EP1861690A1 - Assay by osmotically induced separation and concentration of high-molecular detectable substances and a fluid microsystem for carrying out said assay - Google Patents

Abstract

The invention relates to an assay method and a fluid microsystem for carrying out said assay method, in particular, for carrying out miniaturised affinity tests in a micro-array format. According to said invention, a liquid phase comprising at least one type of detectable high-molecular soluble substance, for example a markable reaction partner or reaction product of an affinity reaction is displaced by the action of a high-molecular osmotic agent on an ultrafilter membrane towards a miniaturised measuring chamber defined thereby. The fraction of the detectable high-molecular substance(s) is concentrated in the measuring chamber, while the dissolved low- molecular components are removed with the solvent through the membrane pores, thereby making it possible to attain a high detection sensitivity.

Description

 Assay with osmotically induced separation and enrichment of high molecular weight substances to be detected and fluidic microsystem for its implementation

The present invention relates to an assay method and a fluidic microsystem for its implementation, which is particularly suitable for miniaturized affinity tests in microarray format.

According to the invention, a liquid phase having at least one high molecular weight soluble substance to be detected, e.g. a labeled reactant or reaction product of an affinity reaction, due to the hydraulic action of a high molecular weight osmoticum on an ultrafilter membrane moved into a limited by this membrane miniaturized measuring chamber. There, the fraction of the high molecular weight substance (s) to be determined concentrates, while low molecular weight dissolved constituents flow away with the solvent through the pores of the membrane. As a result, a high detection sensitivity is achieved. In competitive mode, a positive signal can be obtained or a homogeneous phase assay can be performed. Another advantage is a small amount of time and labor for performing the assay.

Affinity assays are analytical methods based on noncovalent affinity binding. These include the immunoassays or immunoassays that are currently standard procedures in clinical chemistry, diagnostics, environmental analysis and bioprocess control. The basis of each affinity assay is at least one specific bioanalytical recognition reaction, eg the specific binding of an affinity ligand to an antibody. Instead of immune proteins, other affinity receptors, eg lectin ne or aptamers, be used for the selective binding of an analyte.

The inherent specificity and high affinity binding affinity allows selective, highly specific and highly sensitive concentration measurement. With the help of antibodies, for example, picomolar and nanomolar solutions of numerous analytes can be detected. Specific antibodies can be generated against almost all types of chemical compounds, from low molecular weight substances, so-called haptens, to biological or synthetic macromolecules. The methods for preparing and carrying out affinity assays in macroscopic format (reaction volume> 20 microliters) are known in the art and can be read in a variety of publications (eg James P. Gosling "Immunoassays", Practical Approach Series, Oxford University Press, Oxford, England).

An immunoassay or affinity assay usually consists of several stages, i.a. at least one incubation step, at least one washing step and a concentration measurement.

In recent years, modern methods have been developed which simplify the handling of immunoassays and thus make them accessible for new applications, eg the pregnancy self-test in the paper test strip format ("immunochromatography"). Another development direction is to combine a variety of immunoassays in a raster manner on a small test area, the "biochip ^λX or" microarray. "Microarray format immunoassays can detect, for example, a variety of individual analytes from a single blood sample Affinity assays consist of reducing the signal intensity by reducing the amount of reagent. Since the usual affinity receptors (eg, antibodies and antigens) and their binding partners are not directly detectable with the usual detectors, they are labeled with a labeling substance ("label" or "marker") that provides a measurable and analyte-dependent signal. chemically coupled. For miniaturized immunoassays one needs labeling substances of highest specific activity, eg enzymes (eg peroxidase, alkaline phosphatase) or fluorescent dyes with high fluorescence yield. With enzymes, a reinforcing effect is achieved by catalyzing the formation of a signal substance (eg, a colored reaction product). Fluorescent dyes allow the fastest and most direct detection reaction; however, high sensitivity requires powerful and expensive optical devices in fluorescence affinity assays.

High molecular weight antigens such as e.g. Proteins can be detected particularly favorably with a "sandwich" immunoassay. In the most common embodiment, the surface is coated with a specific antibody such that after incubation with a sample solution, the analyte becomes bound to the surface while other sample components are removed in a subsequent washing step. The actual detection takes place after incubation with a second, labeled specific antibody and a further washing step (and optionally a color development reaction in the case of enzyme labels). The dose-effect curve is positive.

The quantitative determination of substances with molecular weights below 1000 daltons is one of the most important applications of immunoassays. Almost exclusively so-called competitive assays are used for this purpose. In these, the (unlabelled) analyte competes with an analog for the free binding sites of an affinity receptor, with either the affinity receptor or the competing analog is labeled. The analog may be a low molecular weight or high molecular weight solute.

In competitive affinity assays for the detection of low-molecular-weight analytes, as a rule, a separation of the labeled molecules depending on the analyte concentration into a soluble and surface-bound fraction takes place. In many cases, the affinity binding of the labeled molecules takes place on a surface occupied by immobile reaction partners. After washing off the soluble fraction, the amount of bound label on the surface is detected. In the absence of the competing analyte, the maximum amount of labeled molecules can be bound to the surface, the amount of bound labeled molecules to the surface is reduced by the specific binding of the analyte to its receptor.

Since the bound amount of the bound labeled substance is detected after a washing operation, an inverse dose-effect curve, i. E. the largest signals are achieved at the lowest analyte concentrations; at very high analyte concentrations, the signal strength is very little dependent on the concentration; at very small concentrations of analyte disturbs the uncertainty of the difference measurement. Due to the principle of difference measurement, the concentration of the labeled reactant must be adjusted to the concentration of the analyte. Since the bound amount of the labeled substance is usually in a very thin layer on the surface, the requirements for the detector are very high for small measuring fields, which may be u.a. affects the costs.

Desirable would be the reversal of the inverse characteristic in competitive assays or a ratiometric measurement of the Amount of the fraction of the labeled molecules formed by the reaction with the analyte. The heterogeneous test system also has the disadvantage that non-specific binding of the labeled molecules to the surface is unavoidable, resulting in a more or less large background signal. It would be of great advantage if competitive affinity assays could be carried out completely in homogeneous liquid phase.

The stated limitations of competitive immunoassays are based primarily on the fact that only the surface-bound part of the labeled binding partner (for example the antibody or antigen) is detected. There is currently a great need for miniaturized immunoassays with positive dose effect and those that can do without the binding of the labeled molecules to a surface.

The object on which the invention is based is thus to provide a new, inexpensive and very sensitive method and a corresponding device for carrying out quantitative, in particular competitive, assays, preferably affinity assays, in single or microarray format. The method according to the invention is intended to offer the possibility of a positive dose effect after a convenient and standardized separation between the fractions of the labeled molecules produced by the reaction with the analyte. It should be feasible without the binding of the labeled molecules to a surface and allow a sensitivity increase over comparable conventional methods.

The object is achieved by an assay according to claim 1 and a fluidic microsystem for its implementation according to claim 11. advantageous Embodiments of the invention are set forth in subclaims 2-10 and 12-16.

The essence of the invention consists in the use of macromolecular osmotics and an ultrafilter membrane for the concentration of high molecular weight substances to be detected and for their separation of potentially interfering low molecular weight substances.

In the case of an affinity assay, these are typically a separation of the fractions of the labeled molecules present as a result of the reaction with the analyte and the concentration of one of the fractions.

Usually, in affinity assays, a sample matrix to be analyzed is mixed with labeled and unlabelled reactants. Subsequently, the labeled reactant is present in affinity binding in different binding forms (e.g., soluble / insoluble or complexed / uncomplexed) due to affinity binding to the analyte or due to competition with the analyte, the ratio of which depends on the concentration of the analyte.

A prerequisite for an affinity assay according to the invention is that the reaction of the analyte with the reactants is carried out so that subsequently only one of the labeled fractions is present in dissolved and at the same time high molecular weight form. For this purpose, experts are aware of several options.

A non-limiting example is the binding of a low molecular weight marker, which is a labeled analog of the analyte, to a receptor, such as an antibody. In this case, after the reaction, depending on the concentration of the analyte, a fraction of the labeled analog is high molecular weight, the other low molecular weight. Further examples are the competition of the analyte with a labeled soluble high molecular weight analog for binding sites on an immobile antibody and the competition of the analyte with an immobilized analog for a labeled soluble antibody. In both cases, labeled macromolecules are present in both dissolved and undissolved form after the reaction, depending on the concentration of the analyte.

The terms "high molecular weight" and "low molecular weight" in the context of this invention are to be understood relative to the pore size of an ultrafilter membrane used according to the invention. This means that a low-molecular-weight fraction can flow through the used ultrafilter membrane relatively unhindered (reflection coefficient <0.5), but the high-molecular-weight fraction does not penetrate through it (reflection coefficient close to the value D).

Suitable ultrafilter membranes in capillary or planar form with molar mass cut-off (based on proteins) between 5000 and 10 ⁶ and molecular size cut-off (based on Stokes diameter) between 1.5 and 50 nm are from numerous manufacturers offered. Typical, but not limiting molecular weight or molecular size ranges for low molecular weight substances are below 5 kDa or below 2 nm, typical molecular size ranges of high molecular weight substances of more than 10 kDa and more than 3 nm. As marked affinity binding partner in various molecular sizes in the range of 0, 3 to 50 nm are commercially available or can be prepared, the choice of the membrane, the critical molecular size for the distinction of the low molecular weight and the high molecular weight fraction can be adapted to the available or selected reactants. If a membrane with a very large molecular size cut-off is used, this has the advantage of high hydraulic permeability, requiring About correspondingly large particles of the labeled high molecular weight reactant or reaction product and the high molecular weight osmoticum.

According to the invention, the liquid phase with the high-molecular-weight soluble fraction to be detected is osmosed by the hydraulic action of a high-molecular osmoticum on an ultrafilter membrane (5) from a larger filling space (2) via a flow path (3) into a smaller measuring space hydraulically connected to it (4) after filling a concentrated solution of the polymeric osmoticum in a suction space (1) (see Figures 1 and 2).

The terms "flow path" and "capillary flow path" are to be understood in the context of this invention as a flow path between the Einfüllraum and the measuring chamber, whose volume makes up only a small part of the volume of the Einfüllraumes.

The high-molecular osmoticum exerts a hydraulic effect on the liquid phase with the labeled reagent on an ultrafilter membrane which separates the suction space from each measuring space. The liquid is moved into the measuring space and its low-molecular components are sucked with the solvent through the pores of the ultrafilter membrane.

In principle, any known high molecular weight substance can be considered as the high molecular weight osmotic agent, in the aqueous solution of which the osmotic potential can be lowered sufficiently (usually by more than 0.1 MPa). Known to be ^¬ sitting in particular concentrated solutions of hydrophilic polymers such as polyethylene oxide or polyvinyl alcohol, the molecules of water-rich tangles represent, independently of their particle a strong osmotic effect, since the concentrated solutions network liquids represent whose osmotic potential loading by the concentration of monomers is true. It is important that at the given concentration, the molecular size of the osmoticum is well above the cut-off of the membrane. This is the case with polyvinyl alcohol or polyethylene oxide, for example, with ultrafiltration membranes with a molecular size cut-off of 5 nra, when the molecular weight is above 20 kDa. For example, polyethylene oxide is well suited as a high molecular weight osmotic agent for the assay of this invention because relatively high osmotic pressures (above 1 MPa) with a reasonable viscosity (<500 mPa s ^-1 ) are achieved with this neutral polymer, which is commercially available in different molecular weights can.

As is known, solutions of high molecular weight polyelectrolytes such as polyacrylate, dextran sulfate, polystyrenesulfonate, poly (diallyldimethylammonium chloride) and the like already exhibit a high osmotic effect in low mass concentration because the electrically sorbed counterions are present in high particle concentration. The hydraulic effect of the polyelectrolytes on an ultrafilter membrane is particularly high when the salt concentration of the aspirated liquid is low compared to the concentration of the polymer-bound ions of the polyelectrolyte solution. A polymeric osmoticum that can be widely used for the invention is high molecular weight sodium dextran sulfate from Pharmacia with an average molecular weight of 500 kDa. It is impermeable to membranes with ultrafiltration properties and a pore size below 20 nm, forms concentrated and osmotically highly effective solutions with relatively low viscosity and is transparent to light in the visible and short wavelength range.

While water and low molecular weight components of the liquid through the pores of the ultrafiltration membrane into the suction chamber at ^¬ transgressed, concentrate the high molecular weight solute to be detected, for example, labeled reactant or reaction products of an affinity reaction, in Measuring room, where their amount can be detected very sensitive, for example, on the basis of radioactivity, optical properties, such as fluorescence or luminescence, magnetic or electrical properties or a catalytic, such as enzymatic, activity of a marker.

The penetration of air into the measuring chamber after complete emptying of the filling chamber can be prevented by adding the polymeric osmoticum in low concentration to the reagent. In this way, the ratio in which the labeled macromolecules are concentrated can be determined.

In the enrichment of the labeled molecules in a small volume with a relatively high layer thickness, there is a significant advantage of the invention, in particular for the development of a miniaturized test array. Another important advantage of the method according to the invention is the convenient separation of the labeled soluble macromolecules or high molecular weight complexes from the other labeled molecules which may be present in excess. The fraction of the labeled molecules to be separated may be prevented from flowing into the measuring chamber by binding to a surface or a particulate affinity sorbent. It can also be low molecular weight and flow with the water through the pores of the ultrafilter membrane into the suction chamber. The former possibility has the advantage of a positive dose effect, the latter allows a homogeneous phase assay and therefore has the advantage that it can be dispensed with the immobilization of an affinity binding partner.

For example, a defined amount of a labeled antibody may be mixed with the sample matrix containing a low molecular weight analyte. Part of the antibody is then bound to the analyte. Then an Af- in excess of the sorbent that competes with the analyte for the antibody. In the liquid phase, only the labeled antibodies associated with the analyte remain. Thereafter, the liquid phase is moved into the measuring chamber. After the solution of the polymeric osmoticum has been introduced into the suction chamber, a large part of the liquid is sucked from the filling space into the measuring chamber, where the labeled antibody concentrates in complex with the analyte. A largely stoichiometric, positive dose effect can be achieved.

A heterogeneous competitive affinity assay with a positive dose effect can also be achieved by the method according to the invention with a labeled particle-bound complex according to the displacement principle. The complex consists of an affinity receptor and a high molecular weight labeled analogue of the analyte,. The unlabelled portion of the complex is physically or covalently bound to the surface, while the labeled portion can be detached by ligand exchange with the analyte. In this case, it is favorable if the analyte has a high affinity for the receptor (e.g., an antibody), while the labeled analog is bound with intermediate affinity, so that a short reaction time and a nearly stoichiometric reaction are to be expected. After the reaction, the labeled soluble reaction products are concentrated in the measuring chamber. Again, a direct dependence of the signal on the concentration of the analyte is achieved.

The invention also enables heterogeneous affinity assays with low molecular weight labeled analogs in which a positive dose-response effect is achieved. For example, the competition of a low molecular weight analyte with a low molecular weight fluorescent analogue on an affinity particle with immobilized antibodies is exploited. The liquid phase contains the fluorescent low molecular weight analogue in a concentration directly dependent on the concentration of the analyte. If the antibody in excess is then added in excess, the marker is converted into a high-molecular-weight complex and can be accumulated in the measurement space. It is also possible that a solution of the unlabelled antibody is introduced into the measurement chamber using capillary forces before adding the sample and the remaining reagent components. After the osmotically induced aspiration of the liquid phase, the soluble fraction of the low molecular weight labeled analog is bound to the antibody and concentrated in the measuring chamber.

If the invention is used for a heterogeneous fluorescence immunoassay with high molecular weight labeled compounds, the measurement of the fluorescence takes place after the immune reaction has taken place in a separate room, the measuring space. If the fluorophore is covalently bound to the macromolecule, fluorescence measurement can be performed in the optimal electrolyte environment for fluorescence, even if it is incompatible with the reaction itself. If e.g. a fluorescein-labeled reactant used, it makes sense to adjust the pH of the solution of the polymeric osmotic agent to about 9. The said fluorophore can be detected in an alkaline medium with a very high fluorescence yield.

The method according to the invention makes it possible to carry out a sensitive competitive affinity assay for a low-molecular-weight analyte in a homogeneous phase. Two reagents are used for this purpose, for example an unmarked affinity receptor for the analyte, as a rule an antibody, and secondly a low molecular weight labeled competitor ligand, for example a conjugate of the analyte with a fluorescent dye. The assay can be carried out in such a way that a limited and defined amount of the antibody is added. next with the analyte and then reacted with a present in excess of the antibody present marked low molecular weight competing ligands. As a result, a dependent on the amount of the analyte portion of the labeled low molecular weight substance is transferred into the high molecular weight complex and can therefore be concentrated in the measuring room. If the concentrations of the analyte and the competitor ligand are well above the respective dissociation constants, the amount of labeled detectable complex will be the difference between the amount of antibody and the amount of analyte. The accuracy of this assay can be increased if paired tests are performed on an array with the sample matrix and blank value. The difference of the signals in this case is positively correlated with the concentration of the analyte.

In the illustrated homogeneous system, a sensitive ratiometric measurement can be performed. If both the low molecular weight competitor ligand and the high molecular weight receptor are labeled and the markers can be quantified separately, for example using different fluorescence spectra, the proportion of singly labeled complexes that are labeled with the analyte can be determined without a parallel approach.

When the invention is used to carry out the homogeneous assay in the illustrated form, it is desirable to completely wash out the low molecular weight labeled competitor ligand. The washing out of the low molecular weight marker fraction from the measuring chamber can be carried out by aspirating an additional unlabelled aqueous solution through the ultrafilter membrane and / or by replacing the osmotically effective polymer solution in the suction chamber. The aspiration of an additional unlabelled aqueous solution also allows complete transfer of the labeled macromolecules into the measuring chamber. It is relieved when between the filling room and The measuring space is a hydrophilic filter with a pore size of about 2 microns or a fixed bed of fine hydrophilic particles, which is permeable to aqueous solutions of colloids and prevents the ingress of air into the measuring space.

The invention also enables miniaturized enzyme-linked immunoassays (ELISA), for example an antibody which is covalently coupled to an enzyme, eg a peroxidase, which is the part of the excess added antibody which complements the analyte. The solution of the high molecular weight osmoticum introduced into the suction space in this case contains a low molecular weight unstained enzyme substrate that crosses the ultrafilter membrane accumulated by ultrafiltration in the measurement space, catalyzes the reaction of the substrate to a colloidal fluorescent or colored substance, which concentrates in the measuring chamber so that, taking advantage of the enzyme reaction in a short time a highly amplified and positively correlated with the analyte concentration rtes signal is generated in a small area.

Similarly, the invention can be used to perform a colloidal Sil ^¬ Bernie derschlages or other colloidal precipitate a sensitive assay based. If, for example, gold nanoparticles are used to label an immunoreagent and are concentrated in the measurement space according to the invention, a precipitation of metallic colloidal silver occurs, depending on the amount of gold particles, if the solution of the osmoticum contains a suitable reducing agent and silver ions.

The fluidic microsystem according to the invention, which is suitable for carrying out the above-described assays, in particular Assuming that it is suitable, but not limited to, at least one measuring chamber (1) and at least one measuring chamber (4) separated from the suction chamber by an ultrafilter membrane (5), each measuring chamber having a flow path (3) whose volume is only one makes up a small part of the volume of the filling space, is connected to a filling space (2) and the volume of the filling space exceeds the volume of the measuring space by a multiple. The volume of a miniaturized measurement space is typically no greater than about 0.2 μl and its surface no greater than about 5 mm ² .

A variant of the fluidic microsystem according to the invention is shown in FIG. It represents an array of numerous miniaturized filling chambers (2) and measuring chambers (4) in a transparent solid. Both chambers are hydraulically connected by a capillary flow path (3). Each measuring chamber is separated from a suction chamber by an ultrafilter membrane. Each measuring chamber is designed as a lumen of the suction space passing through the segment of a capillary membrane with ultrafilter properties, such as a microdialysis fiber. This has the advantage that the labeled soluble macromolecules concentrate in a very small space. They are sensitive in this room due to their fluorescence, luminescence or reactivity detectable. If a dye is accumulated in the measuring chamber by an enzymatically labeled immunoreagent, it can generate a clear signal due to the relatively long light path through the measuring chamber, for example in a simple light microscope. A regenerated cellulose dialysis fiber having an inner diameter of about 0.2 mm, suitable for this purpose, has a transparent, completely impermeable membrane for antibody. Their small cross-section allows microscopic image analysis. With a length of the measuring space of 5 mm, a high color depth or a strong fluorescence signal at high dilution is possible. A particularly high sensitivity in the The fluorescence affinity assay is achieved when the excitation light cross-transverses the hollow fiber lumen and the fluorescence is detected across the cross-section of the hollow fiber. If the excitation is carried out with laser light, the same quantum flow can easily be achieved in numerous measuring chambers in the illustrated arrangement, so that a simultaneous detection in numerous measuring chambers is possible. If an affinity assay is to be carried out in an inhomogeneous phase, a particle filter can be provided between the filling space and the measuring space, with the aid of which the affinity sorbent is prevented from flowing into the measuring space even if the particle size is small.

The illustrated in Figure 1 design of the device according to the invention is not the only possible. For certain applications or for the cost-effective production of the array, it may be favorable if the hollow fiber segments are arranged parallel to the array plane or if instead of capillary membranes planar ultrafilter membranes are used. For example, the array consists of two bodies, between which an ultrafilter membrane is glued. Intake space, filling spaces, flow paths and measuring chambers in this case represent channels or depressions in these bodies.

FIG. 2 shows the array in cross section through a unit of filling spaces (2) and measuring spaces (4). Each measuring room is designed by a narrow vertical channel. The suction chamber (1) represents a wide, perpendicular to the cross-section flow channel in the opposite body. It is separated from the measuring space by the ultrafilter membrane (5).

For the detection of ligands on the basis of their radioactivity with the help of an X-ray film, it is advantageous if a very flat measuring chamber is used, which on one side through the Ultrafilter membrane from the suction chamber and on the other side is bounded by a thin liquid-impermeable membrane, which abuts the X-ray film.

Further design variants of the assays according to the invention, in particular affinity assays, and fluidic microsystems are possible depending on the type of reactants used and / or affinity partners as well as the type of detection methods used and easily realized for the skilled person by modification of the general design principles described.

EXAMPLE

A fluorescently labeled protein was concentrated in the observation field of a fluorescence microscope in a fluidic microsystem according to the invention and the increase in fluorescence intensity was observed with a fluorescence microscope.

The microsystem comprised two microcompartments connected by microdialysis fibers of regenerated cellulose (240 μm diameter). There was an open end of the dialysis fiber capillary membrane in the first chamber, which corresponded to the Einfüllraum, while the second chamber, corresponding to the suction, was traversed by a hollow fiber segment (the measuring chamber), the second open end outside of the chamber with the atmosphere communicated , Thus, the wall of the dialysis fiber formed the ultrafilter membrane, while its cavity provided the flow path and measuring space. After filling each 5 .mu.l of a dilute buffered polymer solution (10 mM sodium phosphate, pH 7, with 0.1% Natriumdextransulfat) into the filling chamber and the suction chamber, the lumen of the capillary membrane (100 nl) filled adhesive. Fluorescein-labeled immunoglobulin was added in high dilution to the loading chamber so that the fluorescence intensity was at the detection limit. Subsequently, the diluted buffered polymer solution in the suction chamber was concentrated by a solution sodium dextran sulfate (20%) with an osmotic pressure of about 30 bar. This made it possible to suck most of the liquid from the filling chamber into the suction chamber in about 30 minutes and to concentrate the fluorescence-labeled immunoglobulin in the lumen of the hollow fiber segment. The fluorescence intensity was now clearly visible in the fluorescence microscope. Since sodium dextran sulfate concentrated in the hollow fiber segment at the same time, the flow was stopped automatically and the hollow fiber lumen did not dry out.

Claims

claims

1. assay, characterized in that the solution of a high molecular weight osmoticum in at least one of an ultrafilter membrane (5) adjacent space, the suction chamber (1), is introduced or flows through it and thereby a liquid phase containing at least one high molecular weight to be detected, from a filling space (2) is moved over a flow path (3) between the filling space and the measuring space, the volume of which makes up only a small part of the volume of the filling space, into a measuring space (4) separated from the suction space by the ultrafilter membrane, wherein the high molecular weight to be detected Concentrated substance in the measuring chamber and is detected there, while low molecular weight components flow through the membrane pores in the suction chamber.

2. An assay according to claim 1, characterized in that the high-molecular osmotic agent is selected from the group consisting of polyvinyl alcohol, polyethylene oxide, polystyrene sulfonate, poly (diallyl dimethyl ammonium chloride), dextran sulfate, e.g. Sodium dextran sulfate, or polyacrylate, e.g. Sodium polyacrylate, is selected.

3. An assay according to claim 1 or 2, characterized in that the detection of the high molecular weight substance in the measuring space by a direct optical interaction, detection of their radioactivity, magnetic or electrical properties o- done by a marker enzymatically or otherwise catalyzed chemical reaction ,

4. An assay according to any one of claims 1 to 3, characterized in that it is an affinity assay and the liquid phase, the reaction partners marked on the basis of a reaction of the analyte with affinity receptors or Contains reaction products in an analyte-dependent concentration, from a filling space (2) over a flow path (3) between the filling and the measuring space whose volume makes up only a small part of the volume of the Einfüllraumes, in a separated by the ultrafilter membrane from the suction chamber measuring space (4) moves is, with high molecular weight labeled reactants or reaction products are concentrated in the measuring chamber and detected there, while the low molecular weight components flow through the membrane pores in the suction chamber.

5. Affinity assay according to claim 4, characterized in that the affinity receptors comprise antibodies, antigens, lectins or aptamers.

6. Affinity assay according to claim 4 or 5, characterized in that the liquid phase contains high molecular weight unlabeled affinity receptors for the analyte and with this competing low molecular weight labeled analogues, of which a part whose size depends on the concentration of the analyte is bound to the receptors and is concentrated in the measuring room.

7. Affinity assay according to one of claims 4 to 6, characterized in that the filling space after the reaction contains immobile reaction partners or analogs of the analyte, in which a part of the labeled molecules is bound as a function of the concentration of the analyte, and these labeled components the flow into the measuring space are hindered.

8. affinity assay according to one of claims 4 to 7, characterized in that the concentrated in the measuring space reactants or reaction products contain fluorophores and the fluorescence yield by electrolytes, which together is added with the osmotically or hydraulically acting polymers in the suction, is reinforced.

9. affinity assay according to one of claims 4 to 7, characterized in that the concentrated in the measuring space reactants or reaction products have an enzyme activity, and enzymatically from a low molecular weight substrate which diffuses from the suction into the measuring space, an analytically well detectable colloidal product, e.g. a dye is formed.

10. affinity assay according to one of claims 4 to 7, characterized in that the concentrated in the measuring space reactants or reaction products contain gold nanoparticles or other catalysts for a precipitation reaction and the solution of high molecular weight osmoticum in the suction chamber silver ions and a reducing agent or other reactants for a Contain by the catalysts induced precipitation reaction.

11. A fluidic microsystem, in particular for carrying out affinity assays, characterized in that it contains at least one suction space (1) and at least one measuring space (4) separated from the suction space by an ultrafilter membrane (5), each measuring space being distributed over a flow path (3). whose volume makes up only a small part of the volume of the filling space, is connected to a filling space and the volume of the filling space exceeds the volume of the measuring space by a multiple.

12. The fluidic microsystem according to claim 11, having a planar ultrafilter membrane lying between two bodies, each measuring chamber being connected through a channel in the planar surface of the one body adjacent to the ultrafilter membrane. pers is formed and the suction chamber consists in a recess in the planar surface of the other body.

13. A fluidic microsystem according to claim 11 or 12, characterized in that the measuring space is applied to an X-ray film.

14. A fluidic microsystem according to claim 11, characterized in that the ultrafilter membrane is a Kapillarmembran and a segment of this Kapillarmembran pervades each suction and here separates a measuring chamber from the suction.

15. A fluidic microsystem according to any one of claims 11 to 14, characterized in that between the filling space and the measuring space a hydrophilic, for air in the water-saturated state is difficult to pass filter for fine particles is provided.

16. A fluidic microsystem according to any one of claims 11 to 15, characterized in that it comprises a plurality of measuring chambers.