WO2000034785A1 - Method and device for carrying out quantitative fluorescent marked affinity tests - Google Patents

Method and device for carrying out quantitative fluorescent marked affinity tests Download PDF

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Publication number
WO2000034785A1
WO2000034785A1 PCT/DE1999/003917 DE9903917W WO0034785A1 WO 2000034785 A1 WO2000034785 A1 WO 2000034785A1 DE 9903917 W DE9903917 W DE 9903917W WO 0034785 A1 WO0034785 A1 WO 0034785A1
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WO
WIPO (PCT)
Prior art keywords
characterized
sample
measuring container
measuring
bottom plate
Prior art date
Application number
PCT/DE1999/003917
Other languages
German (de)
French (fr)
Inventor
Andreas Katerkamp
Markus Meusel
Frank Grawe
Angela Zellmer
Karsten Schult
Dieter Trau
Original Assignee
Institut für Chemo- und Biosensorik Münster E.V.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority to DE19856041.9 priority Critical
Priority to DE1998156041 priority patent/DE19856041A1/en
Application filed by Institut für Chemo- und Biosensorik Münster E.V. filed Critical Institut für Chemo- und Biosensorik Münster E.V.
Publication of WO2000034785A1 publication Critical patent/WO2000034785A1/en

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    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00Investigating or analysing materials by the use of optical means, i.e. using infra-red, visible or ultra-violet light
    • G01N21/62Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64Fluorescence; Phosphorescence
    • G01N21/645Specially adapted constructive features of fluorimeters
    • G01N21/648Specially adapted constructive features of fluorimeters using evanescent coupling or surface plasmon coupling for the excitation of fluorescence
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00Investigating or analysing materials by the use of optical means, i.e. using infra-red, visible or ultra-violet light
    • G01N21/62Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64Fluorescence; Phosphorescence
    • G01N21/6408Fluorescence; Phosphorescence with measurement of decay time, time resolved fluorescence
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00Investigating or analysing materials by the use of optical means, i.e. using infra-red, visible or ultra-violet light
    • G01N21/62Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64Fluorescence; Phosphorescence
    • G01N21/6428Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"

Abstract

The invention relates to a method and device for carrying out quantitative fluorescent marked affinity tests. According to the invention, a fluorescence is provoked by means of an optical evanescent field excitation with a marking substance bound to a chemical or biochemical partner of a general receptor-ligand system. One of the partners of the receptor-ligand system is immobilized on an optically transparent base plate used as a measuring area of a measuring container (2). The intensity of the fluorescent light is measured. According to the invention, the reaction rate should be increased by influencing the transport of substances to the surface, the measuring speed should be decreased, and the measuring precision and sensitivity should be increased. To these ends, a sample which is mixed with a chemical or biochemical substance that is fluorogen-marked according to biochemical assay is poured into the measuring container (2), is subsequently mixed in a circular flowing movement in said measuring container (2), and the intensity of the fluorescent light is measured in a temporally resolved manner according to the affinity test format in order to determine the concentration of at least one chemical or biochemical substance.

Description

Method and apparatus for carrying out quantitative fluorescence-labeled affinity tests

The invention relates to a method and an apparatus for performing quantitative fluorescence labeled affinity tests according to the preamble of claim 1.

Basically, the invention can be used for all ligand-Re Zeptor systems, such as protein-protein interactions, antigen-antibody binding, nucleic acid Hybridis- as chosen protein-DNA interactions, inter alia, be employed.

In making such biochemical affinity tests may be used alone or in parallel run simultaneously different reactions of the reactants contained in each sample or to be used. On the one hand can take place, depends on the reaction rate, among other things on the concentration of the reactants used and the exploitable energy for activating a homogeneous reaction. For example, cause an increase in temperature to increase the reaction rate for a homogeneous reaction.

In parallel, but can take place, which at the other reaction partner with respect to the reaction rate in addition to the activation energy also by mass transfer of the free reaction partner in the sample to the surface immobilized is influenced also a heterogeneous reaction. It follows that at a constant concentration and constant temperature, the reaction rate of the heterogeneous reactions is relevant from the mass transport of the free reactants to the surface on which the respective other partner is immobilized dependent.

The biochemical immune sensors or the appropriate tests are usually based on heterogeneous reactions, so that a targeted influence is made to the above-mentioned mass transfer to the surface, in particular to increase the reaction rate, and consequently to reduce the required measurement time is desired.

In performing such tests the known fact that the measurement of immune complexes formed per unit of time on the surface, such as a measuring cell, a highly sensitive and prone with low error rate measuring method is exploited is.

It is further known that the respective mass transfer to the surface on which the respective other partner of such a complex is immobilized, should take place by a relatively quick process and not by the slow process of diffusion in the implementation of such immunoassays. Under these conditions, namely, corresponding to the rate of immune complexes formed on the surface of a measurement cell, directly to the concentration of the binding partner in the sample volume.

Based on this finding, 196 28 002 Cl a corresponding method and a device suitable for this described in DE, in which a linear flow system is used. In this case the mass transfer is carried out to the surface, whichever is not fusion limited DIF, due to a küvettenförmi- gen receiving area is formed similar to a flow channel and has a small height (50 microns) through which the sample at a flow rate of about 1 s is guided ul /. A larger flow would be desirable, but can not be implemented because you can only work with small sample volumes of about 100 ul, with a measurement time of about 100 s should be achieved.

For the flow of homogeneous reactions, a sample container, according to the solution described there can be used which is separated from the actual measuring site, the so-called cuvette receiving portion arranged reasonable.

it is Proceeding from this, the object of the invention to propose a method and a device in which increased by influencing the mass transfer to the surface, the reaction rate and thus shorten the measuring speed and the measuring accuracy and sensitivity can be increased.

According to the invention this object is achieved with the features of claim 1 for a method and the features of claim 14 for a corresponding device. Advantageous embodiments and developments of the invention will become apparent through use, as stated in the subordinate claims.

According to the invention this case, based on the known optical measurement method, so proceeded that a Evaneszenzfeldanregung is carried out by optical means ways to the intensity of fluorescence to measure the fluorescence generated by quasi-monochromatic light from a light source to a marking substance. In this case, such fluorescence is hervorrufen- the marker substance coupled to a chemical or biochemical partner of a general receptor-ligand system of this receptor-ligand can bind system, at least one of the partner during the execution of such a test on affinity, verwen- det. Before performing such tests at least one of the partners is immobilized on a surface of a measuring container and then, as already mentioned in the introduction, triggered a heterogeneous reaction with fast material transport to the surface.

For carrying out a method according to the invention a measuring container is, the bottom plate consists of a transparent for the excitation light and the fluorescent light material is used, wherein the excitation light is directed at an angle to the transparent base plate, wherein the total reflection at the interface (bottom plate / sample) occurs and thus an evanescent field with a defined penetration depth above the surface of the bottom plate can be produced.

The intensity of the generated fluorescence light can then be measured with a suitable light-sensitive detector, which is arranged below the base plate of such a measuring container.

In the invention, it is important that a labeled with a fluorophore chemical depending on the particular be carried out biochemical assays - or biochemical substance-offset sample is introduced into a metering vessel and after the filling of the measuring container, the sample is placed in the measuring container in a circular flowing motion, so that the reaction rate of the heterogeneous reaction can be accelerated accordingly. For determining the concentration of at least one of chemical or biochemical substances, the increase or the decrease of the intensity of Fluoreszenzlich- tes is then measured.

In order to secure quasi-static conditions during the measurement, the sample should be uniform, that is, with a constant speed and an acceleration equal to zero, at least during the measurement, are set in circular motion.

It is also possible to determine the concentration of the difference in fluorescence intensity at two different points in time.

In carrying out a method according to the invention, it is expedient to wait for the expiration of homogeneous reactions in the sample after the insertion of the respective sample in the measurement container, before the sample is placed in circular flowing motion. This can be time taken into account for the various feasible assay formats easily, since the time required therefor, as likewise mentioned above, only on the concentration of the various reactants and the available energy, that is essentially dependent on the temperature, can be determined. For safety reasons it is also expedient, the actual measurement container until at least one opening, which serves to introduce the sample to form closed on all sides, and this fact in the formation of elements that are to be found to achieve the circular flowing motion of the sample using, must be considered.

Here, at least the drive that this should be comparable turns are arranged outside the actual measuring container. Since the optical detector is to be used for measuring the intensity of the fluorescent light is disposed below the base plate of the measuring container, it is safe expedient, the corresponding rotary drive to the

on the opposite side of the base plate to arrange measuring container.

In a suitable apparatus for performing the method according to the invention, a drive may be used, are used in the electromagnets, permanent magnets or an electromagnetic coil assembly in order to enable the sample, the magnetic bodies are added by utilizing magnetic and electromagnetic forces in circular flowing motion , At least one permanent magnet or an electromagnet stationary arranged for example on a rotor that is rotatably arranged above the measuring container may be attached. Upon rotation of this rotor then magnetic or electromagnetic forces act on the magnetic body contained in the sample in the measuring vessel and the sample is mixed according to the rotational motion in flowing movement. Instead of such a rotor but also a stator can be used, on which an electromagnetic coil assembly is present, the individual spaced-separated coils influenced sequentially by an electronic control, switched on and off, so that almost the same effect can be achieved.

There is also the possibility that container in the measuring range recorded sample to enable mechanical means to a desired circular flowing motion. In this case, for example, can be taken so that the measuring container is closed at the top by an elastic membrane. This membrane can be placed a rotating element, are formed on the projections acting on the membrane, which press the elastic membrane and so reach into the actual sample. so it can upon rotation of the rotating element, a stirring the product be achieved by mechanical means and the measuring container used be kept closed at the same time.

Similar procedure can also be as shovel-shaped elements are present in eigentli- chen measuring container, which are secured to one run through the cover of the measuring container shaft to which a rotational drive can be applied when the sample is to be placed in the desired motion. With an appropriate seal is ensured here that an escape of sample liquid can be prevented.

The actual measurement can be advantageously carried so results in only the increase or the decrease waste (negative slope) of the measured intensity of the measured fluorescent light therefrom and because of the direct proportionality, the concentration of each to be determined chemical or biochemical substance see is determined. In the measurement in this form, it must be performed only within a, each chemical- or biochemical assay format in accordance with predetermined time interval. Thus, the required measurement time can be shortened accordingly.

There is also the possibility to take advantage of only at least two fluorescence intensity signals in a predeterminable time interval from one another and after Dif erenzbildung close to the respective concentration of the corresponding substance.

but the determination of the fluorescence intensity can be measured not only time but also spatially resolved. This has a particularly advantageous when at least one of the partners of the respective receptor-ligand system is immobilized in a radially symmetrical arrangement on the bottom plate of the measuring container. This may be due shall return yet in the description of the embodiments below, find a movable shutter in conjunction with a light-sensitive detector or a linear or planar array of light sensitive detectors, such as CCD rows or CCD-Ar- rays, use. is particularly low, this embodiment, when the bottom plate of the measuring container is radially symmetrical divided into different segments and in the various segments of such different partners are immobilized, so that the concentration of multiple chemical substantially simultaneously - or biochemical substances can be determined.

The filling of the measuring container with the sample can preferably be achieved in various ways, for example by capillary force, by pressure or suction force from the side of the measuring vessel, wherein said combinations of these effects can be utilized.

In many cases it may be advantageous to guide the sample prior to the filling of the measuring container by functional layers or corresponding membranes to reach at the sample, a separation or filtration.

However, it may be carried out taking into account the respective biochemical assays with such layers or membranes and the release of chemical - be achieved or biochemical substances. In other use such layers or membranes may also be for a specific retention of chemical - be exploited or biochemical substances from the sample.

for example, such a membrane may be made of fibrous material, cellulose, nitrocellulose, polypropylene, polycarbosilane nat, polyvinyl difluoride or are made of a hydrogel or of nuclear track or glass fiber membranes. Such membranes are used for example in the dip stick technology and can, for example, the

fulfill function of Conjugate release. The liquid transport of the sample through such a membrane is carried out mainly in translation with capillary force. parts for the separation of cellular blood components and / or for the production of blood plasma can, for example, a membrane which is commercially available under the name plasma SEP used.

Is a device according to the invention under Verwen- fertil such layers or membranes formed as the respectively desired sample pre-treatment can be carried out directly in the apparatus, so that additional work can be avoided, and such a device the final user, which is easy to handle, available are provided.

It is further advantageous to reproach the sample in a sample receiving chamber, which is connected either directly with the measuring container or by at least one functional elle layer or membrane with the measuring container so that the sample from the sample receiving chamber if required in the measuring container, optionally at performing a corresponding sample preparation can pass.

The measuring container may be additionally connected with a sample collection chamber in which an absorbent material is advantageously included, so that the process can take place during the filling benflüssigkeitstransport readily prepared by capillary force. With a known capacity of the absorbent material in the sample collection chamber and a known volume of sample in the sample receiving chamber a defined filling with a desired volume of sample into the actual measuring container can be achieved because the liquid transport, on reaching the Saugkapazitätsgren- ze of the absorbent material ends.

there are many possibilities for the implementation of the various biochemical assays. In the simplest case, the sample (whole blood, plasma or serum, water, urine or other) is treated with a fluorophore labeled reactant. The corresponding corresponding partner is immobilized on the surface of the base plate trained Meßareal. For carrying out, for example, a conventional sandwich Immunoassys for the determination of high molecular weight compounds, the sample, for example, an appropriately labeled detector antibodies is added and immobilized on the surface of the bottom plate on the Meßareal capture antibody provides for binding of each analyte and the detector antibody.

The inventive device can be manufactured considerably simpler and cheaper because of the lower limit of the exploitable volume within the measuring container, compared with that described in DE 196 28 002 C2 flow cell, since a height limit that was required for the convective mass transport in the conventional flow cell is eliminated.

The measuring container should advantageously have a height in the range fillable comprise 0.001 to 50 mm in its interior.

The invention will be explained in more detail with reference to embodiments.

They show:

1 shows flow profiles along two sections in mass transport through a conventional flow cell; 2 shows flow profiles along two sections which can be achieved in the material transport in an inventive device;

Figure 3 schematically illustrates the structure of an example of an inventive device with a rotating member for generating a circular flowing motion of a sample;

4 shows a further embodiment of a device according to the invention with a rotating member with the permanent magnet and the magnetic bodies in the measuring vessel, to produce a circular-shaped flowing movement of the sample;

5 shows another example of a erfindungsgemä- SEN device having a rotating member shaft passage and blades in the measuring vessel, to produce a circular flowing motion of the sample;

6 shows a radially symmetric in various

Segments divided measuring container;

7 shows a construction of a measuring arrangement with the optical elements shown schematically;

Figure 8 shows an example of an inventive apparatus with integrated Probenaufnähme- and sample collection chamber;

9 shows the example shown in Figure 8 with additionally used within the sample collection chamber absorbent material;

Figure 10 is a schematic representation of a possible arrangement of the layers or membranes of a measuring container and a sample collection chamber, wherein the liquid transport a sample for filling using cable pillarkraftwirkung is reachable and

11 shows an example in which a measuring container according to the invention to be used from a functional layer or a membrane is completely enclosed.

In the figure, 1 is a receiving area küvettenförmiger a flow cell, as is known from DE 196 28 002 Cl shown. The mass transfer of the sample liquid takes place translationally through the channel, with quasi-rectangular cross-section, as is indicated by the arrows in the upper diagram. The airfoils correspondingly occurring the indicated sections are shown in the lower left and right images, which is clearly seen that the flow velocity of the sample liquid in the center of the Durchflußmeßkanales (section CD) is the largest, so that precisely in the area in which the a partner of the receptor-ligand system, which is immobilized on the floor of the flow channel to which to connect the other partner, the flow velocity and consequently also the mass transfer is relatively small.

In contrast, can as payments from the representation seen in Figure 2, extends ER- better results when the sample is put in a measuring container 2 in circular flowing motion, with the measurement container 2 advantageously a circular bottom plate 3 on which the corresponding Meßareal is formed having. The circular design of the bottom plate 3 in conjunction with the outer surface of the measuring container 2 causes turbulence can occur only slightly.

In particular, in the lower right hand illustration of Figure 2 is illustrated by the shown flow profile of a circular flowing in a circular measuring container sample, that the sample is moved quasi nit in the center. The tangential flow velocity to achieve a maximum increase in the circular motion in the radially outer direction and then in the direction of the outer edge of the measurement container soft to at least close to zero drops.

The measurement of the fluorescent light, should consequently, as related below. Figure 6 further explains keitsmaximums locally limited in the region of Strömungsgeschwindig-, gene successes of the circularly moving sample.

In the figure 3 a further possibility for the generation of the circular flowing motion of the sample in the measurement container 2 is shown. In this example, magnetic body can be dispensed in the sample to the addition since it is a purely mechanical system. The measuring container 2 is finished in this example above with a resilient flexible membrane. 11 In this membrane 11 can be fitted a circular rotating member 5 to which a plurality of projections 10 are formed, so that the projections 10 and pushing the membrane 11 immerse ing into the Probenflüs-. Is now the rotating element 5 is displaced with a suitable Dreahntrieb in rotation, the desired circular flowing motion can be achieved in the sample in the measuring vessel 2, and it is thereby assured anyway, that the measuring container 2 remains closed.

In the example shown in the Figure 4 example, a measuring container 2 is used again, which is also closed at the bottom with a transparent base plate 3, will return to this later more precisely on, and upwards by a cover. 6 In the measuring container 2 are magnetic body 9, with which the sample can be added after the filling or during the filling contained. The magnetic body may also be located before filling the measuring container. 2 The magnetic bodies 9 are advantageously relatively small in size, and consequently influence the measuring result, if at all, only very slightly. Over the measurement container 2 is a rotating member 5 can be rotated by a not shown rotary drive parallel to the surface of the measuring container 2 exist. In this example, a plurality of permanent magnets 4 are mounted in the same distance from the axis of rotation of the rotating element 5 to the rotating member 5, the magnetic force action is sufficient to transmit the rotary motion of the rotary element 5 to the information contained in the sample magnetic body 9 and consequently also to the test so that it is placed in a circular shape flowing movement. Instead of the permanent magnets 4 but also appropriate electromagnets may be used, for example, can be supplied via flexible leads or sliding contacts with electric energy.

In the figure 5 a further example of an inventive device with a measuring container 2 is shown. Here, in the upward closed with the cover 6 measuring container 2, a mechanical agitation is element 15 was added, in which a shaft is guided through a not shown port sealed in the cover 6 to the outside, to the example, a rotating member 5 or can attack other suitable rotary actuator. The rotating element 5 and the above-mentioned rotational drive can then be arranged outside the measuring container. 2

Shaft and rotating member may constitute integral, which is sealed to the cover of the measuring vessel 2 also an input.

The mechanical stirring member 15 should be arranged so inside the measurement container 2 is fixed kön- NEN, that deterioration of the immobilized on the bottom plate 3 Partners upon rotation of the mechanical stirring member 15 can be avoided.

However, the mechanical stirring means 15 can also consist of a ferromagnetic material, and such as the aforementioned magnetic bodies to be displaced in rotation, on the one hand there is the possibility that the rotating element 5 stationary permanent magnets or electromagnets are present. If now the rotating element 5 in rotation, causing the electromagnetic or magnetic field of the magnets, that the mechanical stirring member 15 also rotate.

but it can also be used in place of the rotating element 5 is a rotating electromagnetic field generating coil arrangement, to cause the rotation of the mechanical stirring member 15th

The rotational movement of the mechanical stirring member 15 achieved by electromagnetic or magnetic force, the whole mechanical stirring member 15 may be within the measuring vessel 2, and therefore un- terhalb the top cover 6, through which a shaft is guided to the outside which consequently no opening, must have to be completed. a bearing for the mechanical stirring element 15 it is only necessary, which can be formed preferably in the center of the circular-shaped bottom plate 3 as well as in the center of the cover 6, for example in the form of a needle bearing.

From Figure 6 is a possible variant of the invention in which is carried a radially symmetrical distribution of Meßareals 21 on the bottom plate 3 in individual measuring points 22 is illustrated. The individual measuring points 22 for the immobilization of various substances can be used so that in conjunction with the time and location-resolved measurement of the fluorescence intensities of a plurality of different substances may also be considered simultaneously. In the center of the observed from above the measuring container 2, a circular region is shown, are arranged in the magnetically diagram body, which is added at the filling with the sample and with which the circular flowing motion of the sample in the measurement container 2, as in the example of Figure 3, can be achieved.

It should in general, as mentioned above, are always measured in areas that are at least in the vicinity of the flow rate maximum.

The maximum is, as illustrated in Figure 6, on the radius R ^, around the center of the measuring container. 2

The center points of the measurement points 22 should therefore also be located on the radius R m. The measuring range is determined by the outer edges of the measuring points 22, the diameter of which from the difference Ra - limited results R 1,.

If the size of the measuring points 22 still be too large to meet the conditions of the highest possible flow rate at the same time, almost the same flow rates, it is simplest to select the image of the fluorescent light on the optical detectors accordingly so that only fluorescent light from an area which satisfies these conditions may be incident on an optical detector or only corresponding light components are taken into account. For the first alternative may be used most simply optical diaphragms, with appropriate openings, corresponding to the moved or may be rotatable. In the second alternative selectively only individual detectors of a detector array can be exploited or taken into account, which for the incident fluorescent light of these conditions are met. It should at flow rates at the maximum of the airfoil (see Figure 2) in the range from 5 to 1000 mm / s and worked measured in regions of laminar flow.

In the figure 7, a measurement setup is shown schematically. In this, a beam 1 of a monochromatic light emitting light source 23, for example, may be a laser light source with a known wavelength on the prismatic shaped region of the bottom plate 3 of a measuring container 2, in a at the interface (bottom plate 3 / sample) total reflection-causing angle directed , Above the interface, that is on the surface of the bottom plate 3 of a partner of a receptor-ligand system is immobilized least zumin- at which the respective other fluorescently labeled partner can bind.

Depending on the number of the connected partner of the re Zeptor ligand system, the fluorescence intensity which is excited with the laser light beam 1 changes.

Below the measurement container 2 is gate 12, an optical Detek- with which the intensity of the fluorescent light can be measured is disposed.

When the measurement container 2 and the detector 12 is disposed here of two optical lenses 13 existing lens system with which the fluorescent light of the different measuring points can be displayed 22 on the light-sensitive detector 12th Above the detector 12 is an aperture 14 in which at least one recess is provided, arranged movable. The aperture 14 can thereby moved so as to be preferably rotated such that the light of a measuring point 22 impinges on the photosensitive detector 12, so that a local allocation of the measured fluorescence intensity at that moment can be achieved for each measurement point the 22nd

In a non-illustrated in Figure 7 example, may conveniently be comparable one consisting of several individual detectors arranged flat member turns, in order to carry out the individual light-sensitive detectors, a spatially resolved measurement, without using a diaphragm.

Conveniently, filters can be 19 and 20 arranged in the light beam paths at once with the

Filter 20 to achieve a quasi-monochromatic fluorescence excitation and to exclude particular with the filter 19 scatter light from the light source 23 from the fluorescent light.

Instead of forming the base plate 3 as a prism, is also not shown herein, the possibility to or up to an optical transmission grating in the bottom plate 3 or introduce so that the light beam is 1 bent so that total reflection of the boundary surface (bottom plate / sample ) entry.

In the figure 8 is an example of a device according to the invention is shown, which can be provided to a user as a compact element.

The sample is in this case filled into a sample-receiving chamber 16 and can fill the actual measuring vessel 2 via a suitable connection. after completion of the measurement can reach a sample collection chamber 17 via another connection, for example the liquid sample liquid or the total sample liquid. The device shown here can be formed otherwise finished, so that undesired leakage of sample liquid can be avoided.

Sample-receiving chamber 16 and the sample collection chamber 17 here are arranged laterally next to the measuring vessel 2, so that the filling and possibly also the distance of the sample is made possible from or to the side of the measuring container. 2 At least the filling should be done in every case from the side, the regardless of whether a sample receiving space 16 existing. Thus, the sample can for example be introduced into einfachster form by means of a conventional syringe into the measuring container 2 by means of a pump.

In the example shown here, a rotating member 5 is indicated which of the at least in the proximity

can be brought measuring container 2 and set in rotation motion, so that the desired circular flowing motion of the sample in the measurement container 2 is thus reached, as has already been described in other examples.

The embodiment of a device according to the invention shown in Figure 9 is substantially identical to that of FIG. 8 It is only additionally ones shown, is that an absorbent material can be inserted into the sample collection chamber 17 18, wherein the suction force can be utilized in conjunction with capillary forces to fill the measuring container. 2 In the figure 10, the possible use of functional layers or membranes is shown. 7 and 8 It can pass into the measuring container 2 by the membrane or layer 7 and 8, the sample, for example, from the sample receiving space 16, wherein a specific sample preparation by appropriate material selection or a specific incubation can be achieved.

In the example shown in Figure 11 as a membrane or layer 8 is used which is used in the center of a circular recess, which may represent the actual measuring container. 2 The measuring container 2 is closed at the top and has a bottom base plate 3, on the surface of a partner of the receptor-ligand system may be immobilized on.

Also such a closed system can obtain the advertising even if the membrane or layer is enclosed liquid-tightly. 8

Claims

claims
1. A method for carrying out quantitative fluorescence-marked affinity tests by means of optical evanescent field excitation, emitting in the light of at least a nearly monochromatic light source of light, causing a bound to a chemical or biochemical partner of a general receptor-ligand system tracer fluorescence wavelength, optically transparent at a bottom plate, as Meßareal a measuring container, on the system is immobilized at least one of the partners of the receptor-ligand, ER drives total reflection and the intensity of the fluorescent light is measured, characterized in that a watch with a depending biochemical assay fluorophore-labeled chemical or biochemical substance filled-offset sample into the measuring container (2) and thereafter in the measuring vessel (2) added in circular flowing motion and the intensity of Fluorszenzlichtes time-resolved, depending on the affinity assay format, for destina- tion of the concentration of at least one chemical or biochemical substance is measured.
2. The method according to claim 1, characterized in that the measuring container (2) loading fills at least one opening with the sample and through which the base plate (3) opposite the closed cover (6, 11) of the measuring container (2) through the sample is placed in a circular flowing motion.
3. The method of claim 1 or 2, characterized in that the sample subsequent to the filling and after the expiration of chemical- or biochemical reactions in the sample volume is placed in circular flowing motion.
4. The method according to any one of claims 1 to 3, characterized in that the increase or the decrease in the intensity of the fluorescent light within at least a predetermined time interval, depending on the affinity assay format to determine the chemical - or biochemical substance (s) is measured.
5. The method according to any one of claims 1 to 3, characterized in that the difference between two predetermined time intervals in measured fluorescence intensity signals, depending on Affinitätstest- format, for determining the chemical - or biochemical substance (s) is used.
6. The method according to any one of claims 1 to 5, characterized in that the sample is treated at least during the measurement in a uniform, unaccelerated circular movement.
7. A method according to any one of claims 1 to 6, characterized in that the sample magnetic body see added and the flowing movement by means of a stationary rotating or time-varying magnetic or electromagnetic field in the immediate vicinity of the measuring container (2) is generated.
8. A method according to any one of claims 1 to 7, characterized in that the flowing movement of the sample with rotating elements (5) mechanically by the bottom plate (3) of the measuring container opposed closed cover (6, 11) is generated through.
9. A method according to any one of claims 1 to 8, characterized in that on the bottom plate (3) of the measuring container (2) is immobilized at least one partner of the receptor-ligand system in a radially symmetrical arrangement, and the fluorescent light is measured in accordance with a spatially resolved manner.
10. A method according to any one of claims 1 to 8, characterized in that the fluorescent - light below the range of the maximum flow rate of the airfoil, the offset in the circular movement of the sample is measured.
11. A method according to any one of claims 1 to 10, characterized in that fluorescent light selectively below the range of a central
Radius R ^ to, is measured to the center of the circular measuring container (2), in which the sample with the maximum flow rate is being moved.
12. The method according to any one of claims 1 to 11, characterized in that the filling of the measuring container (2) from the side force effect by capillary is sufficient ER- pressure or suction force.
13. The method according to claims 1 to 12, characterized in that prior to the filling of the measuring container (2) the sample by at least a functional layer or membrane (7, 8) is guided.
14. The method according to claim 13, characterized in that at least one functional layer or membrane (7, 8) is used for the separation or filtration.
15. The method according to claim 13, characterized in that at least one functional layer or membrane (7, 8) for release of chemical or biochemical
Substances used for carrying out the respective biochemical assays.
16. The method according to claim 13, characterized in that at least one functional layer or membrane (7, 8) is used for a specific retention of chemical or biochemical substances in the sample.
17. The method according to any one of claims 1 to 16, characterized in that the sample in the sample container (2) is filled from the side or is removed to the side.
18. Device for carrying out quantitative fluorescence-labeled affinity tests by means of optical evanescent field excitation, emitting in the light of at least a nearly monochromatic light source of light, causing a bound to a chemical or biochemical partner of a general receptor-ligand system tracer fluorescence wavelength to an optically transparent bottom - plate, as Meßareal a measuring container, on the system is immobilized at least one of the partners of the receptor-ligand directed and the intensity of the fluorescent light is measured by a detector, characterized in that a sample in the measuring container (2) versetzendes in circular flowing motion element (5) outside of the measuring container (2), opposite the base plate (3) is arranged, and the measuring container (2) on which the bottom plate (3) opposite side with a cover (6, 11) is completed.
19. The apparatus according to claim 18, characterized in that the rotating element (5) is mounted at least one permanent magnet or a stationary electromagnet (4).
20. The apparatus according to claim 18, characterized in that a rotating electromagnetic field generating coil arrangement in the immediate vicinity of the measuring container (2) is present.
21. The apparatus of claim 19 or 20, characterized in that the magnetic body (9) in the measuring vessel (2) are located.
22. The device according to claim 18, characterized in that within the measuring container (2), opposite the base plate (3) a mechanical stirrer (15) is present, which in non-continuous contact with the rotating
Element (5) outside the measuring cell (2).
23. The device according to claim 18, characterized in that recorded on the rotating ele- ment (5) outside of the measuring container (2) in the direction of the measuring container in the (2) sample, projections (10) are formed facing.
24. An apparatus according to claim 23, characterized in that the projections (10) to a, the measuring container (2) and the sample to the bottom plate (3) upwards covering elastic membrane (11) act and the membrane (11) in the sample is immersed.
25. The device according to any one of claims 18 to 24, characterized in that the functions as Meßareal bottom plate (3) is circular.
26. The device according to any one of claims 18 to 24, characterized in that at least one partner of a receptor-ligand system is immobilized radially symmetrically on the bottom plate (3).
27. The device 18 to 26, characterized in that between the detector (12) and bottom plate (3), an imaging lens system (13) is arranged according to one of the claims.
28. Device according to one of claims 18 to 27, characterized in that between the detector (12) and bottom plate (3) a movable shutter (14) is arranged.
29. Device according to one of claims 18 to 28, characterized in that the detector (12) is a linear or planar array of light sensitive detectors.
30. The device as claimed in any one of claims 18 to 29, that between the base plate
(3) and detector (12) at least one optical filter (19) is arranged.
31. The device according to any one of claims 18 to 30, characterized in that in the beam path (1) of the light between the light source (23) and bottom plate (3) at least one optical filter (20) is arranged.
32. Device according to one of claims 18 to 31, characterized in that the bottom plate (3) of the measuring container (2) made of a transparent ma- TERIAL is designed in the form of an optical prism.
33. Device according to one of claims 18 to 32, characterized in that an optical on or in the transparent bottom plate (3)
transmission grating is applied or incorporated.
34. The device as claimed in any one of claims 18 to 33, that prior to the measuring vessel
(2) at least one functional layer or membrane (7, 8) is arranged, from which the sample in the measuring container (2) passes.
35. Device according to one of claims 18 to 33, characterized in that the measuring container (2) is provided with a sample receiving space (16), passes from the sample in the measuring container (2), respectively.
36. The apparatus of claim 34 or 35, characterized in that the sample from the sample receiving space (16) by at least one functional layer or membrane (7, 8) in the measuring container (2) passes.
37. Device according to one of claims 18 to 36, characterized in that the measuring container (2) is connected to a sample collection chamber (17).
38. Apparatus according to claim 35, characterized in that in the sample collection chamber (17) an absorbent element (18) is included.
39. Device according to one of claims 35 to 38, characterized in that the sample receiving space (16) and / or sample collection chamber (17) arranged laterally next to the measuring container (2) and connected via an orifice to the measuring vessel (2) is / are.
40. Device according to one of claims 18 to 39, characterized in that the measuring container (2) has a fillable height in the range of 0.001 to 50 mm in its interior.
41. Device according to one of claims 18 to 40, characterized in that the measurement points (22) on the bottom plate (3) below the offset in the circular movement of sample in the maximum range of the flow profile are arranged.
PCT/DE1999/003917 1998-12-04 1999-12-03 Method and device for carrying out quantitative fluorescent marked affinity tests WO2000034785A1 (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
DE19856041.9 1998-12-04
DE1998156041 DE19856041A1 (en) 1998-12-04 1998-12-04 Method and apparatus for carrying out quantitative fluorescence-labeled affinity tests

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WO2000034785A1 true WO2000034785A1 (en) 2000-06-15

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WO (1) WO2000034785A1 (en)

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