charged second region.

9. The analysis system according to one of the preceding claims, wherein each capture construct (100, 1000, 1302) comprises at least a first plurality of capture regions (108a, 108b, 108c, 108d, 108e, 108f, 500, 600, 700, 1002a, 1002b, 1002c, 1002d, 1002e), each capture region comprising at least one affinity capture reagent (400, 502, 602, 702, 704) configured to capture an analyte (402, 504, 604, 706).

10. The analysis system according to one of the preceding claims, wherein each capture construct (100, 1000, 1302) comprises a first optically detectable orientation indicator (104) and a second optically detectable orientation indicator (106).

11. The analysis system according to one of the preceding claims, comprising the capture constructs (100, 1000, 1302).

12. A method for analysing capture constructs (100, 1000, 1302) comprising the following steps: contacting the capture constructs (100, 1000, 1302) with a surface (1304) of an analysing space, the surface (1304) comprising at least first binding moieties (1306) configured to bind the capture constructs (100, 1000, 1302), applying an electric field to the analysing space, and generating an optical readout of the capture constructs (100, 1000, 1302).

13. The method according to claim 12, wherein the capture constructs (100, 1000, 1302) are contacted with target analytes (402, 504, 604, 706) of a biological sample.

14. The method according to one of the preceding claims 12 or 13, wherein prior to generating the optical readout a first set of affinity reporter reagents (300a, 300b, 300c, 300d, 300e, 300f, 506, 606) is introduced to the analysing space.

15. The method according to claim 14, wherein the first set of affinity reporter reagents (300a, 300b, 300c, 300d, 300e, 300f, 506, 606) is removed after generating the optical readout and a second set of affinity reporter reagents (300a, 300b, 300c, 300d, 300e, 300f, 506, 606) is introduced to the analysing space and a further optical readout is generated.