WO2020002350A1 - Système d'essai, son procédé de fabrication et son utilisation - Google Patents

Système d'essai, son procédé de fabrication et son utilisation Download PDF

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Publication number
WO2020002350A1
WO2020002350A1 PCT/EP2019/066871 EP2019066871W WO2020002350A1 WO 2020002350 A1 WO2020002350 A1 WO 2020002350A1 EP 2019066871 W EP2019066871 W EP 2019066871W WO 2020002350 A1 WO2020002350 A1 WO 2020002350A1
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WIPO (PCT)
Prior art keywords
assay
particles
target species
layer
polymer network
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PCT/EP2019/066871
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German (de)
English (en)
Inventor
Anna Herrmann
Stefan RÖDIGER
Uwe Schedler
Peter SCHIERACK
Rainer Haag
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Brandenburgische Technische Universität Cottbus-Senftenberg
PolyAn Gesellschaft zur Herstellung von Polymeren für spezielle Anwendungen und Analytik mbH
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Publication of WO2020002350A1 publication Critical patent/WO2020002350A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form

Definitions

  • 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.
  • 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.
  • 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.
  • microbeads 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.
  • 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.
  • a fluorescence microscope with appropriate software VideoScan
  • 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.
  • the microbeads can be divided into different populations and assigned to the capture probe and thus to the biomarker immobilized on the capture probe.
  • 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.
  • hydrogel materials 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).
  • the object of the invention is to provide an assay based on functionalized particles (microbeads) which overcomes the problems of conventional microbead assays.
  • the assay should be easy to manufacture and use, and should allow for more confident discrimination against populations.
  • 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 A porous three-dimensional polymer network with a predetermined mesh size
  • 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.
  • 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.
  • the immobilization of the particles enables a location-specific assignment, which increases the number of detectable target species.
  • the number of detectable target species is increased if the number k of the polymer network / particle layers is 2 or more.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the assay 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.
  • 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.
  • the particles of different populations differ in their capture probes, so that each population can bind a specific target species.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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:
  • 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.
  • the chain length of the bi-functional polymer linker defines the mesh size (pore size) of the resulting polymer network.
  • 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.
  • 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:
  • 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);
  • FIG. 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);
  • 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.
  • a polymer network / particle layer is also referred to below as detection layer 10.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the polymer network 11 has ionic (cationic or anionic) groups.
  • 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.
  • 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.
  • 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.
  • 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.
  • the polymer network 11 is a hydrogel which comprises a polyethylene glycol (PEG) crosslinked by a dendritic polyglycerol (dPG).
  • PEG polyethylene glycol
  • dPG dendritic polyglycerol
  • 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.
  • the crosslinking reaction proceeds quickly enough to avoid long reaction times.
  • the reaction time of the crosslinking 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.
  • 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.
  • 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.
  • the particles 12 have capture probes 14 which are bound to carrier particles 13.
  • 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.
  • the target species is a nucleic acid
  • the capture probe can be a complementary nucleic acid sequence that hybridizes with the target sequence.
  • 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.
  • 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.
  • the carrier particles 13 consist of a plastic, for example polymethyl methacrylate (PMMA).
  • PMMA polymethyl methacrylate
  • 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.
  • the particles 12 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 comprises k detection layers containing the hydrogel 11 and one or more populations of particles 12.
  • 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 ⁇ m 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.
  • the particles 12 can be arranged near the interface to the layer below or to the carrier 30.
  • Assay 1 according to the invention is produced in layers from bottom to top.
  • 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.
  • 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.
  • the “empty layer” 20 can optionally be generated by merely applying and crosslinking a crosslinkable composition to the top detection layer 10.
  • 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.
  • 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.
  • 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.
  • xy coding can also take place, in particular by arranging different assays 1 in the cavities 32 of a microtiter plate 31.
  • 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.
  • 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.
  • the particles 12 can be equipped with a fluorescence marker so that the target species acts as a fluorescence quencher.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • PEG linker eg PEG 3 kDa, PEG 6 kDa or PEG 10 kDa
  • dPG dendritic polyglycerol
  • 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.
  • 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.
  • 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).
  • 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).
  • 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.
  • 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 TM NucleoLink TM).
  • 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.
  • 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.).

Abstract

L'invention concerne un système d'essai pour l'analyse d'un analysat complexe. Le système d'essai (1) comprenant au moins une couche (10), laquelle comprend : • un réseau polymère (11) tridimensionnel poreux dont la largeur de mailles est préétablie, et • au moins une population de particules (12) incorporées et immobilisées dans le réseau polymère (11) tridimensionnel, comprenant respectivement une particule porteuse (13) et une sonde de capture (14) reliée à la particule porteuse (13) qui peut se lier à une espèce cible (15, 17) de l'analysat. L'immobilisation de la particule (12) au moyen du réseau polymère (11) permet d'augmenter la spécificité du système d'essai et allège l'application. Selon un mode de réalisation préféré, plusieurs couches de ce type sont agencées de manière à être empilées les unes sur les autres, si bien que le système d'essai (1) peut analyser une pluralité d'espèces cibles (15, 17) en une mesure unique.
PCT/EP2019/066871 2018-06-26 2019-06-25 Système d'essai, son procédé de fabrication et son utilisation WO2020002350A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1990014161A1 (fr) * 1989-05-19 1990-11-29 Pb Diagnostic Systems, Inc. Element d'analyse
US20010012537A1 (en) * 1999-07-30 2001-08-09 Anderson Norman G. Dry deposition of materials for microarrays using matrix displacement
WO2003031979A1 (fr) * 2001-10-05 2003-04-17 Surmodics, Inc. Jeux d'echantillons ordonnes de façon aleatoire et procedes de fabrication et d'utilisation
WO2016073336A1 (fr) * 2014-11-03 2016-05-12 Robert Etheredge Chambre de mélange microfluidique à tamis

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1990014161A1 (fr) * 1989-05-19 1990-11-29 Pb Diagnostic Systems, Inc. Element d'analyse
US20010012537A1 (en) * 1999-07-30 2001-08-09 Anderson Norman G. Dry deposition of materials for microarrays using matrix displacement
WO2003031979A1 (fr) * 2001-10-05 2003-04-17 Surmodics, Inc. Jeux d'echantillons ordonnes de façon aleatoire et procedes de fabrication et d'utilisation
WO2016073336A1 (fr) * 2014-11-03 2016-05-12 Robert Etheredge Chambre de mélange microfluidique à tamis

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
EUROPEAN POLYMER JOURNAL, vol. 72, 2015, pages 386 - 412

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