WO2014083474A1

WO2014083474A1 - Capture particle for selectively binding a target molecule

Info

Publication number: WO2014083474A1
Application number: PCT/IB2013/060163
Authority: WO
Inventors: Toon Hendrik Evers
Original assignee: Koninklijke Philips N.V.
Priority date: 2012-11-30
Filing date: 2013-11-15
Publication date: 2014-06-05

Abstract

The invention relates to a capture particle(100), particularly a capture molecule, that comprises two binding domains(BD1, BD2) which can bind to corresponding target domains(E1, E2) on the same target molecule(TM). The binding domains are preferably specific for the respective target domain. Moreover, the capture particle comprises an attachment site(AS) for binding to another component, for example to a surface(15) of a cartridge or to a label particle. The binding domains(BD1, BD2) may particularly be located at the end of molecular strands(111,112), for example of nucleic acids that can be coupled to each other in complementary sections.

Description

CAPTURE PARTICLE FOR SELECTIVELY BINDING A TARGET MOLECULE

FIELD OF THE INVENTION

The invention relates to a capture particle, a method, and a device for selectively binding a given target molecule, for example a molecule of interest in a biological sample.

BACKGROUND OF THE INVENTION

The WO 2008/155716 discloses an optical biosensor in which frustrated total internal reflection (FTIR) of a light beam is detected and evaluated with respect to the amount of target molecules that are specifically bound to capture elements on a detection surface.

SUMMARY OF THE INVENTION

It would be advantageous to have means that allow for an increased accuracy of assays in which target molecules are specifically bound.

This object is addressed by a capture particle according to claim 1 , a method according to claim 13, a device according to claim 14, and a use according to claim 15.

Preferred embodiments are disclosed in the dependent claims.

In a first aspect of the invention, the above object is addressed by a capture particle for binding (or "capturing") a given target molecule, for example a target molecule the presence and/or amount of which in a sample fluid shall be detected. The capture particle comprises (at least) the following components:

A first binding domain that can bind to a first target domain of the target molecule.

A second binding domain that can bind to a second target domain of the same target molecule.

An attachment site (AS) for binding to a component other than the target molecule.

The "capture particle" may in general be any agglomerate of atoms that are more or less firmly bound to each other by physical and/or chemical forces. In particular, the capture particle may be a molecule or a complex in which the atoms are bound to each other via covalent bonds, ionic bonds, hydrogen bonds, and/or van der Waals bonds.

The term "binding domain" shall denote a region, element or component of the capture particle via which binding to the target molecule takes place. Changes in the chemical and/or spatial structure of the binding domain will therefore affect the associated binding to a target molecule and, if the changes are too large, usually eliminate the binding capacity.

The term "target domain" is used here to denote a region on the target molecule that can bind to a corresponding binding domain on the capture particle. The target domain on the target molecule and/or the binding domain on the capture particle may for example be an epitope to which an antibody can bind.

The binding of the attachment site to the component other than the target molecule may be based on the same mechanisms as the described binding between binding domains and target domains. The new term "attachment site" has hence primarily been introduced to serve as a unique reference for a region on the capture particle that binds to another component than the target molecule.

Typical examples of target molecules comprise nucleic acids, proteins, antigens, ligands, lipids, drugs, vitamins, hormones, haptens, carbohydrates, myoglobin, B-type natriuretic peptide, 2,3 C-reactive protein, cardiac markers (e.g. troponin, D-Dimer, procalcitonin, NT proBNP), PTH (parathyroid hormone), oncology markers in blood, melatonin, cellular fragments, cells, viruses, and related compounds.

The aforementioned components are also examples of (possibly intermediate) components the attachment site can bind to. More specifically, the attachment site may be adapted to bind to a particulate label (magnetic particle, fluorescent particle, etc.), a molecular label (fluorescent label, radioactive label etc.), a protein label (e.g. an enzyme like HRP), or a solid phase (e.g. a sensor surface). The binding can be covalent, for example if the attachment site is a particular chemical group that can react with a particular chemical group on the label, surface etc. Alternatively, the binding can be non-covalent, for example if the attachment site is a biotin moiety that can bind to streptavidin, or if the attachment site is an antigen that can be bound by an antibody (in the latter case, a small molecule could preferably be used as an antigen). The attachment site can optionally be or comprise a nucleic acid or a protein (particularly a small peptide), or a derivative thereof. The attachment site can especially be a single stranded nucleic acid (which can bind to another single stranded nucleic acid with a complementary sequence). The described capture particle with its two binding domains for a target molecule has the advantage that it increases the affinity of the capture particle to the target molecule. This in turn will improve the accuracy and sensitivity of many assays that are based on a binding between capture particles and a target molecule.

In a preferred embodiment, the attachment site is unique in the sense that any chemistry or (biochemical) binding reaction targets only this attachment site, and not other sites on the capture particle. To put it differently, a counterpart (label, surface etc.) that can bind to the attachment site cannot bind to any other part of the capture particle. An important aspect about the attachment site is hence that it offers the opportunity to selectively attach it to the counterpart of interest (surface, label, etc.), meaning that it is unique in the capture particle, such that other parts of the capture particle cannot be attached to the same counterpart. To this end, the attachment site may be or comprise a chemical group that is unique and can be selectively reacted with another chemical group on the surface/label. A preferred example is biotin at the attachment site (which can bind to streptavidin on the label/surface).

In general, the first binding domain may be designed such that it can also bind to the second target domain of the target molecule and/or the second binding domain can be designed such that it can also bind to the first target domain on the target molecule. In a preferred embodiment, the binding between binding domains and target domains is however specific in the sense that the first binding domain does substantially not bind to the second target domain and/or that the second binding domain does substantially not bind to the first target domain. In this context, the term "substantially" can be quantified by the requirement that the equilibrium constant of the binding reaction between the first binding domain and the second target domain shall be less than about 20 %, preferably less than about 5%, of the equilibrium constant of the binding reaction between the first binding domain and the first target domain (with an analogous definition for the second binding domain). The first binding domain will then substantially only bind to the first target domain and/or the second binding domain to the second target domain. This significantly increases the specificity with which a target molecule is captured.

The binding behavior discussed in the previous paragraph is typically rooted in the structure of the binding domains and the target domains. Thus the first binding domain may in general have identically or substantially the same structure (defined for example by its chemical formula) as the second binding domain. Additionally or alternatively, the first target domain and the second target domain on the target molecule may have identically or substantially the same structure. In a preferred embodiment, the first binding domain may however be different in structure from the second binding domain and/or the first target domain may be different in structure from the second target domain. Thus a high specificity of the binding domains for "their" target domains can be achieved.

In general, the structure of the capture particle and/or of the target molecule may be such that the target molecule can only bind to either the first binding domain or the second binding domain. In a preferred embodiment, the capture particle is however adapted to the target molecule in such a way that its first binding domain and its second binding domain can simultaneously be bound to the first target domain and the second target domain, respectively, of the same target molecule. This considerably increases the selectivity of the binding between capture particle and target molecule because not only ONE binding domain and target domain must match, but TWO. Accordingly, the risk of an unspecific binding is significantly reduced. Moreover, a coupling between capture particle and target molecule via two (or more) binding domains/target domains is stronger and hence less prone to an undesired breaking than a single binding. Furthermore, it is preferred that the attachment site of the capture particle can be bound to another component, e.g. the surface of a label particle or a detection device, at the same time as bindings between the first and second binding domains and a target molecule exist.

The capture particle may optionally comprise at least one further binding domain that can bind to a third target domain of the target molecule. This at least one further binding domain may particularly have the features of the first and/or the second binding domain discussed above (with other words: the denominations of a binding domain as "first", "second", or "further" binding domain are exchangeable). The further binding domain may for example be designed to essentially not bind to the first and second target domain (e.g. because it is structurally different from the first and second binding domain). Moreover, it is preferred that the first, the second, and at least one of the further binding domains can simultaneously be bound to their respective target domains on the same target molecule, thus further increasing the selectivity and strength of the coupling.

According to a further development of the above mentioned embodiment, the at least one further binding domain is directly connected to both the first and the second binding domain of the capture particle. If the capture particle is for example a complex or molecule, separate molecular/atomic bridges may connect the further binding domain to the first binding domain and the second binding domain, respectively (wherein the first and the second binding domains are preferably directly connected to each other, too). Thus a capture particle with a highly stable geometry and mutually linked binding domains can be achieved.

According to another embodiment, a spacer element may be arranged between the first and the second binding domains. Preferably, this spacer element is substantially rigid. It may for example be composed of a chain of (e.g. covalently bound) atoms. The spacer element shall (by definition) not take part in the binding of the binding domains to their target domains and can hence be designed independently from the binding domains. This allows for example for an adjustment of the size (length) of the spacer element according to given design specifications. The spacer element preferably contains at least one unique connection site at which it is connected to the first and/or the second binding domain (meaning that any chemistry or (biochemical) binding reaction targets only this connection site, and not other sites on the capture particle).

The (spatial) distance between the first binding domain and the second binding domain in the capture particle preferably corresponds to the distance between the associated target domains on the target molecule. With reference to the aforementioned spacer element, this can be achieved by the requirement that the length of the spacer element preferably ranges between about 50 % and about 150 % of the distance that the spacer-connection points assume when both binding domains are bound to the target domains on the target molecule (wherein the "spacer-connection points" are those points on the binding domains where the latter are connected to the spacer element). Most preferably, said distance between the spacer-connection points is substantially identical to the length of the spacer element. Such a matching of distances facilitates or even enables a simultaneous binding of those binding domains to their corresponding target domains. In order to adjust the spatial distance between two binding domains in a capture particle, the above mentioned spacer element between the binding domains can be used and be configured with an appropriate length.

The capture particle comprises an attachment site for binding to another component (other than the target molecule to which the binding domains bind). This other component may for example be a label such as a single fluorophore or a magnetic particle, or it may be the surface of a solid body.

The capture particle may be realized in many different ways. According to one embodiment, it may comprise at least one molecular strand, i.e. a (linear or branched/cross linked) chain of covalently bound atoms. Molecular strands can be produced in various lengths, thus allowing for a well controlled design of the spatial configuration of the capture particle. If for example at least one of the binding domains or the attachment site is coupled to a molecular strand (e.g. positioned at its end or at an intermediate position), its distance from other components of the capture particle can readily be adjusted by an appropriately chosen length of said strand.

According to a further development of the above embodiment, the capture particle may preferably comprise at least two molecular strands that are at least partially bound to each other. Positioning the two binding domains at an end of a first and a second molecular strand, respectively, and binding these molecular strands to each other with a selectable overlap will then for example allow for an adaptation of the distance between the binding domains. In general, a capture particle with at least two molecular strands may optionally comprise sections with single strands and/or double strands (i.e. two strands bound to each other).

The above mentioned molecular strand(s) may preferably comprise nucleic acids and/or derivatives thereof. The buildup of a molecular strand from nucleic acids is an example known from nature (DNA, RNA etc.) that can identically be copied and/or be adapted to novel designs using for example artificial nucleic acids or similar substances. In such a molecular strand, a proper choice of the sequence of nucleic acids can favorably be exploited to incorporate sections that specifically attach to complementary sections in another molecular strand (by hybridization). Thus the spatial configuration of the resulting capture particle can be adjusted in a well controlled manner.

The first binding domain, the second binding domain, and/or the attachment site of the capture particle may preferably comprise at least one element selected from the group consisting of a nucleic acid, a protein structure and/or a derivative thereof. The binding domains and/or the attachment site may for example comprise an antibody component or a nucleic acid strand than can bind to an antigen-component or a

complementary nucleic strand, respectively. Thus a specific binding to many biological target molecules of interest can be achieved.

The capture molecule may further optionally be bound to a label component, for example a fluorescent label, a color label, and/or a magnetic label. The label component may for instance be realized as a particular molecular structure or group and/or as a solid particle that can be covalently bound. Moreover, this binding typically takes place via the attachment site of the capture particle.

In a second aspect, the concerns of the state of the art are addressed by a method for selectively binding a given target molecule, said method comprising the step of exposing the target molecule to a capture particle with a first binding domain that can bind to a first target domain on the target molecule and a second binding domain that can bind to a second target domain on the same target molecule. The capture particle may particularly be a capture particle according to one of the embodiments described above.

In a third aspect, the concerns of the state of the art are addressed by a device for processing a target molecule (or a medium containing the target molecule), said device comprising a capture molecule according to at least one of the embodiments described above. The device may for example be a cartridge, i.e. an exchangeable element or unit in which a sample can be stored, transported, and/or provided to a further apparatus such as a detector. In the device, capture particles may for example be fixed as a coating to a detection surface of a sample chamber, and/or they may be stored in a supply (e.g. as functionalized magnetic beads in a cartridge).

The invention further relates to a system comprising a capture particle according to any of the embodiments described above and the corresponding target molecule (having two or more target domains to which the binding domains of the capture particle can bind).

The capture particle, the method, the device, and the system are different realizations of the same inventive concept, i.e. the provision of two binding domains that can bind to two different target domains of the same target molecule. Explanations and definitions provided for one of these realizations are therefore valid for the other realizations, too. It should further be noted that usually a large number of capture particles and target molecules will simultaneously be present in an application of the method and the device.

The invention further relates to the use of a capture particle and a device of the kind described above for molecular diagnostics, biological sample analysis, chemical sample analysis, food analysis, and/or forensic analysis. Molecular diagnostics may for example be accomplished with the help of magnetic beads or fluorescent particles that are directly or indirectly attached to target molecules.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other aspects of the invention will be apparent from and elucidated with reference to the embodiments described hereinafter.

In the drawings:

Fig. 1 schematically shows a cartridge and a detection apparatus according to an embodiment of the present invention; Fig. 2 schematically illustrates phases of a detection assay taking place in the cartridge of Figure 1;

Fig. 3 schematically shows a target molecule together with a first capture molecule according to an embodiment of the invention, said capture molecule comprising two binding domains at the end of different strands and a stabilization strand;

Fig. 4 shows the components of the capture molecule of Figure 3 separately; Fig. 5 shows a second capture molecule with another arrangement of strands; Fig. 6 shows a third capture molecule with still an another arrangement of strands;

Fig. 7 shows a target molecule and a fourth capture molecule that are coupled via three binding domains and target domains;

Fig. 8 shows a fifth capture molecule with three binding domains that are mutually connected to each other.

Like reference numbers or numbers differing by integer multiples of 100 refer in the Figures to identical or similar components.

DETAILED DESCRIPTION OF EMBODIMENTS

Embodiments of the invention will in the following be described with respect to biosensors based on nanoparticle labels, particularly nanoparticles such as magnetic beads that can be actuated with electromagnetic fields. Typically, the magnetic beads are functionalized with capture molecules that can bind a specific analyte or target molecule. The beads are attracted to the sensor surface, where they can indirectly (by means of a captured target molecule) or directly bind to capture probes printed on the surface. The number of bound beads is directly or inversely related to the amount of target molecules present in the sample. The beads can then be detected using any technique that is more sensitive to beads that are close to the surface. For example, the detection technique may be based on evanescent optical fields, e.g. frustrated total internal reflection (FTIR), as in the

Magnotech® technology developed by the applicant. Another example is the application of dark field microscopy (DFM).

Figure 1 schematically shows a section through a first embodiment of a

(sensor) apparatus 20 that applies the aforementioned FTIR detection technology. The apparatus 20 comprises an accommodation space for an exchangeable cartridge 10. It is used for the detection of target molecules comprised in a sample fluid (e.g. blood) that fills a sample chamber 14 of the cartridge. The cartridge 10 is composed of a transparent base part 13 which borders the processing chamber 14 at its bottom side and which provides a detection surface 15. The side walls of the processing chamber 14 are constituted by an intermediate layer 12, for example a tape into which openings for the processing chamber and associated fluidic channels (not shown) have been cut. The processing chamber 14 is covered at its top side by a (e.g. plastic) cover 11.

At least one detection spot is located on the detection surface 15. As can be seen from Figure 2, this detection spot comprises capture probes 16, for example antibodies, to which certain substances can specifically bind. These substances may particularly be target molecules TM of interest from the sample medium in the sample chamber 14 that are specifically bound to magnetic particles MP via capture molecules 100 on the surface of said magnetic particles.

Figure 1 further shows a magnetic field generator, here comprising a horse-shoe magnet 22 below the detection spot and a top magnet 23 above it. The magnets may individually be controlled for generating a magnetic field in the sample chamber 14 by which the magnetic particles MP can be manipulated.

Figure 1 further indicates a light source 21 for emitting an input light beam LI into the cartridge 10. This input light beam is totally internally reflected at the detection spot and then leaves the cartridge 10 as an output light beam L2 towards a light detector 24. These light beams can be used to detect target molecules of the sample fluid that are specifically bound to magnetic particles MP and the capture probes of the detection spot. Further details of this assay and the optical detection of target molecules by frustrated total internal reflection (FTIR) may be found for example in the WO 2008/115723 Al, which is incorporated into the present text by reference.

Figure 2 illustrates in more detail typical processes taking place in the sample chamber 14 during a sandwich immunoassay using Magnotech® technology. In Figure 2 a), magnetic beads MP coated with primary capture particles or molecules 100 directed against the target molecules TM disperse in the sample liquid and bind the target molecules TM.

In Figure 2 b), top and bottom coils 23, 22 actuate the magnetic particles in a pulsed manner, resulting in binding to the sensor surface where capture probes 16 can bind to the bound target molecule TM.

In Figure 2 c), non-bound beads are removed from the sensor surface and bound beads are detected using an evanescent field.

As can be seen in Figure 2, both the magnetic label particles MP and the sensor surface 15 are functionalized with capture molecules 100, 16. The most important aspects of the assay, sensitivity and selectivity, are highly dependent on the characteristics of the used capture molecules. Insufficient sensitivity and/or selectivity of the assay are often problems in assay development.

In order to address the aforementioned concerns, a novel capture particle is proposed by the invention. Figure 3 illustrates one embodiment of such a capture

particle 100, which is in the following called capture molecule 100 (because its atoms are mostly connected to each other by molecular bindings) and shown when bound to a target molecule TM with two target domains El, E2. The capture molecule 100 comprises the following components:

- A first binding domain BDl that can bind to the first target domain El on the target molecule TM.

At least one second binding domain BD2 that can (simultaneously) bind to the second target domain E2 on the same target molecule TM.

A chemical group or "attachment site" AS for covalent or non-covalent attachment to another component (e.g. a molecule on a surface).

A connecting structure 110 connecting the first binding domain BDl and the at least one second binding domain BD2.

In the shown embodiment, the aforementioned connecting structure 110 comprises a (preferably rigid) spacer element between the binding domains BDl and BD2, preferably with a length w to position the binding domains at an interdomain distance that matches the distance between the corresponding target domains El and E2 on the target molecule TM, thus allowing for the simultaneous binding of the binding domains BDl, BD2 to the respective target domains El and E2

With respect to its molecular design, the capture molecule 100 consists of three essential components that are separately shown in Figure 4. These three principal components are:

A component comprising a first molecular strand 111 with the first binding domain BDl at one end. Here and in the following, it is assumed that the used molecular strands are build up from a sequence of nucleic acids. Potential connection sites between a binding domain and the molecular strand are denoted by capital letters "A", "B", etc., wherein primed letters "A' " etc. refer to the corresponding connection site on a different strand. A connection site may for example be realized by a particular chemical group incorporated into a molecular strand (at its end or at an interior position) and be used for a specific chemical coupling between the strand and the binding domain. This said, it can be noted that the first molecular strand 111 ends with the connection sites A' and C at the end close to the binding domain and the end of the attachment site AS, respectively. The connection site A is connected to a corresponding "spacer-connection point" on the binding domain BDl .

A component comprising a second molecular strand 112 with the second binding domain BD2 at one end. The second molecular strand ends with the connection sites A and B close to the first and the second binding domain, respectively. The connection site B is connected to a corresponding "spacer-connection point" on the binding domain BD2.

A component consisting of a molecular strand 115. This "stabilization strand" 115 ends with the connection sites B' and C at the end close to the second binding domain BS2 and the end of the attachment site AS, respectively.

The sequences of nucleic acids in the mentioned molecular strands 111 , 112, 115 are chosen such that:

a first section starting at C of the first strand 111 hybridizes with a first section starting at C of the stabilization strand 115;

a second section starting at A' of the first strand 111 hybridizes with a first section starting at A of the second strand 112;

a second section starting at B of the second strand 112 hybridizes with a second section starting at B' of the stabilization strand 115.

Thus a T-shaped molecule is achieved with a central connecting structure 110 in which the first and the second binding domains BDl, BD2 are positioned at opposite ends at a fixed distance w. Moreover, an attachment site AS is provided via which the capture molecule 100 can bind to another component, for example to coupling molecules 17 on the surface 15 of the cartridge 10 or on the surface of a magnetic particle MP. Said coupling molecules 17 may comprise a chemical group D via which they can bind to an connection site (here C or C) of the capture molecule 100.

The pairs of

binding domains BDl, BD2 and target domains El, E2;

connection sites A, B, A', B' and associated spacer-connection sites of the binding domains BDl, BD2;

connection sites C, C and coupling molecules 17;

may in general be realized by the same or by similar molecular structures, for example by antibody/antigen pairs, or by complementary nucleic acids. In the shown example, the connecting structure 110 is connected to the binding domains BDl, BD2 and the coupling molecule 17 at the positions indicated by the black spheres, i.e. A', B and C. Connections between separate parts of the connecting structure are indicated with dotted lines.

The capture molecule 100 is multivalent as it contains multiple binding domains that target a single target molecule TM. This 'multivalent effect' predicts an increased affinity for the target molecule.

The capture molecule 100 is also heteromultivalent as both binding domains BDl, BD2 target a different target domain El and E2, respectively, on the same target molecule, wherein it can bind both target domains simultaneously. This results in a further increase in affinity. It also increases the specificity, as it combines the specificity bestowed by both binding domains. The increase in affinity is related to the effective concentration c_eff of one binding domain relative to the other: Ceff = Kintra/Kinter where Ki_nter is the formation constant for the monovalent interaction between a single binding domain and the target, and Ki_ntra describes the equilibrium between the two states of the capture molecule - target complex: one in which the target molecule is bound by one binding domain, and one in which the target molecule is bound by both binding domains.

The effective concentration c_eff is dependent on how effectively the spacer element can position the binding domains apart at the right distance w to bind both target domains on the target molecule at the same time. If the spacer element (here: strand 112) is too short (or long, but very flexible, which still results in a short average distance between the ends of the spacer) or too long, c_eff will be low resulting in a low enhancement of the affinity for the target molecule.

Therefore, the spacer element is ideally relatively rigid, with exactly the correct length w. As double stranded nucleic acids are relatively rigid at typical target domain-binding domain distances (about 5-25 nm) and can be obtained with highly defined lengths, these are a preferred material for the spacer element and/or connecting structure. One further advantage of nucleic acids as material for the connecting structure 110 is that the entire connecting structure can be assembled using different parts with complementary single strands that hybridize to form the structure (indicated by the dotted lines in Figure 3). A further essential feature of the capture molecule 100 is the possibility to attach it to another molecule, for example a label molecule (e.g. a fluorescent label), a particle (e.g. a magnetic particle MP), or a solid surface (e.g. a sensor surface 15). In case of binding to a particle or solid surface, it can be preferred that the connecting structure also provides some distance h between the binding domains and the particle or surface (as indicated in Figure 3).

The capture molecule 100 therefore combines multiple advantages, which make it highly suitable for use in immunoassays: it uses its multivalency to bind to a single target molecule with high affinity and selectivity; it uses a connecting structure to position the binding domains at exactly the right distance for the highest affinity; and it contains a chemical group for easy attachment to a label, particle or surface.

There are many different ways the connecting structure 110 can be assembled, especially when the connecting structure consists of nucleic acids. For example, one single strand 111 of DNA can be coupled to the first binding domain BD1, whereas a second single strand 112 can be coupled to the second binding domain BD2. A third strand 115

("stabilization strand") can then be used to connect the first two strands, provide rigidity to the spacer element and form an attachment site AS to e.g. a surface. As in the example of Figures 3 and 4, it could be preferred that each of the separate strands is used to connect only to one of the binding domains or other molecule (surface). This is advantageous as it offers the highest flexibility of assembling the structure. An essential feature of the separate strands forming the connecting structure 110 in case of using nucleic acids is that each strand contains a sequence of nucleic acids that is complementary to the DNA strand to which it needs to be connected, but unique enough not to hybridize to any of the other sequences used in the strands.

However, many other configurations are possible which can have their advantages. In the example displayed in Figure 5, the DNA between the binding

domains BD1 , BD2 completely consists of only two hybridized single strands 211 and 215 (partially), which can be advantageous to provide optimal stiffness to the DNA.

Not all parts of a connecting structure need to consist of double stranded DNA. This is illustrated in Figure 6 for a capture molecule 300, in which the section of the stabilization strand 315 from the attachment site AS to the second binding domain BD2 is single stranded.

Moreover, the described approach is not limited to bivalent capture molecules. Figure 7 illustrates a capture molecule 400 with multiple (here: three) binding domains BD1, BD2, and BD3 that simultaneously bind to three different target domains El, E2 and E3, respectively, on a target molecule TM.

When more than two binding domains need to be spatially organized, it is preferred that the connecting structure is further stabilized by additional links between the binding domains. An example of this is sketched in Figure 8, which shows a capture molecule 500 with three binding domains BD1, BD2, and BD3 that are mutually linked via associated (double) strands, thus stabilizing their spatial organization.

Further modifications and features of the described embodiments comprise for example the following aspects:

- The binding domains BD1, BD2, etc. can for example consist of (part of) an antibody, but can also comprise nucleic acids, particularly be an RNA aptamer. In this case, the binding domain and part of the connecting structure are actually the same molecule.

To stabilize a connecting structure, two strands forming a duplex can be crosslinked together (cf. Gerrard et al, ACS Nano (2012), 9221-9228; Tagawa et al, "Stabilization of DNA nanostructures by photo-cross-linking", Soft Matter (2011), 10931).

Connection sites (chemical groups) between DNA strands and a binding domain or another molecule do not need to be at the end of a molecular strand, e.g. at the end (3' or 5') of a DNA strand, but can also be incorporated internally.

While Figures 1 and 2 refer to arbitrary capture probes 16 on the sensor surface, these capture probes may particularly be designed as capture particles according to an embodiment of the invention (they may e.g. be realized by one of the shown capture molecules 100-500).

In summary, several embodiments have been described to increase the sensitivity and specificity of immunoassays by using novel capture molecules. These capture molecules comprise multiple binding domains that can bind to different target domains on a single target molecule; and a connecting structure that positions the binding domains at the optimal distance for binding to the target molecule and provides an attachment point to couple to a label molecule or solid surface. The invention can for example be used in in vitro diagnostic (immuno)assays, e.g. with the Magnotech® technology, for the detection of biomarkers in biological samples, such as the detection of cardiac troponin in blood for the diagnosis of acute myocardial infarction.

While the invention has been illustrated and described in detail in the drawings and foregoing description, such illustration and description are to be considered illustrative or exemplary and not restrictive; the invention is not limited to the disclosed embodiments. Other variations to the disclosed embodiments can be understood and effected by those skilled in the art in practicing the claimed invention, from a study of the drawings, the disclosure, and the appended claims. In the claims, the word "comprising" does not exclude other elements or steps, and the indefinite article "a" or "an" does not exclude a plurality. A single processor or other unit may fulfill the functions of several items recited in the claims. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage. A computer program may be stored/distributed on a suitable medium, such as an optical storage medium or a solid-state medium supplied together with or as part of other hardware, but may also be distributed in other forms, such as via the Internet or other wired or wireless

telecommunication systems. Any reference signs in the claims should not be construed as limiting the scope.

Claims

CLAIMS:

1. A capture particle (100-500) for binding a given target molecule (TM), comprising:

a first binding domain (BDl) that can bind to a first target domain (El) of the target molecule;

- a second binding domain (BD2) that can bind to a second target domain (E2) of the target molecule (TM);

an attachment site (AS) for binding to another component.

2. The capture particle (100-500) according to claim 1,

characterized in that a component that can bind to the attachment site (AS) cannot bind to another part of the capture particle.

3. The capture particle (100-500) according to claim 1,

characterized in that the first binding domain (BDl) does substantially not bind to the second target domain (E2), and/or that the second binding domain (BD2) does substantially not bind to the first target domain (El).

4. The capture particle (100-500) according to claim 1,

characterized in that the first binding domain (BDl) and the second binding domain (BD2) can simultaneously be bound to the first target domain (El) and the second target domain (E2), respectively.

5. The capture particle (400, 500) according to claim 1,

characterized in that it comprises at least one further binding domain (BD3) that can bind to a third target domain (E3) of the target molecule (TM).

6. The capture particle (100-500) according to claim 5,

characterized in that all binding domains (BDl, BD2, BD3) are directly connected to each other .

7. The capture particle (100-500) according to claim 1,

characterized in that a spacer element (112, 211) is arranged between the binding domains (BDl, BD2).

8. The capture particle (100-500) according to claim 7,

characterized in that the length (w) of the spacer element (112, 211) ranges between about 50 % and about 150 % of the distance between the spacer-connection points on the binding domains (BDl, BD2) when the latter are bound to the corresponding target domains (E 1 , E2) on the target molecule (TM).

9. The capture particle (100-500) according to claim 1,

characterized in that it comprises at least one molecular strand (111, 112, 115;

211, 212, 215; 311, 312, 315; 411, 412, 413, 415; 511, 512, 513, 515, 516), said molecular strand preferably comprising nucleic acids and/or derivatives thereof.

10. The capture particle (100-500) according to claim 9,

characterized it comprises at least two molecular strands (111, 112, 115; 211,

212, 215; 311, 312, 315; 411, 412, 413, 415; 511, 512, 513, 515, 516) that are at least partially bound to each other.

11. The capture particle (100-500) according to claim 1,

characterized in that the first and/or second binding domain (BDl , BD2) and/or the attachment site (AS) comprises a nucleic acid, a protein structure and/or derivatives thereof.

12. The capture particle (100-500) according to claim 1,

characterized in that it can bind to a label component, particularly a fluorescent label, a color label, and/or a magnetic label (MP).

13. A method for selectively binding a given target molecule (TM),

said method comprising the exposure of the target molecule (TM) to a capture particle (100-500) according to claim 1.

14. A device (10) for processing a target molecule (TM),

said device comprising a capture particle (100-500) according to claim 1.

15. Use of the capture particle (100-500) according to claim 1 or the device according to claim 14 for molecular diagnostics, biological sample analysis, chemical sample analysis, food analysis, and/or forensic analysis.