Classifications

• • 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
  • G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
• • 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
  • G01N33/5436—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals with ligand physically entrapped within the solid phase
• • B—PERFORMING OPERATIONS; TRANSPORTING
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  • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
  • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
  • B01J2219/00277—Apparatus
  • B01J2219/00279—Features relating to reactor vessels
  • B01J2219/00281—Individual reactor vessels
  • B01J2219/00286—Reactor vessels with top and bottom openings
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  • B01J2219/00306—Reactor vessels in a multiple arrangement
  • B01J2219/00313—Reactor vessels in a multiple arrangement the reactor vessels being formed by arrays of wells in blocks
  • B01J2219/00315—Microtiter plates
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
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  • B01J2219/00277—Apparatus
  • B01J2219/00279—Features relating to reactor vessels
  • B01J2219/00306—Reactor vessels in a multiple arrangement
  • B01J2219/00313—Reactor vessels in a multiple arrangement the reactor vessels being formed by arrays of wells in blocks
  • B01J2219/00315—Microtiter plates
  • B01J2219/00317—Microwell devices, i.e. having large numbers of wells
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
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  • B01J2219/00277—Apparatus
  • B01J2219/00351—Means for dispensing and evacuation of reagents
  • B01J2219/00364—Pipettes
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
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  • B01J2219/00277—Apparatus
  • B01J2219/00497—Features relating to the solid phase supports
  • B01J2219/00527—Sheets
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
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  • B01J2219/00277—Apparatus
  • B01J2219/0054—Means for coding or tagging the apparatus or the reagents
  • B01J2219/00572—Chemical means
  • B01J2219/00576—Chemical means fluorophore
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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  • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
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  • B01J2219/00583—Features relative to the processes being carried out
  • B01J2219/00585—Parallel processes
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
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  • B01J2219/00583—Features relative to the processes being carried out
  • B01J2219/00596—Solid-phase processes
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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  • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
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  • B01J2219/00583—Features relative to the processes being carried out
  • B01J2219/00603—Making arrays on substantially continuous surfaces
  • B01J2219/00639—Making arrays on substantially continuous surfaces the compounds being trapped in or bound to a porous medium
  • B01J2219/00641—Making arrays on substantially continuous surfaces the compounds being trapped in or bound to a porous medium the porous medium being continuous, e.g. porous oxide substrates
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
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  • B01J2219/00583—Features relative to the processes being carried out
  • B01J2219/00603—Making arrays on substantially continuous surfaces
  • B01J2219/00659—Two-dimensional arrays
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
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  • B01J2219/00583—Features relative to the processes being carried out
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  • B01J2219/00659—Two-dimensional arrays
  • B01J2219/00662—Two-dimensional arrays within two-dimensional arrays
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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  • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
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  • B01J2219/00583—Features relative to the processes being carried out
  • B01J2219/00603—Making arrays on substantially continuous surfaces
  • B01J2219/00677—Ex-situ synthesis followed by deposition on the substrate
• • B—PERFORMING OPERATIONS; TRANSPORTING
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  • B01J2219/00718—Type of compounds synthesised
  • B01J2219/0072—Organic compounds
  • B01J2219/00722—Nucleotides
• • B—PERFORMING OPERATIONS; TRANSPORTING
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  • B01J2219/00718—Type of compounds synthesised
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  • B01J2219/00725—Peptides
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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  • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
  • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
  • B01J2219/00718—Type of compounds synthesised
  • B01J2219/0072—Organic compounds
  • B01J2219/0074—Biological products
  • B01J2219/00743—Cells

Definitions

• the invention relates to a device for efficiently generating a large number of different substance mixtures of at least two substances and assaying the interaction, especially the joint interaction, of at least two substances with at least one target. Furthermore, the invention relates to a method for assaying the above-mentioned interaction and also to the use of said device and method.
• biological assays aim at identifying from a multitude of compounds, e.g. test substances, single compounds that exert a desired action, e.g. acting as enzyme inhibitors. These compounds may be derived from all commonly used and known sources. Assays are carried out with a single compound per test. The biological activity in terms of the concentration required to exert a half-maximal effect is determined by serial dilution of the test compound into a multitude of separate test containers, mostly in the form of wells in microtiter plates of various formats. Each dilution requires a working step either by the experimentator or by a robotical device.
• the assay for all compounds and their dilutions starts after preparation of all dilution series, either by addition of the target with respect to which the compounds are tested, of an additional compound required to start the reaction or by simultaneous transfer of all samples to the test targets. If timing is critical the starting points of subsets of tests can be delayed by defined intervals that will usually depend on the time required to handle each subset in parallel.
• each test in an assay contains a mixture of compounds (usually equimolar mixtures for all compounds present in a test). These mixtures can either be the outcome of chemical reaction steps carried out in one reaction vessel (e.g. peptide libraries with randomised positions) or generated post-synthesis through mixing of single substances.
• dilution series are performed for the whole mixture of compounds, such that the relative concentrations of the compounds in all tests of a dilution series are kept constant.
• cytostatika in the treatment of cancer, where the optimal selection of a combination is vital for optimal success of chemotherapy.
• cloned tumor-cell assay tumor cells are cultivated in the cell culture, and before therapy different cytostatika combinations are tested. Nevertheless, only a limited number of cytostatika combinations and concentrations of each compound can be tested, being a risk of missing the optimal combination or imposing a too high load of cytostatika on the patient.
• Another field of inventions in which diffusion has been employed as a transport process relates to so called free-format assays in which targets on which a collection of compounds are to be tested are incorporated in a matrix and the test substances are delivered to the matrix by means such as pipetting or contact of a layer carrying the substance collection at defined position with the matrix incorporating the target.
• the substances enter the matrix by means of diffusion.
• the matrix may be present in a dry form and the compounds are applied in solution in the form of drops.
• free-format refers to the fact that the relative positions of the test sites are not determined by a physical separation of test sites as for example wells in a microtiter plate.
• test substances have to be placed far enough apart from each other so that no mixing due to lateral diffusion occurs. If an activity is detected one must be able to assign it to a single active substance based on the position of the assay device where the activity is present.
• Another family of inventions employs diffusive transport to test the permeation of substances across a semipermeable membrane, carrying a monolayer of tissue culture cells.
• diffusive transport to test the permeation of substances across a semipermeable membrane, carrying a monolayer of tissue culture cells.
• One example of such device is given by WO 99/21958. Nevertheless, in this international patent application all test sites have to be physically separated from each other. Diffusion occurs only across the semipermeable membrane, from a single well on one side of the semipermeable membrane to the corresponding well on the other side of the semipermeable membrane.
• the object of the invention is to provide novel devices and methods enabling the efficient generation and testing of substance mixtures in which the concentrations of at least two substances vary at any place of the device where testings are conducted, so that the above-mentioned and other disadvantages are overcome.
• one aspect of the present invention is to provide a device for assaying the interaction, especially the joint interaction, of at least two substances with at least one target, in which mixtures of said substances are generated, comprising a diffusion medium for generating mixtures of said substances by diffusion, and at least one discrete site for immobilizing at least one target with respect to the diffusion medium.
• this diffusion medium can be any medium capable of generating mixtures of substances by diffusion.
• the diffusion medium is a diffusion layer having a considerably greater extension with respect to two of its three dimensions compared to the extension (thickness) of its third dimension.
• the inventive device By using diffusion as the means for generating substance mixtures, for which the concentrations of at least two substances vary at each site where a testing is performed, it is possible with the inventive device to increase dramatically the conditions that can be tested with a limited number of manipulations. It is a characteristic of the present invention that in contrast to the present art, the number of manipulations does not scale with the number of testings performed. By increasing the size of the device or the density of sites where testings are performed, the number of testings scales with the size or the density. However, the number of manipulations to apply test substances to the device remains the same.
• concentration gradients will be established, with highest concentrations at the application site and lowest concentrations at the sites distal from the application site. These concentration gradients will change in a time-dependent manner. The temporal evolution of the concentration gradients will be determined by the diffusion constants of the substances in the respective diffusion medium.
• a substance can be any molecule which is assumed to either a) exert an action on a target, b) modify the response of a target to another substance, c) modify the action of a substance on a target, d) combine with another substance to a whole, and especially with respect to the use of the device and methods in the optimisation of chemical reactions e) catalyze a chemical reaction, f) control a chemical reaction, g) be part of a chemical reaction, or a combination thereof.
• Different substances within one assay can either belong to one class or to a variety of classes. Realisations are also considered in which a substance can be assigned to a class only after the assay has been carried out (e.g. as acting on a target or influencing another substance action on a target by promoting/controlling/being part of a chemical reaction).
• a target is an entity on which a substance or a combination thereof acts or interacts with.
• Targets can be preferably a) purified natural molecules from natural sources or recombinant techniques, especially proteins and nucleic acids, b) prokaryotic and eukaryotic cells, and c) substrates of any kind on which the substances participate in, catalyze or influence chemical reactions.
• One target can contain targets of lesser complexity, e. g. an eukaryotic cell contains a large number of structurally and functionally different molecules that may function as targets with respect to the intention of the assay.
• a device in which the lateral diffusion in two dimensions is decisive for the generation of said mixtures of substances in the diffusion medium.
• the diffusion medium is a three-dimensional diffusion medium in which the generation of said mixtures of substances takes place in all three dimensions.
• This diffusion medium can be e.g. in the form of a cube or other suitable geometry.
• the inventive device is characterized in that the discrete sites in which at least one target is immobilized are part of or are formed by the diffusion medium. If said discrete sites are not part of or are not formed by the diffusion medium, they are preferably part of or are formed by a(n) (separate) assay medium, especially an assay layer.
• This assay medium can reversibly be connected with the device, especially with the diffusion medium for establishing the contact between target(s) and substance mixture(s) necessary for transfer of the substance mixtures to the assay medium.
• the discrete sites can be in the form of cavities (assay cavities), preferably formed in said assay layer.
• assay cavities is used as the general term referring either to physical cavities in the common sense or to places on the device where a read-out is performed.
• Assay cavities can be generated by e.g. physicochemical structuring which is exemplified but not limited to a) hydrophobic ridges separating hydrophilic assay cavities optionally microstructured (as indicated in the next point b)), and b) ridges microstructured with assay solution-repellent microstructures.
• the backside of the assay medium/assay layer may also be coated with assay solution-repellent chemical coating or microstructured with assay solution-repellent microstructure. The latter embodiment is used preferably when contact of the assay medium/assay layer with the diffusion medium/diffusion layer is only transient.
• the discrete sites are arranged in a preferably regular pattern, wherein preferably said discrete sites are for example equally or logarithmically spaced for probing linear or logarithmic dilution patterns with respect to the concentration gradients of substances in the diffusion medium. This will be discussed later with reference to the drawings.
• the discrete sites are separated from the diffusion medium, preferably by a separation medium, especially a separation layer.
• the separation layer can be formed by a semipermeable membrane, wherein preferably said semipermeable membrane is permeable to at least one substance of the substance mixture and substantially impermeable to the targets.
• the separation layer can functionally be replaced by covalent or non-covalent coupling of the targets to an assay medium in which the discrete sites are also preferably formed as cavities, or by covalent or non- covalent coupling of said targets to microspheres located inside said cavities.
• the term "semipermeable” is used according to the invention in that it obstructs assay targets from leaving the assay medium and entering into the diffusion medium, while letting sample (substance) components through.
• the separation medium/layer is obstructing lateral diffusion of solutions and allowing vertical diffusion only. Either by combination of assay medium and separation medium or by integration of both media in one, vertical diffusion of sample (substance/substance mixtures) from the diffusion medium into the assay cavities is only possible at the location of assay cavities.
• the material of the separation medium/layer will be selected by the person skilled in the art. For instance in a biological assay the separation medium/layer may be coated with chemicals that guarantee biocompatibility.
• both assay medium and separation medium are both integrated into one, e.g. by wet-etching techniques from silicon wafers. If a combination of assay medium and diffusion medium already fulfills the requirements of semipermeability and biocompatibility then the separation medium/layer may be omitted. If immobilization of the target is possible in a way that no diffusion of targets in the assay medium occurs while the assay is performed, the functions of the assay medium and diffusion medium may be combined in one single medium, preferably one single layer. In this embodiment, read-outs may be performed for a continuous gradient of assay conditions.
• the device further comprises at least one substance application site for bringing at least two substances (samples) to the assay device and preferably for establishing the necessary contact between sample and diffusion medium.
• substance application sites can be part of or formed by a sample medium, especially a sample application layer.
• the substance application sites can be for example in the form of a fiber or resin-filled hole, a fiber or resin pad, a cavity, a dried substance spot on a solid support and/or a membrane with little (lateral) diffusion.
• any other material with little non-specific interaction with the samples (substances) and form known by the person skilled in the art can be chosen.
• the sample medium can be conne ⁇ ted to or substituted by a microfluidic device comprising ducts or channels connected to inlets and outlets in the diffusion medium.
• the sample medium locally delivers sample (substance) to distinct locations of the diffusion medium.
• the sample medium can be made from any membrane or other suitable material that can be brought into contact with the diffusion medium without causing sample (substance) mixing by processes other than diffusion.
• the substances can be placed on the sample medium in any pattern that is technically compatible with the realisation chosen for the sample medium. As a consequence any kind of overlapping concentration gradients can be formed. These are exemplified but not limited to gradients in which substances diffuse against one another and gradients in which substances diffuse along with one another.
• sample medium can be integrated into the assay medium in form of separate microcavities (microcavities here must be understood in the same broadened sense as assay cavity).
• Substance e.g. can either be soaked into small patches of fiber or resin material present in cavities in the assay medium that - when working the assay - are in contact with the diffusion medium, or dried onto the backside of the assay medium.
• sample medium and diffusion medium can be one joint medium. Substance solutions are spotted locally onto the medium. With this realisation it is advantageous that the sample medium is stored in a dry form or that the substances are linked covalently to the sample medium and released upon beginning of the assay.
• the device comprises further a solid support, preferably for improving the mechanical stability of the assay.
• the device comprises the following sequence of components, namely a solid support, a sample application layer (comprising substance application sites) a diffusion layer, a separation layer, and an assay layer (comprising discrete sites at which target id immobilized).
• each layer can be integrated into each other layer, e.g. sample application layer and assay layer can be integrated into each other or the sample application layer and diffusion layer, respectively.
• a device comprising at least two of the devices as described above is claimed. This allows the working of at least two assays in parallel.
• a method for assaying the interaction, especially the joint interaction, of at least two substances with at least one target.
• This method is characterized in that said mixtures are generated by diffusion of said substances in a diffusion medium, especially a diffusion layer, that interaction of said mixtures takes place with targets substantially immobilized with respect to the diffusion medium at discrete sites, said interaction is detected preferably at a point in time, when the concentrations of at least two substances vary at each site where a testing is performed, and the concentrations of the substances are derived from the positions of the device where the interaction is detected.
• the inventive devices and methods can be used to test different targets in relation to one combination of substances and/or to test the same targets in relation to different combinations of substances. Furthermore, the invention can be used to supply data for establishing models describing the behaviour of a target in response to the combinations of substances in a predictive fashion.
• kits may be provided with which a physician can test a large number of conditions in a standardized way and which are characterized by the fact that with a limited number of manipulations a greater number of conditions can be tested.
• the number of conditions only de- pends on the ratio of the diffusion area and the assay cavities per area. Combinations of antibiotics may be tested in a very similar fashion.
• At least one sample (substance) is not a drug candidate but meant to vary the chemical environment, undergo a chemical reaction with at least one of the drug candidates, or vary the pharmacokinetics or pharmacodynamics or a combination of both of the drug candidates.
• one of the samples (substances) not being a drug candidate may alter the response characteristics of at least one of the targets. If the targets are living cells, at least one of the samples (substances) not being a drug candidate may change the behaviour of living cells.
• These realisations encompass but are not limited to substances that induce cancer, apoptosis, necrosis, cell proliferation, cell differentiation, cell migration, the response of T-lymphocytes to target cells, micropinocytosis, endo- cytosis, phagocytosis, antigen-processing.
• One example for the second class of applications is the assaying of the effect of substances on the activation of T-lymphocytes carried out in the presence of varying concentrations of a cytokine that alters the response characteristic of a T- lymphocyte to a stimulus.
• each sample corresponds to a fraction (either one substance or by itself a group of substances) of all substances isolated from natural sources, preferably plants that in an unknown combination with respect to constituents and relative quantities constitute the active principle.
• the assay device and method serve to identify the optimal recombination of substances constituting the active principle.
• Another field of application may be the so-called solid phase organic chemistry.
• inventive device and method a large number of reaction mixtures can be formed automatically with a limited number of manipulation steps for the preparation of the synthesis that is independent of the number of reaction mixtures to be tested.
• the read out of the interaction of the substance(s) with the target is exemplified but not limited to fluorescence, mass spectrometry, high performance liquid chromatography, reverse PCR, capillary electrophoresis and/or changes of the electric properties of any assay constituent, preferably addressed by one or more microelectrodes. Also any other method of measuring the interaction of the substances with the targets is possible and claimed in accordance with the invention.
• the above-mentioned methods can be further characterized by any feature of the device as defined above.
• the methods and the device as defined above can be used to investigate the interaction of substances to targets like e.g. prokaryotes, eukaryotic cells, cellular organelles, biomolecules from natural or synthetic source and/or synthetic molecules with no correspondence in nature.
• targets like e.g. prokaryotes, eukaryotic cells, cellular organelles, biomolecules from natural or synthetic source and/or synthetic molecules with no correspondence in nature.
• targets e.g. prokaryotes, eukaryotic cells, cellular organelles, biomolecules from natural or synthetic source and/or synthetic molecules with no correspondence in nature.
• targets e.g. prokaryotes, eukaryotic cells, cellular organelles, biomolecules from natural or synthetic source and/or synthetic molecules with no correspondence in nature.
• targets e.g. prokaryotes, eukaryotic cells, cellular organelles, biomolecules from natural or synthetic source and/or synthetic molecules with no correspondence in nature.
• targets e.g. prokary
• the assay can be performed by different modes of operation.
• the targets are present in the assay cavities and the test substance is added to or released from the substance application sites.
• the assay may be read out at any point in time or different time points in a time-resolved manner.
• the interaction of the targets with said substance mixtures is analysed at a point in time, when for at least two substances the equilibrium with respect to the distribution in the diffusion medium has not been reached.
• the concentrations of at least two substances varies for each position where a testing is performed.
• the response of a target may vary for each position of the assay device, even if the read-out is performed at a point in time, when equilibrium with respect to diffusion of the substances has been reached.
• targets closer to the substance application site may have been influenced stronger, due to longer exposure to higher concentrations of the substance.
• the targets are not present in the assay cavities at the beginning of the assay. Only when the substance gradients have been formed the targets are applied to all assay cavities at the same time.
• This operation can be performed by a nanopipetting device or preferably by spraying the target in the form of small droplets into the assay cavities.
• the targets are not exposed to a temporal evolution of substance gradients but get in contact with the substance mixtures, once the concentration gradients for which the read out shall be performed, have been formed.
• One specific embodiment of the assay aims at preserving a gradient at a specific point in time. This may be achieved by providing assay layer and samples (substances) with chemical functionalities that only react with each other upon activation or upon change of the chemical conditions.
• the initiation of a chemical coupling reaction upon activation can be realised by means of so called caging groups as described for example in Methods in Enzymology, Vol. 291.
• the assay layer is functionalised with caged sulfhydryl groups whereas the samples (substances) are modified with maleimido reactive groups.
• a caging group a 2-nitrobenzyI-moiety may be employed that is cleavable by light.
• the sulfhydryl groups will react with the maleimido groups and thereby immobilize the substance and preserve the gradient present at the point in time of illumination.
• caged sulfhydryl groups and maleimido groups any other appropriate combination of caged functionality and reactive group such as caged amino groups and epoxy groups or maleimido groups may be employed. It is also possible to derivatize the samples (substances) with the caged functionalities and derivatize the assay layer with the reactive groups. The initiation of a coupling reaction by a change of chemical conditions is exemplified by derivatizing the assay layer with amino-groups and the samples (substances) with epoxy groups.
• the gradient is formed at chemical conditions at which no reaction takes place.
• this may be achieved by adjusting the pH of the solution to below 6.
• Qf the pH of the solution
• the coupling reaction will take place.
• Such a change of the chemical conditions without perturbance of the gradient may be achieved by spraying a solution with the appropriate pH onto the assay layer.
• an additional functionality (that permits a controlled release of the samples (substances) after immobilization) is provided either as a linker between the assay layer and said reactive group or between the functionality of the sample (substance) reacting with said reactive group and the part of the sample (substance) exerting the activity with respect to the intention of the testing.
• a functionality is exemplified but not limited to an enzymatic cleavage site. If a process other than light was employed for the immobilization of the substance the release of the samples (substances) may occur by light using a photocleavable linker.
• the localization of the reactive groups for immobilization is not restricted to the assay layer but may instead or additionally also take place in the diffusion layer.
• the embodiments, in which the gradients are presented may preferably be combined with the embodiment in which the targets are added once the gradients have been formed.
• the substance mixtures are formed in a diffusion medium/layer, and the targets are in the assay cavities.
• the assay medium/layer is not in contact with the diffusion medium/layer.
• the substance mixtures are transferred into the assay cavities by establishing a transient contact of the assay medium/layer with the diffusion medium/layer.
• the diffusion medium/layer can be used to sequentially transfer the concentration gradient into a large number of different assay media layers.
• the targets are not exposed to a temporal evolution of substance gradients but get in contact with the substance mixtures, once the concentration gradients for which the read out shall be performed, have been formed.
• targets may be added either once the samples (substances) have been immobilized or directly after release of the samples (substances). Due to the preservation of the gradients by immobilization, in these specific embodiments the assay devices may be stored and/or transported with preformed sample (substance) gradients.
• the concentrations of substances at a specific position of the device where an activity is detected can be derived based on the knowledge of the volume of the device that the substances can enter, the position of each substance application site relative to the position of the read-out, the amount of substance applied to the respective substance application site, the diffusion constant of the substance in the diffusion medium and the time over which diffusion took place.
• a reference substance that can be detected for example by fluorescence and does neither interact with the target nor with the substances, can be applied together with the test substance as a reporter to the same substance application site.
• a person skilled in the art may devise a way for deriving the concentration of the test substance from the signal detected from the reporter. For example, the test substance and reporter substance may be added in amounts for which the ratio is known. If the signal detected for the reporter is calibrated for the determination of concentrations, then the concentration of the test substances may be derived directly from the reporter signal. For each test substances a different reporter substance will preferably used, so that the reporter signals do not interfere with each other.
• Figure 1 Side-view of an assay device
• Figure 2 Top-views and side-views of an assay device and working principle
• Figure 3 Side-views of different realisations of assay cavities
• Figure 4 Top-views of assay devices for testing different dilution patterns
• Figure 5 Top-views and side-views of several options for the design of the sample layer
• Figure 6 Top-views and side-views of several options for the design of the diffusion layer
• Figure 7 Top-views and side-views of several options for the design of the separation layer
• Figure 8 Top-view of an assay device consisting out of 4 x 4 single assay devices
• Figure 9 Diffusion driven formation of inverse diagonal substance gradients on silicium nanotiter plates
• Figure 10 Gradient formation in cellular applications
• Figure 11 Enlargement of assay cavities of figure 10
• FIG 1 a general schematic of one possible realisation of an assay device (10) is shown comprising a solid support (11), preferably for improving the mechanical stability of the assay, a sample application layer (12) which may comprise substance application sites for bringing the sample (substance) to the assay device, and especially for establishing the contact between sample and diffusion layer, a diffusion layer (13) where the generation of mixtures of substances by diffusion takes place, a separation layer (14) for separating the diffusion layer from the assay layer, and an assay layer (15) which may comprise target immobilizing sites for immobilizing the target, for instance assay cavities (16).
• a sample application layer which may comprise substance application sites for bringing the sample (substance) to the assay device, and especially for establishing the contact between sample and diffusion layer
• a diffusion layer (13) where the generation of mixtures of substances by diffusion takes place
• a separation layer (14) for separating the diffusion layer from the assay layer
• an assay layer (15) which may comprise target immobilizing sites for immobilizing the target, for instance assay
• all components of said device could reversibly be connected with the other components of the device and/or functionally be replaced by or integrated with any other component, depending on the chosen type of assay.
• the diffusion layer will be soaked with a solution appropriate for the respective testing and assay cavities will be filled with the solution so as to probe the interaction of the substances with targets.
• FIG 2 the working principle of one possible realisation of the inven- tive device is illustrated.
• the components of the device are the same as described in figure 1.
• the samples (substances) are marked in black and grey.
• concentration gradients for sample (substance) A (17, black) and sample (substance) B (18, grey) are built-up by lateral diffusion in the diffusion layer, so that in each assay cavity (16) the sample (substance) concentration represents the underlying concentration gradient.
• the substance mixtures are transferred into the assay layer (15), wherein the targets (19) are immobilized. By this way any kind of overlapping concentration gradients can be formed.
• each assay cavity may be determined by the manufacturing process as well as by the characteristics of the used material.
• the assay cavities can be separated from the surrounding components of the device by a(n) (integrated) separation layer, for in- stance a semipermeable membrane (20) as is illustrated in the middle of figure 3.
• Said semipermeable membrane can be permeable to at least one sample (substance) or to the substance mixtures and substantially impermeable for assay components, and at least for the targets.
• This separation layer can encompass the whole bottom of the assay cavity or just a part of it.
• the distinct assay cavities may be replaced by e.g. realisations in which the sample layer or the dif usion layer integrates the function of the assay cavities (figure 6D). Also these realisations of assay cavities may optionally be identically or differently coated to modify the wettability and the interaction of assay components as well as samples (sub- stances) with the (functionally integrated) assay cavity (21 ).
• Figure 4 shows examples for the testing of different sample (substance) dilution-patterns.
• sample (substance) dilution-patterns Depending on the arrangement of the discrete sites where immobilizing of the targets takes place/a testing is performed dif- ferent dilution patterns can be probed. If for instance the discrete sites are equally/regularly spaced, linear dilution patterns are tested, as is illustrated in the top of figure 4. On the other hand logarithmic dilution patterns are tested if the discrete sites are logarithmically spaced, as is shown in the bottom of figure 4. Depending on the chosen assay, these different dilution patterns can be used to investigate slightest variations of relative and absolute concentrations of compound mixtures in e.g. the optimisation of cancer therapy or optimisation of reaction conditions in the solid phase organic chemistry.
• Figure 5 A shows sample layers with several substance (sample) application sites (22). These substance application sites can be a fiber/resin-filled hole (23) or cavity (24) soaked with sample (substance) in a dry or wet form. It is also possible to spot the sample (substance) so- lution onto a solid support (25) or a fiber/resin pad on a solid support (26) and to dry it. Also a realisation is shown in which the sample (substance) is spotted onto a membrane with little (lateral) diffusion (27) fol- lowed by drying of said sample (substance) on the membrane.
• the sample layer can be connected to or substituted by a micro- fluidic device comprising ducts or channels connected to inlets (•; 28) and outlets (T; 29) in the diffusion layer as is shown in figure 5 B. It is also possible that the function of the sample layer is integrated into the assay layer (figure 5 C (30)) or into the diffusion layer (figure 5 D (31)), e.g. in form of separate micro cavities.
• the diffusion layer can be any layer capable of generating mixtures of substances by diffusion.
• the material has to obstruct convection and to minimize non-specific interaction of sample (substance) components.
• the assay layer can be combined with the diffusion layer or the assay layer can consist out of a suited material to fulfill the function of the diffusion layer (figure 6 B, C, D).
• the boundary of diffusion layer and assay cavity is imperrneable with respect to the target and permeable with respect to the substances and the same component of the device can be used as assay layer/sample application layer/diffusion layer (32).
• FIG. 6 C Another possi- ble realisation for the combination of the assay layer, a sample application layer and the diffusion layer in one component of the device is shown in figure 6 C.
• the targets are only present in distinct sites of the layer (33).
• Realisations are also possible in which the layer of figure 6C is completely functionalised with target (figure 6 D).
• Figure 7 shows several options for the design of the separation medium.
• One possible realisation to separate assay and diffusion layer is the use of a separation layer (14), e.g. a semipermeable membrane (figure 7 A).
• the separation layer can functionally be replaced by immobilizing the targets to the walls of the assay cavities or to supports (e.g. polystyrene beads) placed into the assay cavities that do not enter the diffusion layer (figure 7 B), or by immobilizing targets in a combined assay/diffusion layer as is shown in figure 7 C.
• Figure 8 shows an example for the combination of 16 assay devices (10) used to test different combinations of samples (substances) with respect to one target, several targets with respect to one combination of samples (substances) or a combination thereof.
• the samples (substances) are pipetted into the sample application sites (22), and the individual devices are separated from each other by diffusional barriers (34).
• the individual devices can be designed according to the designs described in figures 1 to 7.
• Figure 9 shows an experiment, in which an inverse diagonal substance gradient was established on a silicium nanotiter plate by diffusion.
• nanotiterplates of 12 x 12 mm size were placed on an agarose bed as diffusion layer. Contact of the assay cavities with the diffusion layer was ensured by the presence of holes in the bottom of the assay cavities.
• Carboxy- tetramethylrhodamine and carboxy-fluorescein solutions were pipetted on 2 x 2 mm pieces of Whatman filter paper serving as sample application layer, dried and then placed onto the diffusion layer. The dimensions and positions of the samples are indicated.
• Figure 10 shows an example for assaying the joint interaction of two substances in a cellular assay.
• Adherent tissue culture cells serving as targets were seeded on a 12 x 12 mm nanotiter plate, placed in a 60 mm tissue culture dish. After two days of cultivation the nanotiter plate was placed on an agarose bed as diffusion layer. Contact of the assay cavities with the diffusion layer was given by holes in the bottom of the assay cavities. The adherent growth of the cells functionally replaced the separation layer. Two times 3 Dl solutions of the DNA dyes Hoechst 33342 (0.1 mM) and propidium iodide (50 Dg/ml) in water were applied on 2 x 2 mm pieces of Whatman filter paper serving as sample applica- tion layer.
• the Hoechst 33342 dye stains the nuclei of all cells, while propidium iodide only stains the nuclei of dead cells with a permeable plasma membrane.
• free propidium iodide has a faint fluorescence, while the Hoechst signal is only visible in the DNA-bound state.
• the test substance show activity, i. e. DNA staining.
• the bright corners of the nanotiter wells in the lower left of the propidium image are due to reflection at the menis- cus of the cell culture medium.
• the bright appearance of the upper surface of the nanotiter plate in the Hoechst 33342 image was caused by the filter set used for detection of fluorescence and does not represent Hoechst 33342 fluo- rescence. This is apparent from the gradient-free homogeneity of this signal over the whole nanotiter plate.
• Figure 11 shows an enlargement of the four lower left assay cavities and a further enlargement of one of those four assay cavities as indicated in the figure 10.
• Each bright spot corresponds to one Hoechst 33342-stained cell nucleus.

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