DE10202713A1 - Detecting biological molecules uses fluorescently labeled and unlabeled antibodies, fluorescence being measured before and after applying electrical or magnetic field - Google Patents

Abstract

Detecting a target substance (A) in a sample comprises feeding a sample into a sensor suspension (10) containing a homogeneous mixture of fluorescently labeled antibodies and unlabeled carrier particles, to which unlabeled antibodies are bound. Fluorescence in a specific region (40) of the suspension is measured, a force applied and fluorescence measured again. A difference between the two measurements indicates the presence of (A). Detecting a target substance in a sample comprises feeding the sample into a sensor suspension (10) which contains a homogeneous mixture of fluorescently labeled antibody molecules and unlabeled carrier particles, to which unlabeled antibodies are bound. The fluorescence in a specific region (40) of the suspension is measured, after which force is applied to it and the fluorescence measured again. A difference between these two measurements indicates the presence of the target substance. An Independent claim is included for apparatus for carrying out the method which comprises a chamber (20) for sensor suspension, electrodes or magnets (30) which produce a field in the suspension and a fluorescence meter (50).

Description

The invention relates to methods for substance detection, in particular by detection of biomolecules, and devices for Implementation of such procedures.

Genetic engineering and pharmacology are different known biomolecular detection tests based on the detection of a Complex formation between an antibody and a corresponding antigen based. A classic immunoassay is for example the ELIZA test. In the ELIZA test, a Solid phase test format realized, in which an aggregation between Antibody and antigen (or: between receptor and substrate) and the marking of this aggregate on the surface of a Solid phase takes place. Before the detection of the bound to the solid phase Complex must first of all the unbound portion of the Antibody used for labeling are removed (see F. Lottsspeich et al. in "Bioanalytics, Spektrum Akademischer Verlag, Heidelberg, Berlin, 1998, page 92). Although the separation is made easier by the solid phase test format Disadvantage in that the ELIZA test is relatively complex and is cumbersome.

Test formats are increasingly being implemented in which one Complex formation between antigens and antibody-laden Particles occur that are movable in a suspension. Also at In this test format, disruptive components must be separated Form of fluorescence-labeled antibodies, but not to Antigens are bound to occur. This separation can can only be avoided if the association between Antibodies and antigen have the properties of the aggregate change that a distinction between the aggregate and its individual components is possible. For example, this will realized with the resonance energy transfer method. Two with different fluorescent dyes labeled antibodies are linked to a common antigen. By a The absorption spectra of both fluorescent dyes overlap there is an energy transfer between the dye molecules that as a wavelength shift in the fluorescence spectra can be registered. A disadvantage of resonance energy transfer However, the method is that both dyes in the antigen Antibody aggregate spatially located so close together must be that an effective energy transfer is possible. This condition is only met with relatively small antigens, since the so-called forester distance to characterize the Energy transfers at approx. 7 nm. By limiting it to small antigens and the often low quantum yields is the Applicability of the resonance energy transfer method limited.

Another known method which does not require the separation of the unbound fluorescence-labeled complements is fluorescence correlation spectroscopy (see, for example, BG Gradl et al. In "BioMethods", vol. 10, 1999, pages 331-351). With this method, the fluorescence is registered in a relatively small observation volume. The observation volume is selected so that the signal of individual molecules can be registered and analyzed. Its size can be estimated from the time behavior of a molecule detected in the observation volume and a distinction can thus be made between aggregates and unbound complements. This method enables extremely high sensitivity. However, it has the disadvantage that the observation volume is necessarily in the µm ³ range and the molecular density in the suspension must be kept low.

The object of the invention is to improve Provide detection methods that overcome the disadvantages of conventional biomolecular recognition tests are overcome and the in particular a high detection sensitivity and an extended Own scope. Methods according to the invention are intended also enable a simplified and fast test procedure. The object of the invention is also to provide detector devices Provide implementation of the procedures.

These tasks are accomplished by methods and devices with the Features solved according to claims 1 and 9. Advantageous embodiments of the invention result from the dependent claims.

The basic idea of the invention is to put a sample into a Introduce sensor suspension that is both fluorescently labeled Antibodies as well as freely suspended, unlabelled carrier particles contains that carry unlabeled antibodies, both the fluorescence-labeled as well as the unlabeled antibodies substance-specific binding sites for at least one target substance form that is to be detected in the sample. The proof the target substance is made by a fluorescence measurement it is recorded whether by linking the searched Target substance with the fluorescence-labeled antibodies and the unlabeled antibodies form fluorescence-labeled aggregates. These units accordingly contain at least one Carrier particle that carries unlabeled antibodies.

The fluorescence of the fluorescence-labeled aggregates is detected by exposure to the sensor suspension external forces that act selectively on the carrier particles, one one or more redistribution of the fluorescent aggregates in at least one detector area and accordingly a one or more locally selective fluorescence measurement in which takes place at least one detector area. The combination according to the invention of the formation of aggregates fluorescence-labeled antibodies, unlabeled antibodies and the Target substance (e.g. antigens) with the collection and detection of the Aggregates in certain detector areas will be advantageous an increased with significantly reduced technological effort Detection sensitivity of detection reached. The Detection sensitivity is related to the sensitivity of the Fluorescence correlation spectroscopy comparable, but without like this small observation volumes or low densities. Another important advantage is the time saved. In the method according to the invention, a separation of the unbound complements are waived. The fast one The procedure is particularly for biochemical and clinical tests significant. According to preferred embodiments Collection of the aggregates in the predetermined detector areas dielectrophoretic, optical or magnetic forces used. Advantageously, these forces are high Selectivity, so that only aggregates that have at least one contain dielectric particles in the detector areas are moved while fluorescent labeled antibodies that are not involved in aggregate formation, remain homogeneous remain distributed in the sensor suspension.

According to further advantageous embodiments of the invention provided that the locally selective fluorescence measurement in the Detector areas by using optical diaphragms or electronic spatial filter (image processing) takes place. Advantageously, with a device technology known per se (Shadowing, mask formation) a highly sensitive fluorescence measurement alternately in at least one homogeneous state in which the sensor suspension with the sample in a force-free state located, and realized in at least one inhomogeneous state be in which the carrier particles or fluorescent Aggregates under the action of the forces mentioned in the minimum a detector area is collected or displaced from it.

According to a particularly advantageous embodiment of the The detection method according to the invention is repeated Fluorescence measurement in the detector area, alternating one Collection or concentration of the aggregates in the detector area and redistribution or homogenization take place. The Redistribution is achieved by an active suspension movement (stirring) and / or a thermal redistribution is realized. To im the latter has the shortest possible measurement times at least one detector area, preferably in at least one Direction a transverse dimension in the sub-mm or µm range, so that after switching off the collecting forces through the aggregates thermal shocks quickly distributed the remaining sensor suspension become.

The method according to the invention advantageously delivers additional information from the kinetics of the fluorescence signal when switching between the different suspension states or measurement phases. If large aggregates form, the Collection and / or redistribution in the homogeneous state run more slowly than when smaller aggregates are formed.

A subject of the invention is also a device for Detection of substances using a suspension chamber said sensor suspension, a field generating device for the collection of aggregates in particular fluorescence-labeled and unlabeled antibodies, particles and at least one Target substance in at least one predetermined detector area the suspension chamber and a fluorescence detector device for locally selective fluorescence measurement in the at least one Detector area includes. The invention Detector device has the particular advantage of a simple structure Components that are inherent in fluidics and in particular fluidic microsystem technology are known. The Detector device can in particular be used as a fluidic microsystem be miniaturized.

According to preferred embodiments of the invention, the Field generating devices electrodes in the sensor chamber arranged with a predetermined geometric orientation are, or a magnetic device.

The invention has the following further advantages. The According to the invention, dielectrophoretic recognition tests are described in Size ranges of the target substances expanded, which so far have not were detectable. There can be very small target substance particles (e.g. macromolecules) in the aggregate dielectric be manipulated. The detection method is a new one Spectroscopy method, which is characterized by an improved signal-noise Ratio. The detection is not specific Target substances set. The results are extremely high Detection speeds in the seconds range.

Further advantages and details of the invention will be apparent from the Description of the accompanying drawings can be seen. Show it:

Fig. 1 and 2 are schematic illustrations of Ausführungsfomen of the inventive method, and

FIGS. 3 to 9 different embodiments of the inventive detector devices.

In the description, reference is made to the application of the invention biomolecular recognition tests referenced by way of example. These are aimed at the detection of biomolecules. Under Biomolecules are generally substances, compounds, Macromolecules or aggregates of biological origin or equivalent more complex materials, such as B. living or dead biological Cells, cell groups or cell components understood. The Biomolecules that are recorded as a target substance in a sample should include, for example, antigens. As signaling Antibodies are used, the substance-specific Form binding sites for the biomolecules. On the one hand labeled antibodies and on the other hand unlabeled antibodies used, the latter being bound to carrier particles. The Marking enables detection with a known one Fluorescence measurement. The carrier particles are general dielectric or magnetic particles synthetic or natural Origin, such as B. commercially available beads or living or dead cells. However, the invention is based on the detection of Biomolecules based on ligand-receptor systems, not limited, but generally for the detection of chemical Applicable substances that selectively target certain substrate molecules tie.

The operating principle of the method according to the invention is explained below with reference to FIGS. 1 and 2. Fig. 1 schematically illustrates a detector device 100 of the invention having a sensor chamber 20 which contains a suspension sensor 10, a field generating device 30, detecting regions 40 and a fluorescence detector 50.

The sensor suspension 10 is formed, for example, by a salt, electrolyte or nutrient solution. As signaling material, it contains fluorescence-labeled antibodies (e.g. antibodies against viruses), which are free and unbound in the sensor suspension, and carrier particles, which are also freely suspended and carry antibodies. In contrast to the free antibodies, the carrier particles and the antibodies coupled to them are unlabeled. When irradiated with excitation light, which triggers fluorescence emission from the fluorescence-labeled antibodies, they show no or negligible fluorescence. For example, beads with a diameter in the range from 20 nm to a few μm are used as carrier particles. The unlabeled antibodies are attached to the beads e.g. B. coupled with streptavidin. Several antibodies can be coupled to a carrier particle that are specifically binding for one or different target substances. Different groups of target particles with different antibodies can also be contained in the sensor suspension.

In order to carry out the detection method, a sample is added to the sensor suspension 10 which is to be tested in relation to one or more target substances. Alternatively, it is also possible for the sensor suspension itself to be formed by the sample and for this to be added the antibodies, some of which are fluorescence-labeled, unbound and some are unlabelled and bound to carrier particles. In a force-free or homogeneous state, the components of the sensor suspension are initially distributed uniformly.

If the sample contains one or more of the target substances sought, aggregation occurs between carrier-particle-coupled, unlabelled antibodies, the target substance and fluorescence-labeled antibodies. The aggregates form a stack or so-called sandwich shape. If the target substance is contained in the sensor suspension, fluorescence-labeled antibodies are bound to carrier particles via the target substance and the unlabeled antibodies. In the homogeneous state, when the fluorescent markers are excited, the sensor suspension delivers a homogeneous fluorescence signal which is distributed evenly over the entire sensor suspension and which is composed of a basic signal from still unbound, fluorescence-labeled antibodies and an aggregate signal from fluorescence-labeled antibodies which are bound to carrier particles via the formation of aggregates. Since the number of fluorescent molecules does not differ from the target substance-free state of the sensor suspension, it is not yet possible to determine from the fluorescence signal whether a target substance is in the sample or not. This becomes possible only when the sensor suspension is brought into an inhomogeneous state by the selective exertion of field forces on the carrier particles and their collection in or displacement from the detector areas 40 .

In general, the detection according to the invention is based on Presence of the aggregate formed on the detection a change in the at least one detector area outgoing fluorescence signal. Depending on the application, a if necessary, multiple evaluation of the increase in fluorescence or reduction and / or the time course of the fluorescence signal at the transition between the field-free (homogeneous) and the field-exposed (inhomogeneous) state of the sensor suspension.

In order to distinguish between the basic and aggregate signals, an electric field is applied to the field generating device 30 in the form of electrodes (see below), under the effect of which the carrier particles are pushed away from the electrodes due to their dielectric properties and into the detector regions 40 between the electrodes Electrodes are moved. The unbound fluorescence-labeled antibodies remain unaffected, they are not shifted. This is illustrated schematically exaggerated in the right part of FIG. 1. In the detector areas 40 there is an increased concentration of carrier particles relative to the previously homogeneous distribution in the field-free state (left partial image). If a target substance is contained in the sample, the number of fluorescent antibodies in the detector regions 40 is increased, since the bound, labeled antibodies also accumulate with the collected carrier particles. In this state, the contribution of the aggregate signal is higher than in the field-free state, while the basic signal remains the same. Overall, there is an increase in the fluorescence signal that is detectable. The principle explained can also be reversed by depleting or emptying the detector regions 40 of carrier particles under the action of the external field forces. In this case the fluorescence signal is reduced.

Fig. 2 shows the time dependence of the fluorescence F (t) illustrated (arbitrary units) in a detector region 40. At time t = 1, the sensor suspension is in a field-free, homogeneous state. The bound or unbound, fluorescent antibodies present in the detector region 40 deliver a fluorescence signal F1. At time t = 2, the field generating device 30 is actuated. The carrier particles collect in the detector regions 40 within an extremely short response time. If the aggregates mentioned are formed, the fluorescence increases to the value F2.

According to the invention, the target substance is detected in the sensor suspension 10 if the first and second fluorescence signals differ in the homogeneous and in the inhomogeneous state. The difference signal F2-F1 is characteristic of the amount of fluorescent antibodies coupled to carrier particles and thus of the concentration of the carrier substance sought in the sensor suspension.

Then the sensor suspension is inhomogeneous, the field effect is switched off until time t = 3. By There is a thermal fluctuation in the sensor suspension Redistribution to the homogeneous state. This process is ongoing slower than the aggregate collection with a certain Time characteristic up to time t = 4. The speed of the Fluorescence decay, which is described with a half-life τ can depend in particular on the size of the aggregates and of the detected target substance particles.

Depending on the application and task of the detector, the occurrence the change in fluorescence (threshold value comparison), the strength of the Fluorescence change F2 - F1 and / or the time constant of the Relaxation kinetics measured. This can be done with a one time Switch on the inhomogeneous state or preferably by a repeated switching between the homogeneous and the inhomogeneous state. This switching can be done with a relative high repetition frequency. The frequency depends on the Diffusion speed and therefore the size of the particles and is in the Hz range, for example. The detection of the Fluorescence signal F with a repetition frequency coordinated log-in amplifier enables a highly sensitive Detection of the target substance.

According to a modification of the method according to the invention groups with different sizes in the sensor suspension Carrier particles may be included. Different antibodies are attached to the carrier particles of different sizes coupled (unmarked) or freely dissolved in the suspension (fluorescence-labeled). When tested a sample with different target substances which bind to the various antibodies can Fluorescence detection for the individual target substances separately be performed. This will be done in repeated attempts initially relatively small field forces exerted on the smaller ones Carrier particles act and none on the larger carrier particles or have a negligible influence. Then the aggregate collection with stronger field forces repeated, which act on all carrier particles, and so on to detect larger carrier particles bound target substances.

Alternatively, it is also possible that the field forces are so weak that not a collection of carrier particles, but a collection or depletion of the freely released, fluorescence-labeled antibodies in the detector areas.

The redistribution of the carrier particles or the antibodies in Transition between the homogeneous and the inhomogeneous state can with different physical or chemical Field forces are effected as below with reference to Embodiments of detector devices according to the invention explained becomes.

In Fig. 3 components of the detector apparatus 1, 100 of FIG. In a schematic perspective view shown. The suspension chamber 20 is formed by a gap 21 which is delimited by plate-shaped bottom and top surfaces 22 , 23 . In the other spatial directions, plate-shaped walls with an inlet and / or outlet are also provided as boundaries, which are not shown for reasons of clarity. Furthermore, spacers with which the gap width is set can also be provided between the bottom and top surfaces 22 , 23 . The bottom and top surfaces 22 , 23 consist, for example, of plastic, glass or semiconductor material. At least the top surface 23 is transparent.

Strip-shaped electrodes 31 are arranged on the inner sides of the bottom and top surfaces 22 , 23 and are connected to a high-frequency generator (not shown). The electrodes 31 generally consist of a conductive, inert material. z. B. platinum or gold. They are arranged opposite one another, one above the other, so that respectively aligned strips 41 remain on the bottom and top surfaces 22 , 23 , which form the boundaries of the detector regions 40 .

The fluorescence detector is formed, for example, by a fluorescence incident light microscope 51 , which is arranged above the transparent cover surface 23 . The fluorescence incident light microscope 51 is set up to excite fluorescence and to measure the emitted fluorescence in the detector regions 40 (see double arrow). The upper electrodes 31 form a masking device 60 , with which the partial areas of the suspension chamber 20 between the opposing electrodes are shaded and the detector areas 40 between laterally adjacent electrodes are left free.

The width of the gap between the bottom and top surfaces 22 , 23 is selected depending on the size of the particles used and corresponds, for example, to 5 to 10 times the particle diameter. The extent of the bottom and top surfaces 22 , 23 is, for example, in the cm to mm range. The width and arrangement of the electrode strips is selected depending on the application. The width and spacing are, for example, 100 µm.

The setting of a suitable width of the detector areas 40 represents an important feature of the invention. In order to obtain a sufficiently rapid redistribution of the carrier particles into the homogeneous state after the field effect has been switched off, the width of the detector areas 40 must be sufficiently small. For a redistribution within approximately 1 min, the width of the detector regions 40 is for carrier particles with a diffusion constant of approx. D = 10 ^-12 m ² / s in the µm range.

The suspension chamber 20 is charged with one of the methods known per se from fluidics by flushing in the sensor suspension or by sucking into the gap 21 under the action of capillary forces. The sample to be examined can already be contained in the sensor suspension before loading the sensor chamber 20 or can be introduced separately into the sensor chamber 20 . The bottom and top surfaces 22 , 23 are formed, for example, by glass plates with the shape of cover glasses, as are used in microscopy.

FIGS. 4 and 5 show modified embodiments of the detector device of the invention. The differences relate to the geometry of the beam path and the design of the masking device. According to FIG. 4 20 electrodes 31 are provided only on the bottom surface 22 of the suspension chamber. The electrodes 31 are subjected to high-frequency electrical fields in such a way that the concentration ratios between free and bound fluorescent antibodies in the suspension chamber 20 temporarily shift. The top surface 23 is made of a transparent material. The masking device 40 (illustrated schematically) is arranged in the beam path of the incident light microscope 51, for example above the objective 52 . The masking device is preferably arranged as a diaphragm at the location of the field diaphragm in front of the photosensitive element 53 (eg photomultiplier). The size of the diaphragm structures of the masking device 40 are selected such that the image structures left free in the image of the suspension chamber 20 have a size of a few μm corresponding to the detection areas 40 . The embodiment according to FIG. 5 is characterized by a laterally arranged light source 54 . The fluorescent light emitted in the detector regions 40 is detected with the light-sensitive element 53 .

The suspension chamber 20 of a detector device according to the invention can alternatively also be formed as a flow chamber, as is illustrated in a schematic plan view in FIG. 6. In the upper part of FIG. 6, the bottom surface 22 is shown with a meandering channel structure 24 , which forms the suspension chamber and through which the sensor suspension flows during the detection (see dashed line). An arrangement of two comb-shaped electrodes 32 , 33 is further provided on the bottom surface 22 , which laterally engage as an electrode finger in the meandering channel structure 24 .

Depending on the operating mode of the detector device and the composition of the sensor suspension, the electrodes 33 , 34 are subjected to a high-frequency AC voltage or a DC voltage. The high-frequency AC voltage is used for the dielectrophoretic shift of the concentration ratios in the suspension chamber, in particular across the flow direction. Analogously, with an anode and a cathode, particles with, for example, pH-dependent net charge, flow direction can be accelerated or slowed down and thus collected in certain partial areas.

The top surface 23 of the sensor chamber 20 illustrated in the lower part of FIG. 6 is at the same time a masking device 60 . It contains diaphragms 61 , which darken predetermined partial areas in particular in the longer, lateral sections of the meandering structure and leave adjacent partial areas 62 free. Under the effect of the electric fields, the originally homogeneously distributed components of the sensor suspension are deflected and transferred electrophoretically into the darkened or uncovered areas. The fluorescence measurement is carried out according to the principles mentioned above.

A further design of the sensor chamber 20 of a detector device according to the invention is illustrated in FIG. 7. For reasons of clarity, only the structured bottom surface 22 of the sensor chamber 20 is shown. The bottom surface 22 has depressions or openings 25 which are directed vertically into the depth. Trenches 26 , in which electrodes (not shown) are arranged, run on the underside of the bottom surface 22 . The trenches 26 can be connected to the openings 25 .

To the sides, the suspension chamber is delimited by walls (not shown), of which at least one wall is transparent for measuring fluorescence. The excitation and measurement of the fluorescence takes place with a horizontal beam path 55 , ie parallel to the flat bottom surface 22 . The suspension chamber can be open at the top.

To carry out the method according to the invention, a sample, such as. B. brought a drop of the solution to be tested into the suspension chamber 20 above the bottom surface 22 . For example, in the direction of arrow 55 , the sensor suspension is illuminated with the sample. A lateral fluorescence measurement is also carried out accordingly. For the transition from the homogeneous to the inhomogeneous state of the sensor suspension, the electrodes in the trenches 26 are subjected to voltages, so that the carrier particles are transferred into the depressions or openings 25 . The fluorescent antibodies, which are associated with carrier particles in the presence of the target substance in the sample, are removed from the fluorescence excitation. The fluorescence is reduced under the effect of the field forces. After switching off the field forces, the thermal return to the homogeneous state takes place. The number of aggregates formed can be determined from the difference between the fluorescence signals. In this embodiment, the function of a masking device is fulfilled by the shape of the bottom surface 22 .

The redistribution of the carrier particles between hidden and non-hidden partial areas under the effect of magnetic field forces is illustrated in FIG. 8. The bottom surface 22 of the suspension chamber 20 is shown with channel-shaped depressions 27 which, analogous to the principle illustrated in FIG. 7, form a shadow region for lateral fluorescence excitation 55 . On the underside of the bottom surface 22 is a switchable magnetic device 35 , for. B. a coil provided.

With this design, the sensor suspension contains magnetic particles (magnetic beads) as carrier particles. When the magnetic device 35 is actuated, the carrier particles with or without attached fluorescent antibodies are drawn into the channels 27. There is no fluorescence excitation in them, so that the fluorescence signal is correspondingly reduced when the aforementioned aggregates are formed. The fluorescence measurement can be carried out vertically above the bottom surface 22 using a fluorescence incident light microscope 51 . A separate masking device is not necessary with this design.

The channels typically have a depth of a few μm so that the magnetic carrier particles can diffuse back sufficiently quickly after the magnetic field has been switched off. A stirring device 70 is provided on the underside of the sensor chamber to accelerate the transition into the homogeneous phase. The stirring device comprises, for example, an ultrasound element in the form of a vibrating platform. When the ultrasound element is actuated, the suspension is swirled and the carrier particles are transferred from the channels 27 into the rest of the suspension chamber.

Another example with a flow sensor chamber is shown in FIG. 9. The bottom surface 22 of the sensor chamber contains at least one channel 28 , one half 28 a with the sensor suspension, which in particular contains carrier particles and that contains the sample, and the other half 28 b is flowed through by a reference solution that contains no antibodies and no sample. With the aid of the electrodes 36 , carrier particles are transferred into the reference flow, in which the fluorescence is measured locally selectively. For this purpose, a masking device in the form of an aperture (not shown) is provided.

The invention can be implemented with the following modifications become. It is possible to transfer the sensor suspension from inhomogeneous to the homogeneous state using the Support field generation device, for example by Field effects are reversed. Furthermore, it is possible instead of an optical masking device, an electronic one To provide masking device. If as a photosensitive Element used for the fluorescence measurement a CCD camera electronic masking of certain Cutouts of the CCD element according to the illustrated Detector areas take place. Alternatively to the electrical or magnetic field effects can also be hydrodynamic, optical or chemical forces work. According to one particularly advantageous embodiment of the invention are the temporary Concentration shifts after a cooling step in the frozen Condition of the sensor suspension examined.

The invention can be used as an alternative to fluorescence measurement for a other measurement method can be designed, then antibodies are used to capture with each Measurement methods adapted, in particular marked.

The in the above description, the drawings and the Features of the invention disclosed in claims can be both individually as well as in any combination for the realization of the invention in its various configurations from Be meaningful.

Claims (16)

1. A method for the detection of at least one target substance sought, which is contained in a sample, with the steps: Introduction of the sample into a sensor suspension ( 10 ) which, in a force-free, homogeneous state, contains fluorescence-labeled antibody molecules and unlabeled, freely suspended carrier particles which carry unlabeled antibody molecules, the labeled and unlabeled antibody molecules having substance-specific binding sites for the target substance, - Fluorescence measurement in the homogeneous state for detecting a first fluorescence signal (F1) of the sensor suspension in at least one predetermined detector area ( 40 ), - exertion of forces, under the effect of which the sensor suspension ( 10 ) is converted into an inhomogeneous state in which the concentration of the carrier particles in the detector area ( 40 ) is changed, - Fluorescence measurement in the inhomogeneous state to detect a second fluorescence signal (F2) of the sensor suspension ( 10 ), and - Detection of the target substance in the sensor suspension ( 10 ) if the first and second fluorescence signals differ in the homogeneous and in the inhomogeneous state (F1, F2). 2. The method according to claim 1, wherein the carrier particles are dielectric or magnetic particles and for Change in the concentration of the carrier particles electrical, optical or magnetic forces are exerted. 3. The method according to claim 1 or 2, wherein the Fluorescence measurements in the homogeneous and inhomogeneous state alternately with a predetermined repetition frequency several times be performed. 4. The method according to claim 3, wherein a frequency sensitive Fluorescence measurement takes place, the frequency of which on the Repetition frequency of the fluorescence measurements is matched. 5. The method according to any one of the preceding claims, at in the case of the occurrence of the target substance, the time course the fluorescence signal between the fluorescence signals (F1, F2) at the transition between the homogeneous and the inhomogeneous Condition is recorded. 6. The method according to any one of the preceding claims, at different types of Carrier particles are contained, which can be selected by different or field forces of different strengths are influenced. 7. The method according to any one of the preceding claims, in which a masking device ( 60 ) is used for fluorescence measurement, which comprises an optical diaphragm or an electronic spatial filter. 8. The method according to any one of the preceding claims, at that in the transition from the inhomogeneous to the homogeneous state Sensor suspension is mixed to the carrier particles to distribute evenly. 9. Detector device ( 100 ), in particular for performing a method according to one of the preceding claims, with a suspension chamber ( 20 ) for receiving a sensor suspension ( 10 ), a field generating device ( 30 ) for enriching or depleting carrier particles in at least one predetermined detector area ( 40 ) of the suspension chamber ( 20 ) and a fluorescence detector device ( 50 ) for locally selective fluorescence measurement in the at least one detector area ( 40 ). 10. A detector device according to claim 9, wherein the field generating device ( 30 ) comprises electrodes ( 31 , 32 , 33 ) or a magnetic device ( 35 ). 11. Detector device according to claim 9 or 10, in which a masking device ( 60 ) is provided which interacts with the fluorescence detector device ( 50 ) for locally selective fluorescence measurement. 12. A detector device according to claim 11, wherein the masking device ( 60 ) is formed by the electrodes of the field generating device ( 30 ). 13. Detector device according to claim 12, wherein the masking device is formed by an aperture in the fluorescence detector device ( 50 ). 14. Detector device according to claim 13, in which the masking device ( 60 ) is formed by an electronic spatial filter which interacts with the fluorescence detector device ( 50 ). 15. Detector device according to claim 9, wherein the suspension chamber forms a flow chamber with at least one channel ( 28 ), the at least one detector area ( 40 ) being provided in partial areas of the channel. 16. Detector device according to claim 9, wherein the suspension chamber ( 20 ) has a bottom surface ( 22 ) with a structure that fulfills the function of the masking device.