DE102005016509A1 - Apparatus for assaying a liquid sample comprises reaction chambers containing immobilized reagents, each connected to an assay chamber so that liquid can be transferred by centrifugal force - Google Patents

Abstract

Apparatus for assaying a liquid sample comprises a sample chamber (4), optionally a diluent chamber (7), a liquid chamber (13) for sequentially receiving various liquids, and reaction chambers (11) to which the sample, optional diluent and various liquids can be passed. An independent claim is also included for a method for assaying a liquid sample for an analyte by performing parallel detection reactions in reaction chambers, where the reaction chambers are sequentially supplied with various liquids from a common liquid chamber.

Description

The The present invention relates to an apparatus and a method for the examination of a sample liquid, in particular by means of the ELISA method.

The present invention themselves with microfluidic systems or devices whose structures a size of about 1 to 1000 microns and / or their cavities each having a volume of about 1 to 1000 ul. The following remarks refer in particular to devices and methods which act capillary, pressure and / or centrifugal forces and in particular for the function are decisive.

Of the The term "ELISA" comes from the English and means "enzyme linked immunosorbent assay ". This term is used in the present invention in the sense of Understand process in which an enzyme to an analyte, in particular linked to a complex of an analyte and an antibody becomes. The enzyme becomes a substrate in a detection reaction in a detection substrate, in particular a fluorescent substance or the like., Modified or converted. By capturing the Detection substrate is a quantitative determination of the analyte in the sample liquid possible. For a high accuracy and a corresponding measuring range to enable becomes common a dilution series the sample liquid examined in this way.

So far The ELISA method is usually manually or automatically - for example by means of Pipette robots - on an open pipetting plate with, for example, 96 open receiving chambers carried out. A sample fluid to be examined is diluted several times in succession in the receiving chambers to different dilution ratios to reach. Subsequently, will the sample liquid with the different dilution ratios pipetted into prepared receiving chambers, in which an analyte in the sample liquid on immobilized antibodies can be tied. After a relatively long reaction time takes place a multiple rinse with a washing liquid. Then an enzyme bound to a detection antibody will be added. The detection antibody binds to the complex of analyte and immobilized antibody. Then are again different washing steps required. Then a substrate added, which is converted by the enzyme into a detection substrate or modified. This detection reaction is very time critical. The stopping of the detection reaction takes place, for example, by adding of acid. The problem is that this can not take place simultaneously in all the reception rooms in which the detection reaction takes place, and that at larger volumes different delays due to diffusion and / or mixing processes may occur. After all For example, the detection substrate is optically, in particular by Fluorescence measurement or the like, determined. From the determined values let yourself determine the concentration of the analyte in the sample fluid. The explained method is very expensive and error prone. In particular, inaccuracies due to the multiplicity add up the individual steps. Furthermore, there is the preparation of the reception rooms for the immobilization of the antibody correspondingly expensive and also with the use of larger quantities of liquid connected. About that In addition, the reactions are due to the large quantities of liquid and accordingly huge Diffusion often very slowly, so that the ELISA method in the usual Form is very time consuming.

Of the Article "Design of a Compact Disk-like Microfluidic Platform for Enzyme-Linked Immunosorbent Assay "by Siyi Lai et al, Analytical Chemistry, Vol. 76, No. 7, April 1, 2004, Pages 1832 to 1837, describes a microfluidic system in Form of a so-called compact disk (CD) for individual steps of the ELISA method. It will be a sample liquid, a washing liquid, a liquid with a detection antibody and a substrate liquid filled into corresponding receiving chambers, which successively through correspondingly different rotation of the CD in a single assigned Reaction chamber are passed to the appropriate reaction. So can individual steps are performed in the microfluidic system. However, the pipetting effort is not significantly reduced, opposite the conventional one ELISA method only avoided the multiple washing steps could become.

As a general rule are already a variety of microfluidic systems in shape known from CDs, where a control of liquid flows through Rotation of the CD, so by centrifugal forces occurs.

WO 03/018198 A1, WO 03/072257 A1 and WO 2004/061414 A2 disclose microfluidic devices in which a liquid, in particular a sample liquid, can be conducted from a receiving chamber into connected chambers and divided into defined individual quantities and / or with one other liquid can be mixed and preferably react. Similar microfluidic systems are also available from US 6,706,519 B1 . US 6,719,682 B2 , US 2004/0203136 A1, WO 00/78455 A1 and WO 01/87485 A2.

Of the present invention is based on the object, a device and a method for assaying a sample liquid indicating a cost-effective, rapid and / or accurate quantitative examination, in particular by means of the ELISA method.

The The above object is achieved by a device according to claim 1 or a method according to claim 7 solved. Advantageous developments are the subject of the dependent claims.

One Aspect of the present invention is a third common Recording chamber for several Provide reaction chambers. In particular, in the receiving chamber several liquids one after the other - so sequential - for example supplied by pipetting become. This will be a common filling hole for the different liquids created.

Preferably the third receiving chamber is in each case - in particular automatically by capillary forces and / or by centrifugal forces - emptied, before the next liquid filled becomes. So can an unwanted mixing of different liquids be avoided in the common receiving chamber. Especially it will be possible to do several or all reaction chambers with minimal effort - in particular with very few pipetting procedures - to prepare properly that is, for example, a reagent such as an antibody or the like in the reaction chambers immobilize. Alternatively or additionally, this allows the reaction chambers associated, common receiving chamber performing the Detection reaction, for example by supplying appropriate liquids with the enzyme, the substrate or the like, with minimal pipetting effort.

Further Advantages, features, characteristics and aspects of the present invention arise from the claims and the following description of preferred embodiments with reference to the drawing. It shows:

1 a not to scale view of a portion of a proposed device according to a first embodiment; and

2 a schematic view of a portion of a proposed device according to a second embodiment.

In the figures are for same or similar Parts the same reference numerals used, with corresponding or Comparable properties and advantages are achieved, even if a repeated description is omitted.

1 shows in a not to scale, very schematic view of a part of a proposed device 1 according to a first embodiment. In the proposed device 1 in particular, it is a microfluidic system, which preferably has the shape of a round disc, for example a compact disc (CD) or the like, and accordingly has an in 1 indicated axis of rotation 2 is rotatable to generate centrifugal forces. However, other configurations and configurations are possible.

The proposed device 1 serves to examine a sample liquid 3 , in particular by means of the ELISA method. The following description is therefore directed essentially to the application or implementation of the ELISA method, wherein, if necessary, additional or alternative measures or method steps can be performed. However, the proposed device 1 or the procedure according to the proposal can in principle also be used for other examinations or procedures.

1 shows the sample liquid 3 immediately after filling in a first, common receiving chamber 4 , Several first dosing chambers 5 are to the first receiving chamber 4 through appropriate channels or the like. Connected and preferably arranged in a row in the circumferential direction.

The sample liquid 3 flows from the first receiving chamber 4 into the connected, first dosing chambers 5 where air and / or excess sample liquid 3 in an optional first collection chamber 6 can continue to flow. 1 shows the device 1 in a state immediately after the filling of the sample liquid 3 in the first receiving chamber 4 So before the sample liquid 3 into the first metering chambers 5 flows.

The device 1 has a second common receiving chamber 7 for receiving a dilution liquid 8th on. To the second receiving chamber 7 are several second metering chambers 9 connected, which in the illustrated embodiment also in a row and at least substantially parallel to the first metering chambers 5 are arranged. The dilution liquid 8th flows into the second metering chambers 9 , Excess dilution liquid 8th may, if necessary, in an optionally provided second collection chamber 10 stream. The 1 shows the device 1 in the state immediately after filling the dilution liquid 8th in the second receiving chamber 7 So before the dilution liquid 8th the second metering chambers 9 and the associated channels and possibly the collection chamber 10 crowded.

The dosing chambers 5 and 9 are preferably formed, for example, by guide elements, not shown for the liquids 3 respectively. 8th in that the metering chambers 5 . 9 and possibly the channels are completely filled without gas or air inclusion. Displaced air can via the preferably open collector chambers 6 . 10 and / or not shown vents, in particular the channels and / or metering chambers 5 . 9 are assigned, escape.

The first and second metering chambers 5 and 9 are reaction chambers 11 assigned in the illustrated embodiment, preferably in a row parallel and / or with respect to the axis of rotation 2 radially outside the first and second metering chambers 5 . 9 are arranged.

The first and second metering chambers 5 . 9 are preferably in pairs one another and one reaction chamber each 11 associated with each pair with the associated reaction chamber 11 by corresponding, in particular radially extending, preferably channel-like connections 12 is fluidically connected.

In the illustrated example, the first metering chambers fill 5 and 9 with the sample liquid 3 or the dilution liquid 8th preferably automatically due to pressure and / or capillary forces. However, other forces, possibly even centrifugal forces, depending on the arrangement and design, alternatively or additionally be used.

Subsequently, by appropriate centrifugal forces (caused by corresponding rotation of the device 1 around the axis of rotation 2 ) in the first metering chambers 5 existing volumes of sample liquid 3 and in the second metering chambers 9 existing volumes of dilution liquid 8th in the respective associated reaction chamber 11 be transferred, with the sample liquid 3 and dilution liquid 8th mix each. However, for the transfer of said volumes into the reaction chambers 11 Additionally or alternatively, other forces, such as pressure forces, capillary forces or the like. Act.

The first dosing chambers 5 and / or second metering chambers 9 vary in their volumes. In fact, the volumes are chosen such that in the reaction chambers 11 different dilution ratios of the sample liquid 3 be achieved.

In particular, both the volumes of the first metering chambers vary 5 as well as the volumes of the second metering chambers 9 , Preferably, a first metering chamber 5 a second metering chamber 9 associated with large volume and vice versa. In the illustrated example, this is achieved in that the volume of the first metering chambers 5 increase or decrease in a circumferential direction and the volumes of the second metering chambers 5 Inversely decrease or increase in this circumferential direction. This allows a dilution series with a large dilution range - ie in particular from a low dilution ratio to a high dilution ratio, for example from 1: 1 to 1: 1000 - and / or a very space-saving, compact arrangement of the metering chambers 5 . 9 with correspondingly small space or space requirement.

Particularly preferred are the sums of the volumes of the pairwise associated first and second metering chambers 5 . 9 at least substantially the same. In this way, in addition to a particularly compact design can be achieved that the individual volumes of differently diluted sample liquid 3 are the same size and the reaction chambers 11 and any other subordinate chambers or the like. Can be uniform in size.

In the previous and in the following description is always on the respective volume of the metering chambers 5 respectively. 9 switched off. To obtain accurate, defined dilution ratios, well-defined volumes are required. Thus at the transfer of the sample liquid 3 and the dilution liquid 8th from the first and second metering chambers 5 and 9 in the associated reaction chambers 11 only defined volumes of liquids 3 . 8th are present and transferred and mixed are not shown valve devices, obstacles or liquid stops, for example, to the compounds 12 towards the channels associated ventilation openings and / or the like. Provided. With regard to the required and / or possible design solutions to ensure defined volumes and / or to provide suitable structures and arrangements for splitting and / or mixing of liquid quantities, reference is made to the aforementioned prior art, which is hereby incorporated in addition as a disclosure.

The above-described "parallel dilution" allows the generation of a dilution series in a single step, so that at most only slight dilution errors occur. In particular, so in the usual chen chen, sequen dilution problems occurring in the summation of individual errors can be avoided.

In every reaction chamber 11 can then run or carried out a desired reaction and in particular several desired reactions, which will be discussed later in more detail. To carry out the ELISA method, the reaction chambers 11 preferably before feeding the diluted sample liquid 3 initially prepared. This preparation takes place in particular before filling the sample liquid 3 in the first receiving chamber 4 and the dilution liquid 8th in the second receiving chamber 7 and will be explained in more detail below.

The device 1 preferably has one, in particular only a single, common receiving chamber 13 for receiving a liquid 14 , in particular sequential uptake of various liquids 14 such as a reaction liquid, a washing liquid, a fixing liquid, a substrate liquid or the like. The reaction chambers 11 are to the third receiving chamber 13 connected, so that in particular by pressure, capillary and / or centrifugal forces into the receiving chamber 13 filled liquid 14 via appropriate channels or the like. In the reaction chambers 11 can flow. Excessive and / or displaced liquid 14 is preferably in an optional collection chamber 15 collected.

In particular, the device 1 formed such that the third receiving chamber 13 first completely emptied or emptied before another liquid 14 the third receiving chamber 13 - For example, by pipetting - is supplied. Emptying the third receiving chamber 13 can be achieved, for example, that after filling the third receiving chamber 13 with a liquid 14 these by capillary forces automatically into the reaction chambers 11 and possibly the collection chamber 15 flows through until the third receiving chamber 13 is completely emptied. Additionally or alternatively this may be due to centrifugal forces, in particular corresponding rotations of the device 1 , or other forces can be achieved.

In addition, the reaction chambers can also 11 If necessary, first be emptied again before a new liquid 14 in the third receiving chamber 13 is filled and this new liquid 14 into the reaction chambers 11 flows. The previous emptying of the reaction chamber 11 is then carried out preferably by centrifugal forces, not shown valve means or the like. To a controlled emptying of the reaction chambers 11 to enable.

To prepare the reaction chamber 11 For the ELISA method, in particular, first of all a liquid 14 with a reagent, preferably an antibody, in the third receiving chamber 13 filled and into the reaction chambers 11 passed to the reagent in the reaction chambers 11 to immobilize, especially the reaction chambers 11 coat with the antibody.

After a certain incubation or reaction time, the reaction chambers become 11 with a washing liquid, the next liquid 14 in the third receiving chamber 13 is filled, rinsed to remove unbound reagent.

With another liquid 14 If necessary, blocking or immobilization of the immobilized reagent or of the immobilized antibodies in the reaction chambers follows 11 ,

After a possibly repeated rinsing with a washing liquid and possibly emptying are then the reaction chambers 11 prepared to the diluted sample liquid 3 - So the sample liquid 3 and the dilution liquid 8th from the associated first and second metering chambers 5 and 9 - to record.

After transferring the sample liquid 3 together with the dilution fluid 8th into the reaction chambers 11 may be the actual detection reaction or a first reaction to examine the sample liquid 3 respectively. One in the sample fluid 3 contained analyte can bind to the immobilized reagent, in particular the immobilized antibody. After a preferably determined reaction time unbound analyte from the reaction chambers 11 rinsed or washed, in particular by a single filling of a washing liquid 14 in the third receiving chamber 13 to the existing fluids 3 . 8th from the reaction chambers 11 to displace, and / or by centrifugal forces or other forces.

Subsequently, another liquid 14 containing, in particular, an enzyme bound to a detection antibody, the reaction chambers 11 fed by this liquid 14 again the third receiving chamber 13 is supplied. The detection antibody is designed to bind, together with the enzyme, to the complexes consisting of the immobilized antibodies and the analyte in the reaction chambers 11 are formed.

Unbound enzymes are then in a washing step by preferably supplying a further washing liquid once 14 from the reaction chambers 11 rinsed.

Finally, a substrate solution becomes a further liquid 14 preferably again via the third receiving chamber 13 the reaction chambers 11 fed. The substrate is released from the enzymes in the reaction chambers 11 is converted or modified in an enzymatic detection reaction, so that a later detectable detection substrate, in particular a fluorescent or other dye or the like., Is formed. On stopping the detection reactions in the reaction chambers 11 and the further investigation will be discussed later.

The feeding of different liquids 14 preferably exclusively via the common third receiving chamber 13 by sequential supply of the liquids 14 takes place, allows a very quick and easy preparation of the reaction chambers 11 and / or guiding the reactions in the reaction chambers 11 , wherein the Pipettieraufwand, the required washing steps and / or the required quantities of liquid compared to the prior art - in particular over the conventional ELISA method in an open pipetting plate - are substantially reduced.

So far, those were in the reaction chambers 11 expiring, already mentioned, in particular enzymatic detection reactions by the addition of acid or the like stopped. This is basically also in the proposed device 1 possible.

Preferably, however, the detection reactions are stopped by means of additionally provided examination chambers 16 by putting in the reaction chambers 11 liquid containing the detection substrate for stopping the detection reactions each in an associated examination chamber 16 is transferred. This transfer preferably takes place for several or all reaction chambers 11 at the same time, so that the detection reactions are stopped at the same time. In particular, said transfer takes place by centrifugal forces by the device 1 is rotated accordingly. However, the transfer is additionally or alternatively by other forces, such as pressure or capillary forces, by means of appropriate valves or the like. Possible.

The said transfer of the liquids from the reaction chambers 11 in which the enzymes and / or other reagents required for the detection reactions are immobilized, into the examination chambers 16 allows a very simple and highly simultaneous stopping of the detection reactions, so that over the prior art, a much more defined process flow and thus a much more accurate determination of the analyte are possible.

After the transfer of the liquids with the detection substrate into the examination chambers 16 may be a sequential examination or detection of the detection substrate in the examination chambers 16 - In particular optically, for example by fluorescence measurement - done. From the values obtained and taking into account the different dilution ratios can then be a very accurate, in particular quantitative determination of the analyte in the sample liquid 3 respectively.

Additionally or alternatively, the reaction chambers 11 also an optional, in 1 Dashed line indicated collecting channel 17 be assigned, for example, the examination chambers 16 and correspond de, preferably radial connections 12 to the reaction chambers 11 connected to emptying the reaction chambers 11 Liquids) from the reaction chambers 11 especially when the reaction chambers 11 by centrifugal forces by corresponding rotation of the device 1 be emptied. These fluids can then pass through the examination chambers 16 through or through, not shown, direct connections or the like. In the collection channel 17 be discharged. Such an emptying of the reaction chambers 11 For example, to remove liquids 3 . 8th and or 14 before feeding a new liquid 14 into the reaction chambers 11 respectively.

With regard to the parallel dilution, it should be noted that preferably in a single dilution step - that is, in a parallel dilution - 3 to 20 , especially about 10 Dilutions or different dilution ratios are generated. Of course, several parallel dilutions can be done simultaneously on the device 1 respectively. Accordingly, the device 1 If necessary, several arrangements, as in 1 shown have.

Hereinafter, a second embodiment of the proposed device 1 and the proposed method based on 2 explained in more detail, the following statements are limited only to significant differences from the first embodiment. Other advantages, aspects and properties thus result in a corresponding manner as in the first embodiment.

In the presentation according to 2 was omitted on the preferably provided curvature in the preferably provided ring structure for placement on a round disc, such as a CD or the like., For a better clarity to enable. Next is the illustration according to 2 also not to scale. In particular, the illustrated lengths, widths, proportions and the like do not correspond to the absolutely necessary or preferred conditions. This is also true in the illustration 1 the case.

In 2 are also no liquids for reasons of simplicity 3 . 8th . 14 shown. However, the relevant statements in the first embodiment and also with regard to the other method sequence apply correspondingly to those in 2 illustrated, second embodiment.

Next is in 2 for simplicity, the optional collection channel 17 omitted.

In the second embodiment, in contrast to the first embodiment after the parallel dilution, a further dilution, ie an under-dilution. This further dilution is in the case of 2 again shown as a parallel dilution. In the illustrated example, only a further dilution of only one already diluted sample liquid from only one reaction chamber takes place 11 , However, if necessary, a lower dilution or further dilution for several or all reaction chambers 11 be provided.

The further parallel dilution takes place essentially corresponding to the parallel dilution already explained above by means of the first and second metering chambers 5 and 9 and the downstream reaction chambers 11 , For additional parallel dilution, therefore, additional first metering chambers are required 5 ' and additional second metering chambers 9 ' as well as additional reaction chambers 11 ' intended. The additional dosing chambers 5 ' and 9 ' preferably have corresponding volume ratios - in particular correspondingly reduced absolute volume - as the first and second metering chambers 5 and 9 on.

The feeding of already diluted sample liquid into the additional first metering chambers 5 ' occurs from the upstream reaction chamber 11 , which in the case of further dilution actually only represents a mixing chamber. The additional second metering chambers 9 ' will turn dilution liquid 8th , in particular the excess dilution liquid 8th supplied for the first dilution, for example via the collection chamber 10 ,

The transfer of the individual liquid volumes in the associated additional reaction chambers 11 ' again, preferably by centrifugal forces. However, as an alternative or in addition, other forces, in particular pressure and / or capillary forces may act, or valves or the like may be used.

However, for the further dilution, another separate or additional dilution liquid can also be separated via an additional receiving chamber (not shown) to the additional second metering chambers 9 ' be supplied.

If only a partial further dilution takes place, as in 2 but are preferably not necessarily those reaction chambers 11 whose contents are not further diluted, each additional reaction chambers 11 ' assigned, in particular to an appropriate extent as the additional dilution serving additional reaction chambers 11 ' are arranged to ensure a simultaneous examination, in particular binding of the analyte to the immobilized reagent for all dilution stages.

Optionally, an additional first collection chamber 6 ' be provided for receiving excess sample liquid 3 to the additional first metering chambers 5 ' connected. Optionally, an additional second collection chamber 10 ' be precluded for receiving excess dilution liquid 8th to the additional second metering chambers 9 ' is arranged.

The diameter of the device 1 or the CD, is preferably about 50 to 250 mm, in particular about 125 mm. The thickness is preferably 1 to 6 mm, in particular about 3 mm. The device 1 is preferably made of a suitable plastic.

The Depth or width of the microstructures, ie in particular the described Chambers, channels, Compounds and the like. Is in the illustrated embodiment preferably 20 to 1000 microns, in particular about 200 microns.

All microstructures are preferably covered by a suitable, not shown or transparent cover. Only the receiving chambers 3 . 7 and 13 , if necessary, the collection chambers 6 . 10 . 15 if necessary, the collection channel 17 and / or other not shown vents or the like. Are open to the outside. Thus, the evaporation losses can be minimized and accordingly work with high accuracy with low volumes of liquid.

The volume of liquids to be used amount per liquid about 10 to 2,000 μl, preferably only about 50 to 200 μl.

The sum of the volumes of the paired first and second metering chambers 5 . 9 is preferably 1 to 100 ul, in particular about 10 ul. The same applies to the volumes of the reaction chambers 11 and the examination chambers 16 , In particular, said sum and the respective volumes of the reaction chambers 11 and the examination chambers 16 equal.

With the proposed device 1 and the proposed method, the ELISA method or another method can be very easily and very quickly and in particular with the use of very small amounts of liquid and thus cost-effective. Furthermore, a minimization of the required pipetting steps or other processes for the supply of liquids is made possible. In particular, a very accurate examination in the form of a precise quantitative determination of an analyte in the sample liquid is made possible.

Claims (16)

Contraption ( 1 ) for the examination of a sample liquid ( 3 ), in particular by means of the ELISA method, with a first receiving chamber ( 4 ) for receiving the sample liquid ( 3 ) and possibly with a second receiving chamber ( 7 ) for receiving a dilution liquid ( 8th ) for the sample liquid ( 3 ), and with a third receiving chamber ( 13 ) for the sequential uptake of different liquids ( 14 ), with several reaction chambers ( 11 ' ) to which the sample liquid ( 3 ), if necessary the dilution liquid ( 8th ) and the other or different liquids ( 14 ) can be fed successively. Device according to claim 1, characterized in that the device ( 1 ) is formed such that the third receiving chamber ( 13 ) each before receiving a new liquid ( 14 ) is emptied, in particular automatically by capillary forces and / or by centrifugal forces. Device according to claim 1 or 2, characterized in that the device ( 1 ) is formed such that a liquid with an antibody, a washing liquid, a fixing liquid, in particular for fixing the antibody in the reaction chamber ( 11 . 11 ' ) a liquid with an enzyme, which is in particular bound to a detection antibody and / or a liquid with a substrate, from the third receiving chamber ( 14 ) are sequentially receivable. Device according to one of the preceding claims, characterized in that several or all reaction chambers ( 11 ) for the sequential uptake of liquids) ( 14 ) by pressure, capillary and / or centrifugal forces to the third receiving chamber ( 13 ) are connected. Device according to one of the preceding claims, characterized in that the device ( 1 ) at least one of the reaction chambers ( 11 ' ) associated collecting chamber ( 15 ) and / or a collection channel ( 17 ) for receiving the liquids ( 3 . 8th . 14 ) having. Device according to one of the preceding claims, characterized in that the device ( 1 ) is designed as a microfluidic system, in particular in the form of a round disc, such as a CD. Method for examining a sample liquid ( 3 ), in particular by means of the ELISA method, wherein in reaction chambers ( 11 . 11 ' ) Detection reactions for detecting an analyte in the sample liquid ( 3 ), whereby the reaction chambers ( 11 . 11 ' ) successively different liquids ( 14 ) via a common third receiving chamber ( 13 ). Method according to claim 7, characterized in that the liquids ( 14 ) by pressure, centrifugal and / or capillary forces the reaction chambers ( 11 . 11 ' ). Process according to Claim 7 or 8, characterized in that the reaction chambers ( 11 . 11 ' ) successively a liquid with an antibody, a washing liquid, a fixing liquid, in particular for fixing an antibody in the reaction chambers ( 11 . 11 ' ), a liquid with an enzyme, which is in particular bound to a detection antibody, and / or a liquid with a substrate are supplied. Method according to one of claims 7 to 9, characterized in that the third receiving chamber ( 13 ) each before receiving a new liquid ( 14 ) is emptied, in particular automatically by capillary forces and / or by centrifugal forces. Method according to one of claims 7 to 10, characterized in that the reaction chambers ( 11 . 11 ' ) before feeding the sample liquid ( 3 ) are provided with an immobilized antibody, in particular coated, in particular wherein the antibody in the reaction chambers ( 11 . 11 ' ) is fixed by means of a fixing liquid. A method according to claim 11, characterized in that in each case after the coating and / or fixing, in particular only once, a washing liquid for rinsing by the Re action chambers ( 11 . 11 ' ). Method according to claim 11 or 12, characterized in that subsequently the sample liquid ( 3 ), preferably with a desired dilution ratio, in particular by pressure, capillary and / or centrifugal forces, in the respective reaction chamber ( 11 . 11 ' ). Process according to claim 13, characterized in that after a certain reaction time and / or binding of an analyte in the sample liquid ( 3 ) to the antibody the reaction chambers ( 11 . 11 ' ), then a detection reagent which binds to the compound of the antibody and analyte, in particular an enzyme, into the reaction chambers ( 11 . 11 ' ) and finally the unbound detection reagent or enzyme is washed out again. A method according to claim 14, characterized in that after washing the unbound detection reagent or enzyme, a substrate in the reaction chambers ( 11 . 11 ' ), which is modified or converted by the detection reagent or enzyme, in particular enzymatically, in the detection reaction into a detection substance. Method according to one of claims 7 to 15, characterized that this Method in a microfluidic system, in particular in form a round disc performed is, in particular wherein for the generation of centrifugal forces to Control of the process flow to determine the microfluidic system Times, for certain periods of time and / or with certain rotational speeds is rotated.