WO2020002350A1

WO2020002350A1 - Assay, method for production thereof, and use thereof

Info

Publication number: WO2020002350A1
Application number: PCT/EP2019/066871
Authority: WO
Inventors: Anna Herrmann; Stefan RÖDIGER; Uwe Schedler; Peter SCHIERACK; Rainer Haag
Original assignee: Brandenburgische Technische Universität Cottbus-Senftenberg; PolyAn Gesellschaft zur Herstellung von Polymeren für spezielle Anwendungen und Analytik mbH
Priority date: 2018-06-26
Filing date: 2019-06-25
Publication date: 2020-01-02
Also published as: DE102018115391A1

Abstract

The invention relates to an assay for the analysis of a complex analyte. The assay (1) comprises at least one layer (10), which comprises: • a porous three-dimensional polymer network (11) having a predetermined mesh size, and • at least one population of particles (12) embedded and immobilised in the three-dimensional polymer network (11), each of the particles comprising a carrier particle (13) and a capture probe (14) bound to the carrier particle (13), which capture probe makes it possible to bind to a target species (15, 17) of the analyte. The immobilisation of the particles (12) by means of the polymer network (11) makes it possible to increase the specificity of the assay and facilitates the application. In a preferred embodiment a plurality of such layers are stacked one above the other, as a result of which the assay (1) can analyse a plurality of target species (15, 17) in a single measurement.

Description

description

Assay, process for its preparation and its use

The invention relates to an in particular multiplexed assay for the analysis of an analyte containing several target species. The invention further relates to a method for producing the assay and its use for analyzing an analyte.

Technological background of the invention

Assays are molecular biological evidence that are used in particular in laboratory medicine and biotechnology. With the help of so-called capture molecules (also called capture probes) target species (also called biomarkers) of an analyte, such as viruses, proteins or nucleic acids, are detected. The evidence is based on molecule-specific affinity interactions, such as the hybridization of specific nucleic acid sequences, antigen-antibody interaction or protein-protein interactions. Multiplexed assays are assays in which several analytes can be examined in a single run from a single sample and, in the best case, quantified. The further development of conventional assays to multiplex assays increases the analytical throughput and allows a higher information density with the same input. This makes multiplex assays an indispensable component in many areas of molecular diagnostics and clinical diagnostics. The predisposition to many diseases such as cancer, Alzheimer's, metabolic diseases or cardiovascular diseases, as well as their outbreak, cause and course often depend on several factors. For example, nucleic acid sequences alone can lead to various changes, such as, for example, single nucleotide polymorphism (SNP, single nucleotide polymorphism), deletion, insertion or gene duplication, all of which can influence gene function. Furthermore, pharmacogenomics is investigating how drugs are metabolized differently by different organisms, which also depends on multiple factors. In addition to nucleic acids, antibody-binding markers can also be used, the multiplexed antibody detection being used, for example, for the research and elucidation of autoimmune diseases. That is why the development towards better multiplex detection systems for the healthcare system is of enormous importance and also economically interesting. The most common two types of multiplex assays are the microarray and the microbead assay.

Microarray describes the arrangement of a large number of microspots on a solid phase, a different capture molecule (capture probe) for a biomarker being immobilized in each of these spots. Since it is known for each xy position which capture probe is there, a large number of biomarkers can be examined from one sample in just one measurement. The biggest problem with the microarrays is their reproducible processing and the high technical effort involved in manufacturing and evaluating them.

Known multiplexed microbead assays, on the other hand, use micrometer-sized particles, the so-called microbeads, on the surface of which a capture probe is immobilized, in suspension. Microbeads have one or more encodings that allow different populations to be distinguished from one another and assigned to the capture probe. In practice, the coding is usually based on a color coding, for example the fluorescence wavelength, and / or the particle size. A flow cytometer is usually used for detection. In order to also be able to examine the microbeads stationary and also to be able to carry out kinetic studies, a fluorescence microscope with appropriate software (VideoScan) can be used for evaluation and discrimination. For the evaluation, the coding of the microbeads is first analyzed, for example the color coding via a first channel of the flow cytometer or of VideoScan and the diameter. In this way, the microbeads can be divided into different populations and assigned to the capture probe and thus to the biomarker immobilized on the capture probe. To examine the sample mixture, the actual detection (and quantification) of the biomarker is then carried out using a further channel. The amount of parameters that can be measured in such assays plays an important role. This amount is limited in microbead-based assays by the parameters size and coding fluorescence of the microbeads. If the difference in the selection parameter between two microbead populations is too small, incorrect assignments can occur. A complex mixture of biomolecules can interfere with detection in a simple microbead assay.

Due to their hydrophilic, biocompatible and easily modifiable properties, hydrogel materials have been of great interest in numerous biotechnological applications in recent years. In particular, the solution-like properties and non-fouling in complex biological samples make hydrogels good substrates for biosensors. Hydrogel coatings and gel dot surfaces have been used in microarrays for nucleic acid assays and immunoassays. Using new microfabrication techniques, encoded particles can be synthesized from hydrogel materials. This has made it possible to develop suspension arrays based on hydrogels. Lithography processes and droplet-based microfluidic technologies enable the generation of hydrogel microbead libraries with unique spectral or graphic codes for the multiplex sensor technology (European Polymer Journal, 2015, 72, pp. 386-412).

Brief description of the invention

The object of the invention is to provide an assay based on functionalized particles (microbeads) which overcomes the problems of conventional microbead assays. In particular, the assay should be easy to manufacture and use, and should allow for more confident discrimination against populations.

These objects are achieved in whole or at least in part by an assay, a method for its production and its use with the features of the independent claims. Preferred embodiments of the invention are the subject of the dependent claims and the present description.

The assay according to the invention for analyzing an analyte which contains a plurality of target species has a number of k layers which extend in the xy plane and are arranged one on top of the other in the z direction, k being an integer greater than or equal to 1. Each of the k layers comprises:

A porous three-dimensional polymer network with a predetermined mesh size, and

• at least one population of particles embedded and immobilized in the polymer network, each comprising a carrier particle and a capture probe bound to the carrier particle, which preferably is able to specifically bind a target species of the analyte.

The assay according to the invention is characterized by various advantageous effects. The particles are fixed in place by the three-dimensional polymer network, which makes handling the assay much easier and the assay can even be prepared and used repeatedly. The polymer network enables the particles to adhere to a large number of different surfaces. In addition, the polymer network allows a mesh size to be set which, although allowing the target species to penetrate the layer, excludes undesired larger constituents, for example cell fragments or the like, which increases the sensitivity and reproducibility. The polymer network thus leads to a filter effect that prevents unwanted components. Furthermore, the immobilization of the particles enables a location-specific assignment, which increases the number of detectable target species. This advantage already exists in the case of a single layer (k = 1), in which the particles are fixed in the xy-plane and thus multiple target species can be detected when different populations are arranged in a location-selective manner within the layer plane. The polymer embedding of the particles enables them to be detected in a location-specific manner and with greater accuracy, and the specificity and sensitivity when measuring a target species in a complex analyte can be improved.

The number of detectable target species is increased if the number k of the polymer network / particle layers is 2 or more. In this case, the z position is opened up as a further spatial parameter that allows an assignment of a particle population. This means that within the Cartesian coordinate system, three-dimensional discrimination of particle populations can take place on the basis of the coordinates x, y, z. Compared to a single polymer network / particle layer (k = 1), the theoretically maximum possible number of discriminable particle population and thus detectable target species increases by a factor k.

The maximum number of polymer network / particle layers is at most limited by the detection system used for the evaluation, which here is in particular a fluorescence microscope, for example an epifluorescence microscope or confocal fluorescence microscope.

The total height of the assay is limited by the available space of the microscope. In one configuration, the number of layers is 2 to 10, in particular 2 to 6.

The particle populations differ in particular in the capture probes which are coupled to the carrier particles. Thus, each population serves as a specific detector for a target species. Since several populations can be immobilized in each layer k, the degree of multiplexing increases by a / c-fold.

The mesh size of the polymer networks present in the layers should be so small that the particles are fixed, i.e. cannot move. On the other hand, the mesh size should be at least so large that the target species contained in this layer Particle population can move as freely as possible within the pores in order to penetrate the layer and to be able to couple to the capture probe.

In a preferred embodiment, the mesh size of the polymer networks of the k layers decreases in the z direction in the direction of gravity, that is to say downwards. In this context in particular, it is preferably provided that the particle population that binds the smallest target species is arranged within the lowest layer. The second lowest layer then contains the particle population that binds the second smallest target species, and so on. In other words, the particle populations are arranged in the order of the size of their target species and that with increasing size of their target species in the z direction from bottom to top. In these configurations, the filter effects of the hydrogel layers are used in a targeted manner so that, in the ideal case, only those target species whose complementary capture probes are arranged in one of the following layers can pass through. This can further improve the specificity of the detection.

In a preferred embodiment of the invention it is provided that an average mesh size of the polymer network is in the range from 1 nm to 1000 nm, in particular 1 nm to 200 nm, preferably 2 nm to 50 nm. As already stated above, the mesh size in this area is small enough to immobilize the particles and optionally exclude certain molecule or particle sizes, and at the same time large enough to let the desired target species pass.

In the context of the present application, the term “three-dimensional polymer network” denotes a polymeric material that is cross-linked via covalent or non-covalent (preferably covalent bonds) and is therefore swellable or swollen in a solvent, so that pores form that form with are filled with the solvent. The three-dimensional polymer network is preferably a hydrogel, which is therefore swellable or swollen in water. Hydrogels are particularly suitable for biological or biomedical applications.

In an advantageous embodiment, in addition to the k polymer network / particle layers, the assay comprises a further layer which is uppermost in terms of gravity and which has a porous polymer network with a predetermined pore size but no particles. In particular, the polymer network of this “empty layer” has the largest mesh size of all existing layers. The mesh size of this uppermost empty layer is dimensioned so that large components of the analyte do not belong to the target species to be detected belong to be excluded. The top layer of this version therefore only has a filter function.

The particles of a layer are preferably arranged in one layer within the xy plane. In this way, the selectivity between adjacent layers is increased.

As already mentioned, the particles of different populations differ in their capture probes, so that each population can bind a specific target species. In addition, the immobilization of the particles according to the invention in the three-dimensional polymer network offers the advantage that, in addition to coding, for example by means of fluorescence and size, spatial coding by the z position (if more than one polymer network / particle layer is present) is introduced. In this way, by appropriately focusing an instrument used in the evaluation (for example a microscope), a respective population can be identified solely on the basis of the position.

In a preferred embodiment of the invention, the particle populations can advantageously be distinguished from one another by one or more further coding properties in order to further improve the discrimination and identification of different populations. In this context, the coding property can be a particle size, in particular a particle diameter and / or a particle diameter distribution; a particle shape; an optical property, in particular a color or fluorescence wavelength; a magnetic property; a particulate material; a particle coating material; a surface structure or a combination of several of these parameters. The coding property is preferably a property of the carrier particles. Such particles are known, for example, from conventional microbe assays.

The particle populations are particularly preferably identifiable by a coding property (ie assignable to a capture probe) and additionally have an indicator property which enables a distinction to be made as to whether the corresponding target species has bound or not. An optical property of the particles is particularly suitable for this purpose, for example an intensity or wavelength of an absorption or emission radiation of the particles, which changes when the target species bind. It is known, for example, to provide the carrier particles with a fluorescent label (fluorescent dye), the target species carrying a quencher so that the fluorescence decreases on binding. Alternatively, the target species can have its own optical property (for example fluorescence) or be provided with a fluorescent label (before or after binding to the capture probe) so that the fluorescence increases upon binding. In all of the above cases, it is not only possible to measure the qualitative binding of the target species, but also to measure the quantity, namely on the basis of the intensity. Such approaches are known from conventional assays.

The layer structure of the present invention is preferably arranged in a vessel or on a carrier. In a particularly preferred embodiment, the carrier has an array of a multiplicity of cavities or spots, an assay according to the invention in each of the cavities or on the spots, each with a number of / extending in the xy plane and in z-plane. Layers arranged on top of one another are arranged as described above.

Such a carrier can be designed as a microtiter plate, the wells of which serve as cavities. The populations of particles of the different cavities preferably differ at least in the capture probes bound to the carrier particles. In this exemplary embodiment, the cavities determine the xy position of the different populations and thus allow their identification.

Another aspect of the invention relates to a method for producing the assay according to the invention, which provides a layered structure. The process includes the following steps:

Application of at least one population of particles to a substrate and

• Applying a crosslinkable composition to the particles, crosslinking the composition while maintaining the three-dimensional polymer network in which the particles are embedded.

These steps are repeated / c-fold, corresponding to the number k of the polymer network / particle layers to be produced.

In an alternative embodiment, a mixture of the at least one particle population and the crosslinkable composition is applied to the substrate and crosslinked to obtain the three-dimensional polymer network in which the particles are embedded.

The composition preferably has a bi-functional polymer linker of a predetermined chain length and a more functional crosslinker. In this way, the chain length of the bi-functional polymer linker defines the mesh size (pore size) of the resulting polymer network. In particular, the chain length of the bi-functional polymer liner of a (/ c + 1) th repetition is chosen so that it is longer than the chain length in the kth repetition. In other words, the chain length (or molecular weight) of the lin- kers with every additional shift. In this way, an assay with increasing mesh size can be produced. To produce a polymer network in the form of a hydrogel from a crosslinked polyethylene glycol, for example, a bi-functionalized polyethylene glycol of a defined chain length (defined molecular weight) can be crosslinked with a dendritic polyglycerol.

Yet another aspect of the present invention relates to the use of the assay according to the invention for analyzing an analyte containing several target species. Use includes the steps:

Applying the analyte to the top layer of the assay in the direction of gravity;

Incubate for a predetermined time; and

• spatially resolved detection of the particles and detection of the target species bound to the particles.

Unless otherwise stated in the individual case, the various embodiments of the invention mentioned in this application can advantageously be combined with one another.

The invention is explained below in exemplary embodiments on the basis of the associated drawings. Show it:

FIG. 1 shows a schematic representation of a multiplex assay with three polymer network / particle layers according to one embodiment of the invention;

FIG. 2 shows a multiplex assay according to the invention in a microtiter plate in a top view (A) and as a side view (B);

3 shows fluorescence images of an assay according to the invention (A) and of particles in suspension (B), in each case 1 h after filling the preparation solution into a cavity (1), after drying overnight at room temperature (2), after resuspension by adding water (3) and after repeated washing, emptying and tapping (4);

FIG. 4 shows a schematic representation of an assay with a polymer network / particle layer for the detection of a Fab fragment in the presence of an antibody according to an embodiment of the invention; FIG. 5 fluorescence intensities of the assay from FIG. 4 after different incubation times;

FIG. 6 shows a schematic representation of an assay with two polymer network / particle layers for the detection of a Fab fragment and a 20-mer oligonucleotide in the presence of an antibody according to an embodiment of the invention;

FIG. 7 fluorescence intensities of the first detection level (left) and second detection level (right) of the assay from FIG. 6 as a function of the incubation time;

FIG. 8 shows a schematic representation of reversible transitions of an assay according to the invention between a fully swollen state (a), a partially swollen state (b) and a dry state (c); and

FIG. 9 swelling of various PEG hydrogels according to FIG. 8 over several drying and swelling cycles.

Figure 1 shows a highly schematic representation of a multiplex assay according to an exemplary embodiment of the present invention. The illustration shows the assay designated overall by 1 in the xz plane, the yz plane being identical in the example shown. The assay 1 comprises a number of k (here k = 3) layers 10, each of which comprises a three-dimensional polymer network 11 and at least one population of particles 12 embedded and immobilized in the polymer network 11. Such a polymer network / particle layer is also referred to below as detection layer 10. In the example shown, an optional further layer 20 is arranged on the top of the three detection layers 10, which is designed as an “empty layer” and as such has a further layer of a three-dimensional polymer network 11, but has no particles. In the exemplary embodiment shown, the polymer networks have a mesh size or pore size which increases from bottom to top, that is to say in the z direction, counter to gravity.

In the example shown, each of the detection layers 10 each comprises a population of particles 12, which are shown enlarged on the right. Each particle 12 of a population has a carrier particle 13 and a capture probe 14 bound to the carrier particle 13, which is capable of specifically binding a target species of an analyte. So every population serves the Detection of another target species. In the example shown, the populations differ in addition to the capture probe 14 also by a coding property which allows the population to be identified by a suitable detection method. The coding property is indicated here by different hatching of the carrier particles 13.

The assay 1 further comprises a vessel or a carrier 30, in or on which the

Layer structure of detection layers 10 and optionally the “empty layer” 20 is arranged.

FIG. 2 shows an assay product 100 according to the invention. The assay product 100 comprises a carrier 30 on which an array of a plurality of assays 1 according to the invention is arranged. In the example shown, the carrier 30 is designed as a so-called microtiter plate 31 with standardized dimensions. The microtiter plate 31 has an array of, for example, 12 × 8 cavities 32, which are also referred to as wells. In each cavity 32, an assay 1 according to the invention comprising a layer structure of detection layer (s) 10 and optionally the “empty layer” 20 is arranged, as shown in FIG. 1, for example. The particles 12 arranged in the individual cavities 32 can belong to different populations and can therefore be sensitive to different target species.

Deviating from FIG. 2, the carrier 30 can also have the shape of a flat disk on which an array of spots, each with an assay 1, is applied.

The components of the assay according to the invention are to be explained in more detail below.

Polvmernetzwerk

In the context of the present invention, polymer network 11 is understood to mean a polymer which is swollen or swellable in a liquid phase, three-dimensionally crosslinked. The polymer network is preferably swollen or swellable in water or an aqueous phase, ie a hydrogel. This enables use for biomedical analyzes. However, polymer networks that are swollen or swellable in organic solvents are also suitable for other applications.

The task of the polymer network is on the one hand to fix the particles. On the other hand, the swollen polymer network must have a porosity that provides a solution-like environment for the diffusion of the target species so that they reach the particles. For this purpose, the polymer network 11 forms pores or meshes, the size of which is defined by the chain lengths of the polymer between the crosslinking points. The mesh size is so small that the particles 12 are fixed in place, so they cannot shift. On the other hand, the mesh size is at least so large that the target species of the particle population contained in this layer 10 can move as freely as possible within the pores in order to be able to penetrate the layer and couple to the capture probe 13. The mean mesh size of the polymer network is in particular in the range from 1 nm to 1000 nm, in particular from 1 nm to 200 nm, preferably 2 nm to 50 nm.

The mesh size of the polymer networks 11 of the k detection layers 10 and of the optional layer 20 can decrease in the z direction in the direction of gravity, that is to say from top to bottom. In this way, a graduated filter effect occurs, wherein only components with decreasing sizes are passed along the path of an analyte applied to the assay, and components whose hydrodynamic diameter exceeds the mesh size are kept at the respective interfaces between two layers 10, 20. For example, the polymer network of the bottom layer can have a small mesh size that can be penetrated by oligonucleotides, but not by larger biomolecules such as proteins or antibodies. The polymer network of a middle layer can only pass through medium-sized molecules, for example a Fab fragment or protein, and exclude larger antibodies. By appropriately selecting the pore sizes, the molecules (target species) to be analyzed can be selectively directed into the respective layers. Since in principle a suitable filter effect can already be achieved by the polymer network 11 of the detection layer 10 arranged at the top, the “empty layer” 20 can also be dispensed with.

In embodiments of the invention, the polymer network 11 has ionic (cationic or anionic) groups. In this way, a filter effect can also be achieved by repelling and thus holding back components of the analyte that are charged the same and allowing components that are charged in the opposite way to be let through. This type of polyarization can also be implemented in combination with the graded mesh size shown above.

The polymer network 11 can be produced from a polymer linker which is crosslinked by means of a crosslinker. The mesh size can be adjusted by a suitable choice of the chain length (or the molecular weight) of the polymer linker and / or the crosslinker. The Crosslinking can be generated in a simple manner by the polymer linker being functionalized with a reactive group at each of its two chain ends and the crosslinker having three or more functional groups which are able to react with the functional groups to form a chemical bond. Depending on the type of functional groups, the coupling reaction can take place spontaneously by mixing the two components, or can be triggered by adding an initiator or by irradiation with a suitable radiation.

From a material point of view, the polymer network 11 should preferably be selected so that it behaves neutrally to the analyte and the particles, that is to say does not react or otherwise interact with any of the components. This property is also called bioorthogonal. At the same time, a suitable choice of the polymer network, depending on the material of the carrier 30 onto which the particles are to be immobilized, enables good adhesion, so that a very stable bond between the substrate and the polymer network is obtained and thus the particles are also fixed with high stability ,

Suitable polymer systems include in particular polyethers, polyalcohols, polyacrylates, polyacylamides (including poly (N-isopropylacrylamide PNIPAM), polyimides (including polyetherimide PEI), agarose, celluloses, modified celluloses (including chitosan), polysaccharides, dextrans, polyvinyl pyrrolidones , Etc.

In an advantageous embodiment, the polymer network 11 is a hydrogel which comprises a polyethylene glycol (PEG) crosslinked by a dendritic polyglycerol (dPG). A suitable pair of functional groups that react spontaneously (without initiator, without thermal excitation and without photoexcitation) is, in particular, cyclooctin and azide. Other pairs of functional groups are, for example, thiol and acrylate, which react with one another via a thiol-ene reaction, ketone / hydroxylamine, or groups that react with one another by means of a Diels-Alder reaction or a Staudinger ligation. In principle, other coupling reactions are possible as well as other gel components. It is preferred that the crosslinking reaction proceeds quickly enough to avoid long reaction times. If the layer is produced from a mixture of crosslinking composition and particles, the reaction time of the crosslinking, on the other hand, should be slow enough so that the particles can sink onto the layer below during gelation. In this way, an arrangement of the particles in a defined plane is ensured, which can be detected sharply by an evaluation instrument, for example in a focal plane of a fluorescence microscope. The reaction time can be varied by varying the concentration the temperature and / or possibly the concentration of an initiator are influenced. Preferably, no by-products should arise and the reaction should preferably take place at room temperature in order to make the preparation quick, easy and economical. The reaction to gel formation should be bioorthogonal in order to prevent diffusing biomolecules from interacting with the network and from getting stuck in the gel unspecifically, making detection more difficult or falsifying.

The type of polymer network 11 can be identical or different for all layers 10, 20. The same polymer system is preferably used for all layers 10, 20, optionally with varying mesh sizes.

particle

The detection particles 12 have the function, on the one hand, of binding a target species as specifically as possible, and, on the other hand, of enabling identification of the target species on the basis of the position data of the particle within the assay and / or another coding property of the particle.

For the purpose of specific binding, the particles 12 have capture probes 14 which are bound to carrier particles 13. In the present case, capture probe is used to denote a molecule or a chemical group which is able to interact as specifically as possible with the respective target species in order to bind them. The capture probe should therefore have a high affinity for the target species and is selected accordingly depending on it. If the target species is a nucleic acid, for example, the capture probe can be a complementary nucleic acid sequence that hybridizes with the target sequence. If the target species is an antibody, the capture probe can be a complementary antigen that interacts with the antibody via protein / protein interactions, or vice versa. In addition, any type of chemical coupling reaction between the capture probe 14 and the target species can be implemented via a suitable choice of the capture probe 14.

The capture probe 14 is bound to the carrier particle 13. The carrier particle 13 can consist of any material and is preferably inert in the respective system. Applicable materials include glasses, metals, plastics, or mixtures or composites of these materials. In exemplary embodiments, the carrier particles 13 consist of a plastic, for example polymethyl methacrylate (PMMA). The carrier particles 13 can have any shape. They are preferably spherical Shape. The size or the diameter of the carrier particles 13 is not limited and can be selected in the range from 100 nm to 1000 pm, in particular in the range from 1000 nm to 100 pm, preferably in the range from 8 pm to 20 pm.

Due to their fixation in the hydrogel 11, the particles 12 can already be identifiable by their position. The identification is given in particular in the z direction by assignment to a specific detection layer 10. In addition, identification via the xy coordinates may also be possible, in particular when arranged in a microtiter plate (see FIG. 2) or by means of another location-specific arrangement of the particles.

Optionally, the particles 12 can also have a coding property that enables or facilitates an assignment of the particles to a specific population and thus to a specific capture probe 14. Suitable coding properties include the particle size, in particular particle diameter and / or particle diameter distribution; the particle shape; an optical property such as color or fluorescent wavelength; a magnetic property; the particulate matter; a particle coating material; the surface structure or a combination of several of these parameters. The coding property is preferably a property of the carrier particle 13.

Particularly preferably, the particles 12 additionally have an indicator property which enables a distinction to be made as to whether the corresponding target species has bound to a population or not. Ideally, the indicator property also allows quantification of the bound target species. An optical property of the particles is particularly suitable for this purpose, for example an intensity or wavelength of an absorption or emission radiation of the particles, which changes when the target species bind. It is known, for example, to provide the carrier particles 13 with a fluorescent label (fluorescent dye), the target species carrying a quencher so that the fluorescence decreases on binding. Alternatively, the target species can have its own optical property (for example fluorescence) or be provided with a fluorescent label (before or after binding to the capture probe), so that the fluorescence increases when bound. In all of the aforementioned cases, it is not only possible to measure the qualitative binding of the target species, but also to measure the quantity, namely on the basis of the intensity. Approaches of this type are known from conventional assays.

In principle, 12 microbeads can be used as particles within the scope of the invention, as are known from conventional microbead assays. Structure of the Assavs

The assay 1 according to the invention comprises k detection layers containing the hydrogel 11 and one or more populations of particles 12. The higher the number k of the layers, the more particle populations can be arranged in the assay and the more target species can be detected. The number k of layers 10 in which the detection is carried out can be tailored to the assay requirements. It is limited by the structure of the analysis instrument, which can reach its limits in the z direction.

A thickness of the layers 10 should at least be dimensioned such that the particles 12, in particular in a single-layer arrangement, are completely embedded. The layer thickness is preferably larger than the (single-layer) particle layer by a predetermined amount. Suitable layer thicknesses of layers 10, 20 are approximately in the range from 100 μm to 5 mm. The total thickness of the layer structure of the assay 1 is thus the sum of all individual layer thicknesses of all detection layers 10 and optionally of the empty layer 20, whereby the layer thicknesses can be the same or different.

The particles 12 of a layer 10 are preferably arranged in one plane, in particular in one layer, in order to enable a possible sharp focus and slight discrimination from an adjacent layer 10. In particular, as shown in FIG. 1, the particles 12 can be arranged near the interface to the layer below or to the carrier 30.

Production of the Assav

Assay 1 according to the invention is produced in layers from bottom to top.

According to a preferred production method, at least a first population of particles 12 is applied, for example sprinkled, onto the substrate 30, in particular onto the bottom of a cavity 32 of a microtiter plate 31. This results in a uniform distribution of the first particle population on the substrate. Subsequently, a crosslinkable composition is applied to the particles 12 and the crosslinking (gelation) is optionally initialized or simply waited, whereby the first (bottom) layer 10 of a polymer network 11 is obtained, which embeds the first particle population and fixes it to the bottom. After sufficient crosslinking (gel formation), a second population of particles is applied to the first layer and a further crosslinkable composition, which after the Crosslinking forms the second polymer layer 10, which embeds the second particle population and fixes it on the first layer. These steps are repeated with further particle populations until the desired number k of detection layers 10 is built up. At the end, the “empty layer” 20 can optionally be generated by merely applying and crosslinking a crosslinkable composition to the top detection layer 10.

In an alternative approach, the particles 12 and the cross-linkable composition are not applied sequentially, but instead a mixture of particles 12 and cross-linkable composition is applied to the support 30 or the layer 10 underneath for each detection layer 10, and the composition is formed of the hydrogel. In this case, it is desirable for the crosslinking reaction to proceed slowly enough to allow the particles to sink onto the substrate 30 or the layer 10 underneath.

The crosslinkable composition contains components which form the polymer network after their crosslinking reaction, as well as a suitable solvent for these components, in particular water or an aqueous phase, and optionally initiators. The crosslinkable components can in principle comprise polymerizable monomers and crosslinkers, so that the crosslinking reaction takes place simultaneously with the polymerization. However, production is preferably carried out from components which have already been prepolymerized (polymer linkers) and are crosslinked using a crosslinker (see explanations on the polymer network above). In this way, the mesh sizes of the individual layers to be produced can be checked more easily.

Application of the Assavs

The assay is used to analyze an analyte that contains several target species by applying the analyte to the top layer (top detection layer 10 or empty layer 20) of the assay 1, for example by pipetting on. Since the particles 12 are fixed in the polymer network / hydrogel 11, mechanical loads such as washing, shaking or pipetting through are also unproblematic.

The system is then allowed to incubate for a predetermined time to allow diffusion of the target species into the respective target layers 10. Due to the solution-like environment of the polymer network, the diffusion is slowed down, which the target species can however, they move freely in those layers in which the gel pores allow. The predetermined time depends in particular on the number k of layers 10 and the sizes of the target species. Incubation times in the range from a few minutes to a few days, in particular 2 hours to 24 hours, have proven successful.

The evaluation then takes place, which comprises a spatially resolved detection of the particles 12 and a determination of the target species bound to the particles 12. The detection of the particles 12 or the instrument used for this purpose is determined by the type of the particles 12, in particular by their coding. If the particles are labeled with fluorescence, detection is carried out using a fluorescence microscope, for example. The spatially resolved detection comprises in particular the z coordinate, for which the focus of the microscope is set on the respective layer 10, so that the detected fluorescence wavelength becomes maximum. This is preferably done using the instrument's autofocus. In addition, xy coding can also take place, in particular by arranging different assays 1 in the cavities 32 of a microtiter plate 31. For this purpose, an xy table on which the assay is arranged is adjusted or by adjusting the optics of the instrument in the xy plane. Since it is known for each coordinate in three-dimensional space which particle population is arranged there, ie which target species can theoretically be present here, it is now only necessary to determine whether a target species and optionally how much of it has bound. In the simplest case, the target species itself can have a measurable property, for example fluoresce itself or carry a fluorescence marker. In this case, the corresponding intensity of the fluorescence is measured and evaluated. The more molecules of the corresponding target species have bound at one location (for example in a z-plane), the higher the fluorescence measured at this location. Alternatively, the particles 12 can be equipped with a fluorescence marker so that the target species acts as a fluorescence quencher. In this case, the more target species has bound, the lower the fluorescence intensity measured at one location. The evaluation is carried out with the aid of software which, on the one hand, recognizes and can assign the fluorescence coding of the microbeads 12. On the other hand, the shell of the microbeads is analyzed in a further channel, the intensity of the fluorescence being dependent on the amount of the bound target molecule. For each level and for each selected microbead population, one obtains a fluorescence intensity that correlates with the amount of target. In order to be able to read out the respective levels, an area in the z direction is specified in which the software automatically focuses the microbeads of a level and the evaluation can be carried out. This z range for the autofocus defines the minimum distance that must lie between the planes k and k _{i + 1} . It depends on the roughness of the surface of a hydrogel layer and thus on the height distribution of the individual microbeads 12 within the level. The more plan the microbeads are, the sharper the software can focus and the more levels can be accommodated in the total layer thickness D. This increases the speed of the analysis.

The methods for detection are essentially known from the methods described in the introduction and can be used analogously. Compared to the known microbead assays, in which the particles are in the form of a suspension in a liquid phase and are evaluated, the assay according to the invention has the advantage that the particles can already be identified by their three-dimensional position. In this way, coding may be dispensed with or the number of coding properties per population may be reduced.

Application Examples

In all of the examples described below, 12 spherical fluorescence-coded microbeads made of PMMA (from PolyAn GmbH, Germany) with average particle diameters of 12 pm and 18 pm were used as particles.

The hydrogels were each crosslinked by cyclooctin homobifunctionalized polyethylene glycol linkers with different molecular weights (PEG linker, eg PEG 3 kDa, PEG 6 kDa or PEG 10 kDa) with a dendritic polyglycerol (dPG , e.g. MW = 1-10 kDa). The crosslinking reaction was carried out in each case at room temperature in the aqueous phase.

Example 1 - Assays for Assay Stability

As described above, the microbeads should not be able to penetrate the meshes of the hydrogel and should be permanently immobilized on the corrugated floor due to the coating with the hydrogel. To demonstrate this, an assay according to the invention was prepared in wells of a cavity strip (Nunc ™ NucleoLink ™) comprising a layer of microbeads embedded in a hydrogel from a PEG network (k = 1).

These assays according to the invention were exposed to various conditions and the arrangement of the microbeads in the following states (1) to (4) on the well bottom was monitored using a fluorescence microscope (see image series in FIG. 3A). As a comparison, the same The same population of microbeads, but in suspension instead of hydrogel, was subjected to the same treatments (see picture series in Fig. 3B).

(1) Preparation solution, 1 h after filling into the well.

(2) Allow to dry, overnight at room temperature.

(3) Resuspension by adding water and repeated pipetting up and down.

(4) After washing, emptying, tapping and washing once (Fig. 3B) or several times (Fig. 3A) with PBS buffer.

The series of images in FIG. 3B shows that the microbeads in the buffer are initially evenly distributed in the well (B1), by drying (B2) aggregates form in the middle of the well. Resuspension (B3) distributes the microbeads evenly again, but in a different arrangement. If the well is emptied and washed once with buffer solution (B4), no microbeads remain in the well. On the other hand, the microbeads covered with hydrogel according to the invention show that the arrangement of the microbeads on the well bottom does not change at any time. Even after repeated washing and tapping, the microbeads remain fixed to the floor by the hydrogel (FIG. 3A).

Example 2 - Filter effect of the assay

The ability of the assay according to the invention to exclude large molecules while smaller ones can diffuse was examined, for example, using an analyte containing a fluorescence-labeled antibody and a comparatively small, also fluorescence-labeled Fab fragment (fragment antigen binding), that is to say an antibody fragment. For this purpose, an assay 1 was produced by applying a microbead population 12 to a well bottom of a microtiter plate and overlaying and immobilizing with the hydrogel 11 (PEG 6 kDa) (see FIG. 4). As a capture probe, the microbeads 12 carried an antibody which, in solution with both components of the analyte, the Fab fragment 15 and the antibody 16, showed an affinity interaction. After the analyte had been added to assay 1, incubation was carried out over a period of 7 days. At the beginning and after 24 h, 48 h and 7 days, the intensities of the fluorescence wavelength of the Fab fragment 15 and of the antibody 16 were read out in the focal plane of the microbeads 12. The result is shown in FIG. 5. It can be seen that even after 7 days, the larger antibody 16 shows only a weakly detectable signal, while the smaller Fab fragment is clearly detectable on the microbeads 12 after only 24 hours. FIG. 4 shows that the smaller Fab molecules 15 can migrate through the network pores of the hydrogel 11, while the larger antibodies 16 hardly penetrate the network. Example 3 - Multiplexer Assay

An assay 1 according to the invention with two detection layers 10i and 10 _{2 was} produced as conceptual proof of the ability for multiplex detection (k = 2, FIG. 6). For this purpose, a first population of microbeads 12-i, provided with a fluorescence-labeled oligonucleotide (capture probe for a complementary oligonucleotide 20mer 17), was overlaid in the first layer 10i with a hydrogel according to the invention (PEG 6 kDa) and immobilized. Microbeads 12 ₂ which carried an antibody were overlaid and immobilized in the second layer 10 ₂ by means of a hydrogel (PEG 10 kDa) with a larger mesh size. The hydrogel also separated the two populations of microbeads 12-i and 12 ₂ from each other. The same assay 1 as shown in Figure 6 was prepared in seven wells of an 8-well strip (Nunc ™ NucleoLink ™). For comparison purposes, the two microbead populations 12-i and 12 _{2 were given} in suspension in the eighth well. 15 ml solution of the target species listed below (alone or in mixtures) were added to the wells prepared in this way and filled up with PBS buffer to a total volume of 45 ml.

(Oligo = 20mer detection oligonucleotide labeled with quencher molecule (17); Fab = Fab fragment labeled with a fluorescent dye molecule (15), Ab = antibody labeled with a fluorescent dye molecule (16), Fluorescent Oligo = arbitrary oligonucleotide sequence labeled with a fluorescent dye molecule)

The corresponding fluorescence intensities of both layers 10-i and 10 ₂ in wells No. 1 to 7 and the fluorescence intensities of the suspension in wells No. 8 were read out over a period of 48 hours using software (VideoScan) (see FIG. 7). In all wells in which the detection oligonucleotide 17 was added, the fluorescence existing on the microbead 12-i was quenched in the first level 10i. The fluorescence on the microbead was also quenched in the wells in which not only the detection oligonucleotide 17 was added alone but also in a mixture with the other fluorescence-labeled molecules 15, 16 (wells 4, 5 and 6) (see FIG 7 left, unfilled symbols). This shows that a clear detection is possible and the background fluorescence of the added solution does not lead to any artifact in the detection. However, the fluorescence is retained in the wells in which the detection oligonucleotide 17 was not added (see FIG. 7, left, filled symbols). Likewise, a linear became in all wells, in which the Fab fragment 15 was added Increase in the fluorescence obtained on the microbead 12 ₂ in the second level 10 ₂ (see FIG. 7 right, unfilled symbols). The much flatter increase compared to the microbeads in suspension (see Fig. 7 right, x-marker, Ab_solution) can be explained by the slowed diffusion due to the mesh of the hydrogel. In all cases, however, in which the antibody (Ab) 16 was added, no increase in fluorescence was observed until the end of the measuring time. In addition, the fact that the fluorescence is retained in the first layer 10i after the addition of the non-specific oligonucleotide (see FIG. 7 left, fluorescent oligo, filled circles) shows that the arbitrary oligonucleotide does not bind non-specifically to the oligonucleotide of the capture probe.

As shown schematically in FIG. 6, very small flexible molecules such as the 20-mer oligonucleotide 17 were able to penetrate the entire assay 1. The small to medium-sized Fab fragment 15, on the other hand, has only penetrated the top layer 10 ₂ , but has not reached the bottom layer 10-i. Large molecules, as shown here using the example of antibody 16, can thus be effectively excluded. The result is a spatially separate, size-selective detection.

Example 4 - Reversibility of swelling

In this experiment, two levels of microbeads were immobilized using a hydrogel layer and spatially separated from one another (see FIG. 8). Hydrogels of different mesh sizes were used (PEG 3 kDa, PEG 6 kDa, PEG 10 kDa). These systems were subjected to multiple drying / swelling cycles for a total of up to 4 weeks, each cycle transitioning the hydrogel from a fully swollen state (Fig. 8a) to a fully dried, collapsed state (Fig. 8c) and re-swelling by adding water included. As shown in FIG. 9, the small-mesh hydrogel PEG 3 kDa was sufficiently reversibly dried over 5 cycles and swelled again. For the two larger pore hydrogels PEG 6 kDa and PEG 10 kDa this could even be shown for 7 cycles. It is therefore possible to reversibly dry and swell the layers separated by the hydrogel without the gel tearing and the microbeads falling through the mesh into deeper layers.

In order to make the assay according to the invention easy and economically manageable, it is advantageous that as few components and specialist knowledge as possible are required for the application. This experiment proves that the assay according to the invention is used as a dried product. can be driven and stored and the user receives a ready-to-use product simply by adding an aqueous solution or water. In the wet, swollen state, the gels are able to form the layers and open their pores for the diffusion of the target species. The dried state of the assay is advantageous for storage and transport from the manufacturer to the user, as the assay is even less susceptible to external influences (temperature, mechanical defects such as cracks, cross-contamination, etc.).

LIST OF REFERENCE NUMBERS

assay

Layer / detection layer

three-dimensional polymer network / hydrogel particles / microbead

carrier particles

capture probe

target species

antibody

target species

top layer / empty layer

Carrier / vessel / substrate

microtiter plate

Cavity / well

Assay Product

Claims

claims

1. Assay (1) for analyzing an analyte containing a plurality of target species (15, 17), where in the assay (1) a number k of layers (10) extending on an xy plane and arranged in the z direction have one another, wherein k is an integer of 1 or greater than 1 and each layer (10) comprises:

• a porous three-dimensional polymer network (11) with a predetermined mesh size, and

• at least one population of particles (12) embedded and immobilized in the polymer network (11), each comprising a carrier particle (13) and a capture probe (14) bound to the carrier particle (13), which contains a target species (15, 17 ) of the analyte.

2. Assay (1) according to claim 1, wherein the particles (12) have different populations of different capture probes (14) which are able to bind different target species (15, 17).

3. Assay (1) according to claim 1 or 2, wherein k is an integer of 2 or greater, in particular a number from 2 to 10.

4. Assay (1) according to claim 3, wherein a mesh size of the polymer networks (1 1) of the k layers (10) decreases in the z direction in the direction of gravity.

5. Assay (1) according to one of the preceding claims, further comprising a further layer (20) which is uppermost in terms of gravity and which has a porous polymer network (11) with a predetermined pore size and no particles (12).

6. Assay (1) according to one of the preceding claims, wherein an average mesh size of the polymer network (1 1) is in the range from 1 nm to 1000 nm.

7. Assay (1) according to one of the preceding claims, wherein the three-dimensional polymer network (11) is a hydrogel.

8. Assay (1) according to one of the preceding claims, wherein the particles (12) of a layer (10) are arranged in one layer within the xy plane.

9. Assay (1) according to one of the preceding claims, wherein the particles (12) of different populations are distinguishable by at least one coding property, the coding property being a particle size, a particle shape, an optical property, a magnetic property, a particle material , is a particle coating material and / or a surface structure.

10. Assay product (100) comprising a carrier (30) on which an array of a plurality of assays (1) according to one of the preceding claims is arranged.

11. A method for producing the assay (1) according to one of claims 1 to 10, with the / c-fold repeating steps:

• Applying at least one population of particles (12) to a carrier (30) and

• Applying a crosslinkable composition to the particles (12), crosslinking the composition while maintaining the three-dimensional polymer network (11) in which the particles (12) are embedded.

12. The method of claim 11, wherein the composition comprises a bi-functional polymer linker of a predetermined chain length and a higher-functional crosslinker.

13. The method of claim 12, wherein k is at least 2 and wherein the chain length of the bi-functional polymer linker of a (/ c + 1) th iteration is longer than in the kth iteration.

14. Use of the assay (1) according to one of claims 1 to 10 for the analysis of an analyte containing several target species (15, 17), with the steps:

• applying the analyte to the top layer (10, 20) of the assay (1) in the direction of gravity;

Incubate for a predetermined time; and

• spatially resolved detection of the particles (12) and determination of the target species (15, 17) bound to the particles (12).

5. Use of the assay (1) according to claim 14, wherein the spatially resolved detection of the particles (12) and the detection of the target species (15, 17) bound to the particles (12) by means of a fluorescence microscope, in particular an epifluorescence microscope or confocal fluorescence microscope.